TERPENE PRODUCTION IN PLANTS
Composition and methods for the modification of the secondary metabolic functions of glandular trichomes in plants, such as tobacco or Cannabis, that control the formation of terpenes that impart specific flavor and aroma characteristics to the plant leaves are provided. Enhanced terpene production and composition are achieved through targeted modification in the biochemical synthesis pathways for menthol and cis-abienol. This application provides novel nucleotide sequences encoding the enzymology for production of these terpenes in tobacco and Cannabis application to their use plants.
This application claims the benefit of U.S. Provisional Patent Application No. 63/145,259, filed Feb. 3, 2021; U.S. Provisional Patent Application No. 63/145,262, filed Feb. 3, 2021; and U.S. Provisional Patent Application No. 63/145,263, filed Feb. 3, 2021, all of which are incorporated by reference herein in their entireties. This application also contains a Sequence Listing, which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Jan. 25, 2022, is named P34829US01_SL.txt and is 743,305 bytes in size as measured in Microsoft Windows®.
FIELDThe present disclosure relates to terpenoid biosynthesis genes and genetic manipulation thereof in plants, including tobacco and Cannabis.
SequencesTable 1 provides nucleic acid sequences and amino acid sequences used in this disclosure.
Plants are capable of producing a wide variety of secondary metabolites, and many secondary metabolites are produced in above-ground structures called glandular trichomes. Terpenes, produced by a variety of plants and by some insects, represent a large and diverse class of organic compounds consisting of isoprene, a five carbon building block. Terpenoids are modified terpenes that contain additional functional groups.
Terpenoids comprise the largest and most diverse family of secondary metabolites present in plants. Terpenoids, which can be cyclic or acyclic, vary in size from five-carbon hemiterpenes to long complex molecules containing thousands of isoprene units. The olefinic backbone of terpenoids is made of multiples of the five-carbon (C) isoprene unit, with the major groups being monoterpenes (10C), sesquiterpenes (15C), and diterpenes (20C). These terpenoids are produced through the condensation of 5-C isoprene units (dimethylallyl diphosphate [DMAPP] and isopentenyl diphosphate [IPP]) most often by the sequential head-to-tail addition of DMAPP to IPP.
Terpenoid levels in plants such as tobacco and Cannabis can be enhanced and modified by targeted manipulation of gene expression of genes in terpene biosynthetic pathways in order to improve flavor and aroma characteristics of downstream plant-based products.
SUMMARYIn one aspect, this disclosure provides a modified tobacco plant, tobacco seed, or part thereof, or a modified Cannabis plant, Cannabis seed, or part thereof, comprising a recombinant nucleic acid molecule comprising a nucleic acid sequence encoding a polypeptide involved in the biosynthesis of at least one terpene operably linked to a heterologous promoter.
In one aspect, this disclosure provides a modified tobacco plant, tobacco seed, or part thereof, or a modified Cannabis plant, Cannabis seed, or part thereof, comprising at least one non-natural mutation in an endogenous gene encoding a polypeptide involved in the biosynthesis of at least one terpene, where the modified tobacco plant or the modified Cannabis plant exhibits increased mRNA expression of the endogenous gene or increased accumulation of the polypeptide as compared to a control tobacco plant or a control Cannabis plant lacking the at least one non-natural mutation when grown under comparable conditions.
In one aspect, this disclosure provides a modified tobacco plant, tobacco seed, or part thereof, or a modified Cannabis plant, Cannabis seed, or part thereof, comprising a recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid encoding at least one small RNA molecule capable of binding to and reducing the expression of a nucleic acid sequence encoding a polypeptide involved in the biosynthesis of at least one terpene.
In one aspect, this disclosure provides a recombinant nucleic acid molecule comprising a nucleic acid sequence encoding a polypeptide involved in the biosynthesis of at least one terpene operably linked to a heterologous promoter.
In one aspect, this disclosure provides a method of producing a modified tobacco plant or a modified Cannabis plant comprising: (a) introducing a recombinant nucleic acid molecule to at least one tobacco cell or Cannabis cell, where the recombinant nucleic acid molecule comprises a nucleic acid sequence encoding a polypeptide involved in terpene biosynthesis operably linked to a heterologous promoter; (b) selecting at least one tobacco cell or Cannabis cell comprising the recombinant nucleic acid molecule; and (c) regenerating a modified tobacco or Cannabis plant from the at least one tobacco or Cannabis cell selected in step (b) where the modified tobacco or Cannabis plant comprises an increased amount of at least one terpene in at least one tissue as compared to a control tobacco plant lacking the recombinant nucleic acid molecule when grown under comparable conditions.
In one aspect, this disclosure provides a method of producing a modified tobacco plant or a modified Cannabis plant comprising: (a) inducing at least one non-natural mutation in at least one tobacco or Cannabis cell in an endogenous gene encoding a polypeptide involved in terpene biosynthesis; (b) selecting at least one tobacco or Cannabis cell comprising the at least one non-natural mutation from step (a); and (c) regenerating a modified tobacco or Cannabis plant from the at least one tobacco or Cannabis cell selected in step (b), where the modified tobacco or Cannabis plant comprises an increased amount of at least one terpene in at least one tissue as compared to a control tobacco or Cannabis plant lacking the at least one non-natural mutation when grown under comparable conditions.
In one aspect, this disclosure provides a method of producing a modified tobacco plant or a modified Cannabis plant comprising: (a) introducing a recombinant DNA construct to at least one tobacco or Cannabis cell, where said recombinant DNA construct comprises a heterologous promoter operably linked to a nucleic acid encoding at least one small RNA molecule capable of binding to and reducing the expression of a nucleic acid sequence encoding a polypeptide involved in the biosynthesis of at least one terpene; (b) selecting at least one tobacco or Cannabis cell comprising the recombinant DNA construct; and (c) regenerating at least one modified tobacco or Cannabis plant from the at least one tobacco or Cannabis cell selected in step (b), where the modified tobacco or Cannabis plant comprises a reduced amount of at least one terpene in at least one tissue as compared to a control tobacco or Cannabis plant lacking the recombinant DNA construct when grown under comparable conditions.
In one aspect, this disclosure provides a method comprising preparing a tobacco product or a Cannabis product using cured tobacco or Cannabis material from a modified tobacco or Cannabis plant, where the modified tobacco or Cannabis plant comprises a recombinant nucleic acid molecule comprising a nucleic acid sequence encoding a polypeptide involved in the biosynthesis of at least one terpene operably linked to a heterologous promoter.
In one aspect, this disclosure provides a method comprising preparing a tobacco product or a Cannabis product using cured tobacco or Cannabis material from a modified tobacco or Cannabis plant, where the modified tobacco or Cannabis plant comprises at least one non-natural mutation in an endogenous gene encoding a polypeptide involved in the biosynthesis of at least one terpene, where the modified tobacco or Cannabis plant exhibits increased mRNA expression of the endogenous gene or increased accumulation of the polypeptide as compared to a control tobacco or Cannabis plant lacking the at least one non-natural mutation when grown under comparable conditions.
In one aspect, this disclosure provides a method comprising preparing a tobacco or Cannabis product using cured tobacco or Cannabis material from a modified tobacco or Cannabis plant, where the modified tobacco or Cannabis plant comprises a recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid molecule encoding at least one small RNA molecule capable of binding to and reducing the expression of a nucleic acid sequence encoding a polypeptide involved in the biosynthesis of at least one terpene.
In one aspect, this disclosure provides a method comprising transforming a tobacco or Cannabis cell with a recombinant nucleic acid molecule, where the recombinant nucleic acid molecule comprises a nucleic acid sequence encoding a polypeptide involved in the biosynthesis of at least one terpene operably linked to a heterologous promoter.
In one aspect, this disclosure provides a method comprising transforming a tobacco or Cannabis cell with a recombinant nucleic acid molecule, where the recombinant nucleic acid molecule comprises a heterologous promoter operably linked to a nucleic acid encoding at least one small RNA molecule capable of binding to and reducing the expression of a nucleic acid sequence encoding a polypeptide involved in the biosynthesis of at least one terpene.
In one aspect, this disclosure provides a method for producing a tobacco plant or a Cannabis plant, the method comprising: (a) crossing at least one tobacco or Cannabis plant of a first tobacco or Cannabis variety with at least one tobacco or Cannabis plant of a second tobacco or Cannabis variety to produce at least one progeny tobacco or Cannabis seed, where the at least one tobacco or Cannabis plant of the first tobacco or Cannabis variety comprises a recombinant nucleic acid molecule comprising a nucleic acid sequence encoding a polypeptide involved in the biosynthesis of at least one terpene operably linked to a heterologous promoter; and (b) selecting for at least one progeny tobacco or Cannabis seed, or a plant germinated therefrom, where the at least one progeny tobacco or Cannabis seed or plant germinated therefrom comprises the recombinant nucleic acid molecule.
In one aspect, this disclosure provides a method for producing a tobacco plant or a Cannabis plant, the method comprising: (a) crossing at least one tobacco or Cannabis plant of a first tobacco or Cannabis variety with at least one tobacco or Cannabis plant of a second tobacco or Cannabis variety to produce at least one progeny tobacco or Cannabis seed, where the at least one tobacco or Cannabis plant of the first tobacco or Cannabis variety comprises at least one non-natural mutation in an endogenous gene encoding a polypeptide involved in the biosynthesis of at least one terpene, where the at least one tobacco or Cannabis plant of the first tobacco or Cannabis variety exhibits increased mRNA expression of the endogenous gene or increased accumulation of the polypeptide as compared to a control tobacco or Cannabis plant of the first tobacco or Cannabis variety lacking the at least one non-natural mutation when grown under comparable conditions; and (b) selecting for at least one progeny tobacco or Cannabis seed, or a plant germinated therefrom, where the at least one progeny tobacco or Cannabis seed or plant germinated therefrom comprises the at least one non-natural mutation.
In one aspect, this disclosure provides for a method for producing a tobacco plant or a Cannabis plat, the method comprising: (a) crossing at least one tobacco or Cannabis plant of a first tobacco or Cannabis variety with at least one tobacco or Cannabis plant of a second tobacco or Cannabis variety to produce at least one progeny tobacco or Cannabis seed, where the at least one tobacco or Cannabis plant of the first tobacco or Cannabis variety comprises a recombinant nucleic acid molecule comprising a heterologous promoter operably linked to a nucleic acid molecule encoding at least one small RNA molecule capable of binding to and reducing the expression of a nucleic acid sequence encoding a polypeptide involved in the biosynthesis of at least one terpene; and (b) selecting for at least one progeny tobacco or Cannabis seed, or a plant germinated therefrom, where the at least one progeny tobacco or Cannabis seed or plant germinated therefrom comprises the recombinant nucleic acid molecule.
Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. One skilled in the art will recognize many methods can be used in the practice of the present disclosure. Indeed, the present disclosure is in no way limited to the methods and materials described. Where a term is provided in the singular, the inventors also contemplate aspects of the disclosure described by the plural of that term, and vice versa. Where there are discrepancies in terms and definitions used in references that are incorporated by reference, the terms used in this application shall have the definitions given herein. Other technical terms used have their ordinary meaning in the art in which they are used, as exemplified by various art-specific dictionaries, for example, “The American Heritage® Science Dictionary” (Editors of the American Heritage Dictionaries, 2011, Houghton Mifflin Harcourt, Boston and New York), the “McGraw-Hill Dictionary of Scientific and Technical Terms” (6th edition, 2002, McGraw-Hill, New York), or the “Oxford Dictionary of Biology” (6th edition, 2008, Oxford University Press, Oxford and New York).
Any references cited herein, including, e.g., all patents, published patent applications, and non-patent publications, are incorporated herein by reference in their entirety.
When a grouping of alternatives is presented, any and all combinations of the members that make up that grouping of alternatives is specifically envisioned. For example, if an item is selected from a group consisting of A, B, C, and D, the inventors specifically envision each alternative individually (e.g., A alone, B alone, etc.), as well as combinations such as A, B, and D; A and C; B and C; etc. The term “and/or” when used in a list of two or more items means any one of the listed items by itself or in combination with any one or more of the other listed items. For example, the expression “A and/or B” is intended to mean either or both of A and B—i.e., A alone, B alone, or A and B in combination. The expression “A, B and/or C” is intended to mean A alone, B alone, C alone, A and B in combination, A and C in combination, B and C in combination, or A, B, and C in combination.
When a range of numbers is provided herein, the range is understood to be inclusive of the edges of the range as well as any number between the defined edges of the range. For example, “between 1 and 10” includes any number between 1 and 10, as well as the number 1 and the number 10
The term “about” is used herein to mean approximately, roughly, around, or in the region of. When the term “about” is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth and is understood to mean plus or minus 10%. For example, “about 100” would include from 90 to 110.
As used herein, the singular form “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a compound” or “at least one compound” may include a plurality of compounds, including mixtures thereof.
As used herein, terms or phrases such as “about,” “at least,” “at least about,” “at most,” “less than,” “greater than,” “within,” or “alike,” when followed by a series of list of numbers of percentages, such terms or phrases are deemed to modify each and every number of percentage in the series or list, regardless whether the adverb, preposition, or other modifier phrase is reproduced prior to each and every member.
Any tobacco plant, or part thereof, provided herein is specifically envisioned for use with any method provided herein. Similarly, any modified tobacco plant, or part thereof, is specifically envisioned for use with any method provided herein. Any Cannabis plant, or part thereof, provided herein is specifically envisioned for use with any method provided herein. Similarly, any modified Cannabis plant, or part thereof, is specifically envisioned for use with any method provided herein.
Any nucleic acid sequence, amino acid sequence, or other composition provided herein is specifically envisioned for use with any method provided herein.
Terpenes represent a large and diverse class of organic compounds consisting of isoprene, a five carbon building block. Terpenes are produced by a variety of plants and by some insects. Terpenes often have a strong odor, and may protect the plants that produce them by deterring herbivores. Terpenoids are modified terpenes that contain additional functional groups.
The largest and most diverse family of secondary metabolite in plants is the terpenoids. Terpenoids are synthesized through the condensation of 5-C isoprene units (dimethylallyl diphosphate [DMAPP] and isopentenyl diphosphate [IPP]) most often by the sequential head-to-tail addition of DMAPP to IPP. The plastidic methylerythritol phosphate (MEP) pathway consists of seven steps that convert pyruvate and glyceraldehyde-3-phosphate into IPP and DMAPP. See
In addition to terpene synthases, trans-isoprenyl diphosphate synthases (IDSs) are among the core enzymes involved in the generation of diverse terpenoids. TPSs and IDSs share a conserved alpha-terpenoid synthase fold and a trinuclear metal cluster for catalysis.
A monoterpene is a cyclic molecule composed of two isoprene units. Monoterpenes are C10 colorless, lipophilic, and structurally diverse (cyclic and acyclic) volatile molecules that impart characteristic aromas and flavors on plants. These molecules also play an important role as chemoattractant between plants and their pollinator species as well as deterrents in plant-plant and plant-herbivore communication and are key components of plant defense. Monoterpene production and accumulation are generally restricted to glandular trichomes, with plants lacking such specialized structures typically accumulating only trace quantities of monoterpenes.
Terpene synthase (TPS) genes can be grouped into seven clades (TPS-a, TPS-b, TPS-c, TPS-d, TPS-e/f, TPS-g, and TPS-h) where TPS-d and TPS-h are specific to gymnosperms and the lycopod Selaginalla moellendorffii and TPS-a, TPS-b, and TPS-g are specific to angiosperm. TPS-a contains mostly sesquiterpene synthases and diterpene synthases. Neophytadiene is an example of an abundant sesquiterpenoid in tobacco. The TPS-b and TPS-g clades comprise mostly monoterpene synthases. The genome of cultivated tomato (S. lycopersicum), a relative of tobacco, contains 44 TPS genes of which 29 appear to be functional or potentially functional. The expression patterns of the tomato TPS genes in leaf and stem trichomes are quantitatively and qualitatively different, consistent with the distinct volatile profiles of these two organs. The tissue-specific differences in gene expression in Solanaceae species is in part responsible for the observed distinct intraspecific and interspecific chemical variability. Many terpene synthases form multiple products from the same substrate.
The aromatic (smell) and organoleptic (taste/flavor) properties of tobacco leaf tissues can be enhanced by targeted genetic manipulation of the biosynthetic pathways that control terpenoid formation. Similarly, Cannabis resin contains a variety of monoterpenes and sesquiterpenes. Two major biosynthetic pathways control terpenoid formation. One pathway controls the formation of menthol and related compounds (see
Targeted overexpression of genes in the menthol biosynthetic pathway can enhance the accumulation of menthol in tobacco plants. The C10 monoterpene (−)-menthol is a characteristic constituent of Mentha x piperita (peppermint), a plant of economic importance for the production of essential oil used various industrial manufacturing and medicinal purposes. In Mentha species, menthol biosynthesis and storage are restricted to the peltate glandular trichomes (oil glands) on the aerial surfaces of the plant.
The biosynthesis of (−)-menthol involves eight enzymatic steps. The formation and subsequent cyclization of the universal monoterpene precursor geranyl diphosphate to the parent olefin (−)-(4S)-limonene is the first committed enzymatic reaction in the menthol biosynthetic pathway.
Genes encoding enzymes of the menthol biosynthetic pathway include geranyl diphosphate synthase (GDP) (SEQ ID NOs: 7, 28, and 49), Limonene synthase (LS) (SEQ ID NOs: 8, 29, and 50), Limonene 3-hydroxylase (L30H) (SEQ ID NOs: 10, 31, and 52), Limonene Synthase chloroplastic isoform X1 (SEQ ID NOs: 165, 181, and 197), Isopiperitenol dehydrogenase (IPD) (SEQ ID NOs: 13, 34, 55, 169, 185, and 201), pulegone reductase (SEQ ID NOs: 14, 35, and 56), menthofuran synthase (MFS) (SEQ ID NOs: 16, 37, and 58) and Menthone reductase, which is alternatively called neomenthol dehydrogenase (NtNMD) (SEQ ID NOs: 15, 36, and 57). See
The second major terpenoid biosynthetic pathway is involved in the formation of labdanoids, such as cis-abienol (see
The biosynthesis of Cis-abienol proceeds in two steps (see
Additional biosynthetic pathways used for targeted aroma enhancement include the cembratrienediol biosynthesis pathway, see
In an aspect this disclosure provides a modified tobacco or Cannabis plant, tobacco or Cannabis seed, or part thereof comprising a recombinant nucleic acid molecule comprising a nucleic acid sequence encoding a polypeptide involved in the biosynthesis of at least one terpene operably linked to a heterologous promoter.
In a further aspect, this disclosure provides a modified tobacco or Cannabis plant, tobacco or Cannabis seed, or part thereof comprising at least one non-natural mutation in an endogenous gene encoding a polypeptide involved in the biosynthesis of at least one terpene, where the modified tobacco or Cannabis plant exhibits increased mRNA expression of the endogenous gene or increased accumulation of the polypeptide as compared to a control tobacco or Cannabis plant lacking the at least one non-natural mutation when grown under comparable conditions.
TerpenesTerpenes are a class of aromatic organic compound produced by plants and some insects. Terpenes are members of a group of volatile unsaturated hydrocarbons found in the essential oils of plants, based on a cyclic molecule having the formula C10H16; and related structures such as, but not limited to, compounds such as sesquiterpene with the formula C15H24, or a simple derivative of such a compound. Terpenes are hydrocarbon molecules that are often used by plants to either directly deter herbivory or to attract predators or parasites of plant herbivores. Non-limiting examples of terpenes include citral, menthol, camphor, salvinorin A, cannabinoids, and curcuminoids.
Several classes of terpenes have been identified in Cannabis, including, but not limited to, myrcene, pinene, limonene, caryophyllene, linalool, terpinolene, camphene, terpineol, phellandrene, carene, humulene, pulegone, and geraniol. Myrcene, specifically beta-myrcene, is a monoterpene and the most common terpene produced by Cannabis. Pinene is a bicyclic monoterpenoid. Two naturally occurring structural isomers of pinene are known: alpha-pinene and beta-pinene. Limonene is a monocyclic monoterpenoid and one of the two major compounds formed from pinene. Caryophyllene, specifically beta caryophyllene, is a sesquiterpene common to many plants besides Cannabis, including cloves, cinnamon leaves, and black pepper. Caryophyllene is the only terpene known to interact with the endocannabinoid system, as beta-caryophyllene is a functional cannabinoid-2 receptor (CB2) agonist. Linalool is a non-cyclic monoterpenoid, a critical precursor in the formation of Vitamin E, and has been isolated from many different plants. Delta-3-carene is a bicyclic monoterpene. Alpha-Terpineol, terpinen-4-ol, and 4-terpineol are three closely related monoterpenoids. Humulene is a sesquiterpene also known as alpha-humulene. Pulegone, a monocyclic monoterpenoid, is a minor component of Cannabis.
In an aspect, a terpene is a terpenoid. Terpenoids (also referred to as isoprenoids) are modified terpenes that contain additional functional groups, which often include oxygen. Terpenoids, which can be cyclic or acyclic, vary in size from five-carbon hemiterpenes to long complex molecules containing thousands of isoprene units. Terpenoids are produced through the condensation of five-carbon isoprene units (e.g., dimethylallyl diphosphate (DMAPP) and isopentenyl diphosphate (IPP)), most often by the sequential head-to-tail addition of DMAPP to IPP. The initial cyclization processes are catalyzed by different terpene synthases and enzyme variation leads to variation in monoterpene structure.
Terpenoids are classified according to the number of isoprene units that comprise the parent terpene. A hemiterpenoid comprises one isoprene unit. A monoterpenoid comprises two isoprene units. A sesquiterpenoid comprises three isoprene units. A diterpenoid comprises four isoprene units. A sesterterpenoid comprises five isoprene units. A triterpenoid comprises six isoprene units. A tetraterpenoid comprises eight isoprene units. A polyterpenoid comprises more than eight isoprene units.
In an aspect, a polypeptide is involved in the biosynthesis of at least one terpene. In an aspect, a polypeptide is involved in the biosynthesis of at least one terpenoid. In an aspect, a polypeptide is involved in the biosynthesis of at least one terpenoid selected from the group consisting of a hemiterpenoid, a monoterpenoid, a sesquiterpenoid, a diterpenoid, a sesterterpenoid, a triterpenoid, a tetraterpenoid, and a polyterpenoid. In an aspect, a polypeptide is involved in the biosynthesis of at least one terpene selected from the group consisting of a hemiterpene, a monoterpene, a sesquiterpene, a diterpene, a sesterterpene, a triterpene, a tetraterpene, and a polyterpene.
As used herein, the term “biosynthesis” refers to the production of a complex molecule (e.g., without being limiting, a terpene or terpenoid) within a plant or plant cell. To be “involved” with the biosynthesis of a compound, a polypeptide can directly interact with a substrate during the biosynthesis of the compound, or the polypeptide can affect the expression (positively or negatively) of a polypeptide that directly interacts with a substrate (e.g., a transcription factor that promotes the expression of an enzyme that converts a substrate to a new form or a repressor that inhibits expression of an enzyme that converts a substrate to a new form). Examples of biosynthetic pathways can be found in
In an aspect, a polypeptide is involved in the biosynthesis of a hemiterpene. In an aspect, a polypeptide is involved in the biosynthesis of a hemiterpenoid. In an aspect, a polypeptide is involved in the biosynthesis of a monoterpene. In an aspect, a polypeptide is involved in the biosynthesis of a monoterpenoid. In an aspect, a polypeptide is involved in the biosynthesis of a sesquiterpene. In an aspect, a polypeptide is involved in the biosynthesis of a sesquiterpenoid. In an aspect, a polypeptide is involved in the biosynthesis of a diterpene. In an aspect, a polypeptide is involved in the biosynthesis of a diterpenoid. In an aspect, a polypeptide is involved in the biosynthesis of a sesterterpene. In an aspect, a polypeptide is involved in the biosynthesis of a sesterterpenoid. In an aspect, a polypeptide is involved in the biosynthesis of a triterpene. In an aspect, a polypeptide is involved in the biosynthesis of a triterpenoid. In an aspect, a polypeptide is involved in the biosynthesis of a tetraterpene. In an aspect, a polypeptide is involved in the biosynthesis of a polyterpenoid. In an aspect, a polypeptide is involved in the biosynthesis of a monoterpene. In an aspect, a polypeptide is involved in the biosynthesis of a polyterpenoid.
Terpene synthase (TPS) genes can be grouped into seven clades: TPS-a, TPS-b, TPS-c, TPS-d, TPS-e/f, TPS-g, and TPS-h. TPS-a, TPS-b, and TPS-g are restricted to angiosperms, and TPS-d and TPS-h are specific to gymnosperms and the lycopod Selaginalla moellendorffii. The TPS-a clade comprises mostly sesquiterpene synthases and diterpene synthases, while the TPS-b and TPS-g clades comprise mostly monoterpene synthases.
Terpene Synthase-Like GenesIn an aspect, a polypeptide involved in the biosynthesis of at least one terpenoid is a TPS-a clade member. In an aspect, a polypeptide involved in the biosynthesis of at least one terpenoid is a TPS-b clade member. In an aspect, a polypeptide involved in the biosynthesis of at least one terpenoid is a TPS-c clade member. In an aspect, a polypeptide involved in the biosynthesis of at least one terpenoid is a TPS-e/f clade member. In an aspect, a polypeptide involved in the biosynthesis of at least one terpenoid is a TPS-g clade member. In an aspect, a polypeptide involved in the biosynthesis of at least one terpenoid is a member of a clade selected from the group consisting of TPS-a, TPS-b, TPS-c, TPS-e/f, and TPS-g.
In an aspect, a terpene is menthol. In an aspect, a terpene is menthol or a related compound. In an aspect, a terpene is a labdanoid. In an aspect, a terpene is cembratrienediol. In an aspect, a terpene is levopimaric acid. In an aspect, a terpene is L-leucine. In an aspect, a terpene is neophytadiene. In an aspect, a labdanoid is cis-abienol. In an aspect, a labdanoid is labdane-diol. In an aspect, a terpene is selected from the group consisting of menthol or a related compound, a labdanoid, cembratrienediol, levopimaric acid, and L-leucine. In an aspect, a terpene is selected from the group consisting of menthol or a related compound, a labdanoid, cembratrienediol, levopimaric acid, L-leucine, and neophytadiene. In an aspect, a terpene is selected from the group consisting of menthol, a labdanoid, cembratrienediol, levopimaric acid, and L-leucine. In an aspect, a terpene is selected from the group consisting of menthol, a labdanoid, cembratrienediol, levopimaric acid, L-leucine, and neophytadiene.
In an aspect, a terpene is myrcene. In an aspect, a terpene is pinene. In an aspect, a terpene is limonene. In an aspect, a terpene is caryophyllene. In an aspect, a terpene is linalool. In an aspect, a terpene is terpinolene. In an aspect, a terpene is camphene. In an aspect, a terpene is terpineol. In an aspect, a terpene is phellandrene. In an aspect, a terpene is carene. In an aspect, a terpene is humulene. In an aspect, a terpene is pulegone. In an aspect, a terpene is geraniol.
As used herein, “menthol” refers to the organic compound having a chemical formula of C10H20O and the International Union of Pure and Applied Chemistry (IUPAC) name 5-Methyl-2-(propan-2-yl)cyclohexan-1-ol. Menthol is also referred to as “(−)-Menthol.” Related compounds of menthol include, but are not limited to, (+)-Menthol, (+)-Isomenthol, (+)-Neomenthol, (+)-Neoisomenthol, (−)-Isomenthol, (−)-Neomethol, and (−)-Neoisomenthol. In an aspect, a related compound of menthol is selected from the group consisting of (+)-Menthol, (+)-Isomenthol, (+)-Neomenthol, (+)-Neoisomenthol, (−)-Isomenthol, (−)-Neomethol, and (−)-Neoisomenthol.
As used herein, “neophytadiene” refers to the organic compound having a chemical formula of C20H38 and the IUPAC name of 7,11,15-trimethyl-3-methylidenehexadec-1-ene.
As used herein, “cembratrienediol” refers to the organic compound having a chemical formula of C20H34O2 and the IUPAC name (1R,3R,4Z,8Z,12S,13Z)-1,5,9-trimethyl-12-propan-2-ylcyclotetradeca-4,8,13-triene-1,3-diol. Cembratrienediol is also referred to as “beta-Cembrenediol.”
As used herein, “levopimaric acid” refers to the organic compound having a chemical formula of C20H30O2 and the IUPAC name (1R,4aR,4bS,10aR)-1,4a-dimethyl-7-propan-2-yl-2,3,4,4b,5,9,10,10a-octahydrophenanthrene-1-carboxylic acid. Levopimaric acid is also referred to as “L-Pimaric acid.” In an aspect, levopimaric acid is involved in diterpene resin acid production.
As used herein, “L-leucine” refers to the amino acid having the chemical formula C6H12NO2 and the IUPAC name (2S)-2-amino-4-methylpentanoic acid.
As used herein, a “labdanoid” refers to a terpenoid derivative of the fundamental parent labdane, a diterpene. A labdane has the chemical formula C20H38 and the IUPAC name (1S,2S,4aS,8aR)-2,5,5,8a-tetramethyl-1-[(3R)-3-methylpentyl]-1,2,3,4,4a,6,7,8-octahydronaphthalene.
A non-limiting example of a labdanoid is cis-abienol. As used herein, “cis-abienol” refers to the organic compound having a chemical formula of C20H34O and the IUPAC name (1R,2R,4aS,8aS)-2,5,5,8a-tetramethyl-1-[(2Z)-3-methylpenta-2,4,-dienyl]-3,4,4a,6,7,8-hexahydro-1H-naphthalen-2-ol.
As used herein, “myrcene” refers to the compound having a chemical formula of C10H16 and the IUPAC name 7-Methyl-3-methylene-octa-1,6-diene. Myrcene, also known as beta-myrcene or alpha-myrcene, belongs to the class of organic compounds known as acyclic monoterpenoids. Thus, myrcene is considered to be an isoprenoid lipid molecule.
As used herein, “pinene” refers to the compound having a chemical formula of C10H16 and the IUPAC name (1S,5S)-2,6,6-Trimethylbicyclo[3.1.1]hept-2-ene ((−)-α-Pinene). (+)-a-Pinene, also known as 2-pinene or acintene a, belongs to the class of organic compounds known as bicyclic monoterpenoids. These are monoterpenoids containing exactly 2 rings, which are fused to each other. Alpha-pinene is a pinene that is bicyclo[3.1.1]hept-2-ene substituted by methyl groups at positions 2, 6 and 6 respectively. It has a role as a plant metabolite.
As used herein, “limonene” refers to the compound having a chemical formula of C10H16 and the IUPAC name 1-methyl-4-(1-methylethenyl)-cyclohexene. Limonene, (+/−)- is a racemic mixture of limonene, a natural cyclic monoterpene.
As used herein, “caryophyllene” or “beta caryophyllene” refers to the organic compound having a chemical formula of C15H24 and the IUPAC name (1R,4E,9S)-4,11,11-trimethyl-8-methylidenebicyclo[7.2.0]undec-4-ene. Beta-Caryophyllene is classified as a sesquiterpenoid. (−)-beta-caryophyllene is a beta-caryophyllene in which the stereocenter adjacent to the exocyclic double bond has S configuration, while the remaining stereocenter has R configuration. It is the most commonly occurring form of beta-caryophyllene, occurring in many essential oils.
As used herein, “linalool” refers to the compound having a chemical formula of C10H18O and the IUPAC name 3,7-dimethylocta-1,6-dien-3-ol. Beta-linalool, also known as 3,7-Dimethyl-1,6-octadien-3-ol or 2,6-dimethylocta-2,7-dien-6-ol, is classified as an acyclic monoterpenoid.
As used herein, “terpinolene” refers to the organic compound having a chemical formula of C10H16 and the IUPAC name 1-methyl-4-propan-2-ylidenecyclohexene. Terpinolene, also known as alpha-terpinolene or isoterpinene, is classified as a menthane monoterpenoid. These are monoterpenoids with a structure based on an o-, m-, or p-menthane backbone.
As used herein, “camphene” refers to the organic compound having a chemical formula of C10H16 and the IUPAC name 2,2-dimethyl-3-methylidenebicyclo[2.2.1]heptane. Camphene is classified as a bicyclic monoterpenoid, containing two rings which are fused to each other. Camphene is a monoterpene with a bicyclic skeleton that is bicyclo[2.2.1]heptane substituted by geminal methyl groups at position 2 and a methylidene group at position 3.
As used herein, “terpineol” refers to the monoterpene alcohol of which there are four isomers: alpha-terpineol, beta-terpineol, gamma-terpineol, and terpinen-4-ol. Beta-terpineol and gamma-terpineol differ only by the location of the double bond. Terpineol is usually a mixture of these isomers with alpha-terpineol as the major constituent. As used herein, “alpha terpineol” refers to the organic compound having a chemical formula of C10H18O and the IUPAC name 2-(4-methylcyclohex-3-en-1-yl)propan-2-ol.
As used herein, “phellandrene” refers to the cyclic monoterpenes “alpha-phellandrene” or “beta-phellandrene.” As used herein, “alpha-phellandrene” refers to the organic compound having a chemical formula of C10H16 and the IUPAC name 2-methyl-5-propan-2-ylcyclohexa-1,3-diene. Alpha-phellandrene is also known as alpha-phellandren or dihydro-p-cymene. As used herein, “beta-phellandrene” refers to the organic compound having a chemical formula of C10H16 and the IUPAC name 3-methylidene-6-propan-2-ylcyclohexene. Beta-phellandrene is also known as beta-phellandren or 2-p-menthadiene.
As used herein, “carene” or “alpha carene” refers to the organic compound having a chemical formula of C10H16 and the IUPAC name 3,7,7-trimethylbicyclo[4.1.0]hept-3-ene. Alpha-Carene, also known as 3-carene or delta-car-3-ene, is classified as a bicyclic monoterpenoid.
As used herein, “humulene” refers to the organic compound having a chemical formula of C15H24 and the IUPAC name (1E,4E,8E)-2,6,6,9-tetramethylcycloundeca-1,4,8-triene.
As used herein, “pulegone” refers to the organic compound having a chemical formula of C10H16O and the IUPAC name (5R)-5-methyl-2-propan-2-ylidenecyclohexan-1-one. (+)-Pulegone, also known as D-pulegone or cis-isopulegone, is classified as a menthane monoterpenoid.
As used herein, “geraniol” refers to the organic compound having a chemical formula of C10H18O and the IUPAC name (2E)-3,7-dimethylocta-2,6-dien-1-ol. Geraniol is an acyclic monoterpenoid, and is also referred to as 2E-Geraniol, (e)-geraniol, or geranyl alcohol.
In an aspect, a polypeptide is geranylgeranyl diphosphate synthase. In an aspect, a polypeptide is 8-hydroxy-copalyl diphosphate synthase. In an aspect, a polypeptide is cis-abienol synthase. In an aspect, a polypeptide is cembratrienol synthase 2a. In an aspect, a polypeptide is levopimaradiene synthase. In an aspect, a polypeptide is 2-isopropylmalate synthetase. In an aspect, a polypeptide is 2-oxoisovalerate dehydrogenase. In an aspect, a polypeptide is geranyl diphosphate synthase. In an aspect, a polypeptide is limonene synthase. In an aspect, a polypeptide is limonene 3-hydroxylase. In an aspect, a polypeptide is isopiperitenol dehydrogenase. In an aspect, a polypeptide is pulegone reductase. In an aspect, a polypeptide is menthofuran synthase. In an aspect, a polypeptide is neomenthol dehydrogenase. In an aspect, a polypeptide is geranylgeranyl pyrophosphate synthase. In an aspect, a polypeptide is kolavenyl diphosphate synthase. In an aspect, a polypeptide is solanesyl diphosphate synthase. In an aspect, a polypeptide is terpene synthase. In an aspect, a polypeptide is 2-alkenal reductase. In an aspect, a polypeptide is germacrene synthase. In an aspect, a polypeptide is cytochrome P450. In an aspect, a polypeptide is a carveol dehydrogenase. In an aspect, a polypeptide is a MYB61 transcription factor.
In an aspect, a polypeptide is selected from the group consisting of geranyl diphosphate synthase (GDP), limonene synthase (LS), limonene 3-hydroxylase (L30H), isopiperitenol dehydrogenase (IPD), pulegone reductase, menthofuran synthase, and GGPPS neomenthol dehydrogenase (NtNMD).
In an aspect, a polypeptide is selected from the group consisting of geranylgeranyl diphosphate synthase (GGPPS2), 8-hydroxy-copalyl diphosphate synthase, and cis-abienol synthase.
In an aspect, a polypeptide is selected from the group consisting of geranylgeranyl diphosphate synthase, 8-hydroxy-copalyl diphosphate synthase, cis-abienol synthase, cembratrienol synthase 2a, levopimaradiene synthetase, 2-isopropylmalate synthetase, 2-oxoisovalerate dehydrogenase, geranyl diphosphate synthase, limonene synthase, limonene 3-hydroxylase, isopiperitenol dehydrogenase, pulegone reductase, menthofuran synthase, neomenthol dehydrogenase, phylloplanin, and premnaspirodiene oxygenase.
In an aspect, a polypeptide is selected from the group consisting of geranylgeranyl pyrophosphate synthase, kolavenyl diphosphate synthase, solanesyl diphosphate synthase, terpene synthase, limonene synthase, isopiperitenol dehydrogenase, 2-alkenal reductase, germacrene synthase, 2-isopropylmalate synthase, and MYB61.
In an aspect, a polypeptide is selected from the group consisting of geranylgeranyl diphosphate synthase, 8-hydroxy-copalyl diphosphate synthase, cis-abienol synthase, cembratrienol synthase 2a, levopimaradiene synthetase, 2-isopropylmalate synthetase, 2-oxoisovalerate dehydrogenase, geranyl diphosphate synthase, limonene synthase, limonene 3-hydroxylase, isopiperitenol dehydrogenase, pulegone reductase, menthofuran synthase, neomenthol dehydrogenase, phylloplanin, premnaspirodiene oxygenase, geranylgeranyl pyrophosphate synthase, kolavenyl diphosphate synthase, solanesyl diphosphate synthase, terpene synthase, 2-alkenal reductase, germacrene synthase, and MYB61.
As a non-limiting example, SEQ ID NOs: 45 and 3 are representative examples of amino acid and nucleic acid sequences, respectively, for 8-hydroxy-copalyl diphosphate synthase. As a non-limiting example, SEQ ID NOs: 59 and 17 are representative examples of amino acid and nucleic acid sequences, respectively, for cembratrienol synthase 2a. As a non-limiting example, SEQ ID NOs: 60 and 18 are representative examples of amino acid and nucleic acid sequences, respectively, for levopimaradiene synthase. As a non-limiting example, SEQ ID NOs: 61 and 207 are representative examples of amino acid sequences for 2-isopropylmalate synthase. As a non-limiting example, SEQ ID NOs: 61 and 174 are representative examples of nucleic acid sequences for 2-isopropylmalate synthase.
As a non-limiting example, SEQ ID NOs: 160 and 158 are representative examples of amino acid and nucleic acid sequences, respectively, for premnaspirodiene oxygenase (Gene id g89817). As a non-limiting example, SEQ ID NOs: 157 and 155 are representative examples of amino acid and nucleic acid sequences, respectively, for phylloplanin (Gene id g62191). As a non-limiting example, SEQ ID NOs: 194 and 164 are representative amino acid and nucleic acid sequences, respectively, for a terpene synthase.
In an aspect, geranylgeranyl diphosphate synthase is a component of the cis-abienol biosynthesis pathway. As a non-limiting example, SEQ ID NOs: 43 and 1 are representative examples of amino acid and nucleic acid sequences, respectively, for geranylgeranyl diphosphate synthase. In an aspect, geranylgeranyl pyrophosphate synthase is a component of the cis-abienol biosynthesis pathway. As a non-limiting example, SEQ ID NOs: 44 and 191 are non-limiting examples of amino acid sequences for geranylgeranyl pyrophosphate synthase. As a non-limiting example, SEQ ID NOs: 2 and 161 are representative examples of nucleic acid sequences for geranylgeranyl pyrophosphate synthase.
In an aspect, 2-oxoisovalerate dehydrogenase is a component of the L-leucine biosynthesis pathway in plants. As a non-limiting example, SEQ ID NOs: 62 and 20 are representative examples of amino acid and nucleic acid sequences, respectively, for dehydrogenase subunit alpha (Gene id g50844). As a non-limiting example, SEQ ID NOs: 63 and 21 are representative examples of amino acid and nucleic acid sequences, respectively, for 2-oxoisovalerate dehydrogenase subunit alpha (Gene id g63865).
In an aspect, cis-abienol synthase is a component of the cis-abienol biosynthesis pathway. As a non-limiting example, SEQ ID NOs: 147 and 119 are representative examples of amino acid and nucleic acid sequences, respectively, for cis-abienol synthase. In an aspect, a cis-abienol synthase is selected from the group consisting of cis-abienol synthase ISOFORM1 and cis-abienol synthase ISOFORM 2. In an aspect, a cis-abienol synthase is selected from the group consisting of cis-abienol synthase ISOFORM1, cis-abienol synthase ISOFORM 2, and Ent-kaurene synthase. In an aspect, a cis-abienol synthase is cis-abienol synthase ISOFORM 1. In an aspect, a cis-abienol synthase is cis-abienol synthase ISOFORM 2. In an aspect, cis-abienol synthase is ent-kaurene synthase. As a non-limiting example, SEQ ID NOs: 46 and 4 are representative examples of amino acid and nucleic acid sequences, respectively, for cis-abienol synthase ISOFORM 1. As a non-limiting example, SEQ ID NOs: 47 and 5 are representative examples of amino acid and nucleic acid sequences, respectively, for cis-abienol synthase ISOFORM 2. As a non-limiting example, SEQ ID NOs: 48 and 6 are representative examples of amino acid and nucleic acid sequences, respectively, for ent-kaurene synthase.
I an aspect, kolavenyl diphosphate synthase catalyzes the first reaction of salvinorin A biosynthesis, which is the formation of (−)-kolavenyl diphosphate ((−)-KPP). (−)-KPP is subsequently dephosphorylated to result in (−)-kolavenol. As a non-limiting example, SEQ ID NOs: 192 and 162 are representative examples of amino acid and nucleic acid sequences, respectively, for kolavenyl diphosphate synthase.
In an aspect, NAD(P)-dependent oxidoreductases catalyze NADPH-dependent reduction of a wide range of substrates. In an aspect, an oxidoreductase protein contains an oxidoreductase domain. In an aspect, SDR family oxidoreductase shares a conserved oxidoreductase domain with identified [mammalian] carbonyl reductases. In an aspect, an example of a protein containing a SDR family oxidoreductase conserved domain is uncharacterized protein LOC115704491. As a non-limiting example, SEQ ID NOs: 201 and 171 are representative examples of amino acid and nucleic acid sequences, respectively, of uncharacterized protein LOC115704491.
In an aspect, another example of an enzyme in the oxidoreductase family is 2-alkenal reductase. Substrates of 2-alkenal reductase are n-alkanal, NAD+, and NADP+. 2-alkenal reductase is also called NADPH-dependent alkenal/one oxidoreductase. As a non-limiting example, SEQ ID NOs: 200 and 170 are representative examples of amino acid and nucleic acid sequences, respectively, for 2-alkenal reductase.
In an aspect, cytochromes P450 (CYP) are a large enzyme family that catalyze the hydroxylation of diverse terpene substrates. In an aspect, premnaspirodiene oxygenase (PSO) (SEQ ID NOs: 158 and 160) catalyzes the hydroxylation of diverse sesquiterpene substrates. Hydroxylation reactions catalyzed by various cytochrome P450s are regio-specific and stereo-specific. As a non-limiting example, SEQ ID NOs: 31-33, 37, 159, 166-168, and 172 are representative examples of nucleic acid sequences for cytochrome P450. As a non-limiting example, SEQ ID NOs: 52-54, 58, 160, 196-198, and 202 are representative examples of amino acid sequences for cytochrome P450.
In an aspect, germacrene D synthase is a sesquiterpene synthase that catalyzes the formation of germacrene D. In an aspect, a germacrene D synthase is (−)-germacrene D synthase. As a non-limiting example, SEQ ID NOS: 203 and 173 are representative examples of amino acid and nucleic acid sequences, respectively, for (−)-germacrene D synthase.
In an aspect, a modified plant, seed, or plant part comprising a recombinant nucleic acid provided herein comprises an increased amount of at least one terpene as compared to a control plant, seed, or plant part lacking the recombinant nucleic acid molecule when grown under comparable conditions. In an aspect, a modified tobacco or Cannabis plant, tobacco or Cannabis seed, or tobacco plant part comprising a recombinant nucleic acid provided herein comprises an increased amount of at least one terpene as compared to a control tobacco or Cannabis plant, tobacco or Cannabis seed, or tobacco or Cannabis plant part lacking the recombinant nucleic acid molecule when grown under comparable conditions.
In an aspect, an increased amount of at least one terpene comprises an increase of at least 0.5%. In an aspect, an increased amount of at least one terpene comprises an increase of at least 1%. In an aspect, an increased amount of at least one terpene comprises an increase of at least 2%. In an aspect, an increased amount of at least one terpene comprises an increase of at least 3%. In an aspect, an increased amount of at least one terpene comprises an increase of at least 4%. In an aspect, an increased amount of at least one terpene comprises an increase of at least 5%. In an aspect, an increased amount of at least one terpene comprises an increase of at least 10%. In an aspect, an increased amount of at least one terpene comprises an increase of at least 12.5%. In an aspect, an increased amount of at least one terpene comprises an increase of at least 15%. In an aspect, an increased amount of at least one terpene comprises an increase of at least 17.5%. In an aspect, an increased amount of at least one terpene comprises an increase of at least 20%. In an aspect, an increased amount of at least one terpene comprises an increase of at least 25%. In an aspect, an increased amount of at least one terpene comprises an increase of at least 30%. In an aspect, an increased amount of at least one terpene comprises an increase of at least 40%. In an aspect, an increased amount of at least one terpene comprises an increase of at least 50%. In an aspect, an increased amount of at least one terpene comprises an increase of at least 60%. In an aspect, an increased amount of at least one terpene comprises an increase of at least 70%. In an aspect, an increased amount of at least one terpene comprises an increase of at least 80%. In an aspect, an increased amount of at least one terpene comprises an increase of at least 90%. In an aspect, an increased amount of at least one terpene comprises an increase of at least 100%. In an aspect, an increased amount of at least one terpene comprises an increase of at least 150%. In an aspect, an increased amount of at least one terpene comprises an increase of at least 200%. In an aspect, an increased amount of at least one terpene comprises an increase of at least 250%. In an aspect, an increased amount of at least one terpene comprises an increase of at least 500%.
In an aspect, an increased amount of at least one terpene comprises an increase of between 0.5% and 500%. In an aspect, an increased amount of at least one terpene comprises an increase of between 0.5% and 250%. In an aspect, an increased amount of at least one terpene comprises an increase of between 0.5% and 100%. In an aspect, an increased amount of at least one terpene comprises an increase of between 0.5% and 75%. In an aspect, an increased amount of at least one terpene comprises an increase of between 0.5% and 50%. In an aspect, an increased amount of at least one terpene comprises an increase of between 0.5% and 25%. In an aspect, an increased amount of at least one terpene comprises an increase of between 0.5% and 10%. In an aspect, an increased amount of at least one terpene comprises an increase of between 0.5% and 5%. In an aspect, an increased amount of at least one terpene comprises an increase of between 0.5% and 500%. In an aspect, an increased amount of at least one terpene comprises an increase of between 5% and 250%. In an aspect, an increased amount of at least one terpene comprises an increase of between 5% and 100%. In an aspect, an increased amount of at least one terpene comprises an increase of between 5% and 50%. In an aspect, an increased amount of at least one terpene comprises an increase of between 25% and 500%. In an aspect, an increased amount of at least one terpene comprises an increase of between 25% and 250%. In an aspect, an increased amount of at least one terpene comprises an increase of between 50% and 100%. In an aspect, an increased amount of at least one terpene comprises an increase of between 100% and 500%.
Terpenes may be extracted using any method known in the art, for example, solvent extraction. See, for example, Jiang et al., Curr Protoc Plant Biol., 1:345-358 (2016). Solventless extraction methods are also known in the art. See, for example, Yang and Xie, Science Direct, 162: 135-139 (2006).
The amount of terpenes in a plant can be measured using any method known in the art, including, without being limiting, gas chromatography mass spectrometry (GC-MS), Nuclear Magnetic Resonance Spectroscopy, and liquid chromatography-linked mass spectrometry. See The Handbook of Plant Metabolomics, edited by Weckwerth and Kahl, (Wiley-Blackwell) (May 28, 2013). In an aspect, an amount of at least one terpene refers to the concentration of that terpene in the tissue sampled.
In an aspect, a modified tobacco plant or Cannabis plant provided herein further comprises a recombinant nucleic acid molecule comprising a nucleic acid sequence encoding a polypeptide operably linked to a heterologous promoter, where the polypeptide causes an increase in average trichome density as compared to a control tobacco plant or Cannabis plant lacking the transgene or modification when grown under comparable conditions. Non-limiting examples of genes that can be overexpressed to increase average trichome density include NtGIS, NtMYB86, NbGIS, Cannabis MYB61, Cannabis GIS3, and Cannabis non-specific lipid-transfer protein 1-like.
CannabinoidsCannabinoids are naturally occurring compounds found in Cannabis plants. Many cannabinoids are concentrated in a resin produced in glandular trichomes, and at least 113 cannabinoids are known. Two main cannabinoids are tetrahydrocannabinol (THC) and cannabidiol (CBD), though over 100 additional cannabinoids have been identified. CBD composes approximately 40% of the plant resin extract.
Cannabinoids exert their effects by interacting with specific cannabinoid receptors. The two main types of cannabinoid receptors in humans are cannabinoid receptor type 1 (CB1) and cannabinoid receptor type 2 (CB2).
In an aspect, a heterologous polynucleotide is involved in the biosynthesis of a cannabinoid. There are several main classes of natural cannabinoids. Cannabis plant-derived cannabinoids are termed phytocannabinoids. All classes derive from cannabigerol-type (CBG) compounds and differ mainly in the way this precursor is cyclized.
In an aspect, a cannabinoid is selected from the group consisting of a cannabigerol-type (CBG) cannabinoid, a cannabichromene-type (CBC) cannabinoid, a cannabidiol-type (CBD) cannabinoid, a tetrahydrocannabinol-type (THC) cannabinoid, a cannabinol-type (CBN) cannabinoid, a cannabinodiol-type (CBDL) cannabinoid, a cannabielsoin-type (CBE) cannabinoid, an iso-tetrahydrocannabinol-type (iso-THC) cannabinoid, a cannabicyclol-type (CBL) cannabinoid, and a cannabitriol-type (CBT) cannabinoid.
Cannabigerol (CBG) is one of the major phytocannabinoids present in Cannabis sativa. CBG is the non-acidic form of cannabigerolic acid, the parent molecule from which other cannabinoids are synthesized. Cannabichromene (CBC) is a non-psychoactive cannabinoid. CBC is also called cannabichrome, cannanbichromene, pentylcannabichromene, or cannabinochromene.
Cannabidiol (CBD) is a naturally occurring compound found in the resinous flower of Cannabis. CBD is a major phytocannabinoid, accounting for up to 40% of the Cannabis plant's extract, and binds to a wide variety of physiological targets of the endocannabinoid system. Tetrahydrocannabinol (THC) is also known as Delta-9-tetrahydrocannabinol. Some Cannabis plants contain very little THC, and are considered “industrial hemp.”
Cannabinol (CBN) is a mildly psychoactive cannabinoid found only in trace amounts in Cannabis, and is mostly found in aged Cannabis. THC breaks down into CBN after prolonged periods of time. The degradation of THC to CBN can be accelerated by exposing dried plant matter to oxygen and heat. Cannabinodiol (CBDL) is a fully-aromatized extract of CBD. CBDL is a psychoactive cannabinoid, present in Cannabis sativa at low concentrations.
Cannabielsoin (CBE) is a non-psychoactive cannabinoid metabolite derived from CBD. Cannabicyclol (CBL) is a non-psychoactive cannabinoid found in Cannabis. Cannabichromene degrades into CBL through natural irradiation or under acidic conditions. Cannabitriol CBT is a cannabinoid that is not always detectable in Cannabis, but when detectable, CBT is often present in low concentrations. CBT is structurally similar to THC. There are currently nine known types of CBT.
In an aspect, a Cannabis-derived cannabinoid, or a phytocannabinoid, is selected from the group consisting of tetrahydrocannabinol, tetrahydrocannabinolic acid, cannabidiol, cannabidiolic acid, cannabinol, cannabigerol, cannabichromene, cannabicyclol, cannabivarin, tetrahydrocannabivarin, cannabidivarin, cannabichromevarin, cannabigerovarin, cannabigerol monomethyl ether, cannabielsoin, and cannabicitran.
As used herein, “tetrahydrocannabinol” (“THC”) refers to the organic compound having a chemical formula of C21H30O2 and the IUPAC name of (−)-(6aR,10aR)-6,6,9-trimethyl-3-pentyl-6a,7,8,10a-tetrahydro-6H-benzo[c]chromen-1-ol.
As used herein, “tetrahydrocannabinolic acid” (“THCA” or “2-COOH-THC”) refers to the organic compound having a chemical formula of C22H30O4.
As used herein, “cannabidiol” (“CBD”) refers to the organic compound having a chemical formula of C21H30O2, a molecular weight of 314.469 g/mol, and the IUPAC name of 2-[(1R,6R)-3-methyl-6-(prop-1-en-2-yl)cyclohex-2-en-1-yl]-5-pentylbenzene-1,3-diol. It is a 21-carbon terpenophenolic compound, which is formed following decarboxylation from a cannabidiolic acid precursor, although it can also be produced synthetically. CBD is normally taken to refer to the naturally occurring (−)-enantiomer. (+) CBD has been synthesized.
As used herein, “cannabidiolic acid” (“CBDA”) refers to the organic compound having a chemical formula of C22H30O4 and the IUPAC name of 2,4-dihydroxy-3-[(1R,6R)-3-methyl-6-prop-1-en-2-ylcyclohex-2-en-1-yl]-6-pentylbenzoic acid.
In plants, THC and CBD are derived from their acidic precursors THCA and CBDA, respectively, which are both derived from cannabigerolic acid (CBGA). The final biosynthesis step differs, with THCA synthase producing THCA and CBDA synthase producing CBDA, respectively, from CBGA. Subsequent decarboxylation of THCA and CBDA via light exposure, heating, or aging, results in the biosynthesis of THC or CBD. THCA synthase catalyzes oxidative cyclization of the monoterpene moiety of CBGA to form THCA. CBDA synthase catalyze oxidative cyclization of the monoterpene moiety of CBGA to form CBDA. THC is generated from THCA by non-enzymatic decarboxylation. CBD is generated from CBDA by non-enzymatic decarboxylation.
As used herein, “cannabinol” refers to the organic compound having a chemical formula of C21H26O2 and the IUPAC name of 6,6,9-trimethyl-3-pentylbenzo[c]chromen-1-ol.
As used herein, “cannabigerol” refers to the organic compound having a chemical formula of C21H32O2 and the IUPAC name of 2-[(2E)-3,7-dimethylocta-2,6-dienyl]-5-pentylbenzene-1,3-diol.
As used herein, “cannabichromene” refers to the organic compound having a chemical formula of C21H30O2 and the IUPAC name of 2-methyl-2-(4-methylpent-3-enyl)-7-pentylchromen-5-ol.
As used herein, “cannabicyclol” refers to the organic compound having a chemical formula of C21H30O2 and the IUPAC name of 9,13,13-trimethyl-5-pentyl-8-oxatetracyclo[7.4.1.0.0]tetradeca-2,4,6-trien-3-ol.
As used herein, “cannabivarin” refers to the organic compound having a chemical formula of C19H22O2 and the IUPAC name of 6,6,9-trimethyl-3-propylbenzo[c]chromen-1-ol.
As used herein, “tetrahydrocannabivarin” refers to the organic compound having a chemical formula of C19H26O2 and the IUPAC name of (6aR,10aR)-6,6,9-trimethyl-3-propyl-6a,7,8,10a-tetrahydrobenzo[c]chromen-1-ol.
As used herein, “cannabidivarin” refers to the organic compound having a chemical formula of C19H26O2 and the IUPAC name of 2-[(1R,6R)-3-methyl-6-prop-1-en-2-ylcyclohex-2-en-1-yl]-5-propylbenzene-1,3-diol.
As used herein, “cannabichromevarin” refers to the organic compound having a chemical formula of C19H26O2 and the IUPAC name of 2-methyl-2-(4-methylpent-3-enyl)-7-propylchromen-5-ol.
As used herein, “cannabigerovarin” refers to the organic compound having a chemical formula of C19H28O2 and the IUPAC name of 2-[(2E)-3,7-dimethylocta-2,6-dienyl]-5-propylbenzene-1,3-diol.
As used herein, “cannabigerol monomethyl ether” refers to the organic compound having a chemical formula of C22H28O2 and the IUPAC name of 1-methoxy-6,6,9-trimethyl-3-pentylbenzo[c]chromene.
As used herein, “cannabielsoin” refers to the organic compound having a chemical formula of C21H30O3 and the IUPAC name of (5aS,6S,9R,9aR)-6-methyl-3-pentyl-9-prop-1-en-2-yl-7,8,9,9a-tetrahydro-5aH-dibenzofuran-1,6-diol.
As used herein, “cannabicitran” refers to the organic compound having a chemical formula of C21H30O2 and the IUPAC name of 1,5,5-trimethyl-9-pentyl-6,15-dioxatetracyclo[9.3.1.0.0]pentadeca-7(12),8,10-triene.
PromotersAs commonly understood in the art, the term “promoter” refers to a DNA sequence that contains an RNA polymerase binding site, a transcription start site, and/or a TATA box and assists or promotes the transcription and expression of an associated transcribable polynucleotide sequence and/or gene (or transgene). A promoter can be synthetically produced, varied, or derived from a known or naturally occurring promoter sequence or other promoter sequence. A promoter can also include a chimeric promoter comprising a combination of two or more heterologous sequences. A promoter of the present application can thus include variants of promoter sequences that are similar in composition, but not identical to, other promoter sequence(s) known or provided herein.
In one aspect, this disclosure provides a recombinant nucleic acid molecule comprising a nucleic acid sequence encoding a polypeptide involved in the biosynthesis of at least one terpene operably linked to a heterologous promoter. In one aspect, this disclosure provides a recombinant nucleic acid molecule comprising a nucleic acid sequence encoding a polypeptide involved in the biosynthesis of at least one terpene operably linked to a heterologous promoter. In one aspect, this disclosure provides a recombinant nucleic acid molecule comprising a nucleic acid sequence encoding a polypeptide involved in the biosynthesis of at least one terpene operably linked to a heterologous promoter.
It is appreciated in the art that a fragment of a promoter sequence can function to drive transcription of an operably linked nucleic acid molecule. For example, without being limiting, if a 1000 bp promoter is truncated to 500 bp, and the 500 bp fragment is capable of driving transcription, the 500 bp fragment is referred to as a “functional fragment.”
In an aspect, a heterologous promoter comprises a nucleic acid sequence at least 90% identical to SEQ ID NOs: 104-111, 206-208, or a functional fragment thereof. In an aspect, a heterologous promoter comprises a nucleic acid sequence at least 92.5% identical to SEQ ID NOs: 104-111, 206-208, or a functional fragment thereof. In an aspect, a heterologous promoter comprises a nucleic acid sequence at least 95% identical to SEQ ID NOs: 104-111, 206-208, or a functional fragment thereof. In an aspect, a heterologous promoter comprises a nucleic acid sequence at least 96% identical to SEQ ID NOs: 104-111, 206-208, or a functional fragment thereof. In an aspect, a heterologous promoter comprises a nucleic acid sequence at least 97% identical to SEQ ID NOs: 104-111, 206-208, or a functional fragment thereof. In an aspect, a heterologous promoter comprises a nucleic acid sequence at least 98% identical to SEQ ID NOs: 104-111, 206-208, or a functional fragment thereof. In an aspect, a heterologous promoter comprises a nucleic acid sequence at least 99% identical to SEQ ID NOs: 104-111, 206-208, or a functional fragment thereof. In an aspect, a heterologous promoter comprises a nucleic acid sequence at least 99.9% identical to SEQ ID NOs: 104-111, 206-208, or a functional fragment thereof. In an aspect, a heterologous promoter comprises a nucleic acid sequence 100% identical to SEQ ID NOs: 104-111, 206-208, or a functional fragment thereof.
Promoters that drive expression in all or most tissues of the plant are referred to as “constitutive” promoters. In an aspect, a constitutive promoter is selected from the group consisting of a Cauliflower Mosaic Virus 35S promoter, a ubiquitin promoter, an actin promoter, an opine promoter, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and an alcohol dehydrogenase promoter.
Promoters that drive expression during certain periods or stages of development are referred to as “developmental” promoters.
Promoters that drive enhanced expression in certain tissues of an organism relative to other tissues of the organism are referred to as “tissue-preferred” promoters. Thus, a “tissue-preferred” promoter causes relatively higher or preferential expression in a specific tissue(s) of a plant, but with lower levels of expression in other tissue(s) of the plant. As a non-limiting example, a trichome tissue-preferred promoter exhibits higher activity in trichomes, but may also exhibit activity, albeit at lower levels, in additional tissues such as stem, leaves, and floral tissues. A “tissue-specific” promoter causes expression only in a specific tissue. As a non-limiting example, a trichome tissue-specific promoter drives expression only in trichomes. In an aspect, a tissue-specific promoter is a trichome tissue-specific promoter. In another aspect, a tissue-preferred promoter is a trichome tissue-preferred promoter.
In an aspect, a promoter is selected from the group consisting of a cyclase promoter, a premnaspirodiene oxygenase (PSO) promoter, and a ribulose biphosphate carboxylase promoter.
In an aspect, this disclosure provides a modified tobacco plant, tobacco seed, or part thereof, where a tissue-preferred promoter comprises a trichome-preferred promoter. In an aspect, this disclosure provides a modified tobacco plant, tobacco seed, or part thereof, where a tissue-specific promoter comprises a trichome-specific promoter. In an aspect, this disclosure provides a modified tobacco plant, tobacco seed, or part thereof where a tissue-preferred promoter or tissue-specific promoter comprises a trichome-preferred promoter or a trichome-specific promoter. In an aspect, a trichome-specific promoter sequence is selected from the group consisting of SEQ ID NOs: 104-111 and 206-208. In an aspect, a trichome-preferred promoter sequence is selected from the group consisting of SEQ ID NOs: 104-111 and 206-208.
An “inducible” promoter is a promoter that initiates transcription in response to an environmental stimulus such as heat, cold, drought, light, or other stimuli, such as wounding or chemical application.
In an aspect, a promoter provided herein is a constitutive promoter. In another aspect, a promoter provided herein is an inducible promoter. In a further aspect, a promoter provided herein is a developmental promoter. In another aspect, a promoter is a tissue-preferred or tissue-specific promoter. In a further aspect, a promoter is selected from the group consisting of a constitutive promoter, a tissue-preferred promoter, a tissue-specific promoter, and an inducible promoter.
In an aspect, this disclosure provides a heterologous promoter. In another aspect, this disclosure provides a promoter that is operably linked to a heterologous polynucleotide. In another aspect, this disclosure provides a polynucleotide sequence that is operably linked to a heterologous promoter.
As used herein, “operably linked” refers to a functional linkage between two or more elements. For example, an operable linkage between a polynucleotide of interest and a regulatory sequence (e.g., a promoter) is a functional link that allows for expression of the polynucleotide of interest. Operably linked elements may be contiguous or non-contiguous. In an aspect, a promoter provided herein is operably linked to a heterologous nucleic acid molecule.
PlantsIn an aspect, this disclosure provides a modified tobacco or Cannabis plant, tobacco or Cannabis seed, or part thereof comprising a recombinant nucleic acid molecule comprising a nucleic acid sequence encoding a polypeptide involved in the biosynthesis of at least one terpene operably linked to a heterologous promoter.
In an aspect, this disclosure provides a modified tobacco or Cannabis plant, tobacco or Cannabis seed, or part thereof comprising at least one non-natural mutation in an endogenous gene encoding a polypeptide involved in the biosynthesis of at least one terpene, where the modified tobacco or Cannabis plant exhibits increased mRNA expression of the endogenous gene or increased accumulation of the polypeptide as compared to a control tobacco or Cannabis plant lacking the at least one non-natural mutation when grown under comparable conditions.
In an aspect, a plant provided herein is a modified plant. In an aspect, a seed provided herein is a modified seed. In an aspect, a plant part provided herein is a modified plant part. As used herein, “modified,” in the context of a plant, seed, or plant part, refers to a plant, seed, or plant part, comprising a genetic alteration introduced for certain purposes and beyond natural polymorphisms. Without being limiting, a modified plant, seed, or plant part comprises a recombinant nucleic acid molecule. In another aspect, a modified plant, seed, or plant part comprises a genetic modification. In an aspect, a modified plant, seed, or plant part is transgenic.
Cannabis cultivars range from plants grown to produce Cannabis for recreational purposes to plants produced to use hemp fiber derived from the stems of the plant (e.g., industrial hemp). In cultivars utilized for recreational purposes, THC quantity exceeds CBD quantity in the dried female inflorescences used for smoking and oral administration. Industrial hemp cultivars produce substantially lower levels of THC and higher levels of CBD.
The plant genus Cannabis encompasses the species C. sativa, C. indica, and C. ruderalis. Another classification scheme applied to Cannabis is a chemotaxonomic classification system. There are five chemotaxonomic types of Cannabis: one with high levels of THC, one with high levels of CBD, one that has similar levels of THC and CBD, one with high levels of CBG, and one with low to no detectable cannabinoids.
In addition to genetic characteristics, cultivated Cannabis plants are influenced by environmental conditions and production technology during their life cycle. A study evaluating the effects of ambient temperature and humidity, soil temperature and precipitation on the content of THC and CBD in industrial hemp noted that these agroclimatic conditions have differing effects on THC and CBD. For example, CBD content is positively affected by soil temperature and ambient temperature, but negatively influenced by precipitation. See, for example, Sikora, V., et al., 2011, Genetika, 43(3):449-456.
In an aspect, a plant is a tobacco plant. In an aspect, a plant is a Nicotiana plant. In an aspect, a tobacco plant is a Nicotiana tabacum plant.
In an aspect, a Nicotiana plant, seed, or plant part is selected from the group consisting of Nicotiana tabacum, Nicotiana amplexicaulis PI 271989; Nicotiana benthamiana PI 555478; Nicotiana bigelovii PI 555485; Nicotiana debneyi; Nicotiana excelsior PI 224063; Nicotiana glutinosa PI 555507; Nicotiana goodspeedii PI 241012; Nicotiana gossei PI 230953; Nicotiana hesperis PI 271991; Nicotiana knightiana PI 555527; Nicotiana maritima PI 555535; Nicotiana megalosiphon PI 555536; Nicotiana nudicaulis PI 555540; Nicotiana paniculata PI 555545; Nicotiana plumbaginfolia PI 555548; Nicotiana repanda PI 555552; Nicotiana rustica; Nicotiana suaveolens PI 230960; Nicotiana sylvestris PI 555569; Nicotiana tomentosa PI 266379; Nicotiana tomentosiformis; and Nicotiana trigonophylla PI 555572.
In an aspect, a plant is a Cannabis plant. In an aspect, a plant is a Cannabis plant. In an aspect, a Cannabis plant is a Cannabis sativa plant. In an aspect, a Cannabis plant is a Cannabis indica plant. In an aspect, a Cannabis plant is a Cannabis ruderalis plant. In an aspect, a Cannabis plant is selected from the group consisting of Cannabis sativa, Cannabis indica, and Cannabis ruderalis.
In an aspect, a Cannabis plant may be placed into chemotype or chemotaxonomic grouping based on ratios of THC/CBD. In an aspect, a ratio of THC/CBD is based on relative levels of THC and CBD within a given Cannabis plant. A Cannabis plant with a THC/CBD ratio of greater than 1.0 has a high level of THC. A Cannabis plant with a THC/CBD ratio of approximately 1.0 has similar levels of THC and CBD. A Cannabis plant with a THC/CBD ratio of less than 1.0 has a high level of CBD.
In an aspect, a Cannabis plant is placed within a chemotaxonomic type comprising high levels of THC, relative to other assessed cannabinoids. In an aspect, a Cannabis plant is placed within a chemotaxonomic type comprising high levels of CBD, relative to other assessed cannabinoids. In an aspect, a Cannabis plant is placed within a chemotaxonomic type comprising similar levels of THC and CBD. In an aspect, a Cannabis plant is placed within a chemotaxonomic type comprising high levels of CBG, relative to other assessed cannabinoids. In an aspect, a Cannabis plant is placed within a chemotaxonomic type comprising low to no detectable cannabinoids.
In an aspect, a Cannabis plant, plant part, or seed comprises low to no detectable levels of THC. In an aspect, a Cannabis plant, plant part, or seed comprises low to no detectable levels of CBD. In an aspect, a Cannabis plant, plant part, or seed comprises low to no detectable levels of CBG.
In an aspect, a seed is a tobacco seed. In an aspect, a seed is a Nicotiana seed. In an aspect, a tobacco seed is a Nicotiana tabacum seed.
In an aspect, a seed is a Cannabis seed. In an aspect, a seed is a Cannabis seed. In an aspect, a Cannabis seed is a Cannabis sativa seed. In an aspect, a Cannabis seed is a Cannabis indica seed. In an aspect, a Cannabis seed is a Cannabis ruderalis seed. In an aspect, a Cannabis seed is selected from the group consisting of Cannabis sativa, Cannabis indica, and Cannabis ruderalis.
In an aspect, a Cannabis seed is placed within a chemotaxonomic type comprising high levels of THC, relative to other assessed cannabinoids. In an aspect, a Cannabis seed is placed within a chemotaxonomic type comprising high levels of CBD, relative to other assessed cannabinoids. In an aspect, a Cannabis seed is placed within a chemotaxonomic type comprising similar levels of THC and CBD. In an aspect, a Cannabis seed is placed within a chemotaxonomic type comprising high levels of CBG, relative to other assessed cannabinoids. In an aspect, a Cannabis seed is placed within a chemotaxonomic type comprising low to no detectable cannabinoids.
In an aspect, a plant part is a tobacco plant part. In an aspect, a plant part is a Nicotiana plant part. In an aspect, a tobacco plant part is a Nicotiana tabacum plant part.
In an aspect, a plant part is a Cannabis plant part. In an aspect, a plant part is a Cannabis plant part. In an aspect, a Cannabis plant part is a Cannabis sativa plant part. In an aspect, a Cannabis plant part is a Cannabis indica plant part. In an aspect, a Cannabis plant part is a Cannabis ruderalis plant part. In an aspect, a Cannabis plant part is selected from the group consisting of Cannabis sativa, Cannabis indica, and Cannabis ruderalis.
In an aspect, a Cannabis plant part is placed within a chemotaxonomic type comprising high levels of THC, relative to other assessed cannabinoids. In an aspect, a Cannabis plant part is placed within a chemotaxonomic type comprising high levels of CBD, relative to other assessed cannabinoids. In an aspect, a Cannabis plant part is placed within a chemotaxonomic type comprising similar levels of THC and CBD. In an aspect, a Cannabis plant part is placed within a chemotaxonomic type comprising high levels of CBG, relative to other assessed cannabinoids. In an aspect, a Cannabis plant part is placed within a chemotaxonomic type comprising low to no detectable cannabinoids.
In an aspect, a plant part provided includes, but is not limited to, a leaf, a stem, a root, a trichome, a seed, a flower, pollen, an anther, an ovule, a pedicel, a fruit, a meristem, a cotyledon, a hypocotyl, a pod, an embryo, endosperm, an explant, a callus, a tissue culture, a shoot, a cell, and a protoplast. In an aspect, a plant part does not include a seed. In an aspect, this disclosure provides plant cells, tissues, and organs that are not reproductive material and do not mediate the natural reproduction of the plant. In another aspect, this disclosure also provides plant cells, tissues, and organs that are reproductive material and mediate the natural reproduction of the plant. In another aspect, this disclosure provides plant cells, tissues, and organs that cannot maintain themselves via photosynthesis. In another aspect, this disclosure provides somatic plant cells. Somatic cells, contrary to germline cells, do not mediate plant reproduction.
Cells, tissues and organs can be from seed, fruit, leaf, cotyledon, hypocotyl, meristem, embryos, endosperm, root, shoot, stem, trichome, pod, flower, inflorescence, stalk, pedicel, style, stigma, receptacle, petal, sepal, pollen, anther, filament, ovary, ovule, pericarp, phloem, vascular tissue. In another aspect, this disclosure provides a plant chloroplast. In a further aspect, this disclosure provides epidermal cells, stomata cell, leaf or root hairs, a storage root, or a tuber. In another aspect, this disclosure provides a tobacco protoplast. In another aspect, this disclosure provides a Cannabis protoplast.
Skilled artisans understand that tobacco and Cannabis plants naturally reproduce via seeds, not via asexual reproduction or vegetative propagation. In an aspect, this disclosure provides plant endosperm.
This disclosure provides cells from plants provided herein.
As used herein, a “progeny plant” or “progeny seed” can be from any filial generation, e.g., F1, F2, F3, F4, F5, F6, F7, etc.
In an aspect, a tobacco plant, seed, or plant part, is of a tobacco variety selected from the group consisting of a flue-cured variety, a bright variety, a Burley variety, a Virginia variety, a Maryland variety, a dark variety, a Galpão variety, an Oriental variety, and a Turkish variety.
In an aspect, a tobacco cell is of a tobacco variety selected from the group consisting of a flue cured variety, a bright variety, a Burley variety, a Virginia variety, a Maryland variety, a dark variety, a Galpão variety, an Oriental variety, and a Turkish variety.
In an aspect, a tobacco leaf is of a tobacco variety selected from the group consisting of a flue cured variety, a bright variety, a Burley variety, a Virginia variety, a Maryland variety, a dark variety, a Galpão variety, an Oriental variety, and a Turkish variety.
In an aspect, a cured tobacco leaf or plant part is of a tobacco variety selected from the group consisting of a flue cured variety, a bright variety, a Burley variety, a Virginia variety, a Maryland variety, a dark variety, a Galpão variety, an Oriental variety, and a Turkish variety. Skilled artisans further understand that cured tobacco does not constitute a living organism and is not capable of growth or reproduction
Flue-cured tobaccos (also called “Virginia” or “bright” tobaccos) amount to approximately 40% of world tobacco production. Flue-cured tobaccos are often also referred to as “bright tobacco” because of the golden-yellow to deep-orange color it reaches during curing. Flue-cured tobaccos have a light, bright aroma and taste. Flue-cured tobaccos are generally high in sugar and low in oils. Major flue-cured tobacco growing countries are Argentina, Brazil, China, India, Tanzania and the United States of America. In one aspect, tobacco plants, seeds, or plant parts provided herein are of a flue-cured tobacco variety selected from the group consisting of the varieties listed in Table 2, and any variety essentially derived from any one of the foregoing varieties. See WO 2004/041006 A1. In a further aspect, tobacco plants, seeds, or plant parts provided herein are in a flue-cured variety selected from the group consisting of K326, K346, and NC196.
Air-cured tobaccos include “Burley,” Maryland,” and “dark” tobaccos. The common factor linking air-cured tobaccos is that curing occurs primarily without artificial sources of heat and humidity. Burley tobaccos are light to dark brown in color, high in oil, and low in sugar. Burley tobaccos are typically air-cured in barns. Major Burley growing countries include Argentina, Brazil, Italy, Malawi, and the United States of America.
Maryland tobaccos are extremely fluffy, have good burning properties, low nicotine and a neutral aroma. Major Maryland growing countries include the United States of America and Italy.
In one aspect, tobacco plants, seeds, or plant parts provided herein are of a Burley tobacco variety selected from the group consisting of the tobacco varieties listed in Table 3, and any variety essentially derived from any one of the foregoing varieties. In a further aspect, tobacco plants, seeds, or plant parts provided herein are in a Burley variety selected from the group consisting of TN 90, KT 209, KT 206, KT212, and 89 4488.
In another aspect, tobacco plants, seeds, or plant parts provided herein are of a Maryland tobacco variety selected from the group consisting of the tobacco varieties listed in Table 4, and any variety essentially derived from any one of the foregoing varieties.
Dark air-cured tobaccos are distinguished from other tobacco types primarily by its curing process, which gives dark air-cured tobacco its medium-brown to dark-brown color and a distinct aroma. Dark air-cured tobaccos are mainly used in the production of chewing tobacco and snuff. In one aspect, tobacco plants, seeds, or plant parts provided herein are of a dark air-cured tobacco variety selected from the group consisting of Sumatra, Jatim, Dominican Cubano, Besuki, One sucker, Green River, Virginia sun-cured, and Paraguan Passado, and any variety essentially derived from any one of the foregoing varieties.
Dark fire-cured tobaccos are generally cured with low-burning wood fires on the floors of closed curing barns. Dark fire-cured tobaccos are typically used for making pipe blends, cigarettes, chewing tobacco, snuff, and strong-tasting cigars. Major growing regions for dark fire-cured tobaccos are Tennessee, Kentucky, and Virginia in the United States of America. In one aspect, tobacco plants, seeds, or plant parts provided herein are of a dark fire-cured tobacco variety selected from the group consisting of the tobacco varieties listed in Table 5, and any variety essentially derived from any one of the foregoing varieties.
Oriental tobaccos are also referred to as Greek, aroma and Turkish tobaccos due to the fact that they are typically grown in eastern Mediterranean regions such as Turkey, Greece, Bulgaria, Macedonia, Syria, Lebanon, Italy, and Romania. The small plant size, small leaf size, and unique aroma properties of Oriental tobacco varieties are a result of their adaptation to the poor soil and stressful climatic conditions in which they have been developed. In one aspect, tobacco plants, seeds, or plant parts provided herein are of an Oriental tobacco variety selected from the group consisting of the tobacco varieties listed in Table 6, and any variety essentially derived from any one of the foregoing varieties.
In an aspect, tobacco plants, seeds, or plant parts provided herein are of a cigar tobacco variety selected from the group consisting of the tobacco varieties listed in Table 7, and any variety essentially derived from any one of the foregoing varieties.
In an aspect, tobacco plants, seeds, or plant parts provided herein are of a tobacco variety selected from the group consisting of the tobacco varieties listed in Table 8, and any variety essentially derived from any one of the foregoing varieties.
In an aspect, a tobacco plant or plant part is from a variety selected from the group consisting of the tobacco varieties listed in Table 2, Table 3, Table 4, Table 5, Table 6, Table 7, and Table 8. In another aspect, a tobacco plant or plant part is from a variety listed in Table 2. In another aspect, a tobacco plant or plant part is from a variety listed in Table 3. In another aspect, a tobacco plant or plant part is from a variety listed in Table 4. In another aspect, a tobacco plant or plant part is from a variety listed in Table 5. In another aspect, a tobacco plant or plant part is from a variety listed in Table 6. In another aspect, a tobacco plant or plant part is from a variety listed in Table 7. In another aspect, a tobacco plant or plant part is from a variety listed in Table 8.
In an aspect, a tobacco seed is from a variety selected from the group consisting of the tobacco varieties listed in Table 2, Table 3, Table 4, Table 5, Table 6, Table 7, and Table 8. In another aspect, a tobacco seed is from a variety listed in Table 2. In another aspect, a tobacco seed is from a variety listed in Table 3. In another aspect, a tobacco seed is from a variety listed in Table 4. In another aspect, a tobacco seed is from a variety listed in Table 5. In another aspect, a tobacco seed is from a variety listed in Table 6. In another aspect, a tobacco seed is from a variety listed in Table 7. In another aspect, a tobacco seed is from a variety listed in Table 8.
In an aspect, a tobacco cell is from a variety selected from the group consisting of the tobacco varieties listed in Table 2, Table 3, Table 4, Table 5, Table 6, Table 7, and Table 8. In another aspect, a tobacco cell is from a variety listed in Table 2. In another aspect, a tobacco cell is from a variety listed in Table 3. In another aspect, a tobacco cell is from a variety listed in Table 4. In another aspect, a tobacco cell is from a variety listed in Table 5. In another aspect, a tobacco cell is from a variety listed in Table 6. In another aspect, a tobacco cell is from a variety listed in Table 7. In another aspect, a tobacco cell is from a variety listed in Table 8.
All foregoing mentioned specific varieties of flue-cured, dark air-cured, Burley, Maryland, dark fire-cured, cigar, or Oriental type are listed only for exemplary purposes. Any additional flue-cured, dark air-cured, Burley, Maryland, dark fire-cured, cigar, or Oriental varieties are also contemplated in the present application.
In an aspect, a plant or variety provided herein is an inbred plant or variety. As used herein, an “inbred” variety is a variety that has been bred for genetic homogeneity.
As used herein, a “hybrid” is created by crossing two plants from different varieties or species, such that the progeny comprises genetic material from each parent. Skilled artisans recognize that higher order hybrids can be generated as well. For example, a first hybrid can be made by crossing Variety C with Variety D to create a C×D hybrid, and a second hybrid can be made by crossing Variety E with Variety F to create an E×F hybrid. The first and second hybrids can be further crossed to create the higher order hybrid (C×D)×(E×F) comprising genetic information from all four parent varieties. In an aspect, a plant or seed provided herein is a hybrid plant or seed.
In an aspect, a tobacco plant provided herein is an inbred tobacco plant. In an aspect, a tobacco seed provided herein is an inbred tobacco seed. In an aspect, a tobacco plant provided herein is a hybrid tobacco plant. In another aspect, a tobacco seed provided herein is a hybrid tobacco seed.
In an aspect, a Cannabis plant provided herein is an inbred Cannabis plant. In an aspect, a Cannabis seed provided herein is an inbred Cannabis seed. In an aspect, a Cannabis plant provided herein is a hybrid Cannabis plant. In another aspect, a Cannabis seed provided herein is a hybrid Cannabis seed.
Unless specified otherwise, all comparisons to control plants require similar growth conditions or comparable growth conditions for the two plants being compared. As used herein, “grown under comparable conditions,” “similar growth conditions” or “comparable growth conditions” refer to similar environmental conditions and/or agronomic practices for growing and making meaningful comparisons between two or more plant genotypes so that neither environmental conditions nor agronomic practices would contribute to or explain any difference observed between the two or more plant genotypes. Environmental conditions include, for example, light, temperature, water (humidity), and nutrition (e.g., nitrogen and phosphorus). Agronomic practices include, for example, seeding, clipping, undercutting, transplanting, topping, and suckering. See Chapters 4B and 4C of Tobacco, Production, Chemistry and Technology, Davis & Nielsen, eds., Blackwell Publishing, Oxford (1999), pp 70-103. See, also, Amaducci et al. Field Crops Research, 107:161-169 (2008). As used herein, a “control plant” refers to a plant of identical, or nearly identical, genetic makeup as the modified plant being compared, except for the non-natural mutation or recombinant DNA construct provided herein that was introduced to the modified plant.
In an aspect, a first plant variety and a second plant variety are the same variety. In an aspect, a first plant variety and a second plant variety are two different varieties. In an aspect, a second plant variety comprises a recombinant nucleic acid molecule.
In an aspect, a first plant variety is heterozygous for a recombinant nucleic acid molecule. In an aspect, a first plant variety is hemizygous for a recombinant nucleic acid molecule. In an aspect, a first plant variety is homozygous for a recombinant nucleic acid molecule. In an aspect, a second plant variety is heterozygous for a recombinant nucleic acid molecule. In an aspect, a second plant variety is hemizygous for a recombinant nucleic acid molecule. In an aspect, a second plant variety is homozygous for a recombinant nucleic acid molecule. In an aspect, a progeny seed, or a plant germinated therefrom, is heterozygous for a recombinant nucleic acid molecule. In an aspect, a progeny seed, or a plant germinated therefrom, is hemizygous for a recombinant nucleic acid molecule. In an aspect, a progeny seed, or a plant germinated therefrom, is homozygous for a recombinant nucleic acid molecule.
In an aspect, a first plant variety is a tobacco plant variety. In an aspect, a second plant variety is a tobacco plant variety.
In an aspect, a first plant variety is a Cannabis plant variety. In an aspect, a second plant variety is a Cannabis plant variety.
As used herein, the term “crossing” refers to the deliberate mating of two plants. In an aspect, crossing comprises pollination and/or fertilization of a first plant by a second plant. The two plants being crossed can be distantly related, closely related, or identical. In an aspect, the two plants being crossed are both modified plants. In an aspect, the two plants being crossed are of the same variety. In an aspect, the two plants being crossed are of two different varieties. In an aspect, one of the two plants being crossed is male sterile. In an aspect, one of the two plants being crossed is female sterile. In an aspect, at least one of the two plants being crossed is a hybrid tobacco plant. In an aspect, at least one of the two plants being crossed is a hybrid Cannabis plant. In an aspect, at least one of the two plants being crossed is a modified plant.
In an aspect, a plant of a first variety is the male parent in a crossing step. In an aspect, a plant of a first variety is the female parent in a crossing step. In an aspect, a plant of a second variety is the male parent in a crossing step. In an aspect, a plant of a second variety is the female parent in a crossing step.
In an aspect, a plant or variety provided herein is male sterile. In another aspect, a plant or variety provided herein is cytoplasmic male sterile (CMS). Male sterile plants can be produced by any method known in the art. Methods of producing male sterile tobacco are described in Wernsman, E. A., and Rufty, R. C. 1987. Chapter Seventeen. Tobacco. Pages 669-698 in: Cultivar Development. Crop Species. W. H. Fehr (ed.), MacMillan Publishing Go., Inc., New York, N.Y. 761 pp. See also kush[dot]com/blog/feminized-Cannabis-seeds-all-you-need-to-know, which describes processes such as rodelization and spraying female Cannabis plants with colloidal silver every day after flowering to reduce ethylene production.
In another aspect, a plant or variety provided herein is female sterile. As a non-limiting example, female sterile plants can be made by mutating the STIGI gene. See, for example, Goldman et al. 1994, EMBO Journal 13:2976-2984.
In an aspect, this disclosure provides a method of producing a modified tobacco plant comprising: (a) introducing a recombinant nucleic acid molecule to at least one tobacco cell, where the recombinant nucleic acid molecule comprises a nucleic acid sequence encoding a polypeptide involved in terpene biosynthesis operably linked to a heterologous promoter; (b) selecting at least one tobacco cell comprising the recombinant nucleic acid molecule; and (c) regenerating a modified tobacco plant from the at least one tobacco cell selected in step (b) where the modified tobacco plant comprises an increased amount of at least one terpene in at least one tissue as compared to a control tobacco plant lacking the recombinant nucleic acid molecule when grown under comparable conditions.
In an aspect, this disclosure provides a method of producing a modified Cannabis plant comprising: (a) introducing a recombinant nucleic acid molecule to at least one Cannabis cell, where the recombinant nucleic acid molecule comprises a nucleic acid sequence encoding a polypeptide involved in terpene biosynthesis operably linked to a heterologous promoter; (b) selecting at least one Cannabis cell comprising the recombinant nucleic acid molecule; and (c) regenerating a modified Cannabis plant from the at least one Cannabis cell selected in step (b) where the modified Cannabis plant comprises an increased amount of at least one terpene in at least one tissue as compared to a control Cannabis plant lacking the recombinant nucleic acid molecule when grown under comparable conditions.
TransformationNumerous methods for introducing a recombinant DNA construct to a plant cell are known in the art, which can be used according to methods of the present application to produce a transgenic plant cell and plant. Any suitable method or technique for transformation of a plant cell known in the art can be used according to present methods. Effective methods for transformation of plants include bacterially mediated transformation, such as Agrobacterium-mediated or Rhizobium-mediated transformation and microprojectile bombardment-mediated transformation. See, e.g., Feeney and Punja, In Vitro Cell. and Dev. Biol.—Plant, 39:578-585 (2003). A variety of methods are known in the art for transforming explants with a transformation vector via bacterially mediated transformation or microprojectile bombardment and then subsequently culturing, etc., those explants to regenerate or develop transgenic plants. Other methods for plant transformation, such as microinjection, electroporation, vacuum infiltration, pressure, sonication, silicon carbide fiber agitation, polyethylene glycol (PEG)-mediated transformation, etc., are also known in the art. Transgenic plants produced by these transformation methods can be chimeric or non-chimeric for the transformation event depending on the methods and explants used.
Methods of transforming plant cells are well known by persons of ordinary skill in the art. For instance, specific instructions for transforming plant cells by microprojectile bombardment with particles coated with recombinant DNA (e.g., biolistic transformation) are found in U.S. Pat. Nos. 5,550,318; 5,538,880 6,160,208; 6,399,861; and 6,153,812 and Agrobacterium-mediated transformation is described in U.S. Pat. Nos. 5,159,135; 5,824,877; 5,591,616; 6,384,301; 5,750,871; 5,463,174; and 5,188,958, all of which are incorporated herein by reference. Additional methods for transforming plants can be found in, for example, Compendium of Transgenic Crop Plants (2009) Blackwell Publishing. Any appropriate method known to those skilled in the art can be used to transform a tobacco or Cannabis cell with any of the nucleic acid molecules provided herein.
In an aspect, this disclosure provides a method comprising transforming a tobacco or Cannabis cell with a recombinant nucleic acid molecule, where the recombinant nucleic acid molecule comprises a nucleic acid sequence encoding a polypeptide involved in the biosynthesis of at least one terpene operably linked to a heterologous promoter.
In an aspect, this disclosure provides a method for producing a tobacco or Cannabis plant, the method comprising: (a) crossing at least one tobacco or Cannabis plant of a first tobacco or Cannabis variety with at least one tobacco or Cannabis plant of a second tobacco or Cannabis variety to produce at least one progeny tobacco or Cannabis seed, where the at least one tobacco or Cannabis plant of the first tobacco or Cannabis variety comprises a recombinant nucleic acid molecule comprising a nucleic acid sequence encoding a polypeptide involved in the biosynthesis of at least one terpene operably linked to a heterologous promoter; and (b) selecting for at least one progeny tobacco or Cannabis seed, or a plant germinated therefrom, where the at least one progeny tobacco or Cannabis seed or plant germinated therefrom comprises the recombinant nucleic acid molecule.
In an aspect, this disclosure provides a method for producing a tobacco or Cannabis plant, the method comprising: (a) crossing at least one tobacco or Cannabis plant of a first tobacco or Cannabis variety with at least one tobacco or Cannabis plant of a second tobacco or Cannabis variety to produce at least one progeny tobacco or Cannabis seed, where the at least one tobacco or Cannabis plant of the first tobacco or Cannabis variety comprises at least one non-natural mutation in an endogenous gene encoding a polypeptide involved in the biosynthesis of at least one terpene, where the at least one tobacco or Cannabis plant of the first tobacco or Cannabis variety exhibits increased mRNA expression of the endogenous gene or increased accumulation of the polypeptide as compared to a control tobacco or Cannabis plant of the first tobacco or Cannabis variety lacking the at least one non-natural mutation when grown under comparable conditions; and (b) selecting for at least one progeny tobacco or Cannabis seed, or a plant germinated therefrom, where the at least one progeny tobacco or Cannabis seed or plant germinated therefrom comprises the at least one non-natural mutation.
In an aspect, a method of introducing a nucleic acid molecule to a tobacco or Cannabis cell comprises Agrobacterium-mediated transformation. In another aspect, a method of introducing a nucleic acid molecule to a tobacco or Cannabis cell comprises PEG-mediated transformation. In another aspect, a method of introducing a nucleic acid molecule to a tobacco or Cannabis cell comprises biolistic transformation. In another aspect, a method of introducing a nucleic acid molecule to a tobacco or Cannabis cell comprises liposome-mediated transfection (lipofection). In another aspect, a method of introducing a nucleic acid molecule to a tobacco or Cannabis cell comprises lentiviral transfection.
Lipofection is described in e.g., U.S. Pat. Nos. 5,049,386, 4,946,787; and 4,897,355) and lipofection reagents are sold commercially (e.g., Transfectam™ and Lipofectin™). Cationic and neutral lipids that are suitable for efficient receptor-recognition lipofection of polynucleotides include those of International Publication WO 1991/017424 and International Publication WO 1991/016024. Delivery can be to cells (e.g. in vitro or ex vivo administration) or target tissues (e.g. in vivo administration).
Any tobacco cell from which a fertile tobacco plant can be regenerated is contemplated as a useful recipient cell for practice of this disclosure. Similarly, any Cannabis cell from which a fertile Cannabis plant can be regenerated is contemplated as a useful recipient cell for practice of this disclosure.
In an aspect, a recombinant DNA construct is introduced to a tobacco cell. In an aspect, a recombinant DNA construct is introduced to a tobacco protoplast cell. In another aspect, a recombinant DNA construct is introduced to a tobacco callus cell. In an aspect, a recombinant DNA construct is introduced to a tobacco cell selected from the group consisting of a seed cell, a fruit cell, a leaf cell, a cotyledon cell, a hypocotyl cell, a meristem cell, an embryo cell, an endosperm cell, a root cell, a shoot cell, a stem cell, a flower cell, an inflorescence cell, a stalk cell, a pedicel cell, a style cell, a stigma cell, a receptacle cell, a petal cell, a sepal cell, a pollen cell, an anther cell, a filament cell, an ovary cell, an ovule cell, a pericarp cell, and a phloem cell.
In an aspect, a recombinant DNA construct is introduced to a Cannabis cell. In an aspect, a recombinant DNA construct is introduced to a Cannabis protoplast cell. In another aspect, a recombinant DNA construct is introduced to a Cannabis callus cell. In an aspect, a recombinant DNA construct is introduced to a Cannabis cell selected from the group consisting of a seed cell, a fruit cell, a leaf cell, a cotyledon cell, a hypocotyl cell, a meristem cell, an embryo cell, an endosperm cell, a root cell, a shoot cell, a stem cell, a flower cell, an inflorescence cell, a stalk cell, a pedicel cell, a style cell, a stigma cell, a receptacle cell, a petal cell, a sepal cell, a pollen cell, an anther cell, a filament cell, an ovary cell, an ovule cell, a pericarp cell, and a phloem cell.
Callus can be initiated from various tissue sources, including, but not limited to, immature embryos or parts of embryos, seedling apical meristems, microspores, and the like. Those cells which are capable of proliferating as callus can serve as recipient cells for transformation. Practical transformation methods and materials for making transgenic plants of this disclosure (e.g., various media and recipient target cells, transformation of immature embryos, and subsequent regeneration of fertile transgenic plants) are disclosed, for example, in U.S. Pat. Nos. 6,194,636 and 6,232,526 and U.S. Patent Application Publication 2004/0216189, all of which are incorporated herein by reference.
In an aspect, this disclosure provides a method of producing a modified tobacco or Cannabis plant comprising: (a) introducing a recombinant DNA construct to at least one tobacco or Cannabis cell, where said recombinant DNA construct comprises a heterologous promoter operably linked to a nucleic acid encoding at least one small RNA molecule capable of binding to and reducing the expression of a nucleic acid sequence encoding a polypeptide involved in the biosynthesis of at least one terpene; (b) selecting at least one tobacco or Cannabis cell comprising said recombinant DNA construct; and (c) regenerating at least one modified tobacco or Cannabis plant from said at least one tobacco or Cannabis cell selected in step (b), where the modified tobacco or Cannabis plant comprises a reduced amount of at least one terpene in at least one tissue as compared to a control tobacco or Cannabis plant lacking the recombinant DNA construct when grown under comparable conditions.
In an aspect, this disclosure provides a method comprising transforming a tobacco or Cannabis cell with a recombinant nucleic acid molecule, where the recombinant nucleic acid molecule comprises a heterologous promoter operably linked to a nucleic acid encoding at least one small RNA molecule capable of binding to and reducing the expression of a nucleic acid sequence encoding a polypeptide involved in the biosynthesis of at least one terpene.
In an aspect, this disclosure provides a method for producing a tobacco or Cannabis plant, the method comprising: (a) crossing at least one tobacco or Cannabis plant of a first tobacco or Cannabis variety with at least one tobacco or Cannabis plant of a second tobacco or Cannabis variety to produce at least one progeny tobacco or Cannabis seed, where the at least one tobacco or Cannabis plant of the first tobacco or Cannabis variety comprises a recombinant nucleic acid molecule comprising a heterologous promoter operably linked to a nucleic acid encoding at least one small RNA molecule capable of binding to and reducing the expression of a nucleic acid sequence encoding a polypeptide involved in the biosynthesis of at least one terpene; and selecting for at least one progeny tobacco or Cannabis seed, or a plant germinated therefrom, where the at least one progeny tobacco or Cannabis seed or plant germinated therefrom comprises the recombinant nucleic acid molecule.
MutationsWithout being limiting, a modified plant can comprise a non-natural mutation or a recombinant DNA construct. In an aspect, a modified tobacco plant comprises a non-natural mutation. In another aspect, a modified tobacco plant comprises a recombinant DNA construct. In another aspect, a modified tobacco plant comprises a genetic modification. In an aspect, a modified Cannabis plant comprises a non-natural mutation. In another aspect, a modified Cannabis plant comprises a recombinant DNA construct. In another aspect, a modified Cannabis plant comprises a genetic modification.
In an aspect, this disclosure provides a method of producing a modified tobacco or Cannabis plant comprising: (a) inducing at least one non-natural mutation in at least one tobacco or Cannabis cell in an endogenous gene encoding a polypeptide involved in terpene biosynthesis; (b) selecting at least one tobacco or Cannabis plant comprising the at least one non-natural mutation from step (a); and (c) regenerating a modified tobacco or Cannabis plant from the at least one tobacco or Cannabis cell selected in step (b), where the modified tobacco or Cannabis plant comprises an increased amount of at least one terpene in at least one tissue as compared to a control tobacco or Cannabis plant lacking the at least one non-natural mutation when grown under comparable conditions.
As used herein, a “mutation” refers to an inheritable genetic modification introduced into a gene to alter the expression or activity of a product encoded by a reference sequence of the gene. A mutation in a certain gene, such as, for example, limonene synthase (LS), is referred to as an LS mutant. Such a modification can be in any sequence region of a gene, for example, in a promoter, 5′ untranslated region (UTR), exon, intron, 3′-UTR, or terminator region. In an aspect, a mutation reduces, inhibits, or eliminates the expression or activity of a gene product. In another aspect, a mutation increases, elevates, strengthens, or augments the expression or activity of a gene product.
In an aspect, mutations are not natural polymorphisms that exist in a particular tobacco variety or cultivar. In an aspect, a mutation is a “non-natural” or “non-naturally occurring” mutation. As used herein, a “non-natural” or “non-naturally occurring” mutation refers to a non-spontaneous mutation generated via human intervention, and does not correspond to a spontaneous mutation generated without human intervention. Non-limiting examples of human intervention include mutagenesis (e.g., chemical mutagenesis, ionizing radiation mutagenesis) and targeted genetic modifications (e.g., CRISPR-based methods, TALEN-based methods, zinc finger-based methods). Non-natural mutations and non-naturally occurring mutations do not include spontaneous mutations that arise naturally (e.g., via aberrant DNA replication in a germ line of a plant).
In an aspect, mutations are not natural polymorphisms that exist in a particular tobacco variety or cultivar. It will be appreciated that, when identifying a mutation, the reference DNA sequence should be from the same variety of tobacco. For example, if a modified tobacco plant comprising a mutation is from the variety TN90, then the endogenous reference sequence must be the endogenous TN90 sequence, not a homologous sequence from a different tobacco variety (e.g., K326). Similarly, if a modified tobacco cell comprising a mutation is a TN90 cell, then the endogenous reference sequence must be the endogenous TN90 sequence, not a homologous sequence from a tobacco cell from a different tobacco variety (e.g., K326).
In an aspect, mutations are not natural polymorphisms that exist in a particular Cannabis species, variety or cultivar. It will be appreciated that, when identifying a mutation, the reference DNA sequence should be from the same species, variety, or cultivar of Cannabis. For example, if a modified Cannabis plant comprising a mutation is a strain of C. indica, then the endogenous reference sequence must be the corresponding endogenous C. indica sequence, not a homologous sequence from a different Cannabis species or strain (e.g. a sequence from C. sativa or a different strain of C. indica). Similarly, if a modified Cannabis cell comprising a mutation is a cell from C. indica, then the endogenous reference sequence must be the corresponding endogenous C. indica sequence, not a homologous sequence from a Cannabis cell from a different Cannabis variety or species (e.g., a Sativa strain).
In an aspect, a tobacco plant, tobacco seed, or part thereof, is homozygous for at least one non-natural mutation. In another aspect, a tobacco plant, tobacco seed, or part thereof, is heterozygous for at least one non-natural mutation. In an aspect, a tobacco plant, tobacco seed, or part thereof is hemizygous for at least one non-natural mutation. In another aspect, a tobacco plant, tobacco seed, or part thereof, is homozygous for an introduced recombinant DNA construct. In another aspect, a tobacco plant, tobacco seed, or part thereof, is hemizygous for an introduced recombinant DNA construct. In a further aspect, a tobacco plant, tobacco seed, or part thereof, is heterozygous for an introduced recombinant DNA construct.
In an aspect, a Cannabis plant, Cannabis seed, or part thereof, is homozygous for at least one non-natural mutation. In another aspect, a Cannabis plant, Cannabis seed, or part thereof, is heterozygous for at least one non-natural mutation. In an aspect, a Cannabis plant, Cannabis seed, or part thereof is hemizygous for at least one non-natural mutation. In another aspect, a Cannabis plant, Cannabis seed, or part thereof, is homozygous for an introduced recombinant DNA construct. In another aspect, a Cannabis plant, Cannabis seed, or part thereof, is hemizygous for an introduced recombinant DNA construct. In a further aspect, a Cannabis plant, Cannabis seed, or part thereof, is heterozygous for an introduced recombinant DNA construct.
In an aspect, a tobacco seed, or the plant germinated therefrom, is heterozygous for the recombinant nucleic acid molecule. In an aspect, a tobacco seed, or the plant germinated therefrom, is hemizygous for the recombinant nucleic acid molecule. In an aspect, a tobacco seed, or the plant germinated therefrom, is homozygous for the recombinant nucleic acid molecule.
In an aspect, a Cannabis seed, or the plant germinated therefrom, is heterozygous for the recombinant nucleic acid molecule. In an aspect, a Cannabis seed, or the plant germinated therefrom, is hemizygous for the recombinant nucleic acid molecule. In an aspect, a Cannabis seed, or the plant germinated therefrom, is homozygous for the recombinant nucleic acid molecule.
In an aspect, a mutation provided herein creates a dominant allele of the mutated locus. Dominant alleles are alleles that mask the contribution of a second allele at the same locus. A dominant allele can be a “dominant negative allele” or a “dominant positive allele.” Dominant negative alleles, or antimorphs, are alleles that act in opposition to normal allelic function. A dominant negative allele typically does not function normally and either directly inhibits the activity of a wild-type protein (e.g., through dimerization) or inhibits the activity of a second protein that is required for the normal function of the wild-type protein (e.g., an activator or a downstream component of a pathway). For example, a dominant negative allele abrogates or reduces the normal function of an allele in a heterozygous or homozygous state. Dominant positive alleles can increase normal gene function (e.g., a hypermorph) or provide new functions for a gene (e.g., a neomorph). A semi-dominant allele occurs when penetrance of a linked phenotype in individuals heterozygous for the allele is less than that which is observed in individuals homozygous for the allele.
In an aspect, a mutation provided herein creates a dominant negative allele of the mutated locus. In another aspect, a mutation provided herein creates a dominant positive allele of a mutated locus.
As used herein, “inducing” a mutation refers to generating a mutation in a polynucleotide sequence via human intervention. Many suitable methods for inducing mutations in tobacco and Cannabis are known in the art. Non-limiting examples of such methods include use of chemical mutagens, use of irradiation, use of nucleases, use of transposons, and use of Agrobacterium. In an aspect, inducing a mutation comprises the use of an agent selected from the group consisting of a chemical mutagen, irradiation, a transposon, Agrobacterium, and a nuclease.
In an aspect, inducing a mutation comprises the use of a chemical mutagen. In an aspect, a chemical mutagen comprises ethyl methanesulfonate (EMS).
In another aspect, inducing a mutation comprises the use of irradiation. In an aspect, irradiation comprises gamma rays, X-rays, ionizing radiation, or fast neutrons.
In an aspect, inducing a mutation comprises the use of a transposon. In another aspect, inducing a mutation comprises the use of Agrobacterium.
In a further aspect, inducing a mutation comprises the use of a nuclease. In an aspect, a nuclease is selected from the group consisting of a meganuclease, a zinc-finger nuclease, a transcription activator-like effector nuclease, a CRISPR/Cas9 nuclease, a CRISPR/Cpf1 nuclease, a CRISPR/CasX nuclease, a CRISPR/CasY nuclease, and a Csm1 nuclease. In an aspect, inducing a mutation comprises the use of a CRISPR/Cas9 nuclease. In an aspect, inducing a mutation comprises the use of a CRISPR/Cpf1 nuclease. In an aspect, inducing a mutation comprises the use of a CRISPR/CasX nuclease. In an aspect, inducing a mutation comprises the use of a CRISPR/CasY nuclease. In an aspect, inducing a mutation comprises the use of a Csm1 nuclease.
Several types of mutations are known in the art. In an aspect, a mutation comprises an insertion. An “insertion” refers to the addition of one or more nucleotides or amino acids to a given polynucleotide or amino acid sequence, respectively, as compared to an endogenous reference polynucleotide or amino acid sequence. In another aspect, a mutation comprises a deletion. A “deletion” refers to the removal of one or more nucleotides or amino acids to a given polynucleotide or amino acid sequence, respectively, as compared to an endogenous reference polynucleotide or amino acid sequence. In another aspect, a mutation comprises a substitution. A “substitution” refers to the replacement of one or more nucleotides or amino acids to a given polynucleotide or amino acid sequence, respectively, as compared to an endogenous reference polynucleotide or amino acid sequence. In another aspect, a mutation comprises an inversion. An “inversion” refers to when a segment of a polynucleotide or amino acid sequence is reversed end-to-end. A “duplication” refers to when a segment of a polynucleotide or amino acid sequence is repeated. The repeated segment can immediately follow the original segment, or it can be separated from the original segment by one or more nucleotides or amino acids. In an aspect, a mutation provided herein comprises a mutation selected from the group consisting of an insertion, a deletion, a substitution, a duplication, and an inversion.
In an aspect, a non-natural mutation comprises a mutation selected from the group consisting of a substitution, a deletion, an insertion, a duplication, and an inversion of one or more nucleotides relative to an endogenous nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-42, 112-139, 155-156, 158-159, and 161-190.
In an aspect, a non-natural mutation comprises a mutation selected from the group consisting of a substitution, a deletion, an insertion, a duplication, and an inversion of one or more nucleotides relative to an endogenous nucleic acid sequence encoding an amino acid sequence selected from the group consisting of SEQ ID NOs: 43-63, 140-153, 157, 160, and 191-205.
In an aspect, a non-natural mutation comprises at least one mutation selected from the group consisting of a nonsense mutation, a missense mutation, a frameshift mutation, a splice-site mutation, and any combinations thereof. As used herein, a “nonsense mutation” refers to a mutation to a nucleic acid sequence that introduces a premature stop codon to an amino acid sequence by the nucleic acid sequence. As used herein, a “missense mutation” refers to a mutation to a nucleic acid sequence that causes a substitution within the amino acid sequence encoded by the nucleic acid sequence. As used herein, a “frameshift mutation” refers to an insertion or deletion to a nucleic acid sequence that shifts the frame for translating the nucleic acid sequence to an amino acid sequence. A “splice-site mutation” refers to a mutation in a nucleic acid sequence that causes an intron to be retained for protein translation, or, alternatively, for an exon to be excluded from protein translation. Splice-site mutations can cause nonsense, missense, or frameshift mutations.
Mutations in coding regions of genes (e.g., exonic mutations) can result in a truncated protein or polypeptide when a mutated messenger RNA (mRNA) is translated into a protein or polypeptide. In an aspect, this disclosure provides a mutation that results in the truncation of a protein or polypeptide. As used herein, a “truncated” protein or polypeptide comprises at least one fewer amino acid as compared to an endogenous control protein or polypeptide. For example, if endogenous Protein A comprises 100 amino acids, a truncated version of Protein A can comprise between 1 and 99 amino acids. In an aspect, a non-natural mutation results in a truncation of a polypeptide.
Without being limited by any scientific theory, one way to cause a protein or polypeptide truncation is by the introduction of a premature stop codon in an mRNA transcript of an endogenous gene. In an aspect, this disclosure provides a mutation that results in a premature stop codon in an mRNA transcript of an endogenous gene. As used herein, a “stop codon” refers to a nucleotide triplet within an mRNA transcript that signals a termination of protein translation. A “premature stop codon” refers to a stop codon positioned earlier (e.g., on the 5′-side) than the normal stop codon position in an endogenous mRNA transcript. Without being limiting, several stop codons are known in the art, including “UAG,” “UAA,” “UGA,” “TAG,” “TAA,” and “TGA.”
In an aspect, a mutation provided herein comprises a null mutation. In an aspect, at least one non-natural mutation provided herein comprises a null mutation. As used herein, a “null mutation” refers to a mutation that confers a complete loss-of-function for a protein encoded by a gene comprising the mutation, or, alternatively, a mutation that confers a complete loss-of-function for a small RNA encoded by a genomic locus. A null mutation can cause lack of mRNA transcript production, a lack of small RNA transcript production, a lack of protein function, or a combination thereof.
A mutation provided herein can be positioned in any part of an endogenous gene. In an aspect, a mutation provided herein is positioned within an exon of an endogenous gene. In another aspect, a mutation provided herein is positioned within an intron of an endogenous gene. In a further aspect, a mutation provided herein is positioned within a 5′-UTR of an endogenous gene. In still another aspect, a mutation provided herein is positioned within a 3′-UTR of an endogenous gene. In yet another aspect, a mutation provided herein is positioned within a promoter of an endogenous gene. In yet another aspect, a mutation provided herein is positioned within a terminator of an endogenous gene. In an aspect, a non-natural mutation provided herein comprises a mutation in a sequence region selected from the group consisting of a promoter, a 5′-UTR, a 3′-UTR, an exon, an intron, and a terminator.
The screening and selection of mutagenized tobacco plants can be through any methodologies known to those having ordinary skill in the art. Examples of screening and selection methodologies include, but are not limited to, Southern analysis, PCR amplification for detection of a polynucleotide, Northern blots, RNase protection, primer-extension, RT-PCR amplification for detecting RNA transcripts, Sanger sequencing, Next Generation sequencing technologies (e.g., Illumina, PacBio, Ion Torrent, 454, and Oxford nanopore) enzymatic assays for detecting enzyme or ribozyme activity of polypeptides and polynucleotides, and protein gel electrophoresis, Western blots, immunoprecipitation, and enzyme-linked immunoassays to detect polypeptides. Other techniques such as in situ hybridization, enzyme staining, and immunostaining, also can be used to detect the presence or expression of polypeptides and/or polynucleotides. Methods for performing all of the referenced and similar techniques are known.
Reduced Expression/ActivityIn an aspect, a small RNA molecule reduces the expression of any nucleic acid sequence to which it is capable of binding. In another aspect, a non-natural mutation provided herein reduces the expression of the mutated nucleic acid sequence as compared to the non-mutated nucleic acid sequence in a control plant grown under comparable conditions.
Reduced expression of an endogenous nucleic acid sequence can be measured using any suitable method known in the art. Non-limiting examples of measuring expression include quantitative reverse transcriptase polymerase chain reaction (qRT-PCR), digital PCR, RNA blot (e.g., a Northern blot), RNA sequencing. Differences in expression can be described as an absolute quantification or a relative quantification. See, for example, Livak and Schmittgen, Methods, 25:402-408 (2001). If an endogenous nucleic acid sequence encodes a protein, changes in expression can be inferred by examining the accumulation of the encoded protein. Non-limiting examples of measuring protein accumulation include Western blots and enzyme-linked immunosorbent assays (ELISAs).
In an aspect, a reduction in expression is measured using qRT-PCR. In another aspect, a reduction in expression is measured using an RNA blot. In another aspect, a reduction in expression is measured using RNA sequencing. In an aspect, a reduction in expression is measured using digital PCR. In a further aspect, a reduction in expression is measured using a Western blot. In yet a further aspect, a reduction in expression is measured using an ELISA.
In an aspect, a non-natural mutation in a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-42, 112-139, 155-156, 158-159, and 161-190 results in a reduced level of expression of the nucleic acid sequence as compared to the nucleic acid sequence lacking the non-natural mutation in a control plant grown under comparable conditions. In an aspect, a non-natural mutation in a nucleic acid sequence encoding an amino acid sequence selected from the group consisting of SEQ ID NOs: 43-63, 140-153, 157, 160, and 191-205 results in a reduced level of expression of the nucleic acid sequence as compared to the nucleic acid sequence lacking the non-natural mutation in a control plant grown under comparable conditions.
In an aspect, a reduction in expression comprises a reduction of at least 1% as compared to expression in the same tissue of a control plant grown under comparable conditions. In another aspect, a reduction in expression comprises a reduction of at least 5% as compared to expression in the same tissue of a control plant grown under comparable conditions. In another aspect, a reduction in expression comprises a reduction of at least 10% as compared to expression in the same tissue of a control plant grown under comparable conditions. In another aspect, a reduction in expression comprises a reduction of at least 25% as compared to expression in the same tissue of a control plant grown under comparable conditions. In another aspect, a reduction in expression comprises a reduction of at least 50% as compared to expression in the same tissue of a control plant grown under comparable conditions. In another aspect, a reduction in expression comprises a reduction of at least 75% as compared to expression in the same tissue of a control plant grown under comparable conditions. In another aspect, a reduction in expression comprises a reduction of at least 90% as compared to expression in the same tissue of a control plant grown under comparable conditions. In another aspect, a reduction in expression comprises a reduction of at least 95% as compared to expression in the same tissue of a control plant grown under comparable conditions.
In an aspect, a reduction in expression comprises a reduction of between 1% and 99% as compared to expression in the same tissue of a control plant grown under comparable conditions. In another aspect, a reduction in expression comprises a reduction of between 1% and 90% as compared to expression in the same tissue of a control plant grown under comparable conditions. In another aspect, a reduction in expression comprises a reduction of between 1% and 75% as compared to expression in the same tissue of a control plant grown under comparable conditions. In another aspect, a reduction in expression comprises a reduction of between 1% and 50% as compared to expression in the same tissue of a control plant grown under comparable conditions. In another aspect, a reduction in expression comprises a reduction of between 1% and 25% as compared to expression in the same tissue of a control plant grown under comparable conditions. In another aspect, a reduction in expression comprises a reduction of between 25% and 90% as compared to expression in the same tissue of a control plant grown under comparable conditions. In another aspect, a reduction in expression comprises a reduction of between 50% and 90% as compared to expression in the same tissue of a control plant grown under comparable conditions. In another aspect, a reduction in expression comprises a reduction of between 25% and 75% as compared to expression in the same tissue of a control plant grown under comparable conditions. In an aspect, a reduction in expression comprises a statistically significant reduction as compared to expression in the same tissue of a control plant grown under comparable conditions.
One of ordinary skill in the art would recognize that any level of reduction is envisioned, so long as the level of reduction has been determined to be statistically significant using an accepted statistical hypothesis test. As a non-limiting example, a Student's t-test is one statistical hypothesis test that can be used to determine if a reduction in expression between a modified plant and a control plant is statistically significant. As used herein, “statistically significant” refers to a p-value of less than or equal to 0.05.
In an aspect, a non-natural mutation results in a reduced level of activity by a protein or polypeptide encoded by a nucleic acid sequence provided herein as compared to the activity of a control plant grown under comparable conditions. In another aspect, a non-natural mutation in an endogenous nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-42, 112-139, 155-156, 158-159, and 161-190 reduces the level of activity by a protein or polypeptide encoded by the nucleic acid sequence as compared to activity of a protein or polypeptide encoded by the endogenous nucleic acid sequence in a control tobacco or Cannabis plant when grown under comparable conditions, where the nucleic acid sequence lacks the non-natural mutation in the control tobacco or Cannabis plant. In another aspect, a non-natural mutation in an endogenous nucleic acid sequence, where the endogenous nucleic acid sequence encodes an amino acid sequence selected from the group consisting of SEQ ID NOs: 43-63, 140-153, 157, 160, and 191-205 reduces the level of activity by a protein or polypeptide encoded by the nucleic acid sequence as compared to activity of a protein or polypeptide encoded by the endogenous nucleic acid sequence in a control tobacco or Cannabis plant when grown under comparable conditions, where the nucleic acid sequence lacks the non-natural mutation in the control tobacco or Cannabis plant.
As used herein, when referring to a protein or polypeptide, “activity” refers to the ability to carry out an enzymatic function. As a non-limiting example, activity of a dehydrogenase protein or polypeptide refers to the ability of a dehydrogenase to remove a hydrogen group from an organic molecule substrate. Without being limited by any scientific theory, if a mutated dehydrogenase protein has reduced activity as compared to a non-mutated control dehydrogenase protein, the mutated dehydrogenase (a) may be unable to remove a hydrogen group from any organic molecule substrate; (b) only be able to remove a hydrogen group from an organic molecule substrate at a reduced rate as compared to a non-mutated dehydrogenase protein; (c) only be able to remove hydrogen groups from substrates that are not suitable substrates for a non-mutated dehydrogenase protein; or (d) a combination of (b) and (c). Conversely, also without being limited by any scientific theory, if a mutated dehydrogenase protein has increased activity as compared to a non-mutated control dehydrogenase protein, the mutated dehydrogenase may be able to (a) remove a hydrogen group from an organic molecule substrate at an increased rate as compared to a non-mutated dehydrogenase protein; (b) be able to remove hydrogen groups from substrates that are not suitable substrates for a non-mutated dehydrogenase; or (c) both (a) and (b). The activity of a dehydrogenase can be measured using techniques standard in the art. For example, see Yu et al., Enzyme Microb. Technol., 49:272-276 (2011); Seely and Pegg, J. Biol. Chem., 258:2496-2500 (1983); and Mizusaki et al., Plant and Cell Physiology, 14:103-110 (1973).
Increased Expression/ActivityIn an aspect, a non-natural mutation results in increased expression of a nucleic acid sequence. In an aspect, a non-natural mutation results in an increased level of expression of said nucleic acid sequence as compared to expression of said nucleic acid sequence in the same tissue of a control tobacco or Cannabis plant when grown under comparable conditions, where said nucleic acid sequence lacks the at least one non-natural mutation in said control tobacco or Cannabis plant.
In an aspect, this disclosure provides a tobacco plant comprising increased expression of a sequence at least 70% identical to a sequence selected from the group consisting of SEQ ID NOs: 1-21, 112-125, 155, 158, and 176-190. In an aspect, this disclosure provides a tobacco plant comprising increased expression of a sequence at least 75% identical to a sequence selected from the group consisting of SEQ ID NOs: 1-21, 112-125, 155, 158, and 176-190. In an aspect, this disclosure provides a tobacco plant comprising increased expression of a sequence at least 80% identical to a sequence selected from the group consisting of SEQ ID NOs: 1-21, 112-125, 155, 158, and 176-190. In an aspect, this disclosure provides a tobacco plant comprising increased expression of a sequence at least 85% identical to a sequence selected from the group consisting of SEQ ID NOs: 1-21, 112-125, 155, 158, and 176-190. In an aspect, this disclosure provides a tobacco plant comprising increased expression of a sequence at least 90% identical to a sequence selected from the group consisting of SEQ ID NOs: 1-21, 112-125, 155, 158, and 176-190. In an aspect, this disclosure provides a tobacco plant comprising increased expression of a sequence at least 92.5% identical to a sequence selected from the group consisting of SEQ ID NOs: 1-21, 112-125, 155, 158, and 176-190. In an aspect, this disclosure provides a tobacco plant comprising increased expression of a sequence at least 95% identical to a sequence selected from the group consisting of SEQ ID NOs: 1-21, 112-125, 155, 158, and 176-190. In an aspect, this disclosure provides a tobacco plant comprising increased expression of a sequence at least 96% identical to a sequence selected from the group consisting of SEQ ID NOs: 1-21, 112-125, 155, 158, and 176-190. In an aspect, this disclosure provides a tobacco plant comprising increased expression of a sequence at least 97% identical to a sequence selected from the group consisting of SEQ ID NOs: 1-21, 112-125, 155, 158, and 176-190. In an aspect, this disclosure provides a tobacco plant comprising increased expression of a sequence at least 98% identical to a sequence selected from the group consisting of SEQ ID NOs: 1-21, 112-125, 155, 158, and 176-190. In an aspect, this disclosure provides a tobacco plant comprising increased expression of a sequence at least 99% identical to a sequence selected from the group consisting of SEQ ID NOs: 1-21, 112-125, 155, 158, and 176-190. In an aspect, this disclosure provides a tobacco plant comprising increased expression of a sequence at least 99.5% identical to a sequence selected from the group consisting of SEQ ID NOs: 1-21, 112-125, 155, 158, and 176-190. In an aspect, this disclosure provides a tobacco plant comprising increased expression of a sequence 100% identical to a sequence selected from the group consisting of SEQ ID NOs: 1-21, 112-125, 155, 158, and 176-190.
In an aspect, this disclosure provides a Cannabis plant comprising increased expression of a sequence at least 70% identical to a sequence selected from the group consisting of SEQ ID NOs: 22-42, 126-139, 156, 159, and 161-175. In an aspect, this disclosure provides a Cannabis plant comprising increased expression of a sequence at least 75% identical to a sequence selected from the group consisting of SEQ ID NOs: 22-42, 126-139, 156, 159, and 161-175. In an aspect, this disclosure provides a Cannabis plant comprising increased expression of a sequence at least 80% identical to a sequence selected from the group consisting of SEQ ID NOs: 22-42, 126-139, 156, 159, and 161-175. In an aspect, this disclosure provides a Cannabis plant comprising increased expression of a sequence at least 85% identical to a sequence selected from the group consisting of SEQ ID NOs: 22-42, 126-139, 156, 159, and 161-175. In an aspect, this disclosure provides a Cannabis plant comprising increased expression of a sequence at least 90% identical to a sequence selected from the group consisting of SEQ ID NOs: 22-42, 126-139, 156, 159, and 161-175. In an aspect, this disclosure provides a Cannabis plant comprising increased expression of a sequence at least 92.5% identical to a sequence selected from the group consisting of SEQ ID NOs: 22-42, 126-139, 156, 159, and 161-175. In an aspect, this disclosure provides a Cannabis plant comprising increased expression of a sequence at least 95% identical to a sequence selected from the group consisting of SEQ ID NOs: 22-42, 126-139, 156, 159, and 161-175. In an aspect, this disclosure provides a Cannabis plant comprising increased expression of a sequence at least 96% identical to a sequence selected from the group consisting of SEQ ID NOs: 22-42, 126-139, 156, 159, and 161-175. In an aspect, this disclosure provides a Cannabis plant comprising increased expression of a sequence at least 97% identical to a sequence selected from the group consisting of SEQ ID NOs: 22-42, 126-139, 156, 159, and 161-175. In an aspect, this disclosure provides a Cannabis plant comprising increased expression of a sequence at least 98% identical to a sequence selected from the group consisting of SEQ ID NOs: 22-42, 126-139, 156, 159, and 161-175. In an aspect, this disclosure provides a Cannabis plant comprising increased expression of a sequence at least 99% identical to a sequence selected from the group consisting of SEQ ID NOs: 22-42, 126-139, 156, 159, and 161-175. In an aspect, this disclosure provides a Cannabis plant comprising increased expression of a sequence at least 99.5% identical to a sequence selected from the group consisting of SEQ ID NOs: 22-42, 126-139, 156, 159, and 161-175. In an aspect, this disclosure provides a Cannabis plant comprising increased expression of a sequence 100% identical to a sequence selected from the group consisting of SEQ ID NOs: 22-42, 126-139, 156, 159, and 161-175.
In an aspect, this disclosure provides a tobacco plant comprising increased expression of a sequence at least 70% identical to a sequence selected from the group consisting of SEQ ID NOs: 22-42, 126-139, 156, 159, and 161-175. In an aspect, this disclosure provides a tobacco plant comprising increased expression of a sequence at least 75% identical to a sequence selected from the group consisting of SEQ ID NOs: 22-42, 126-139, 156, 159, and 161-175. In an aspect, this disclosure provides a tobacco plant comprising increased expression of a sequence at least 80% identical to a sequence selected from the group consisting of SEQ ID NOs: 22-42, 126-139, 156, 159, and 161-175. In an aspect, this disclosure provides a tobacco plant comprising increased expression of a sequence at least 85% identical to a sequence selected from the group consisting of SEQ ID NOs: 22-42, 126-139, 156, 159, and 161-175. In an aspect, this disclosure provides a tobacco plant comprising increased expression of a sequence at least 90% identical to a sequence selected from the group consisting of SEQ ID NOs: 22-42, 126-139, 156, 159, and 161-175. In an aspect, this disclosure provides a tobacco plant comprising increased expression of a sequence at least 92.5% identical to a sequence selected from the group consisting of SEQ ID NOs: 22-42, 126-139, 156, 159, and 161-175. In an aspect, this disclosure provides a tobacco plant comprising increased expression of a sequence at least 95% identical to a sequence selected from the group consisting of SEQ ID NOs: 22-42, 126-139, 156, 159, and 161-175. In an aspect, this disclosure provides a tobacco plant comprising increased expression of a sequence at least 96% identical to a sequence selected from the group consisting of SEQ ID NOs: 22-42, 126-139, 156, 159, and 161-175. In an aspect, this disclosure provides a tobacco plant comprising increased expression of a sequence at least 97% identical to a sequence selected from the group consisting of SEQ ID NOs: 22-42, 126-139, 156, 159, and 161-175. In an aspect, this disclosure provides a tobacco plant comprising increased expression of a sequence at least 98% identical to a sequence selected from the group consisting of SEQ ID NOs: 22-42, 126-139, 156, 159, and 161-175. In an aspect, this disclosure provides a tobacco plant comprising increased expression of a sequence at least 99% identical to a sequence selected from the group consisting of SEQ ID NOs: 22-42, 126-139, 156, 159, and 161-175. In an aspect, this disclosure provides a tobacco plant comprising increased expression of a sequence at least 99.5% identical to a sequence selected from the group consisting of SEQ ID NOs: 22-42, 126-139, 156, 159, and 161-175. In an aspect, this disclosure provides a tobacco plant comprising increased expression of a sequence 100% identical to a sequence selected from the group consisting of SEQ ID NOs: 22-42, 126-139, 156, 159, and 161-175.
In an aspect, this disclosure provides a Cannabis plant comprising increased expression of a sequence at least 70% identical to a sequence selected from the group consisting of SEQ ID NOs: 1-21, 112-125, 155, 158, and 176-190. In an aspect, this disclosure provides a Cannabis plant comprising increased expression of a sequence at least 75% identical to a sequence selected from the group consisting of SEQ ID NOs: 1-21, 112-125, 155, 158, and 176-190. In an aspect, this disclosure provides a Cannabis plant comprising increased expression of a sequence at least 80% identical to a sequence selected from the group consisting of SEQ ID NOs: 1-21, 112-125, 155, 158, and 176-190. In an aspect, this disclosure provides a Cannabis plant comprising increased expression of a sequence at least 85% identical to a sequence selected from the group consisting of SEQ ID NOs: 1-21, 112-125, 155, 158, and 176-190. In an aspect, this disclosure provides a Cannabis plant comprising increased expression of a sequence at least 90% identical to a sequence selected from the group consisting of SEQ ID NOs: 1-21, 112-125, 155, 158, and 176-190. In an aspect, this disclosure provides a Cannabis plant comprising increased expression of a sequence at least 92.5% identical to a sequence selected from the group consisting of SEQ ID NOs: 1-21, 112-125, 155, 158, and 176-190. In an aspect, this disclosure provides a Cannabis plant comprising increased expression of a sequence at least 95% identical to a sequence selected from the group consisting of SEQ ID NOs: 1-21, 112-125, 155, 158, and 176-190. In an aspect, this disclosure provides a Cannabis plant comprising increased expression of a sequence at least 96% identical to a sequence selected from the group consisting of SEQ ID NOs: 1-21, 112-125, 155, 158, and 176-190. In an aspect, this disclosure provides a Cannabis plant comprising increased expression of a sequence at least 97% identical to a sequence selected from the group consisting of SEQ ID NOs: 1-21, 112-125, 155, 158, and 176-190. In an aspect, this disclosure provides a Cannabis plant comprising increased expression of a sequence at least 98% identical to a sequence selected from the group consisting of SEQ ID NOs: 1-21, 112-125, 155, 158, and 176-190. In an aspect, this disclosure provides a Cannabis plant comprising increased expression of a sequence at least 99% identical to a sequence selected from the group consisting of SEQ ID NOs: 1-21, 112-125, 155, 158, and 176-190. In an aspect, this disclosure provides a Cannabis plant comprising increased expression of a sequence at least 99.5% identical to a sequence selected from the group consisting of SEQ ID NOs: 1-21, 112-125, 155, 158, and 176-190. In an aspect, this disclosure provides a Cannabis plant comprising increased expression of a sequence 100% identical to a sequence selected from the group consisting of SEQ ID NOs: 1-21, 112-125, 155, 158, and 176-190.
In another aspect, this disclosure provides increased expression of a sequence encoding a protein at least 70% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 43-63, 140-153, 157, 160, and 191-205. In another aspect, this disclosure provides increased expression of a sequence encoding a protein at least 75% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 43-63, 140-153, 157, 160, and 191-205. In another aspect, this disclosure provides increased expression of a sequence encoding a protein at least 80% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 43-63, 140-153, 157, 160, and 191-205. In another aspect, this disclosure provides increased expression of a sequence encoding a protein at least 85% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 43-63, 140-153, 157, 160, and 191-205. In another aspect, this disclosure provides increased expression of a sequence encoding a protein at least 90% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 43-63, 140-153, 157, 160, and 191-205. In another aspect, this disclosure provides increased expression of a sequence encoding a protein at least 92.5% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 43-63, 140-153, 157, 160, and 191-205. In another aspect, this disclosure provides increased expression of a sequence encoding a protein at least 95% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 43-63, 140-153, 157, 160, and 191-205. In another aspect, this disclosure provides increased expression of a sequence encoding a protein at least 96% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 43-63, 140-153, 157, 160, and 191-205. In another aspect, this disclosure provides increased expression of a sequence encoding a protein at least 97% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 43-63, 140-153, 157, 160, and 191-205. In another aspect, this disclosure provides increased expression of a sequence encoding a protein at least 98% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 43-63, 140-153, 157, 160, and 191-205. In another aspect, this disclosure provides increased expression of a sequence encoding a protein at least 99% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 43-63, 140-153, 157, 160, and 191-205. In another aspect, this disclosure provides increased expression of a sequence encoding a protein at least 99.5% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 43-63, 140-153, 157, 160, and 191-205. In another aspect, this disclosure provides increased expression of a sequence encoding a protein 100% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 43-63, 140-153, 157, 160, and 191-205.
In an aspect, an increased level of expression comprises an increase of at least 1% as compared to expression in the same tissue of a control plant grown under comparable conditions. In an aspect, an increased level of expression comprises an increase of at least 5% as compared to expression in the same tissue of a control plant grown under comparable conditions. In another aspect, an increased level of expression comprises an increase of at least 10% as compared to expression in the same tissue of a control plant grown under comparable conditions. In another aspect, an increased level of expression comprises an increase of at least 25% as compared to expression in the same tissue of a control plant grown under comparable conditions. In another aspect, an increased level of expression comprises an increase of at least 50% as compared to expression in the same tissue of a control plant grown under comparable conditions. In another aspect, an increased level of expression comprises an increase of at least 75% as compared to expression in the same tissue of a control plant grown under comparable conditions. In another aspect, an increased level of expression comprises an increase of at least 100% as compared to expression in the same tissue of a control plant grown under comparable conditions. In another aspect, an increased level of expression comprises an increase of at least 200% as compared to expression in the same tissue of a control plant grown under comparable conditions. In another aspect, an increased level of expression comprises an increase of at least 500% as compared to expression in the same tissue of a control plant grown under comparable conditions.
In an aspect, a non-natural mutation results in an increased level of activity by a protein or polypeptide encoded by said nucleic acid sequence as compared to activity of a protein or polypeptide encoded by said nucleic acid sequence in a control tobacco plant or a control Cannabis plant when grown under comparable conditions, where said nucleic acid sequence lacks the at least one non-natural mutation in said control tobacco or Cannabis plant.
Nucleic Acids and Amino AcidsAs used herein, “heterologous” refers to a sequence (nucleic acid or amino acid) that originates from a foreign species, or, if from the same species, is substantially modified from its native form in composition and/or genomic locus by deliberate human intervention. The term also is applicable to nucleic acid constructs, also referred to herein as “polynucleotide constructs.” In this manner, a “heterologous” nucleic acid construct is intended to mean a construct that originates from a foreign species, or, if from the same species, is substantially modified from its native form in composition and/or genomic locus by deliberate human intervention. Heterologous nucleic acid constructs include, but are not limited to, recombinant nucleotide constructs that have been introduced into a plant or plant part thereof, for example, via transformation methods or subsequent breeding of a transgenic plant with another plant of interest. It will be appreciated that an endogenous promoter can be considered heterologous to an operably linked endogenous gene if the endogenous promoter and endogenous gene are not naturally operably linked (e.g., human intervention is required to put them in operable linkage). As used herein, an “endogenous” nucleic acid sequence refers to a nucleic acid sequence that occurs naturally in the genome of an organism.
In an aspect, a heterologous polynucleotide comprises a gene. In an aspect, a heterologous polynucleotide encodes a small RNA molecule or a precursor thereof. In an aspect, a heterologous polynucleotide encodes a polypeptide.
As used herein, a “gene” refers to a polynucleotide that can produce a functional unit (e.g., without being limiting, for example, a polypeptide, or a small RNA molecule). A gene can comprise a promoter, an enhancer sequence, a leader sequence, a transcriptional start site, a transcriptional stop site, a polyadenylation site, one or more exons, one or more introns, a 5′-UTR, a 3′-UTR, or any combination thereof. A “gene sequence” can comprise a polynucleotide sequence encoding a promoter, an enhancer sequence, a leader sequence, a transcriptional start site, a transcriptional stop site, a polyadenylation site, one or more exons, one or more introns, a 5′-UTR, a 3′-UTR, or any combination thereof. In one aspect, a gene encodes a small RNA molecule or a precursor thereof. In another aspect, a gene encodes a polypeptide.
In an aspect, a gene comprises a nucleic acid sequence at least 70% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-21, 112-125, 155, 158, and 176-190. In an aspect, a gene comprises a nucleic acid sequence at least 75% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-21, 112-125, 155, 158, and 176-190. In an aspect, a gene comprises a nucleic acid sequence at least 80% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-21, 112-125, 155, 158, and 176-190. In an aspect, a gene comprises a nucleic acid sequence at least 85% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-21, 112-125, 155, 158, and 176-190. In an aspect, a gene comprises a nucleic acid sequence at least 90% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-21, 112-125, 155, 158, and 176-190. In an aspect, a gene comprises a nucleic acid sequence at least 92.5% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-21, 112-125, 155, 158, and 176-190. In an aspect, a gene comprises a nucleic acid sequence at least 95% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-21, 112-125, 155, 158, and 176-190. In an aspect, a gene comprises a nucleic acid sequence at least 96% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-21, 112-125, 155, 158, and 176-190. In an aspect, a gene comprises a nucleic acid sequence at least 97% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-21, 112-125, 155, 158, and 176-190. In an aspect, a gene comprises a nucleic acid sequence at least 98% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-21, 112-125, 155, 158, and 176-190. In an aspect, a gene comprises a nucleic acid sequence at least 99% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-21, 112-125, 155, 158, and 176-190. In an aspect, a gene comprises a nucleic acid sequence at least 99.5% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-21, 112-125, 155, 158, and 176-190. In an aspect, a gene comprises a nucleic acid sequence 100% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-21, 112-125, 155, 158, and 176-190.
In an aspect, a gene comprises a nucleic acid sequence at least 70% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 22-42, 126-139, 156, 159, and 161-175. In an aspect, a gene comprises a nucleic acid sequence at least 75% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 22-42, 126-139, 156, 159, and 161-175. In an aspect, a gene comprises a nucleic acid sequence at least 80% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 22-42, 126-139, 156, 159, and 161-175. In an aspect, a gene comprises a nucleic acid sequence at least 85% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 22-42, 126-139, 156, 159, and 161-175. In an aspect, a gene comprises a nucleic acid sequence at least 90% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 22-42, 126-139, 156, 159, and 161-175. In an aspect, a gene comprises a nucleic acid sequence at least 92.5% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 22-42, 126-139, 156, 159, and 161-175. In an aspect, a gene comprises a nucleic acid sequence at least 95% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 22-42, 126-139, 156, 159, and 161-175. In an aspect, a gene comprises a nucleic acid sequence at least 96% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 22-42, 126-139, 156, 159, and 161-175. In an aspect, a gene comprises a nucleic acid sequence at least 97% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 22-42, 126-139, 156, 159, and 161-175. In an aspect, a gene comprises a nucleic acid sequence at least 98% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 22-42, 126-139, 156, 159, and 161-175. In an aspect, a gene comprises a nucleic acid sequence at least 99% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 22-42, 126-139, 156, 159, and 161-175. In an aspect, a gene comprises a nucleic acid sequence at least 99.5% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 22-42, 126-139, 156, 159, and 161-175. In an aspect, a gene comprises a nucleic acid sequence 100% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 22-42, 126-139, 156, 159, and 161-175.
In an aspect, a gene comprises a nucleic acid sequence encoding an amino acid sequence at least 70% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 43-63, 140-153, 157, 160, and 191-205. In an aspect, a gene comprises a nucleic acid sequence encoding an amino acid sequence at least 75% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 43-63, 140-153, 157, 160, and 191-205. In an aspect, a gene comprises a nucleic acid sequence encoding an amino acid sequence at least 80% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 43-63, 140-153, 157, 160, and 191-205. In an aspect, a gene comprises a nucleic acid sequence encoding an amino acid sequence at least 85% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 43-63, 140-153, 157, 160, and 191-205. In an aspect, a gene comprises a nucleic acid sequence encoding an amino acid sequence at least 90% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 43-63, 140-153, 157, 160, and 191-205. In an aspect, a gene comprises a nucleic acid sequence encoding an amino acid sequence at least 92.5% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 43-63, 140-153, 157, 160, and 191-205. In an aspect, a gene comprises a nucleic acid sequence encoding an amino acid sequence at least 95% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 43-63, 140-153, 157, 160, and 191-205. In an aspect, a gene comprises a nucleic acid sequence encoding an amino acid sequence at least 96% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 43-63, 140-153, 157, 160, and 191-205. In an aspect, a gene comprises a nucleic acid sequence encoding an amino acid sequence at least 97% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 43-63, 140-153, 157, 160, and 191-205. In an aspect, a gene comprises a nucleic acid sequence encoding an amino acid sequence at least 98% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 43-63, 140-153, 157, 160, and 191-205. In an aspect, a gene comprises a nucleic acid sequence encoding an amino acid sequence at least 99% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 43-63, 140-153, 157, 160, and 191-205. In an aspect, a gene comprises a nucleic acid sequence encoding an amino acid sequence at least 99.9% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 43-63, 140-153, 157, 160, and 191-205. In an aspect, a gene comprises a nucleic acid sequence encoding an amino acid sequence 100% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 43-63, 140-153, 157, 160, and 191-205.
In an aspect, a polypeptide comprises an amino acid sequence at least 70% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 43-63, 140-153, 157, 160, and 191-205. In an aspect, a polypeptide comprises an amino acid sequence at least 75% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 43-63, 140-153, 157, 160, and 191-205. In an aspect, a polypeptide comprises an amino acid sequence at least 80% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 43-63, 140-153, 157, 160, and 191-205. In an aspect, a polypeptide comprises an amino acid sequence at least 85% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 43-63, 140-153, 157, 160, and 191-205. In an aspect, a polypeptide comprises an amino acid sequence at least 90% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 43-63, 140-153, 157, 160, and 191-205. In an aspect, a polypeptide comprises an amino acid sequence at least 92.5% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 43-63, 140-153, 157, 160, and 191-205. In an aspect, a polypeptide comprises an amino acid sequence at least 95% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 43-63, 140-153, 157, 160, and 191-205. In an aspect, a polypeptide comprises an amino acid sequence at least 96% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 43-63, 140-153, 157, 160, and 191-205. In an aspect, a polypeptide comprises an amino acid sequence at least 97% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 43-63, 140-153, 157, 160, and 191-205. In an aspect, a polypeptide comprises an amino acid sequence at least 98% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 43-63, 140-153, 157, 160, and 191-205. In an aspect, a polypeptide comprises an amino acid sequence at least 99% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 43-63, 140-153, 157, 160, and 191-205. In an aspect, a polypeptide comprises an amino acid sequence at least 99.9% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 43-63, 140-153, 157, 160, and 191-205. In an aspect, a polypeptide comprises an amino acid sequence 100% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 43-63, 140-153, 157, 160, and 191-205.
The terms “percent identity” or “percent identical” as used herein in reference to two or more nucleotide or amino acid sequences is calculated by (i) comparing two optimally aligned sequences (nucleotide or amino acid) over a window of comparison (the “alignable” region or regions), (ii) determining the number of positions at which the identical nucleic acid base (for nucleotide sequences) or amino acid residue (for proteins and polypeptides) occurs in both sequences to yield the number of matched positions, (iii) dividing the number of matched positions by the total number of positions in the window of comparison, and then (iv) multiplying this quotient by 100% to yield the percent identity. If the “percent identity” is being calculated in relation to a reference sequence without a particular comparison window being specified, then the percent identity is determined by dividing the number of matched positions over the region of alignment by the total length of the reference sequence. Accordingly, for purposes of the present application, when two sequences (query and subject) are optimally aligned (with allowance for gaps in their alignment), the “percent identity” for the query sequence is equal to the number of identical positions between the two sequences divided by the total number of positions in the query sequence over its length (or a comparison window), which is then multiplied by 100%.
When percentage of sequence identity is used in reference to amino acids it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the molecule. When sequences differ in conservative substitutions, the percent sequence identity can be adjusted upwards to correct for the conservative nature of the substitution. Sequences that differ by such conservative substitutions are said to have “sequence similarity” or “similarity.”
For optimal alignment of sequences to calculate their percent identity, various pair-wise or multiple sequence alignment algorithms and programs are known in the art, such as ClustalW or Basic Local Alignment Search Tool® (BLAST™), etc., that can be used to compare the sequence identity or similarity between two or more nucleotide or amino acid sequences. Although other alignment and comparison methods are known in the art, the alignment and percent identity between two sequences (including the percent identity ranges described above) can be as determined by the Clustal W algorithm, see, e.g., Chenna et al., “Multiple sequence alignment with the Clustal series of programs,” Nucleic Acids Research 31: 3497-3500 (2003); Thompson et al., “Clustal W: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice,” Nucleic Acids Research 22: 4673-4680 (1994); Larkin M A et al., “Clustal W and Clustal X version 2.0,” Bioinformatics 23: 2947-48 (2007); and Altschul et al. “Basic local alignment search tool.” J. Mol. Biol. 215:403-410 (1990), the entire contents and disclosures of which are incorporated herein by reference.
The terms “percent complementarity” or “percent complementary” as used herein in reference to two nucleotide sequences is similar to the concept of percent identity but refers to the percentage of nucleotides of a query sequence that optimally base-pair or hybridize to nucleotides a subject sequence when the query and subject sequences are linearly arranged and optimally base paired without secondary folding structures, such as loops, stems or hairpins. Such a percent complementarity can be between two DNA strands, two RNA strands, or a DNA strand and a RNA strand. The “percent complementarity” can be calculated by (i) optimally base-pairing or hybridizing the two nucleotide sequences in a linear and fully extended arrangement (i.e., without folding or secondary structures) over a window of comparison, (ii) determining the number of positions that base-pair between the two sequences over the window of comparison to yield the number of complementary positions, (iii) dividing the number of complementary positions by the total number of positions in the window of comparison, and (iv) multiplying this quotient by 100% to yield the percent complementarity of the two sequences. Optimal base pairing of two sequences can be determined based on the known pairings of nucleotide bases, such as G-C, A-T, and A-U, through hydrogen binding. If the “percent complementarity” is being calculated in relation to a reference sequence without specifying a particular comparison window, then the percent identity is determined by dividing the number of complementary positions between the two linear sequences by the total length of the reference sequence. Thus, for purposes of the present application, when two sequences (query and subject) are optimally base-paired (with allowance for mismatches or non-base-paired nucleotides), the “percent complementarity” for the query sequence is equal to the number of base-paired positions between the two sequences divided by the total number of positions in the query sequence over its length, which is then multiplied by 100%.
The use of the term “polynucleotide” or “nucleic acid molecule” is not intended to limit the present disclosure to polynucleotides comprising deoxyribonucleic acid (DNA). For example, ribonucleic acid (RNA) molecules are also envisioned. Those of ordinary skill in the art will recognize that polynucleotides and nucleic acid molecules can comprise ribonucleotides and combinations of ribonucleotides and deoxyribonucleotides. Such deoxyribonucleotides and ribonucleotides include both naturally occurring molecules and synthetic analogues. The polynucleotides of the present disclosure also encompass all forms of sequences including, but not limited to, single-stranded forms, double-stranded forms, hairpins, stem-and-loop structures, and the like. In an aspect, a nucleic acid molecule provided herein is a DNA molecule. In another aspect, a nucleic acid molecule provided herein is an RNA molecule. In an aspect, a nucleic acid molecule provided herein is single-stranded. In another aspect, a nucleic acid molecule provided herein is double-stranded. A nucleic acid molecule can encode a polypeptide or a small RNA.
As used herein, a “recombinant nucleic acid molecule” refers to a nucleic acid molecule formed by laboratory methods of genetic recombination, such as, without being limiting, molecular cloning. Similarly, a “recombinant DNA construct” refers to a DNA molecule formed by laboratory methods of genetic recombination.
In one aspect, this disclosure provides a recombinant nucleic acid molecule comprising a nucleic acid sequence encoding a polypeptide involved in the biosynthesis of at least one terpene operably linked to a heterologous promoter. In one aspect, this disclosure provides a recombinant nucleic acid molecule comprising a nucleic acid sequence encoding a polypeptide involved in the biosynthesis of at least one terpene operably linked to a heterologous promoter. In one aspect, this disclosure provides a recombinant nucleic acid molecule comprising a nucleic acid sequence encoding a polypeptide involved in the biosynthesis of at least one terpene operably linked to a heterologous promoter.
Nucleic acids can be isolated using techniques routine in the art. For example, nucleic acids can be isolated using any method including, without limitation, recombinant nucleic acid technology, and/or the polymerase chain reaction (PCR). General PCR techniques are described, for example in PCR Primer: A Laboratory Manual, Dieffenbach & Dveksler, Eds., Cold Spring Harbor Laboratory Press, 1995. Recombinant nucleic acid techniques include, for example, restriction enzyme digestion and ligation, which can be used to isolate a nucleic acid. Isolated nucleic acids also can be chemically synthesized, either as a single nucleic acid molecule or as a series of oligonucleotides. Non-limiting examples of primers that can be used to amplify specific candidate genes provided in this disclosure are provided in Table 1 (SEQ ID NOs: 64-103).
In an aspect, this disclosure provides methods of detecting mRNA expression of an endogenous gene encoding a polypeptide involved in the biosynthesis of at least one terpene. Messenger RNA (mRNA) levels can be measured by techniques standard in the art, such as reverse transcription polymerase chain reaction (RT-PCR) and microarray analysis. RNA can be reverse transcribed to cDNA (complementary DNA) by methods known in the art. Synthesized cDNA may be amplified by methods known in the art, such as real-time PCR and digital PCR for relative or absolute quantification of mRNA transcript targets.
As used herein, the term “polypeptide” refers to a chain of at least two covalently linked amino acids. Polypeptides can be encoded by polynucleotides provided herein. Proteins provided herein can be encoded by nucleic acid molecules provided herein. Proteins can comprise polypeptides provided herein. As used herein, a “protein” refers to a chain of amino acid residues that is capable of providing structure or enzymatic activity to a cell.
Polypeptides can be purified from natural sources (e.g., a biological sample) by known methods such as DEAE ion exchange, gel filtration, and hydroxyapatite chromatography. A polypeptide also can be purified, for example, by expressing a nucleic acid in an expression vector. In addition, a purified polypeptide can be obtained by chemical synthesis. The extent of purity of a polypeptide can be measured using any appropriate method, e.g., column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.
In one aspect, this disclosure provides methods of detecting recombinant nucleic acids and polypeptides in plant cells. Without being limiting, nucleic acids also can be detected using hybridization. Hybridization between nucleic acids is discussed in detail in Sambrook et al. (1989, Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.). mRNA expression can be localized in tissue samples using in situ hybridization.
In an aspect, this disclosure provides methods for detecting accumulation of a polypeptide in a modified tobacco plant as compared to a control tobacco plant lacking at least one non-natural mutation when grown under comparable conditions. Increased accumulation of a polypeptide in a modified plant is determined relative to detection of the same polypeptide in a control plant lacking the non-natural mutation, and grown under comparable conditions.
Polypeptides can be detected using antibodies. Techniques for detecting polypeptides using antibodies include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations and immunofluorescence. An antibody provided herein can be a polyclonal antibody or a monoclonal antibody. An antibody having specific binding affinity for a polypeptide provided herein can be generated using methods well known in the art. An antibody provided herein can be attached to a solid support such as a microtiter plate using methods known in the art.
Detection (e.g., of an amplification product, of a hybridization complex, of a polypeptide) can be accomplished using detectable labels. The term “label” is intended to encompass the use of direct labels as well as indirect labels. Detectable labels include enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials.
In an aspect, this disclosure provides a modified tobacco or Cannabis plant, tobacco or Cannabis seed, or part thereof, comprising a recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid encoding at least one small RNA molecule capable of binding to and reducing the expression of a nucleic acid sequence encoding a polypeptide involved in the biosynthesis of at least one terpene.
In an aspect, this disclosure provides a small RNA molecule, or a precursor thereof. As used herein, a “small RNA molecule” refers to a non-coding RNA molecule of between 16 nucleotides and 50 nucleotides in length. In an aspect, a small RNA molecule comprises between 16 nucleotides and 40 nucleotides. In another aspect, a small RNA molecule comprises between 16 nucleotides and 30 nucleotides. In another aspect, a small RNA molecule comprises between 18 nucleotides and 50 nucleotides. In another aspect, a small RNA molecule comprises between 18 nucleotides and 40 nucleotides. In another aspect, a small RNA molecule comprises between 18 nucleotides and 30 nucleotides. In another aspect, a small RNA molecule comprises between 18 nucleotides and 25 nucleotides. In another aspect, a small RNA molecule comprises between 20 nucleotides and 28 nucleotides. In another aspect, a small RNA molecule comprises between 20 nucleotides and 24 nucleotides. In another aspect, a small RNA molecule comprises between 21 nucleotides and 23 nucleotides. In another aspect, a small RNA molecule comprises 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides.
In an aspect, a small RNA molecule is at least 75% identical or complementary to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-42, 112-139, 155-156, 158-159, and 161-190. In an aspect, a small RNA molecule is at least 80% identical or complementary to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-42, 112-139, 155-156, 158-159, and 161-190. In an aspect, a small RNA molecule is at least 85% identical or complementary to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-42, 112-139, 155-156, 158-159, and 161-190. In an aspect, a small RNA molecule is at least 90% identical or complementary to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-42, 112-139, 155-156, 158-159, and 161-190. In an aspect, a small RNA molecule is at least 92.5% identical or complementary to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-42, 112-139, 155-156, 158-159, and 161-190. In an aspect, a small RNA molecule is at least 95% identical or complementary to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-42, 112-139, 155-156, 158-159, and 161-190. In an aspect, a small RNA molecule is at least 97.5% identical or complementary to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-42, 112-139, 155-156, 158-159, and 161-190. In an aspect, a small RNA molecule is at least 99% identical or complementary to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-42, 112-139, 155-156, 158-159, and 161-190. In an aspect, a small RNA molecule is 100% identical or complementary to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-42, 112-139, 155-156, 158-159, and 161-190.
In an aspect, a small RNA molecule is at least 75% identical or complementary to a polynucleotide encoding a polypeptide selected from the group consisting of SEQ ID NOs: 43-63, 140-153, 157, 160, and 191-205. In an aspect, a small RNA molecule is at least 80% identical or complementary to a polynucleotide encoding a polypeptide selected from the group consisting of SEQ ID NOs: 43-63, 140-153, 157, 160, and 191-205. In an aspect, a small RNA molecule is at least 85% identical or complementary to a polynucleotide encoding a polypeptide selected from the group consisting of SEQ ID NOs: 43-63, 140-153, 157, 160, and 191-205. In an aspect, a small RNA molecule is at least 90% identical or complementary to a polynucleotide encoding a polypeptide selected from the group consisting of SEQ ID NOs: 43-63, 140-153, 157, 160, and 191-205. In an aspect, a small RNA molecule is at least 92.5% identical or complementary to a polynucleotide encoding a polypeptide selected from the group consisting of SEQ ID NOs: 43-63, 140-153, 157, 160, and 191-205. In an aspect, a small RNA molecule is at least 95% identical or complementary to a polynucleotide encoding a polypeptide selected from the group consisting of SEQ ID NOs: 43-63, 140-153, 157, 160, and 191-205. In an aspect, a small RNA molecule is at least 97.5% identical or complementary to a polynucleotide encoding a polypeptide selected from the group consisting of SEQ ID NOs: 43-63, 140-153, 157, 160, and 191-205. In an aspect, a small RNA molecule is at least 99% identical or complementary to a polynucleotide encoding a polypeptide selected from the group consisting of SEQ ID NOs: 43-63, 140-153, 157, 160, and 191-205. In an aspect, a small RNA molecule is 100% identical or complementary to a polynucleotide encoding a polypeptide selected from the group consisting of SEQ ID NOs: 43-63, 140-153, 157, 160, and 191-205.
In an aspect, a small RNA provided herein comprises a nucleic acid sequence at least 88.7% identical or complementary to at least 18 contiguous nucleotides of a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-42, 112-139, 155-156, 158-159, and 161-190. In another aspect, a small RNA provided herein comprises a nucleic acid sequence at least 94.3% identical or complementary to at least 18 contiguous nucleotides of a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-42, 112-139, 155-156, 158-159, and 161-190. In another aspect, a small RNA provided herein comprises a nucleic acid sequence 100% identical or complementary to at least 18 contiguous nucleotides of a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-42, 112-139, 155-156, 158-159, and 161-190. In another aspect, a small RNA provided herein comprises a nucleic acid sequence at least 85% identical or complementary to at least 20 contiguous nucleotides of a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-42, 112-139, 155-156, 158-159, and 161-190. In another aspect, a small RNA provided herein comprises a nucleic acid sequence at least 90% identical or complementary to at least 20 contiguous nucleotides of a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-42, 112-139, 155-156, 158-159, and 161-190. In another aspect, a small RNA provided herein comprises a nucleic acid sequence at least 95% identical or complementary to at least 20 contiguous nucleotides of a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-42, 112-139, 155-156, 158-159, and 161-190. In another aspect, a small RNA provided herein comprises a nucleic acid sequence 100% identical or complementary to at least 20 contiguous nucleotides of a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-42, 112-139, 155-156, 158-159, and 161-190.
In an aspect, a small RNA molecule is selected from the group consisting of a double-stranded RNA, a small interfering RNA (siRNA), a trans-acting siRNA, and a microRNA (miRNA).
miRNAs are generally of between about 19 to about 25 nucleotides (commonly about 20-24 nucleotides in plants), that guide cleavage in trans of target transcripts, negatively regulating the expression of genes involved in various regulation and development pathways. In some cases, miRNAs serve to guide in-phase processing of siRNA primary transcripts.
Many microRNA genes (MIR genes) have been identified and made publicly available in a database (“miRBase,” available online at microrna[dot]sanger[dot]ac[dot]uk/sequences; also see Griffiths-Jones et al. (2003) Nucleic Acids Res., 31:439-441). MIR genes have been reported to occur in intergenic regions, both isolated and in clusters in the genome, but can also be located entirely or partially within introns of other genes (both protein-coding and non-protein-coding). For a review of miRNA biogenesis, see Kim (2005) Nature Rev. Mol. Cell. Biol., 6:376-385. Transcription of MIR genes can be, at least in some cases, under promotional control of a MIR gene's own promoter. The primary transcript, termed a “pri-miRNA,” can be quite large (several kilobases) and can be polycistronic, containing one or more pre-miRNAs (fold-back structures containing a stem-loop arrangement that is processed to the mature miRNA) as well as the usual 5′ “cap” and polyadenylated tail of an mRNA.
Maturation of a mature miRNA from its corresponding precursors (pri-miRNAs and pre-miRNAs) differs significantly between animals and plants. For example, in plant cells, microRNA precursor molecules are believed to be largely processed to the mature miRNA entirely in the nucleus, whereas in animal cells, the pri-miRNA transcript is processed in the nucleus by the animal-specific enzyme Drosha, followed by export of the pre-miRNA to the cytoplasm where it is further processed to the mature miRNA. Mature miRNAs in plants are typically 21 nucleotides in length.
Transgenic expression of miRNAs (whether a naturally occurring sequence or an artificial sequence) can be employed to regulate expression of the miRNA's target gene or genes. Inclusion of a miRNA recognition site in a transgenically expressed transcript is also useful in regulating expression of the transcript. Recognition sites of miRNAs have been validated in all regions of an mRNA, including the 5′ untranslated region, coding region, and 3′ untranslated region, indicating that the position of the miRNA target site relative to the coding sequence may not necessarily affect suppression. Because miRNAs are important regulatory elements in eukaryotes, transgenic suppression of miRNAs is useful for manipulating biological pathways and responses. Finally, promoters of MIR genes can have very specific expression patterns (e.g., cell-specific, tissue-specific, temporally specific, or inducible), and thus are useful in recombinant constructs to induce such specific transcription of a DNA sequence to which they are operably linked. Various utilities of miRNAs, their precursors, their recognition sites, and their promoters are described in detail in U.S. Patent Application Publication 2006/0200878 A1, incorporated by reference herein. Non-limiting examples of these utilities include: (1) the expression of a native miRNA or miRNA precursor sequence to suppress a target gene; (2) the expression of an artificial miRNA or miRNA precursor sequence to suppress a target gene; (3) expression of a transgene with a miRNA recognition site, where the transgene is suppressed when the mature miRNA is expressed; (4) expression of a transgene driven by a miRNA promoter.
Designing an artificial miRNA sequence can be as simple as substituting sequence that is complementary to the intended target for nucleotides in the miRNA stem region of the miRNA precursor, as demonstrated by Zeng et al. (2002) Mol. Cell, 9:1327-1333. One non-limiting example of a general method for determining nucleotide changes in the native miRNA sequence to produce the engineered miRNA precursor includes the following steps: (a) Selecting a unique target sequence of at least 18 nucleotides specific to the target gene, e.g., by using sequence alignment tools such as BLAST (see, for example, Altschul et al. (1990) J. Mol. Biol., 215:403-410; Altschul et al. (1997) Nucleic Acids Res., 25:3389-3402), for example, of both tobacco cDNA and genomic DNA databases, to identify target transcript orthologues and any potential matches to unrelated genes, thereby avoiding unintentional silencing of non-target sequences; (b) Analyzing the target gene for undesirable sequences (e.g., matches to sequences from non-target species), and score each potential 19-mer segment for GC content, Reynolds score (see Reynolds et al. (2004) Nature Biotechnol., 22:326-330), and functional asymmetry characterized by a negative difference in free energy (“.DELTA..DELTA.G” or “ΔΔG”) (see Khvorova et al. (2003) Cell, 115:209-216). Preferably 19-mers are selected that have all or most of the following characteristics: (1) a Reynolds score>4, (2) a GC content between about 40% to about 60%, (3) a negative ΔΔG, (4) a terminal adenosine, (5) lack of a consecutive run of 4 or more of the same nucleotide; (6) a location near the 3′ terminus of the target gene; (7) minimal differences from the miRNA precursor transcript. Positions at every third nucleotide in an siRNA have been reported to be especially important in influencing RNAi efficacy and an algorithm, “siExplorer” is publicly available at rna[dot]chem[dot]t[dot]u-tokyo[dot]ac[dot]jp/siexplorer.htm (see Katoh and Suzuki (2007) Nucleic Acids Res., 10.1093/nar/gkl1120); (c) Determining the reverse complement of the selected 19-mers to use in making a modified mature miRNA. The additional nucleotide at position 20 is preferably matched to the selected target sequence, and the nucleotide at position 21 is preferably chosen to either be unpaired to prevent spreading of silencing on the target transcript or paired to the target sequence to promote spreading of silencing on the target transcript; and (d) transforming the artificial miRNA into a plant.
In an aspect, a vector can be used for overexpression and knockdown, e.g., of candidate terpene biosynthesis genes. A non-limiting example of a vector is pGWB551. In an aspect, a vector comprises a nucleic acid sequence at least 90% identical to SEQ ID NO: 154. In an aspect, a vector comprises a nucleic acid sequence at least 95% identical to SEQ ID NO: 154. In an aspect, a vector comprises a nucleic acid sequence 100% identical to SEQ ID NO: 154.
ProductsIn an aspect, this disclosure provides plant material from any plant or plant part provided herein. In an aspect, this disclosure provides Cannabis material from any Cannabis plant or Cannabis plant part provided herein. In an aspect, this disclosure provides cured plant material from any plant or plant part provided herein. In an aspect, this disclosure provides cured tobacco material from any tobacco plant or tobacco plant part provided herein. In an aspect, this disclosure provides cured Cannabis material from any Cannabis plant or Cannabis plant part provided herein.
In an aspect, this disclosure provides a method comprising preparing a tobacco product or a Cannabis product using cured tobacco material or Cannabis material from a modified tobacco or Cannabis plant, where the modified tobacco or Cannabis plant comprises a recombinant nucleic acid molecule comprising a nucleic acid sequence encoding a polypeptide involved in the biosynthesis of at least one terpene operably linked to a heterologous promoter. In an aspect, this disclosure provides a method comprising preparing a tobacco product or a Cannabis product using cured tobacco material or Cannabis material from a modified tobacco or Cannabis plant, where the modified tobacco or Cannabis plant comprises at least one non-natural mutation in an endogenous gene encoding a polypeptide involved in the biosynthesis of at least one terpene, where the modified tobacco or Cannabis plant exhibits increased mRNA expression of the endogenous gene or increased accumulation of the polypeptide as compared to a control tobacco or Cannabis plant lacking the at least one non-natural mutation when grown under comparable conditions.
In an aspect, cured tobacco plant material is made by a curing process selected from the group consisting of flue curing, air curing, fire curing, and sun curing. In an aspect, cured tobacco material is made by a curing process selected from the group consisting of flue curing, air curing, fire curing, and sun curing.
“Curing” is the aging process that reduces moisture and brings about the destruction of chlorophyll giving tobacco leaves a golden color and by which starch is converted to sugar. Cured tobacco therefore has a higher reducing sugar content and a lower starch content compared to harvested green leaf. In one aspect, tobacco plants or plant components provided herein can be cured using conventional means, e.g., flue-cured, barn-cured, fire-cured, air-cured or sun-cured. See, for example, Tso (1999, Chapter 1 in Tobacco, Production, Chemistry and Technology, Davis & Nielsen, eds., Blackwell Publishing, Oxford) for a description of different types of curing methods. Cured tobacco is usually aged in a wooden drum (e.g., a hogshead) or cardboard cartons in compressed conditions for several years (e.g., two to five years), at a moisture content ranging from 10% to about 25%. See, U.S. Pat. Nos. 4,516,590 and 5,372,149. Cured and aged tobacco then can be further processed. Further processing includes conditioning the tobacco under vacuum with or without the introduction of steam at various temperatures, pasteurization, and fermentation.
Following Cannabis plant harvest, Cannabis plant material is typically dried, prior to curing. Cannabis can be dried in a dark room with temperatures kept between about 15° C. and about 22° C. and humidity between 45% and 55%, with a fan for gentle air circulation. An initial drying cycle ranges between 5 days and 15 days. Dried Cannabis can then be cured to increase flavor, aroma, and potency. Length of Cannabis curing is strain-dependent. For example, some Cannabis strains benefit from 2 to 3 weeks of curing, other Cannabis strains benefit from 4 to 8 weeks of curing, while other Cannabis strains benefit from 6 months or more of curing.
Information regarding the harvesting of burley and dark tobacco varieties can be found in the 2019-2020 Burley and Dark Tobacco Production Guide (December 2018) published by the University of Kentucky, The University of Tennessee, Virginia Tech, and North Carolina State University, which is incorporated herein by reference in its entirety.
In an aspect, cured tobacco material comprises tobacco material selected from the group selected from cured leaf material, cured stem material, cured bud material, cured flower material, and cured root material. In an aspect, cured tobacco material comprises cured leaf material, cured stem material, or both. In an aspect, cured tobacco material comprises cured leaf material. In an aspect, cured tobacco material comprises cured stem material.
In an aspect, Cannabis material comprises Cannabis material selected from the group selected from leaf material, stem material, bud material, flower material, root material, hairy roots, cell culture, and callus. In an aspect, Cannabis material comprises leaf material, stem material, or both. In an aspect, Cannabis material comprises leaf material. In an aspect, Cannabis material comprises stem material.
In an aspect, cured tobacco material comprises flue-cured tobacco material. In an aspect, cured tobacco material comprises air-cured tobacco material. In an aspect, cured tobacco material comprises fire-cured tobacco material. In an aspect, cured tobacco material comprises sun-cured tobacco material. In an aspect, cured tobacco material provided herein is selected from the group consisting of air-cured tobacco material, fire-cured tobacco material, sun-cured tobacco material, and flue-cured tobacco material. In an aspect, cured tobacco material is from a tobacco variety selected from the group consisting of a flue-cured variety, a bright variety, a Burley variety, a Virginia variety, a Maryland variety, a dark variety, an Oriental variety, and a Turkish variety.
In an aspect, cured tobacco leaf provided herein is selected from the group consisting of air-cured tobacco leaf, fire-cured tobacco leaf, sun-cured tobacco leaf, and flue-cured tobacco leaf. In an aspect, cured tobacco leaf is from a tobacco variety selected from the group consisting of a flue-cured variety, a bright variety, a Burley variety, a Virginia variety, a Maryland variety, a dark variety, an Oriental variety, and a Turkish variety.
Fermentation typically is characterized by high initial moisture content, heat generation, and a 10 to 20% loss of dry weight. See, for example, U.S. Pat. Nos. 4,528,993, 4,660,577, 4,848,373, 5,372,149; U.S. Publication No. 2005/0178398; and Tso (1999, Chapter 1 in Tobacco, Production, Chemistry and Technology, Davis & Nielsen, eds., Blackwell Publishing, Oxford). Cured, aged, and fermented tobacco can be further processed (e.g., cut, shredded, expanded, or blended). See, for example, U.S. Pat. Nos. 4,528,993; 4,660,577; and 4,987,907. Cured, aged, and fermented Cannabis can be further processed (e.g., cut, shredded, expanded, or blended). In an aspect, this disclosure provides fermented tobacco material from any tobacco plant, or part thereof, provided herein. In another aspect, this disclosure provides fermented tobacco material from any modified tobacco plant, or part thereof, provided herein. In an aspect, this disclosure provides fermented Cannabis material from any Cannabis plant, or part thereof, provided herein. In another aspect, this disclosure provides fermented Cannabis material from any modified Cannabis plant, or part thereof, provided herein.
Tobacco material obtained from the tobacco lines, varieties or hybrids of the present disclosure can be used to make tobacco products. As used herein, “tobacco product” is defined as any product made or derived from tobacco that is intended for human use or consumption. In an aspect, this disclosure provides a tobacco product comprising plant material from a tobacco plant provided herein. In another aspect, this disclosure provides a tobacco product comprising plant material from a modified tobacco plant provided herein. In another aspect, this disclosure provides a tobacco product comprising cured tobacco material. In another aspect, this disclosure provides a tobacco product comprising fermented tobacco material. In another aspect, this disclosure provides a tobacco product comprising a tobacco blend.
Cannabis material obtained from the Cannabis lines, strains, varieties or hybrids of the present disclosure can be used to make Cannabis products. As used herein, “Cannabis product” is defined as any product made or derived from Cannabis that is intended for human use or consumption. In an aspect, this disclosure provides a Cannabis product comprising plant material from a Cannabis plant provided herein. In another aspect, this disclosure provides a Cannabis product comprising plant material from a modified Cannabis plant provided herein. In another aspect, this disclosure provides a Cannabis product comprising Cannabis material. In another aspect, this disclosure provides a Cannabis product comprising cured Cannabis material. In another aspect, this disclosure provides a Cannabis product comprising fermented Cannabis material. In another aspect, this disclosure provides a Cannabis product comprising a Cannabis blend.
Tobacco products include, without limitation, cigarette products (e.g., cigarettes and bidi cigarettes), cigar products (e.g., cigar wrapping tobacco and cigarillos), pipe tobacco products, products derived from tobacco, tobacco-derived nicotine products, smokeless tobacco products (e.g., moist snuff, dry snuff, and chewing tobacco), films, chewables, tabs, shaped parts, gels, consumable units, insoluble matrices, hollow shapes, reconstituted tobacco, expanded tobacco, and the like. See, e.g., U.S. Patent Publication No. US 2006/0191548.
As used herein, “cigarette” refers a tobacco product having a “rod” and “filler”. The cigarette “rod” includes the cigarette paper, filter, plug wrap (used to contain filtration materials), tipping paper that holds the cigarette paper (including the filler) to the filter, and all glues that hold these components together. The “filler” includes (1) all tobaccos, including but not limited to reconstituted and expanded tobacco, (2) non-tobacco substitutes (including but not limited to herbs, non-tobacco plant materials and other spices that may accompany tobaccos rolled within the cigarette paper), (3) casings, (4) flavorings, and (5) all other additives (that are mixed into tobaccos and substitutes and rolled into the cigarette).
In an aspect, a tobacco product comprises reconstituted tobacco. In another aspect, this disclosure provides reconstituted tobacco comprising cured tobacco material. As used herein, “reconstituted tobacco” refers to a part of tobacco filler made from tobacco dust and other tobacco scrap material, processed into sheet form and cut into strips to resemble tobacco. In addition to the cost savings, reconstituted tobacco is very important for its contribution to cigarette taste from processing flavor development using reactions between ammonia and sugars.
In an aspect, a Cannabis product comprises reconstituted Cannabis. In another aspect, this disclosure provides reconstituted Cannabis comprising Cannabis material. As used herein, “reconstituted Cannabis” refers to a part of Cannabis filler made from Cannabis dust and other Cannabis scrap material, processed into sheet form and cut into strips to resemble Cannabis.
In an aspect, a tobacco product comprises expanded tobacco. As used herein, “expanded tobacco” refers to a part of tobacco filler which is processed through expansion of suitable gases so that the tobacco is “puffed” resulting in reduced density and greater filling capacity. It reduces the weight of tobacco used in cigarettes.
In an aspect, a Cannabis product comprises expanded Cannabis. As used herein, “expanded Cannabis” refers to a part of Cannabis filler which is processed through expansion of suitable gases so that the Cannabis is “puffed” resulting in reduced density and greater filling capacity.
Tobacco products derived from plants of the present disclosure also include cigarettes and other smoking articles, particularly those smoking articles including filter elements, where the rod of smokable material includes cured tobacco within a tobacco blend. In an aspect, a tobacco product of the present disclosure is selected from the group consisting of a kretek, a bidi cigarette, a cigarillo, a non-ventilated recess filter cigarette, a vented recess filter cigarette, a cigar, snuff, pipe tobacco, cigar tobacco, cigarette tobacco, chewing tobacco, leaf tobacco, hookah tobacco, shredded tobacco, and cut tobacco.
Cannabis products derived from plants of the present disclosure also include cigarettes and other smoking articles, particularly those smoking articles including filter elements, where the rod of smokable material includes cured Cannabis within a Cannabis blend. In an aspect, a Cannabis product of the present disclosure is selected from the group consisting of a kretek, a cigarillo, a cigar, snuff, pipe Cannabis, cigar Cannabis, cigarette Cannabis, chewing Cannabis, hookah Cannabis, shredded Cannabis, and cut Cannabis.
In an aspect, a tobacco product of the present disclosure is selected from the group consisting of a cigarette, a heated tobacco product, a kretek, a bidi cigarette, a cigar, a cigarillo, a non-ventilated cigarette, a vented recess filter cigarette, pipe tobacco, snuff, snus, chewing tobacco, moist smokeless tobacco, fine cut chewing tobacco, long cut chewing tobacco, pouched chewing tobacco product, gum, a tablet, a lozenge, and a dissolving strip.
In an aspect, a Cannabis product of the present disclosure is selected from the group consisting of a cigarette, a heated Cannabis product, a kretek, a cigar, a cigarillo, pipe Cannabis, snuff, snus, chewing Cannabis, moist smokeless Cannabis, fine cut chewing Cannabis, long cut chewing Cannabis, pouched chewing Cannabis product, gum, a tablet, a lozenge, and a dissolving strip.
In an aspect, a Cannabis product of the present disclosure is a consumable product. In a further aspect, a consumable product is an edible product. In an aspect, a consumable product is an oil.
In an aspect, a tobacco product of the present disclosure is a smokeless tobacco product. In an aspect, a smokeless tobacco product is selected from the group consisting of loose leaf chewing tobacco, plug chewing tobacco, moist snuff, nasal snuff, dry snuff, and snus.
In an aspect, a Cannabis product of the present disclosure is a smokeless Cannabis product. In an aspect, a smokeless Cannabis product is selected from the group consisting of chewing Cannabis, moist snuff, dry snuff, and snus.
Smokeless tobacco or Cannabis products are not combusted and include, but not limited to, chewing tobacco, moist smokeless tobacco, moist smokeless Cannabis, snus, and dry snuff. Chewing tobacco or Cannabis is coarsely divided tobacco or Cannabis leaf that is typically packaged in a large pouch-like package and used in a plug or twist. Moist smokeless tobacco or Cannabis is a moist, more finely divided tobacco or Cannabis that is provided in loose form or in pouch form and is typically packaged in round cans and used as a pinch or in a pouch placed between an adult tobacco or Cannabis consumer's cheek and gum. Snus is a heat-treated smokeless tobacco or Cannabis. Dry snuff is finely ground tobacco or Cannabis that is placed in the mouth or used nasally.
In yet another aspect, a tobacco or Cannabis product of the present disclosure is selected from the group consisting of an electronically heated cigarette, an e-cigarette, an electronic vaporing device.
In an aspect, a tobacco or Cannabis product of the present disclosure can be a blended tobacco or Cannabis product.
In another aspect, this disclosure provides a tobacco blend comprising cured tobacco material. A tobacco blend can comprise any combination of cured tobacco, uncured tobacco, fermented tobacco, unfermented tobacco, expanded tobacco, and reconstituted tobacco.
In another aspect, this disclosure provides a Cannabis blend comprising Cannabis material. A Cannabis blend can comprise any combination of fermented Cannabis, unfermented Cannabis, expanded Cannabis, and reconstituted Cannabis.
In an aspect, a tobacco blend comprises at least 5% cured tobacco by weight. In an aspect, a tobacco blend comprises at least 10% cured tobacco by weight. In an aspect, a tobacco blend comprises at least 15% cured tobacco by weight. In an aspect, a tobacco blend comprises at least 20% cured tobacco by weight. In an aspect, a tobacco blend comprises at least 25% cured tobacco by weight. In an aspect, a tobacco blend comprises at least 30% cured tobacco by weight. In an aspect, a tobacco blend comprises at least 35% cured tobacco by weight. In an aspect, a tobacco blend comprises at least 40% cured tobacco by weight. In an aspect, a tobacco blend comprises at least 45% cured tobacco by weight. In an aspect, a tobacco blend comprises at least 50% cured tobacco by weight. In an aspect, a tobacco blend comprises at least 55% cured tobacco by weight. In an aspect, a tobacco blend comprises at least 60% cured tobacco by weight. In an aspect, a tobacco blend comprises at least 65% cured tobacco by weight. In an aspect, a tobacco blend comprises at least 70% cured tobacco by weight. In an aspect, a tobacco blend comprises at least 75% cured tobacco by weight. In an aspect, a tobacco blend comprises at least 80% cured tobacco by weight. In an aspect, a tobacco blend comprises at least 85% cured tobacco by weight. In an aspect, a tobacco blend comprises at least 90% cured tobacco by weight. In an aspect, a tobacco blend comprises at least 95% cured tobacco by weight.
In an aspect, a Cannabis blend comprises at least 5% Cannabis by weight. In an aspect, a Cannabis blend comprises at least 10% Cannabis by weight. In an aspect, a Cannabis blend comprises at least 15% Cannabis by weight. In an aspect, a Cannabis blend comprises at least 20% Cannabis by weight. In an aspect, a Cannabis blend comprises at least 25% Cannabis by weight. In an aspect, a Cannabis blend comprises at least 30% Cannabis by weight. In an aspect, a Cannabis blend comprises at least 35% Cannabis by weight. In an aspect, a Cannabis blend comprises at least 40% Cannabis by weight. In an aspect, a Cannabis blend comprises at least 45% Cannabis by weight. In an aspect, a Cannabis blend comprises at least 50% Cannabis by weight. In an aspect, a Cannabis blend comprises at least 55% Cannabis by weight. In an aspect, a Cannabis blend comprises at least 60% Cannabis by weight. In an aspect, a Cannabis blend comprises at least 65% Cannabis by weight. In an aspect, a Cannabis blend comprises at least 70% Cannabis by weight. In an aspect, a Cannabis blend comprises at least 75% Cannabis by weight. In an aspect, a Cannabis blend comprises at least 80% Cannabis by weight. In an aspect, a Cannabis blend comprises at least 85% Cannabis by weight. In an aspect, a Cannabis blend comprises at least 90% Cannabis by weight. In an aspect, a Cannabis blend comprises at least 95% Cannabis by weight.
In an aspect, a tobacco blend comprises at least 5% cured tobacco by volume. In an aspect, a tobacco blend comprises at least 10% cured tobacco by volume. In an aspect, a tobacco blend comprises at least 15% cured tobacco by volume. In an aspect, a tobacco blend comprises at least 20% cured tobacco by volume. In an aspect, a tobacco blend comprises at least 25% cured tobacco by volume. In an aspect, a tobacco blend comprises at least 30% cured tobacco by volume. In an aspect, a tobacco blend comprises at least 35% cured tobacco by volume. In an aspect, a tobacco blend comprises at least 40% cured tobacco by volume. In an aspect, a tobacco blend comprises at least 45% cured tobacco by volume. In an aspect, a tobacco blend comprises at least 50% cured tobacco by volume. In an aspect, a tobacco blend comprises at least 55% cured tobacco by volume. In an aspect, a tobacco blend comprises at least 60% cured tobacco by volume. In an aspect, a tobacco blend comprises at least 65% cured tobacco by volume. In an aspect, a tobacco blend comprises at least 70% cured tobacco by volume. In an aspect, a tobacco blend comprises at least 75% cured tobacco by volume. In an aspect, a tobacco blend comprises at least 80% cured tobacco by volume. In an aspect, a tobacco blend comprises at least 85% cured tobacco by volume. In an aspect, a tobacco blend comprises at least 90% cured tobacco by volume. In an aspect, a tobacco blend comprises at least 95% cured tobacco by volume.
In an aspect, a Cannabis blend comprises at least 5% Cannabis by volume. In an aspect, a Cannabis blend comprises at least 10% Cannabis by volume. In an aspect, a Cannabis blend comprises at least 15% Cannabis by volume. In an aspect, a Cannabis blend comprises at least 20% Cannabis by volume. In an aspect, a Cannabis blend comprises at least 25% Cannabis by volume. In an aspect, a Cannabis blend comprises at least 30% Cannabis by volume. In an aspect, a Cannabis blend comprises at least 35% Cannabis by volume. In an aspect, a Cannabis blend comprises at least 40% Cannabis by volume. In an aspect, a Cannabis blend comprises at least 45% Cannabis by volume. In an aspect, a Cannabis blend comprises at least 50% Cannabis by volume. In an aspect, a Cannabis blend comprises at least 55% Cannabis by volume. In an aspect, a Cannabis blend comprises at least 60% Cannabis by volume. In an aspect, a Cannabis blend comprises at least 65% Cannabis by volume. In an aspect, a Cannabis blend comprises at least 70% Cannabis by volume. In an aspect, a Cannabis blend comprises at least 75% Cannabis by volume. In an aspect, a Cannabis blend comprises at least 80% Cannabis by volume. In an aspect, a Cannabis blend comprises at least 85% Cannabis by volume. In an aspect, a Cannabis blend comprises at least 90% Cannabis by volume. In an aspect, a Cannabis blend comprises at least 95% Cannabis by volume.
In an aspect, this disclosure provides a method comprising preparing a tobacco or Cannabis product using cured tobacco or Cannabis material from a modified tobacco or Cannabis plant, where the modified tobacco or Cannabis plant comprises a recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid encoding at least one small RNA molecule capable of binding to and reducing the expression of a nucleic acid sequence encoding a polypeptide involved in the biosynthesis of at least one terpene.
EMBODIMENTSThe following examples of non-limiting embodiments are envisioned:
- 1. A modified tobacco or Cannabis plant, tobacco or Cannabis seed, or part thereof comprising a recombinant nucleic acid molecule comprising a nucleic acid sequence encoding a polypeptide involved in the biosynthesis of at least one terpene operably linked to a heterologous promoter.
- 2. A modified tobacco or Cannabis plant, tobacco or Cannabis seed, or part thereof comprising at least one non-natural mutation in an endogenous gene encoding a polypeptide involved in the biosynthesis of at least one terpene, where the modified tobacco or Cannabis plant exhibits increased mRNA expression of the endogenous gene or increased accumulation of the polypeptide as compared to a control tobacco or Cannabis plant lacking the at least one non-natural mutation when grown under comparable conditions.
- 3. A modified tobacco or Cannabis plant, tobacco or Cannabis seed, or part thereof, comprising a recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid encoding at least one small RNA molecule capable of binding to and reducing the expression of a nucleic acid sequence encoding a polypeptide involved in the biosynthesis of at least one terpene.
- 4. The modified tobacco or Cannabis plant, tobacco or Cannabis seed, or part thereof of any one of embodiments [0059]-3, where the polypeptide is selected from the group consisting of geranyl diphosphate synthase (GDP), limonene synthase (LS), limonene 3-hydroxylase (L30H), isopiperitenol dehydrogenase (IPD), pulegone reductase, menthofuran synthase (MFS), geranylgeranyl diphosphate synthase (GGPPS), neomenthol dehydrogenase (NtNMD), phylloplanin, premnaspirodiene oxygenase (PSO), kolavenyl diphosphate synthase (KPS), solanesyl phosphate synthase 3, terpene synthase 10-like, and germacrene D synthase.
- 5. The modified tobacco or Cannabis plant, tobacco or Cannabis seed, or part thereof of any one of embodiments [0059]-3, where the at least one terpene is menthol or a related compound.
- 6. The modified tobacco or Cannabis plant, tobacco or Cannabis seed, or part thereof of any one of embodiments [0059]-3, where the polypeptide is selected from the group consisting of geranylgeranyl diphosphate synthase (GGPPS2), 8-hydroxy-copalyl diphosphate synthase, and cis-abienol synthase.
- 7. The modified tobacco or Cannabis plant, tobacco or Cannabis seed, or part thereof of embodiment 6, where the cis-abienol synthase is selected from the group consisting of cis-abienol synthase ISOFORM1 and cis-abienol synthase ISOFORM2.
- 8. The modified tobacco or Cannabis plant, tobacco or Cannabis seed, or part thereof of any one of embodiments [0059]-3, where the at least one terpene is a labdanoid.
- 9. The modified tobacco or Cannabis plant, tobacco or Cannabis seed, or part thereof of any one of embodiments [0059]-3, where the at least one terpene is neophytadiene.
- 10. The modified tobacco or Cannabis plant, tobacco or Cannabis seed, or part thereof of embodiment 8, where the labdanoid is cis-abienol.
- 11. The modified tobacco or Cannabis plant, tobacco or Cannabis seed, or part thereof of any one of embodiments [0059]-3, where the polypeptide is cembratrienol synthase 2a.
- 12. The modified tobacco or Cannabis plant, tobacco or Cannabis seed, or part thereof of any one of embodiments [0059]-3, where the at least one terpene is cembratrienediol.
- 13. The modified tobacco or Cannabis plant, tobacco or Cannabis seed, or part thereof of any one of embodiments [0059]-3, where the polypeptide is levopimaradiene synthetase.
- 14. The modified tobacco or Cannabis plant, tobacco or Cannabis seed, or part thereof of any one of embodiments [0059]-3, where the at least one terpene is levopimaric acid.
- 15. The modified tobacco or Cannabis plant, tobacco or Cannabis seed, or part thereof of any one of embodiments [0059]-3, where the polypeptide is selected from the group consisting of 2-isopropylmalate synthetase (IPS), 2-oxoisovalerate dehydrogenase, and 2-alkenal reductase.
- 16. The modified tobacco or Cannabis plant, tobacco or Cannabis seed, or part thereof of any one of embodiments [0059]-3, where the at least one terpene is L-leucine.
- 17. The modified tobacco or Cannabis plant, tobacco or Cannabis seed, or part thereof of embodiment [0059], where the nucleic acid sequence comprises a sequence at least 70% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-21, 112-125, 155, 158, and 176-190.
- 18. The modified tobacco or Cannabis plant, tobacco or Cannabis seed, or part thereof of embodiment 2, where the endogenous gene comprises a sequence at least 70% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 22-42, 126-139, 156, 159, and 161-175.
- 19. The modified tobacco or Cannabis plant, tobacco or Cannabis seed, or part thereof of any one of embodiments [0059]-3, where the polypeptide comprises an amino acid sequence at least 70% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 43-63, 140-153, 157, 160, and 191-205.
- 20. The modified tobacco or Cannabis plant, tobacco or Cannabis seed, or part thereof of any one of embodiments [0059]-3, where the heterologous promoter comprises a constitutive promoter.
- 21. The modified tobacco or Cannabis plant, tobacco or Cannabis seed, or part thereof of any one of embodiments [0059]-3, where the heterologous promoter comprises a tissue-preferred promoter or tissue-specific promoter.
- 22. The modified tobacco or Cannabis plant, tobacco or Cannabis seed, or part thereof of embodiment 21, where the tissue-preferred promoter or tissue-specific promoter comprises a trichome-preferred promoter or a trichome-specific promoter.
- 23. The modified tobacco or Cannabis plant, tobacco or Cannabis seed, or part thereof of any one of embodiments [0059]-3, where the heterologous promoter comprises a nucleic acid sequence at least 90% identical to SEQ ID NOs: 104-111, 206-208, or a functional fragment thereof.
- 24. The modified tobacco or Cannabis plant, tobacco or Cannabis seed, or part thereof of embodiment [0059], where the modified tobacco or Cannabis plant comprises an increased amount of the at least one terpene as compared to a control tobacco or Cannabis plant lacking the recombinant nucleic acid molecule when grown under comparable conditions.
- 25. The modified tobacco or Cannabis plant, tobacco or Cannabis seed, or part thereof of embodiment 2, where the modified tobacco or Cannabis plant comprises an increased amount of the at least one terpene as compared to the control tobacco or Cannabis plant.
- 26. The modified tobacco or Cannabis plant, tobacco or Cannabis seed, or part thereof of embodiment 24 or 25, where the increased amount of the at least one terpene comprises an increase of at least 5% as compared to the control tobacco or Cannabis plant.
- 27. The modified tobacco plant, tobacco seed, or part thereof of any one of embodiments [0059]-3, where the tobacco plant, tobacco seed, or part thereof is of a tobacco variety selected from the group consisting of a flue-cured variety, a bright variety, a Burley variety, a Virginia variety, a Maryland variety, a dark variety, a Galpão variety, an Oriental variety, and a Turkish variety.
- 28. The modified tobacco or Cannabis plant, tobacco or Cannabis seed, or part thereof of any one of embodiments [0059]-3, where the tobacco or Cannabis plant is male sterile or cytoplasmically male sterile.
- 29. The modified tobacco or Cannabis plant, tobacco or Cannabis seed, or part thereof, of embodiment 2, where the modified tobacco or Cannabis plant, tobacco or Cannabis seed, or part thereof is homozygous for the at least one non-natural mutation.
- 30. The modified tobacco or Cannabis plant, tobacco or Cannabis seed, or part thereof, of embodiment 2, where the modified tobacco or Cannabis plant, tobacco or Cannabis seed, or part thereof is heterozygous for the at least one non-natural mutation.
- 31. The modified tobacco or Cannabis plant, tobacco or Cannabis seed, or part thereof, of embodiment 2, where the at least one non-natural mutation comprises a mutation selected from the group consisting of an insertion, a deletion, a substitution, a duplication, and an inversion.
- 32. The modified tobacco or Cannabis plant, tobacco or Cannabis seed, or part thereof, of embodiment 2, where the at least one non-natural mutation comprises at least one mutation selected from the group consisting of a nonsense mutation, a missense mutation, a frameshift mutation, a splice-site mutation, and any combination thereof.
- 33. The modified tobacco or Cannabis plant, tobacco or Cannabis seed, or part thereof, of embodiment 2, where the at least one non-natural mutation comprises a null mutation.
- 34. The modified tobacco or Cannabis plant, tobacco or Cannabis seed, or part thereof, of embodiment 2, where the at least one non-natural mutation results in a truncation of the polypeptide.
- 35. The modified tobacco or Cannabis plant, tobacco or Cannabis seed, or part thereof, of embodiment 2, where the at least one non-natural mutation comprises a mutation in a sequence region selected from the group consisting of a promoter, a 5′-untranslated region (UTR), an exon, an intron, a 3′ -UTR, and a terminator.
- 36. Cured tobacco material or Cannabis material from the modified tobacco or Cannabis plant, tobacco or Cannabis seed, or part thereof of any one of embodiments [0059]-35.
- 37. The cured tobacco material of embodiment 36, where the cured tobacco material is made by a curing process selected from the group consisting of flue curing, air curing, fire curing, and sun curing.
- 38. A tobacco product comprising the cured tobacco material or the Cannabis material of embodiment 36 or 37, where the tobacco or Cannabis product is selected from the group consisting of a kretek, a bidi cigarette, a cigarillo, a non-ventilated recess filter cigarette, a vented recess filter cigarette, a cigar, snuff, pipe tobacco, pipe Cannabis, cigar tobacco, cigar Cannabis, cigarette tobacco, cigarette Cannabis, chewing tobacco, leaf tobacco, hookah tobacco, hookah Cannabis, shredded tobacco, shredded Cannabis, cut tobacco, and cut Cannabis.
- 39. The tobacco or Cannabis product of embodiment 38, where the tobacco or Cannabis product is a smokeless tobacco or Cannabis product.
- 40. The tobacco or Cannabis product of embodiment 39, where the smokeless tobacco or Cannabis product is selected from the group consisting of loose leaf chewing tobacco, loose leaf chewing Cannabis, plug chewing tobacco, plug chewing Cannabis, moist snuff, nasal snuff, dry snuff, and snus.
- 41. A reconstituted tobacco or Cannabis comprising the cured tobacco material or the Cannabis material of embodiment 36 or 37.
- 42. A fermented tobacco or a fermented Cannabis comprising the cured tobacco material or the Cannabis material of embodiment 36 or 37.
- 43. The tobacco or Cannabis product of embodiment 38, where the tobacco or Cannabis product comprises reconstituted tobacco or reconstituted Cannabis.
- 44. The tobacco or Cannabis product of embodiment 38, where the tobacco or Cannabis product comprises fermented tobacco or fermented Cannabis.
- 45. A recombinant nucleic acid molecule comprising a nucleic acid sequence encoding a polypeptide involved in the biosynthesis of at least one terpene operably linked to a heterologous promoter.
- 46. A method of producing a modified tobacco or Cannabis plant comprising: (a) introducing a recombinant nucleic acid molecule to at least one tobacco or Cannabis cell, where the recombinant nucleic acid molecule comprises a nucleic acid sequence encoding a polypeptide involved in terpene biosynthesis operably linked to a heterologous promoter; (b) selecting at least one tobacco or Cannabis cell comprising the recombinant nucleic acid molecule; and (c) regenerating a modified tobacco or Cannabis plant from the at least one tobacco or Cannabis cell selected in step (b) where the modified tobacco or Cannabis plant comprises an increased amount of at least one terpene in at least one tissue as compared to a control tobacco or Cannabis plant lacking the recombinant nucleic acid molecule when grown under comparable conditions.
- 47. A method of producing a modified tobacco or Cannabis plant comprising: (a) inducing at least one non-natural mutation in at least one tobacco or Cannabis cell in an endogenous gene encoding a polypeptide involved in terpene biosynthesis; (b) selecting at least one tobacco or Cannabis plant comprising the at least one non-natural mutation from step (a); and (c) regenerating a modified tobacco or Cannabis plant from the at least one tobacco or Cannabis cell selected in step (b), where the modified tobacco or Cannabis plant comprises an increased amount of at least one terpene in at least one tissue as compared to a control tobacco or Cannabis plant lacking the at least one non-natural mutation when grown under comparable conditions.
- 48. A method of producing a modified tobacco or Cannabis plant comprising: (a) introducing a recombinant DNA construct to at least one tobacco or Cannabis cell, where said recombinant DNA construct comprises a heterologous promoter operably linked to a nucleic acid encoding at least one small RNA molecule capable of binding to and reducing the expression of a nucleic acid sequence encoding a polypeptide involved in the biosynthesis of at least one terpene; (b) selecting at least one tobacco or Cannabis cell comprising said recombinant DNA construct; and (c) regenerating at least one modified tobacco or Cannabis plant from said at least one tobacco or Cannabis cell selected in step (b), where the modified tobacco or Cannabis plant comprises a reduced amount of at least one terpene in at least one tissue as compared to a control tobacco or Cannabis plant lacking the recombinant DNA construct when grown under comparable conditions.
- 49. A method comprising preparing a tobacco or Cannabis product using cured tobacco material from a modified tobacco or Cannabis plant, where the modified tobacco or Cannabis plant comprises a recombinant nucleic acid molecule comprising a nucleic acid sequence encoding a polypeptide involved in the biosynthesis of at least one terpene operably linked to a heterologous promoter.
- 50. A method comprising preparing a tobacco or Cannabis product using cured tobacco or Cannabis material from a modified tobacco or Cannabis plant, where the modified tobacco or Cannabis plant comprises at least one non-natural mutation in an endogenous gene encoding a polypeptide involved in the biosynthesis of at least one terpene, where the modified tobacco or Cannabis plant exhibits increased mRNA expression of the endogenous gene or increased accumulation of the polypeptide as compared to a control tobacco or Cannabis plant lacking the at least one non-natural mutation when grown under comparable conditions.
- 51. A method comprising preparing a tobacco or Cannabis product using cured tobacco or Cannabis material from a modified tobacco or Cannabis plant, where the modified tobacco or Cannabis plant comprises a recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid encoding at least one small RNA molecule capable of binding to and reducing the expression of a nucleic acid sequence encoding a polypeptide involved in the biosynthesis of at least one terpene.
- 52. A method comprising transforming a tobacco or Cannabis cell with a recombinant nucleic acid molecule, where the recombinant nucleic acid molecule comprising a nucleic acid sequence encoding a polypeptide involved in the biosynthesis of at least one terpene operably linked to a heterologous promoter.
- 53. A method comprising transforming a tobacco or Cannabis cell with a recombinant nucleic acid molecule, where the recombinant nucleic acid molecule comprises a heterologous promoter operably linked to a nucleic acid encoding at least one small RNA molecule capable of binding to and reducing the expression of a nucleic acid sequence encoding a polypeptide involved in the biosynthesis of at least one terpene.
- 54. A method for producing a tobacco or Cannabis plant, the method comprising: (a) crossing at least one tobacco or Cannabis plant of a first tobacco variety with at least one tobacco or Cannabis plant of a second tobacco or Cannabis variety to produce at least one progeny tobacco or Cannabis seed, where the at least one tobacco or Cannabis plant of the first tobacco or Cannabis variety comprises a recombinant nucleic acid molecule comprising a nucleic acid sequence encoding a polypeptide involved in the biosynthesis of at least one terpene operably linked to a heterologous promoter; and (b) selecting for at least one progeny tobacco or Cannabis seed, or a plant germinated therefrom, where the at least one progeny tobacco or Cannabis seed or plant germinated therefrom comprises the recombinant nucleic acid molecule.
- 55. A method for producing a tobacco or Cannabis plant, the method comprising: (a) crossing at least one tobacco or Cannabis plant of a first tobacco or Cannabis variety with at least one tobacco plant of a second tobacco or Cannabis variety to produce at least one progeny tobacco or Cannabis seed, wherein the at least one tobacco or Cannabis plant of the first tobacco or Cannabis variety comprises a recombinant nucleic acid molecule comprising a heterologous promoter operably linked to a nucleic acid encoding at least one small RNA molecule capable of binding to and reducing the expression of a nucleic acid sequence encoding a polypeptide involved in the biosynthesis of at least one terpene; and (b) selecting for at least one progeny tobacco seed, or a plant germinated therefrom, wherein the at least one progeny tobacco or Cannabis seed or plant germinated therefrom comprises the recombinant nucleic acid molecule.
- 56. A method for producing a tobacco or Cannabis plant, the method comprising: (a) crossing at least one tobacco or Cannabis plant of a first tobacco variety with at least one tobacco or Cannabis plant of a second tobacco or Cannabis variety to produce at least one progeny tobacco or Cannabis seed, where the at least one tobacco or Cannabis plant of the first tobacco variety comprises at least one non-natural mutation in an endogenous gene encoding a polypeptide involved in the biosynthesis of at least one terpene, where the at least one tobacco or Cannabis plant of the first tobacco or Cannabis variety exhibits increased mRNA expression of the endogenous gene or increased accumulation of the polypeptide as compared to a control tobacco or Cannabis plant of the first tobacco or Cannabis variety lacking the at least one non-natural mutation when grown under comparable conditions; and (b) selecting for at least one progeny tobacco or Cannabis seed, or a plant germinated therefrom, where the at least one progeny tobacco or Cannabis seed or plant germinated therefrom comprises the at least one non-natural mutation.
- 57. The method of any one of embodiments 54-56, where the first tobacco variety and the second tobacco variety are the same tobacco variety, or the first Cannabis variety and the second Cannabis variety are the same Cannabis variety.
- 58. The method of any one of embodiments 54-55, where the at least one tobacco or Cannabis plant of a second tobacco or Cannabis variety comprises the recombinant nucleic acid molecule.
- 59. The method of any one of embodiments 54-55, where the at least one progeny tobacco or Cannabis seed, or the plant germinated therefrom, is heterozygous for the recombinant nucleic acid molecule.
- 60. The method of any one of embodiments 54-55, where the at least one progeny tobacco or Cannabis seed, or the plant germinated therefrom, is homozygous for the recombinant nucleic acid molecule.
- 61. The method of embodiment 56, where the at least one tobacco or Cannabis plant of a second tobacco or Cannabis variety comprises the at least one non-natural mutation.
- 62. The method of embodiment 56, where the at least one progeny tobacco or Cannabis seed, or the plant germinated therefrom, is heterozygous for the at least one non-natural mutation.
- 63. The method of embodiment 56, where the at least one progeny tobacco or Cannabis seed, or the plant germinated therefrom, is homozygous for the at least one non-natural mutation.
Having now generally described the disclosure, the same will be more readily understood through reference to the following examples that are provided by way of illustration, and are not intended to be limiting of the present disclosure, unless specified.
EXAMPLES Example 1. Identification of Candidate Trichome/Terpenoid GenesProteins that carry out the biosynthetic steps for producing for several terpenes (see
Expression profiles of the candidate genes identified in Example 1 are examined in tobacco trichomes. See
Expression profiles of the candidate genes identified in Example 1 are examined in flowers and leaves from tobacco cultivars TN90 and K326 and N. benthamiana at various stages of tobacco plant development.
Neomenthol dehydrogenase (NMD; SEQ ID NO: 15) and cis-abienol (ABS) isoforms 1 (SEQ ID NO: 4) and 2 (SEQ ID NO: 5) transcript abundance is performed using RNA sequencing at different stages of plant development in TN90 and K326 tobacco cultivars.
Approximately 22 to 25 plants per variety are grown in two rows. The experiment is triplicated with randomized design. Because flowering time varies from plant to plant, all plants are topped when at least 50% of the plants start to flower. Plants close to uniform sizes are selected for sampling at different time points. Assessed plant samples include flower (before topping) and leaf at a variety of post topping stages (e.g., 1 day post-topping; 3 days post-topping; 1 week post-topping; 2 weeks post-topping; 3 weeks post-topping; and harvest stage). See
Candidate genes identified in Example 1 encoding enzymes in the cis-abienol biosynthetic pathway (e.g., geranylgeranyl diphosphate synthase (GGPPS2) (SEQ ID NOs: 1 and 113), 8-hydroxy-copalyl diphosphate synthase (SEQ ID NOs: 3 and 115), cis-abienol synthase isoform 1 (SEQ ID NO:4), and cis-abienol synthase isoform 2 (SEQ ID NO: 5)) are cloned into constructs.
Separate transformation vectors comprising one each of SEQ ID NOs: 1-21, 112-125, 155, 158, and 176-190 under the control of a CaMV 35S promoter are constructed using methods known in the art and vector pGWB551 (SEQ ID NO: 154). A representative vector construct used to transform tobacco or Cannabis plants is depicted in
Each vector construct generated is separately transformed into tobacco or Cannabis cells in separate experiments, using methods standard in the art. Briefly, vectors are introduced into tobacco or Cannabis leaf discs via Agrobacterium-mediated transformation. See, for example, Mayo et al., Nat. Protoc., 1:1105-1111 (2006); and Horsch et al., Science, 227:1229-1231 (1985).
Representative images of calli from control and transgenic tobacco lines expressing GFP in different backgrounds (Nb=N. benthamiana; TN90=tobacco cultivar ‘TN90’) are provided in
Tobacco genotypes (TN90 and K326) and N. benthamiana plants are transformed with vector constructs as described in Example 4 are grown in Magenta™ GA-7 boxes and leaf discs are cut and placed into Petri plates. Agrobacterium tumefaciens cells comprising a transformation vector are collected by centrifuging a 20 mL cell suspension in a 50 mL centrifuge tube at 3500 RPM for 10 minutes. The supernatant is removed, and the Agrobacterium tumefaciens cell pellet is re-suspended in 40 mL liquid re-suspension medium. Tobacco leaves, avoiding the midrib, are cut into eight 0.6 cm discs with a #15 razor blade and placed upside down in a Petri plate. A thin layer of Murashige & Skoog (MS) with B5 vitamin liquid re-suspension medium is added to the Petri plate and the leaf discs are poked uniformly with a fine point needle. About 25 mL of the Agrobacterium tumefaciens suspension is added to the Petri plate and the leaf discs are incubated in the suspension for 10 minutes.
Leaf discs are transferred to co-cultivation Petri plates (½ MS medium) and discs are placed upside down in contact with filter paper overlaid on the co-cultivation TOM medium (MS medium with 30 g/L sucrose; 0.1 mg/L indole-3-acetic acid; and 1 mg/L 6-benzyl aminopurine (BAP)). The Petri plate is sealed with parafilm prior to incubation in dim light (between 60 and 80 mE/ms) with photoperiods of 18 hours light, 6 hours dark, at 24° C. for two days.
After incubation, leaf discs are transferred to regeneration/selection TOM Hyg medium Petri plates (TOM medium plus 200 mg/L cefotaxime and 50 mg/L hygromycin). Leaf discs are sub-cultured bi-weekly to fresh TOM K medium in dim light (between 60 and 80 mE/ms) with photoperiods of 18 hours light, 6 hours dark, at 24° C. until shoots become excisable. Shoots from leaves are removed with forceps and inserted in MS rooting medium (MS Gamborg B5, 3 g/L sucrose and 7 g/L dextrose) with 50 mg/L hygromycin and 200 mg/L cefotaxime. Shoots on MS rooting medium with 50 mg/L hygromycin and 200 mg/L cefotaxime are incubated under light (approximately 60-80 mE/ms) with photoperiods of 18 hours light, 6 hours dark, at 24° C. to induce rooting.
When plantlets comprising both shoots and roots grow large enough (e.g., over half the height of a Magenta™ GA-7 box), they are transferred to Jiffy peat pellet for acclimatization in the growth room. Once established, seedlings are transferred to a greenhouse for further growth, breeding, and analysis.
Example 6. Confirming Expression of Terpene Biosynthesis Genes in Modified Tobacco PlantsRNA is isolated from young leaves of modified tobacco plants (To generation) generated in Example 5 during the vegetative stage of growth. RNA is also isolated from control tobacco plants lacking the recombinant nucleic acid constructs grown under comparable conditions. Total RNA is isolated using TRIzol™, and cDNA is synthesized using methods known in the art. Terpenoid gene expression in different transgenic lines of each gene construct is quantified via quantitative real-time PCR (qRT-PCR). Primers specific for candidate genes are provided in Table 1 (SEQ ID NOs: 64-103).
Gene expression of NtNMD (SEQ ID NO: 15), NtaABS (SEQ ID NO: 4), and NtABS (SEQ ID NO: 5) in modified tobacco plants is quantified using qRT-PCR and compared to NtNMD, NtaABS, and NtABS expression in control tobacco plants. Relative gene expression of cis-abienol synthase isoform 1 (aABS) (SEQ ID NO: 4) and cis-abienol synthase isoform 2 (ABS) (SEQ ID NO: 5) are calculated for T0 lines in transgenic tobacco plants. See
Quantitative metabolic profile analysis is performed using T0 transgenic plants generated in Example 5 during the vegetative growth stage. Leaf samples from young or old leaves, separately, are ground in liquid nitrogen for solvent extraction of terpenes. Ground leaves are mixed with 60:40 hexane: ethyl acetate solvent mixture (v/v) supplemented with heptadecanol as an internal standard and then incubated overnight in a shaker. The solvent extract is concentrated in a refrigerated SpeedVac and placed into a silica column. The column is washed with hexane and allowed to flow through collection tubes. Samples are aliquoted from the flow through and used for gas chromatography mass spectrometry (GC-MS) metabolite analysis.
Elevated levels of neophytadiene are observed in transgenic tobacco plants overexpressing NtNMD (SEQ TD NO: 15). See
Neomenthol, as detected by GC-MS, hyperaccumulates in transgenic tobacco plants overexpressing NtNMD (SEQ TD NO: 15), compared to TN9 control plants. See Table 9.
Enhanced volatile flavor compounds, as detected by GC-MS, accumulate in transgenic tobacco plants overexpressing NtABS (SEQ TD NO: 5) and NtaABS (SEQ TD NO: 4). See Table 10.
Mutations are produced in each of the genes identified in Example 1 by specifically editing SEQ ID NOs: 1-42 112-139, 155-156, 158-159, and 161-190, separately, in the tobacco or Cannabis genomes. Tobacco or Cannabis protoplasts are transfected using polyethylene glycol (PEG) with plasmids encoding a CRISPR protein or a CRISPR protein and specific guide RNA (gRNA) targeting individual genes at desired positions.
Transfected protoplasts are then immobilized in 1% agarose beads and subjected to tissue culture. When calli grow to approximately 1 millimeter in diameter, they are spread on TOM2 plates. Calli are screened for mutations (e.g., insertions or deletions (indels)) at the target positions using fragment analysis. Candidates, showing size shifts compared to wildtype control, are selected for further culture and the consequent shoots are tested by fragment analysis again to confirm the presence of mutations.
Modified tobacco or Cannabis plants (To generation) are grown as described in Example 5. Then, terpenoid levels are measured as described in Example 7.
Example 9. Knockdown of Candidate Genes Using Small RNA Molecules in Tobacco and CannabisReducing the expression of genes identified in Example 1 is tested for their effect on the levels of specific terpenoids.
Separate transformation vectors comprising an artificial miRNA designed to reduce the transcription or translation of one each of SEQ ID NOs: 1-42, 112-139, 155-156, 158-159, and 161-190 driven by CaMV 35S are constructed.
Modified tobacco plants (To generation) are grown as described in Example 5. Then, terpenoid levels are measured as described in Example 7.
Example 10. Expressing Terpenoid Biosynthesis Genes with Trichome-Specific PromotersTrichome-specific promoters are used to drive the expression of genes involved in terpenoid biosynthesis. Constructs are produced as described in Example 4, with each trichome-specific promoter (e.g., SEQ ID NOs: 104-111 and 206-208, see Table 1) driving the expression of neomenthol dehydrogenase (NtNMD; SEQ ID NO: 15) or one of two isoforms of cis-abienol synthase (NtaABS(Isoform 1; SEQ ID NO: 4) and NtABS(Isoform 2; SEQ ID NO: 5) or a homolog thereof, for example, cis-abienol synthase (e.g. SEQ ID NO: 119), in separate constructs.
Each of the constructs are separately transformed into tobacco or Cannabis cells and modified tobacco or Cannabis plants are regenerated as described in Example 5. Modified tobacco or Cannabis plants are tested for terpenes as provided in Example 7.
Claims
1. A modified tobacco or Cannabis plant, tobacco or Cannabis seed, or part thereof comprising a recombinant nucleic acid molecule comprising a nucleic acid sequence encoding a polypeptide involved in the biosynthesis of at least one terpene operably linked to a heterologous promoter.
2. A modified tobacco or Cannabis plant, tobacco or Cannabis seed, or part thereof comprising at least one non-natural mutation in an endogenous gene encoding a polypeptide involved in the biosynthesis of at least one terpene, wherein the modified tobacco or Cannabis plant exhibits increased mRNA expression of the endogenous gene or increased accumulation of the polypeptide as compared to a control tobacco or Cannabis plant lacking the at least one non-natural mutation when grown under comparable conditions.
3. A modified tobacco or Cannabis plant, tobacco or Cannabis seed, or part thereof, comprising a recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid molecule comprising a nucleic acid sequence encoding at least one small RNA molecule capable of binding to and reducing the expression of a nucleic acid sequence encoding a polypeptide involved in the biosynthesis of at least one terpene.
4. The modified tobacco or Cannabis plant, tobacco or Cannabis seed, or part thereof of claim 1, wherein the polypeptide is selected from the group consisting of geranyl diphosphate synthase (GDP), limonene synthase (LS), limonene 3-hydroxylase (L30H), isopiperitenol dehydrogenase (IPD), pulegone reductase, menthofuran synthase (MFS), geranylgeranyl diphosphate synthase (GGPPS), neomenthol dehydrogenase (NtNMD), phylloplanin, premnaspirodiene oxygenase (PSO), kolavenyl diphosphate synthase (KPS), solanesyl phosphate synthase 3, terpene synthase 10-like, germacrene D synthase, geranylgeranyl diphosphate synthase (GGPPS2), 8-hydroxy-copalyl diphosphate synthase, cis-abienol synthase, cembratrienol synthase 2a, levopimaradiene synthetase, 2-isopropylmalate synthetase (IPS), 2-oxoisovalerate dehydrogenase, and 2-alkenal reductase.
5. The modified tobacco or Cannabis plant, tobacco or Cannabis seed, or part thereof of claim 1, wherein the at least one terpene is selected from the group consisting of menthol or a related compound, a labdanoid, neophytadiene, cembratrienediol, levopimaric acid, and L-leucine.
6. (canceled)
7. The modified tobacco or Cannabis plant, tobacco or Cannabis seed, or part thereof of claim 4, wherein the cis-abienol synthase is selected from the group consisting of cis-abienol synthase ISOFORM1 and cis-abienol synthase ISOFORM2.
8. (canceled)
9. (canceled)
10. The modified tobacco or Cannabis plant, tobacco or Cannabis seed, or part thereof of claim 5, wherein the labdanoid is cis-abienol.
11.-16. (canceled)
17. The modified tobacco or Cannabis plant, tobacco or Cannabis seed, or part thereof of claim 1, wherein the nucleic acid sequence comprises a sequence at least 70% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-21, 112-125, 155, 158, and 176-190.
18. The modified tobacco or Cannabis plant, tobacco or Cannabis seed, or part thereof of claim 2, wherein the endogenous gene comprises a sequence at least 70% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 22-42, 126-139, 156, 159, and 161-175.
19. The modified tobacco or Cannabis plant, tobacco or Cannabis seed, or part thereof of claim 1, wherein the polypeptide comprises an amino acid sequence at least 70% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 43-63, 140-153, 157, 160, and 191-205.
20. The modified tobacco or Cannabis plant, tobacco or Cannabis seed, or part thereof of claim 1, wherein the heterologous promoter comprises a constitutive promoter.
21. The modified tobacco or Cannabis plant, tobacco or Cannabis seed, or part thereof of claim 1, wherein the heterologous promoter comprises a tissue-preferred promoter or tissue-specific promoter.
22. The modified tobacco or Cannabis plant, tobacco or Cannabis seed, or part thereof of claim 21, wherein the tissue-preferred promoter or tissue-specific promoter comprises a trichome-preferred promoter or a trichome-specific promoter.
23. The modified tobacco or Cannabis plant, tobacco or Cannabis seed, or part thereof of claim 1, wherein the heterologous promoter comprises a nucleic acid sequence at least 90% identical to SEQ ID NOs: 104-111, 206-208, or a functional fragment thereof.
24. The modified tobacco or Cannabis plant, tobacco or Cannabis seed, or part thereof of claim 1, wherein the modified tobacco or Cannabis plant comprises an increased amount of the at least one terpene as compared to a control tobacco or Cannabis plant lacking the recombinant nucleic acid molecule when grown under comparable conditions.
25. (canceled)
26. The modified tobacco or Cannabis plant, tobacco or Cannabis seed, or part thereof of claim 24, wherein the increased amount of the at least one terpene comprises an increase of at least 5% as compared to the control tobacco or Cannabis plant.
27. The modified tobacco plant, tobacco seed, or part thereof of claim 1, wherein the tobacco plant, tobacco seed, or part thereof is of a tobacco variety selected from the group consisting of a flue-cured variety, a bright variety, a Burley variety, a Virginia variety, a Maryland variety, a dark variety, a Galpão variety, an Oriental variety, and a Turkish variety.
28. The modified tobacco or Cannabis plant, tobacco or Cannabis seed, or part thereof of claim 1, wherein the tobacco or Cannabis plant is male sterile or cytoplasmically male sterile.
29.-35. (canceled)
36. Cured tobacco material or Cannabis material from the modified tobacco or Cannabis plant, tobacco or Cannabis seed, or part thereof of claim 1.
37. The cured tobacco material of claim 36, wherein the cured tobacco material is made by a curing process selected from the group consisting of flue curing, air curing, fire curing, and sun curing.
38.-63. (canceled)
Type: Application
Filed: Feb 2, 2022
Publication Date: Aug 4, 2022
Inventors: Chengalrayan KUDITHIPUDI (Midlothian, VA), Yanxin SHEN (Henrico, VA), Dongmei XU (Glen Allen, VA), Michael Paul TIMKO (Charlottesville, VA), Roel RABARA (Charlottesville, VA)
Application Number: 17/591,033
