USE OF PLASTID TRANSIT PEPTIDES DERIVED FROM GLAUCOCYSTOPHYTES
Compositions and methods for targeting polypeptides to plastids are provided. Compositions comprise plastid transit peptides as well as nucleotide sequences encoding such transit peptides and variants thereof. Compositions further comprise DNA constructs comprising a nucleotide sequence encoding the plastid transit peptide operably linked to a nucleotide sequence encoding a polypeptide of interest. These DNA constructs find use in expression and targeting of the polypeptide of interest to a plastid. Compositions also comprise expression cassettes, vectors, transformed plants, transformed plant cells, and stably transformed plant seeds wherein a polypeptide of interest is targeted to a plastid by the plastid transit peptide of the invention.
This application claims priority from U.S. Provisional Application No. 61/024,844, filed Jan. 30, 2008. This application also claims priority from U.S. Provisional Application No. 61/047,692, filed Apr. 24, 2008.
REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLYThe official copy of the sequence listing is submitted concurrently with the specification as a text file via EFS-Web, in compliance with the American Standard Code for Information Interchange (ASCII), with a file name of “Sequence Listing 71999.txt”, a creation date of Jan. 10, 2009, and a size of 7.25 KB. The sequence listing filed via EFS-Web is part of the specification and is hereby incorporated in its entirety by reference herein.
FIELD OF THE INVENTIONThe present invention relates to the genetic modification of plants, particularly to the transport of polypeptides of interest to plant plastids.
BACKGROUND OF THE INVENTIONThe plastid contains it own genome. However, during evolution many of the plastid genes were transferred to the nuclear genome. Thus, a mechanism for the transport of the nuclear encoded plastid proteins back to the plastid was developed. The proteins are transported through the use of transit peptide sequences located in the N-terminus of the transported proteins. These peptides direct the proteins to the plastid and can be subsequently recognized by specific proteases which can lead to removal of the transit peptide.
A fundamental problem in cell biology is the precise and efficient targeting of proteins synthesized by cytoplasmic ribosomes to their appropriate intracellular locations. This is especially true for transgenic higher plants where the transgene product is needed in an appropriate cellular organelle. The present invention provides plastid transit peptides that efficiently transport a heterologous polypeptide into the chloroplast of transgenic higher plants. Surprisingly, this can be accomplished using a cyanelle transit peptide derived from a Glaucocystophyte.
BRIEF SUMMARY OF THE INVENTIONCompositions and methods for targeting polypeptides to chloroplasts are provided. Compositions comprise transit peptides as well as nucleotide sequences encoding a transit peptide (i.e., plastid targeting peptide or transport peptide) and variants thereof. Compositions further comprise DNA constructs comprising a nucleotide sequence encoding the transit peptide operably linked to a nucleotide sequence encoding a heterologous polypeptide. These DNA constructs find use in expression and targeting of the heterologous polypeptide to a plastid. Compositions also comprise expression cassettes, vectors, transformed plants, transformed plant cells, and stably transformed plant seeds comprising a polynucleotide encoding a heterologous polypeptide which is targeted to a chloroplast by the plastid transit peptide of the invention.
DETAILED DESCRIPTION OF THE INVENTIONThe plastid targeting or transport peptides of the invention mediate targeting, localization, or transport of operably linked polypeptides into plastids. Plastids are organelles found in plants and algae. Plastids are responsible for photosynthesis, storage of products such as starch, and for the synthesis of many classes of molecules such as fatty acids, terpenes, and other molecules which are needed as cellular building blocks. Plastids have the ability to differentiate, or redifferentiate, into several forms depending upon their role in the cell. Undifferentiated proplastids may develop into any of the following plastids, including, chloroplasts, chromoplasts, leucoplasts, amyloplasts, statoliths, elaioplasts, and proteinoplasts.
A “chloroplast transit peptide” or “plastid transport peptide” or “plastid transit peptide” or “plastid targeting peptide” or “transit peptide” or “targeting peptide” is necessary and sufficient to facilitate the import of a protein into the plastid of its native host cell. The plastid may be a cyanelle or a chloroplast. Transit peptides are located at the N-terminal end of the proteins imported into the plastids. The transit peptide facilitates co-translational or post-translational transport of an operably linked polypeptide into a plastid.
These transit peptides generally comprise between 40 and 100 amino acids. Studies indicate that transit peptides contain common characteristics. These include: they are virtually devoid of negatively charged amino acids, such as aspartic acid, glutamic acid, asparagine or glutamine; the N-terminal region is devoid of charged amino acids, and of amino acids such as glycine or proline; their central region contains a very high proportion of basic or hydroxylated amino acids, such as serine or threonine; and, their C-terminal region is rich in arginine and has the ability to form an amphipathic beta-sheet secondary structure.
According to one authority (Cline and Henry, Annu Rev Cell Dev Biol 12: 1-26 (1996)) chloroplast transit peptides from higher plants share the following characteristics: (1) They have superficially similar properties to mitochondrial transit peptides. That is they are rich in hydroxylated residues and poor in acidic residues. (2) They are 30-120 residues long. (3) The N-terminal 10-15 amino acids are devoid of glycine, proline and charged residues. (4) The variable, middle region is rich in serine, threonine, lysine and arginine. (5) The C-proximal region contains the loosely conserved sequence (Ile/Val-x-Ala/Cys*Ala) for proteolytic processing. (6) There is no extended sequence conservation or conserved secondary structural motifs (7) They, theoretically, adopt a predominantly random coil conformation.
Several computational approaches exist which use the above features to predict chloroplast targeting sequences from higher plants. Computational tools include PSORT (Nakai and Kanehisa, Proteins 11(2): 95-110 (1991); Horton et al., Proceedings of the 4th Annual Asia Pacific Bioinformatics Conference APBC06, Taipei, Taiwan pp. 39-48 (2006); http://www.psort.org), ChloroP (Emanuelsson et al., J Mol Biol 300: 1005-1016 (1999); http://www.cbs.dtu.dk/services/ChloroP/) or TargetP (Nielsen et al., Protein Engineer 10:1-6 (1997); Emanuelsson et al., J Mol Biol 300: 1005-1016 (2000); http://www.cbs.dtu.dk/services/TargetP/).
Glaucocystophytes are a family of algae that contain cyanelles as a photosynthetic organelle. Several genera exist within the Glaucocystophytes including Cyanophora, Glaucocystis and Gloeochaete. Cyanelles are in essence primitive plastids. Unlike other eukaryotic plastids, cyanelles have a peptidoglycan layer which is believed to be a relic of the endosymbiotic origin of plastids from cyanobacteria. Glaucocystophytes contain the photosynthetic pigment chlorophyll a. Along with red algae and cyanobacteria they harvest light via phycobilisomes, structures consisting largely of phycobiliproteins. The green algae and land plants have lost that pigment.
Amongst all of the platids, Glaucocystophyte cyanelles are the closest relatives to the free-living cyanobacteria. The presence of the peptidioglycan layer in the cell wall of the cyanelles is likely to be a key component of the protein import machinery of the cyanelle. See Steiner and Loffelhardt, Molecular Membrane Biology 22(1-2): 123-132 (2005) for a review of cyanelle protein translocation. The chloroplasts of higher plants have lost the peptidioglycan layer and thus differ in their membrane structure from cyanelles. It was further demonstrated that the import machinery of cyanelles and higher plants differ as the cyanelle targeted protein, ferredoxin-NADP+-oxidoreductase, was imported into isolated pea and spinach chloroplasts (Steiner and Loffelhardt, Trends Plant Sci 7: 72-77 (2002)); however, higher plant chloroplast targeted proteins were not functional with cyanelles (Steiner and Loffelhardt Mol. Memb. Biol. 22:123-132 (2005)).
Cyanelle transit peptide sequences differ from chloroplast stroma targeting peptides in containing in their N-terminal domain an invariant phenylalanine residue which is crucial for import by the cyanelle. See, for example, Steiner and Loffelhardt Trends Plant Sci. 7:72-77 (2002); and Steiner and Loffelhardt Mol. Memb. Biol. 22:123-132 (2005). The invariant phenylalanine residue is unique to cyanelles and the rhodoplast (red algae plastids) and is not a feature of the plastid transit peptide in the green lineage organisms (green algae, Euglena, diatoms and higher plants). The cyanelle transit sequences correspond to stroma-targeting peptides from higher plants in their amino acid composition, in their positive net charge and in their domain structure. The N-termini of cyanelle transit peptides show a short consensus sequence. See, Steiner et al. (2005) The Plant J. 44:646-652 (2005). While not bound to any mechanism of action, it is believed that the N-terminal phenylalanine residue is only necessary for importation into cyanelles, thus, this residue may be modified in the practice of the present invention and still effectively transport a polypeptide into a higher plant chloroplast.
Taken together, the above data raises doubts on the ability of cyanelle targeting sequences to direct heterologous proteins to the chloroplasts of higher plants. While the Cyanophora paradoxa pre-FNR molecule was able to enter isolated chloroplasts from pea, it does not necessarily follow that the FNR transit peptide when attached to a heterologous protein would be able to interact with the higher plant secretory system to effectively target a heterologous protein to the chloroplast. Surprisingly, the instant application describes methods for targeting heterologous proteins to chloroplasts in higher plants using cyanelle transit peptides.
In one embodiment of the invention, the transit peptide is from a Glaucocystophyte which when operably linked to a heterologous protein and expressed in a transgenic higher plant, targets the heterologous protein to the chloroplast. The Glaucocystophyte is selected from the group consisting of Cyanophora, Glaucocystis, Gloeochaete and Cyanophora paradoxa.
Another embodiment of the invention is a method of targeting a heterologous protein to the chloroplast of a transgenic higher plant comprising the steps of operably linking a transit peptide from a Glaucocystophyte to a heterologous protein and generating a transgenic plant comprising the heterologous protein wherein the heterologous protein is detected in the chloroplast of the transgenic plant. The Glaucocystophyte is selected from the group consisting of Cyanophora, Glaucocystis, Gloeochaete and Cyanophora paradoxa.
The compositions comprise nucleotide sequences encoding a transit peptide as well as variants thereof. In one embodiment the transit peptide comprises the amino acid sequence set forth in SEQ ID NO:2 or fragments and variants thereof as well as the nucleotide sequence set forth in SEQ ID NO:1 and variants thereof. Compositions further comprise DNA constructs comprising a nucleotide sequence encoding the transit peptide operably linked to a nucleotide sequence encoding a heterologous polypeptide. These DNA constructs find use in expression and targeting of the heterologous polypeptide to a plastid. Compositions also comprise expression cassettes, vectors, transformed plants, transformed plant cells, and stably transformed plant seeds wherein a heterologous polypeptide is targeted to a plastid by the transit peptide of the invention.
In another embodiment of the invention, the transit peptide is from a Glaucocystophyte which when operably linked to a heterologous protein and expressed in a transgenic higher plant, targets the heterologous protein to the chloroplast. Transit peptides are derived from the nucleic acid sequence encoding a protein which is targeted to the cyanelle of the Glaucocystophyte. Transit peptides can be found in genes known to be encoded in the nucleus of the host cell but upon translation to be targeted to the photosynthetic organelle of the host cell and can be selected from the group consisting of ribulose bisphosphate carboxylase/oxygenase, 5-enolpyruvyl-shikimate-3-phosphate synthase, acetolactate synthase, chloroplast ribosomal protein CS17, Cs protein, ferredoxin, plastocyanin, ribulose bisphosphate carboxylase activase, tryptophan synthase, acyl carrier protein, plastid chaperonin-60, cyochrome C552, 22-kDA heat shock protein, 33-kDA oxygen-evolving enhancer protein 1, ATP synthase gamma subunit, ATP synthase omega subunit, chlorophyll-a/b-binding proteinII-1, oxygen-evolving enhancer protein 2, oxygen-evolving enhancer protein 3, photosystem I P21, photosystem I P28, photosystem I P30, photosystem I P35, photosystem I P37, glycerol-3-phosphate acyltransferases, chlorophyll a/b binding protein, CAB2 protein, hydroxymethyl-bilane synthase, pyruvate-orthophosphate dikinase, CAB3 protein, plastid ferritin, ferritin, early light-inducible protein, glutamate-1-semialdehyde aminotransferase, protochlorophyllide reductase, starch-granule-bound amylase synthase, light-harvesting chlorophyll a/b-binding protein of photosystem II, major pollen allergen Lol p 5a, plastid ClpB ATP dependent protease, superoxide dismutase, ferredoxin NADP oxidoreductase, 28-kDa ribonucleotoprotein, 31-kDa ribonucleoprotein, 33-kDa ribonucleoprotein, acetolacate synthase, ATP synthase CFO subunit 1, 2, 3, or 4; cytochrome f, ADP-glucose pyrophosphorylase, glutamine synthase, glutamine synthase 2, carbonic anhydrase, GapA protein, heat shock protein hsp 21, phosphate translocator, plastid CIpA ATP dependent protease, plastid ribosomal protein CL24, plastid ribosomal protein CL9, plastid ribosomal protein PsCL18, plastid ribosomal protein PsCL25, DAHP synthase, starch phosphorylase, root acyl carrier protein 11, betaine-aldehyde dehydrogase, GapB protein, glutamine synthase 2, phosphoribulokinase, nitrite reductase, ribosomal protein L12, ribosomal protein L13, ribosomal protein L21, ribosomal protein L35, ribosomal protein L40, triose phosphate-3-phosphoglyerate phosphate translocator, ferredoxin dependent glutamate synthase, glyceraldehyde 3 phosphate dehydrogenase, NADP dependent malic enzyme and NADP malate dehydrogenase. Table 1 identifies additional nuclear encoded genes containing plastid targeting sequences. In one embodiment of the invention the transit peptide is from Cyanophora pardoxa, particularly from C. pardoxa ferredoxin-NADP+-oxidoreductase and comprises the amino acid sequence set forth in SEQ ID NO:2.
The transit peptides of the invention include variant or modified sequences. Such sequences have altered amino acid sequences yet retain the ability to transport a linked polypeptide into a plastid. While methods are known for substituting amino acid residues in polypeptide sequences, it is recognized that serine and threonine have been identified as abundant in transit peptides in comparison to the entire chloroplast targeting protein. Aspartic acid and glutamic acid have been reported as under represented in transit peptides. Such considerations as well as those discussed above may be taken into account when constructing variant transit peptides. However, as detailed below, one of skill can assay whether a variant transit peptide is capable of effecting transport of an operably linked polypeptide by assaying for the presence of the operably linked polypeptide in the plastid.
As used herein, the term “nucleic acid molecule” is intended to include DNA molecules (e.g., cDNA or genomic DNA) and RNA molecules (e.g., mRNA) and analogs of the DNA or RNA generated using nucleotide analogs. The nucleic acid molecule can be single-stranded or double-stranded, but preferably is double-stranded DNA.
By a “crop plant” or “higher plant” is intended any plant that is cultivated for the purpose of producing plant material that is sought after by man for either oral consumption, or for utilization in an industrial, pharmaceutical, or commercial process. The invention may be applied to any of a variety of plants, including, but not limited to maize, wheat, rice, barley, soybean, cotton, sorghum, oats, tobacco, Miscanthus grass, Switch grass, trees, beans in general, rape/canola, alfalfa, flax, sunflower, safflower, millet, rye, sugarcane, sugar beet, cocoa, tea, Brassica, cotton, coffee, sweet potato, flax, peanut, clover; vegetables such as lettuce, tomato, cucurbits, cassava, potato, carrot, radish, pea, lentils, cabbage, cauliflower, broccoli, Brussels sprouts, peppers, and pineapple; tree fruits such as citrus, apples, pears, peaches, apricots, walnuts, avocado, banana, nut and coconut; and flowers such as orchids, carnations, roses, and the like.
As used herein, the term “plant part” or “plant tissue” includes plant cells, plant protoplasts, plant cell tissue cultures from which plants can be regenerated, plant calli, plant clumps, and plant cells that are intact in plants or parts of plants such as embryos, pollen, ovules, seeds, leaves, flowers, branches, fruit, kernels, ears, cobs, husks, stalks, roots, root tips, anthers, and the like.
The article “a” and “an” are used herein to refer to one or more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one or more element. Throughout the specification the word “comprising,” or variations such as “comprises” or “comprising,” will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.
Transit Peptide CompositionsAs indicated, the compositions of the invention include transit peptides capable of transporting operably linked polypeptides into plastids. The term “peptide” broadly refers to an amino acid chain that includes naturally occurring amino acids, synthetic amino acids, genetically encoded amino acids, non-genetically encoded amino acids, and combinations thereof. Peptides can include both L-form and D-form amino acids.
Representative non-genetically encoded amino acids include but are not limited to 2-aminoadipic acid; 3-aminoadipic acid; β-aminopropionic acid; 2-aminobutyric acid; 4-aminobutyric acid (piperidinic acid); 6-aminocaproic acid; 2-aminoheptanoic acid; 2-aminoisobutyric acid; 3-aminoisobutyric acid; 2-aminopimelic acid; 2,4-diaminobutyric acid; desmosine; 2,2′-diaminopimelic acid; 2,3-diaminoproprionic acid; N-ethylglycine; N-ethylasparagine; hydroxylysine; allo-hydroxylysine; 3-hydroxyproline; 4-hydroxyproline; isodesmosine; allo-isoleucine; N-methylglycine (sarcosine); N-methylisoleucine; N-methylvaline; norvaline; norleucine; and ornithine.
Representative derivatized amino acids include, for example, those molecules in which free amino groups have been derivatized to form amine hydrochlorides, p-toluene sulfonyl groups, carbobenzoxy groups, t-butyloxycarbonyl groups, chloroacetyl groups or formyl groups. Free carboxyl groups can be derivatized to form salts, methyl and ethyl esters or other types of esters or hydrazides. Free hydroxyl groups can be derivatized to form O-acyl or O-alkyl derivatives. The imidazole nitrogen of histidine can be derivatized to form N-im-benzylhistidine.
The plastid transit peptide is generally fused N-terminal to the polypeptide to be transported into a plastid. It is recognized that additional amino acid residues may be N-terminal to the plastid transit peptide. The plastid transit peptide is generally cleaved from the polypeptide of interest upon localization to the plastid. The position of cleavage may vary slightly between plant species.
The transit peptides of the invention include the transit peptide from a Cyanophora ferredoxin-NADP+-oxidoreductase, particularly the transit peptide from a Cyanophora pardoxa ferredoxin-NADP+-oxidoreductase, more particularly the amino acid sequence set forth in SEQ ID NO:2. Biologically active variants of the peptides of the invention are also encompassed by the present invention. Such variants should retain the ability to transport an operatively linked polypeptide into a cellular plastid. Preferably, the variant has at least the same activity as the native molecule. The ability to transport polypeptides into a plastid can be measured by methods in the art. For example, the nucleotide sequence encoding the transit peptide can be linked to a reporter gene such as chloramphenicol acetyl transferase (CAT) and β-glucuronidase (GUS) and introduced into a plant. Analysis of CAT and GUS activities in the subcellular fractions of the transformed plant indicate whether the transit peptide is capable of targeting the reporter proteins to the plastids. See, for example, Silva-Filho et al. (1997) J Biol Chem 272:15264-15269 and US Patent Application No. 20080168580.
Suitable biologically active variants can be fragments and derivatives. By “fragment” is intended a peptide consisting of only a part of the intact transit peptide sequence and structure, and can be a C-terminal deletion or N-terminal deletion of amino acids or deletions at both the C- and N-terminal ends. By “derivatives” is intended any suitable modification of a transit peptide or peptide fragment encompassing any change in amino acid residues, so long as the transport activity is retained. For example, without being limited, the invariant phenylalanine residue on the cyanelle transit peptide can be used to identify cyanelle transit peptide sequences; however, this residue may be changed to another amino acid to create a derivative that is also functional in transgenic higher plants to target heterologous proteins to chloroplasts.
Peptide variants will generally have at least 70%, preferably at least 80%, more preferably about 90% to 95% or more, about 96%, about 97%, and most preferably about 98%, about 99% or more amino acid sequence identity to the amino acid sequence of the reference peptide molecule. A variant may differ by as few as 5, 4, 3, 2, or even 1 amino acid residue. Methods for determining identity between sequences are well known in the art. See, for example, the ALIGN program (Dayhoff (1978) in Atlas of Protein Sequence and Structure 5:Suppl. 3 (National Biomedical Research Foundation, Washington, D.C.) and programs in the Wisconsin Sequence Analysis Package, Version 8 (available from Genetics Computer Group, Madison, Wis.), for example, the GAP program. For purposes of optimal alignment of the two sequences, the contiguous segment of the amino acid sequence of the variant may have additional amino acid residues or deleted amino acid residues with respect to the amino acid sequence of the reference molecule. The contiguous segment used for comparison to the reference amino acid sequence will comprise at least twenty (20) contiguous nucleotides, and may be about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 15, about 20, about 25, about 30, about 40 or more nucleotides. Corrections for increased sequence identity associated with inclusion of gaps in the variant's amino acid sequence can be made by assigning gap penalties. Methods of sequence alignment are well known in the art.
When considering percentage of amino acid sequence identity, some amino acid residue positions may differ as a result of conservative amino acid substitutions, which do not affect properties of protein function. In these instances, percent sequence identity may be adjusted upwards to account for the similarity in conservatively substituted amino acids. Such adjustments are well known in the art. See, for example, Meyers and Miller (1988) Computer Applic. Biol. Sci. 4:11-17.
For example, preferably, conservative amino acid substitutions may be made. A “nonessential” amino acid residue is a residue that can be altered without altering the biological activity, whereas an “essential” amino acid residue is required for biological activity. A “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). See, for example, Sambrook J., and Russell, D. W. (2001) Molecular Cloning. A Laboratory Manual. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.) and Innis, et al. (1990) PCR Protocols: A Guide to Methods and Applications (Academic Press, NY).
The peptides of the invention can be subject to various changes, substitutions, insertions, and deletions where such changes provide for certain advantages in its use. Thus, the term “peptide” encompasses any of a variety of forms of peptide derivatives including, for example, amides, conjugates with proteins, cyclone peptides, polymerized peptides, conservatively substituted variants, analogs, fragments, chemically modified peptides, and peptide mimetics. Any peptide that has desired transport characteristics can be used in the practice of the present invention.
By “transports,” “targets,” or “transfers” is intended that the transit peptides of the invention are capable of transporting, transferring or carrying polypeptides expressed in the nucleus of the cell into a cellular plastid, for example, without limitation, a chloroplast. In some embodiments, the transit peptides of the invention are capable of transporting 100%, at least 90%, at least 80%, at least 70%, at least 60%, at least 50% of an operably linked polypeptide into a plastid.
“Isolated” means altered “by the hand of man” from its natural state; i.e., that, if it occurs in nature, it has been changed or removed from its original environment, or both. For example, a naturally occurring polynucleotide or a polypeptide naturally present in a living animal in its natural state is not “isolated,” but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is “isolated”, as the term is employed herein. For example, with respect to polynucleotides, the term isolated means that it is separated from the chromosome and cell in which it naturally occurs. A sequence is also isolated if separated from the chromosome and cell in which it naturally occurs in but inserted into a genetic context, chromosome, or cell in which it does not naturally occur.
One aspect of the invention pertains to isolated nucleic acid molecules comprising nucleotide sequences encoding transit peptides or biologically active portions thereof. As used herein, the term “nucleic acid molecule” is intended to include DNA molecules (e.g., cDNA or genomic DNA) and RNA molecules (e.g., mRNA) and analogs of the DNA or RNA generated using nucleotide analogs. The nucleic acid molecule can be single-stranded or double-stranded, but preferably is double-stranded DNA.
Nucleic acid molecules that are fragments of these transit peptide encoding nucleotide sequences are also encompassed by the present invention. By “fragment” is intended a portion of the nucleotide sequence encoding a transit peptide. Nucleic acid molecules that are fragments of a transit peptide nucleotide sequence comprise at least about 15, about 20, about 50, about 75, about 100, about 125, about 150, about 175, about 180, about 185, about 190, about 195 contiguous nucleotides. By “contiguous” nucleotides is intended nucleotide residues that are immediately adjacent to one another.
The skilled artisan will further appreciate that changes can be introduced by mutation into the nucleotide sequences of the invention thereby leading to changes in the amino acid sequence of the encoded transit peptides, without altering the transport. Thus, variant isolated nucleic acid molecules can be created by introducing one or more nucleotide substitutions, additions, or deletions into the corresponding nucleotide sequence disclosed herein, such that one or more amino acid substitutions, additions or deletions are introduced into the encoded peptide. Mutations can be introduced by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. Such variant nucleotide sequences are also encompassed by the present invention.
The compositions of the invention can be used to identify and isolate corresponding transit peptides from other organisms. In one method, the sequence can be used in hybridization assays to find sequences with substantial homology to the sequences of the invention. As used herein, the term “hybridization” is used in reference to the pairing of complementary (including partially complementary as discussed above) polynucleotide strands. Hybridization and the strength of hybridization (i.e., the strength of the association between polynucleotide strands) is impacted by many factors well known in the art including the degree of complementarity between the polynucleotides; stringency of the conditions involved that are affected by such conditions as the concentration of salts; the melting temperature (Tm) of the formed hybrid; the presence of other components; the molarity of the hybridizing strands; and, the G:C content of the polynucleotide strands.
Thus, isolated sequences that are capable of transporting polypeptides into a plastid and which hybridize under stringent conditions to the sequences disclosed herein, or to fragments thereof, are encompassed by the present invention. Such sequences will be at least about 40% to 50% homologous, about 60%, 65%, or 70% homologous, and even at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homologous with the disclosed sequence[s]. That is, the sequence identity of sequences may range, sharing at least about 40% to 50%, about 60%, 65%, or 70%, and even at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity.
As used herein, “Tm” and “melting temperature” are interchangeable terms which are the temperature at which 50% of a population of double-stranded polynucleotide molecules becomes dissociated into single strands. The equation for calculating the Tm of polynucleotides is well known in the art. For example, the Tm may be calculated by the following equation: Tm=69.3+0.41×(G+C) %−650/L, wherein L is the length of the probe in nucleotides. The Tm of a hybrid polynucleotide may also be estimated using a formula adopted from hybridization assays in 1 M salt, and commonly used for calculating Tm for PCR primers: [(number of A+T)×2° C.+(number of G+C)×4° C.], see, for example, Newton et al. (1997) PCR 2nd Ed. (Springer-Verlag, New York). Other more sophisticated computations exist in the art, which take structural as well as sequence characteristics into account for the calculation of Tm. A calculated Tm is merely an estimate; the optimum temperature is commonly determined empirically.
Typically, stringent conditions will be those in which the salt concentration is less than about 1.5 M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30° C. for short probes (e.g., 10 to 50 nucleotides) and at least about 60° C. for long probes (e.g., greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. Exemplary low stringency conditions include hybridization with a buffer solution of 30 to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulphate) at 37° C., and a wash in 1× to 2×SSC (20×SSC=3.0 M NaCl/0.3 M trisodium citrate) at 50 to 55° C. Exemplary moderate stringency conditions include hybridization in 40 to 45% formamide, 1.0 M NaCl, 1% SDS at 37° C., and a wash in 0.5× to 1×SSC at 55 to 60° C. Exemplary high stringency conditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37° C., and a wash in 0.1×SSC at 60 to 65° C. Optionally, wash buffers may comprise about 0.1% to about 1% SDS. Duration of hybridization is generally less than about 24 hours, usually about 4 to about 12 hours. The duration of the wash time will be at least a length of time sufficient to reach equilibrium.
Corresponding sequences can also be identified by PCR reactions. See, Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.). See also Innis et al., eds. (1990) PCR Protocols: A Guide to Methods and Applications (Academic Press, New York); Innis and Gelfand, eds. (1995) PCR Strategies (Academic Press, New York); and Innis and Gelfand, eds. (1999) PCR Methods Manual (Academic Press, New York). The primers of the present invention can be prepared using techniques known in the art, including, but not limited to, cloning and digestion of the appropriate sequences and direct chemical synthesis.
Chemical synthesis methods that can be used to make the primers of the present invention, include, but are not limited to, the phosphotriester method described by Narang et al., Methods in Enzymology, 68:90 (1979), the phosphodiester method disclosed by Brown et al., Methods in Enzymology, 68:109 (1979), the diethylphosphoramidate method disclosed by Beaucage et al., Tetrahedron Letters, 22:1859 (1981) and the solid support method described in U.S. Pat. No. 4,458,066. The use of an automated oligonucleotide synthesizer to prepare synthetic oligonucleotide primers of the present invention is also contemplated herein. Additionally, if desired, the primers can be labeled using techniques known in the art and described below.
Plant Expression CassettesAs discussed, the plastid transit peptides of the invention operably linked to a polypeptide of interest transports the linked polypeptide into a plastid. Thus, when assembled into a DNA construct and used to transform a plant, the transit peptide directs a polypeptide of interest to plastids in the transformed plant cells, plants, and seeds after expression. Thus, the compositions of the invention also comprise nucleic acid sequences for transformation and expression and accumulation of a polypeptide of interest in the plastids of a plant cell of interest. The nucleic acid sequences may be present in DNA constructs or expression cassettes. “Expression cassette” as used herein means a nucleic acid molecule capable of directing expression of a particular nucleotide sequence in an appropriate host cell, comprising a promoter operatively linked to the nucleotide sequence of interest (i.e., a nucleotide sequence encoding a polypeptide of interest) which is operatively linked to termination signals. It also typically comprises sequences required for proper translation of the nucleotide sequence. The expression cassette comprising the nucleotide sequence of interest may be chimeric, meaning that at least one of its components is heterologous with respect to at least one of its other components. The expression cassette may also be one that is naturally occurring but has been obtained in a recombinant form useful for heterologous expression. Typically, however, the expression cassette is heterologous with respect to the host, i.e., the particular DNA sequence of the expression cassette does not occur naturally in the host cell and must have been introduced into the host cell or an ancestor of the host cell by a transformation event. The expression of the nucleotide sequence in the expression cassette may be under the control of a constitutive promoter or of an inducible promoter that initiates transcription only when the host cell is exposed to some particular external stimulus. Additionally, the promoter can also be specific to a particular tissue or organ or stage of development.
The present invention encompasses the transformation of plants with expression cassettes capable of directing expression and accumulation of a polypeptide of interest in the plastids of a plant cell. The expression cassette will include in the 5′-3′ direction of transcription, a transcriptional and translational initiation region (i.e., a promoter), a polynucleotide encoding a plastid transit peptide, and a polynucleotide encoding a polypeptide of interest. The expression cassette may optionally comprise a transcriptional and translational termination region (i.e. termination region) functional in plants.
In addition to the polynucleotide sequence encoding the plastid transit peptide, the construct may further comprise additional regulatory elements to facilitate transcription, translation, or transport of the polypeptide of interest. The regulatory sequences of the expression construct are operably linked to the polynucleotide of interest. By “operably linked” is intended a functional linkage between a regulatory element and a second sequence wherein the regulatory element initiates and/or mediates transcription, translation, or translocation of the DNA sequence corresponding to the second sequence. Generally, operably linked means that the nucleotide sequences being linked are contiguous. The regulatory elements include promoters, enhancers, and signal sequences useful for targeting cytoplasmically-synthesized proteins to plastids of the plant cell. In one embodiment, the construct comprises, in the 5′ to 3′ direction of transcription, a transcriptional and translational initiation region (i.e., a promoter), a polynucleotide encoding a plastid transit peptide, and a polynucleotide encoding a polypeptide of interest.
Any promoter capable of driving expression in the plant of interest may be used in the practice of the invention. The promoter may be native or analogous or foreign or heterologous to the plant host. The terms “heterologous” and “exogenous” when used herein to refer to a nucleic acid sequence (e.g. a DNA or RNA sequence) or a gene, refer to a sequence that originates from a source foreign to the particular host cell or, if from the same source, is modified from its original form. Thus, a heterologous gene in a host cell includes a gene that is endogenous to the particular host cell but has been modified through, for example, the use of DNA shuffling. The terms also include non-naturally occurring multiple copies of a naturally occurring DNA sequence. Thus, the terms refer to a DNA segment that is foreign or heterologous to the cell, or homologous to the cell but in a position within the host cell nucleic acid in which the element is not ordinarily found. Exogenous DNA segments are expressed to yield exogenous polypeptides.
A “homologous” nucleic acid (e.g. DNA) sequence is a nucleic acid (e.g. DNA or RNA) sequence naturally associated with a host cell into which it is introduced.
The choice of promoters to be included depends upon several factors, including, but not limited to, efficiency, selectability, inducibility, desired expression level, and cell- or tissue-preferential expression. It is a routine matter for one of skill in the art to modulate the expression of a sequence by appropriately selecting and positioning promoters and other regulatory regions relative to that sequence.
Some suitable promoters initiate transcription only, or predominantly, in certain cell types. Thus, as used herein a cell type- or tissue-preferential promoter is one that drives expression preferentially in the target tissue, but may also lead to some expression in other cell types or tissues as well. Methods for identifying and characterizing promoter regions in plant genomic DNA include, for example, those described in the following references: Jordano, et al., Plant Cell, 1:855-866 (1989); Bustos, et al., Plant Cell, 1:839-854 (1989); Green, et al., EMBO J. 7, 4035-4044 (1988); Meier, et al., Plant Cell, 3, 309-316 (1991); and Zhang, et al., Plant Physiology 110: 1069-1079 (1996).
Promoters active in photosynthetic tissue in order to drive transcription in green tissues such as leaves and stems are of particular interest for the present invention. Most suitable are promoters that drive expression only or predominantly in such tissues. The promoter may confer expression constitutively throughout the plant, or differentially with respect to the green tissues, or differentially with respect to the developmental stage of the green tissue in which expression occurs, or in response to external stimuli.
Examples of such promoters include the ribulose-1,5-bisphosphate carboxylase (RbcS) promoters such as the RbcS promoter from eastern larch (Larix laricina), the pine cab6 promoter (Yamamoto et al. (1994) Plant Cell Physiol. 35:773-778), the Cab-1 gene promoter from wheat (Fejes et al. (1990) Plant Mol. Biol. 15:921-932), the CAB-1 promoter from spinach (Lubberstedt et al. (1994) Plant Physiol. 104:997-1006), the cab1R promoter from rice (Luan et al. (1992) Plant Cell 4:971-981), the pyruvate orthophosphate dikinase (PPDK) promoter from corn (Matsuoka et al. (1993) Proc Natl Acad Sci USA 90:9586-9590), the tobacco Lhcb1*2 promoter (Cerdan et al. (1997) Plant Mol. Biol. 33:245-255), the Arabidopsis thaliana SUC2 sucrose-H+ symporter promoter (Truernit et al. (1995) Planta 196:564-570), and thylakoid membrane protein promoters from spinach (psaD, psaF, psaE, PC, FNR, atpC, atpD, cab, rbcS. Other promoters that drive transcription in stems, leafs and green tissue are described in U.S. Patent Publication No. 2007/0006346, herein incorporated by reference in its entirety.
A maize gene encoding phosphoenol carboxylase (PEPC) has been described by Hudspeth & Grula (Plant Molec Biol 12: 579-589 (1989)). Using standard molecular biological techniques the promoter for this gene can be used to drive the expression of any gene in a green tissue-specific manner in transgenic plants.
In some other embodiments of the present invention, inducible promoters may be desired. Inducible promoters drive transcription in response to external stimuli such as chemical agents or environmental stimuli. For example, inducible promoters can confer transcription in response to hormones such as giberellic acid or ethylene, or in response to light or drought.
A variety of transcriptional terminators are available for use in expression cassettes. These are responsible for the termination of transcription beyond the transgene and correct mRNA polyadenylation. The termination region may be native with the transcriptional initiation region, may be native with the operably linked DNA sequence of interest, may be native with the plant host, or may be derived from another source (i.e., foreign or heterologous to the promoter, the DNA sequence of interest, the plant host, or any combination thereof). Appropriate transcriptional terminators are those that are known to function in plants and include the CAMV 35S terminator, the tml terminator, the nopaline synthase terminator and the pea rbcs E9 terminator. These can be used in both monocotyledons and dicotyledons. In addition, a gene's native transcription terminator may be used.
In some embodiments, the expression cassette will comprise a selectable marker gene for the selection of transformed cells. Selectable marker genes are utilized for the selection of transformed cells or tissues.
Numerous sequences have been found to enhance gene expression from within the transcriptional unit and these sequences can be used in conjunction with the genes of this invention to increase their expression in transgenic plants.
Various intron sequences have been shown to enhance expression, particularly in monocotyledonous cells. For example, the introns of the maize Adhl gene have been found to significantly enhance the expression of the wild-type gene under its cognate promoter when introduced into maize cells. Intron 1 was found to be particularly effective and enhanced expression in fusion constructs with the chloramphenicol acetyltransferase gene (Callis et al., Genes Develop. 1: 1183-1200 (1987)). In the same experimental system, the intron from the maize bronze 1 gene had a similar effect in enhancing expression. Intron sequences have been routinely incorporated into plant transformation vectors, typically within the non-translated leader.
A number of non-translated leader sequences derived from viruses are also known to enhance expression, and these are particularly effective in dicotyledonous cells. Specifically, leader sequences from Tobacco Mosaic Virus (TMV, the “W-sequence”), Maize Chlorotic Mottle Virus (MCMV), and Alfalfa Mosaic Virus (AMV) have been shown to be effective in enhancing expression (e.g. Gallie et al. Nucl. Acids Res. 15: 8693-8711 (1987); Skuzeski et al. Plant Molec. Biol. 15: 65-79 (1990)). Other leader sequences known in the art include but are not limited to: picornavirus leaders, for example, EMCV leader (Encephalomyocarditis 5′ noncoding region) (Elroy-Stein, O., Fuerst, T. R., and Moss, B. PNAS USA 86:6126-6130 (1989)); potyvirus leaders, for example, TEV leader (Tobacco Etch Virus) (Allison et al., 1986); MDMV leader (Maize Dwarf Mosaic Virus); Virology 154:9-20); human immunoglobulin heavy-chain binding protein (BiP) leader, (Macejak, D. G., and Samow, P., Nature 353: 90-94 (1991); untranslated leader from the coat protein mRNA of alfalfa mosaic virus (AMV RNA 4), (Jobling, S. A., and Gehrke, L., Nature 325:622-625 (1987); tobacco mosaic virus leader (TMV), (Gallie, D. R. et al., Molecular Biology of RNA, pages 237-256 (1989); and Maize Chlorotic Mottle Virus leader (MCMV) (Lommel, S. A. et al., Virology 81:382-385 (1991). See also, Della-Cioppa et al., Plant Physiology 84:965-968 (1987).
As indicated the plastid transit peptide is operably linked to a polypeptide to be transported into the plastid. Polypeptides for traits of interest include agronomic traits that primarily are of benefit to a seed company, a grower, or a grain processor, for example, herbicide resistance, virus resistance, bacterial pathogen resistance, insect resistance, nematode resistance, and fungal resistance. See, e.g., U.S. Pat. Nos. 5,569,823; 5,304,730; 5,495,071; 6,329,504; and 6,337,431. Traits of interest may also be one that increases plant vigor or yield (including traits that allow a plant to grow at different temperatures, soil conditions and levels of sunlight and precipitation), or one that allows identification of a plant exhibiting a trait of interest (e.g., selectable marker gene, seed coat color, etc.). A plethora of genes useful for generating plants with desired secondary traits are available in the art.
Herbicide resistance genes of interest include those encoding precursors of acetolactase synthetase (ALS), see, for example, U.S. Pat. No. 5,013,659; mutated acetolactate synthetase; 3-enolpyruvylshikimate-5-phosphate synthetase (EPSP synthetase), see, for example, U.S. Pat. Nos. 4,971,908 and 6,225,114; enzymes that modify a physiological process that occurs in a plastid including photosynthesis, fatty acid, amino acid, oil, arotenoid, terpenoid, and starch; etc. Other genes of interest include but are not limited to those encoding zeaxanthin epoxidase, choline monooxygenase, ferrochelatase, omega-3 fatty acid desaturase, glutamine synthetase, starch modifying enzymes, essential amino acids, provitamin A, hormones, Bt toxin proteins, and the like. Nucleotide sequences for such polypeptides are available in the art.
PlantsPlants useful in the present invention include plants that are transgenic for at least a polynucleotide encoding a plastid-targeted polypeptide of interest. Such plants will comprise a polynucleotide encoding a transit peptide operably linked to a polynucleotide encoding a polypeptide of interest. One of skill in the art will recognize that the polypeptide sequences of interest may be associated with or contributing to one or more secondary trait(s) of interest. These polypeptides are cytoplasmically-expressed, and targeted to a plastid.
The type of plant selected depends on a variety of factors, including for example, the downstream use of the harvested plant material, amenability of the plant species to transformation, and the conditions under which the plants will be grown, harvested, and/or processed. One of skill will further recognize that additional factors for selecting appropriate plant varieties for use in the present invention include high yield potential, good stalk strength, resistance to specific diseases, drought tolerance, rapid dry down and grain quality sufficient to allow storage and shipment to market with minimum loss.
It is further contemplated that the constructs of the invention may be introduced into plant varieties having improved properties suitable or optimal for a particular downstream use. Plants useful in the present invention include, but are not limited to, monocotyledonous and dicotyledonous plants, particularly crop plants such as maize, soybean, wheat, rice, Brassica, and the like. Examples of plant species of interest include, but are not limited to, corn (Zea mays), Brassica sp. (e.g., B. napus, B. rapa, B. juncea), particularly those Brassica species useful as sources of seed oil, alfalfa (Medicago sativa), rice (Oryza sativa), rye (Secale cereals), sorghum (Sorghum bicolor, Sorghum vulgare), millet (e.g., pearl millet (Pennisetum glaucum), proso millet (Panicum miliaceum), foxtail millet (Setaria italica), finger millet (Eleusine coracana)), sunflower (Helianthus annuus), safflower (Carthamus tinctorius), wheat (Triticum aestivum), soybean (Glycine max), tobacco (Nicotiana tabacum), potato (Solanum tuberosum), cotton (Gossypium barbadense, Gossypium hirsutum), sweet potato (Ipomoea batatus), cassava (Manihot esculenta), coffee (Coffea spp.), coconut (Cocos nucifera), pineapple (Ananas comosus), citrus trees (Citrus spp.), cocoa (Theobroma cacao), tea (Camellia sinensis), banana (Musa spp.), avocado (Persea americana), fig (Ficus casica), guava (Psidium guajava), mango (Mangifera indica), olive (Olea europaea), papaya (Carica papaya), cashew (Anacardium occidentale), macadamia (Macadamia integrifolia), almond (Prunus amygdalus), sugar beets (Beta vulgaris), sugarcane (Saccharum spp.), oats, barley, vegetables, ornamentals, and conifers.
Vegetables include tomatoes (Lycopersicon esculentum), lettuce (e.g., Lactuca sativa), green beans (Phaseolus vulgaris), lima beans (Phaseolus limensis), peas (Lathyrus spp.), and members of the genus Cucumis such as cucumber (C. sativus), cantaloupe (C. cantalupensis), and musk melon (C. melo). Ornamentals include azalea (Rhododendron spp.), hydrangea (Macrophylla hydrangea), hibiscus (Hibiscus rosasanensis), roses (Rosa spp.), tulips (Tulipa spp.), daffodils (Narcissus spp.), petunias (Petunia hybrida), carnation (Dianthus caryophyllus), poinsettia (Euphorbia pulcherrima), and chrysanthemum.
Plants of interest include cauliflower (Brassica oleracea), artichoke (Cynara scolvmus), and safflower (Carthamus, e.g. tinctorius); fruits such as apple (Malus, e.g. domesticus), banana (Musa, e.g. acuminata), berries (such as the currant, Ribes, e.g. rubrum), cherries (such as the sweet cherry, Prunus, e.g. avium), cucumber (Cucumis, e.g. sativus), grape (Vitis, e.g. vinifera), lemon (Citrus limon), melon (Cucumis melo), nuts (such as the walnut, Juglans, e.g. regia; peanut, Arachis hypoaeae), orange (Citrus, e.g. maxima), peach (Prunus, e.g. persica), pear (Pyra, e.g. communis), pepper (Solanum, e.g. capsicum), plum (Prunus, e.g. domestica), strawberry (Fragaria, e.g. moschata), tomato (Lycopersicon, e.g. esculentum); leafs, such as alfalfa (Medicago, e.g. sativa), sugar cane (Saccharum), cabbages (such as Brassica oleracea), endive (Cichoreum, e.g. endivia), leek (Allium, e.g. porrum), lettuce (Lactuca, e.g. sativa), spinach (Spinacia e.g. oleraceae), tobacco (Nicotiana, e.g. tabacum); roots, such as arrowroot (Maranta, e.g. arundinacea), beet (Beta, e.g. vulgaris), carrot (Daucus, e.g. carota), cassava (Manihot, e.g. esculenta), turnip (Brassica, e.g. rapa), radish (Raphanus, e.g. sativus) yam (Dioscorea, e.g. esculenta), sweet potato (Ipomoea batatas); seeds, such as bean (Phaseolus, e.g. vulgaris), pea (Pisum, e.g. sativum), soybean (Glycine, e.g. max), wheat (Triticum, e.g. aestivum), barley (Hordeum, e.g. vulgare), corn (Zea, e.g. mays), rice (Oryza, e.g. sativa); grasses, such as Miscanthus grass (Miscanthus, e.g., giganteus) and switchgrass (Panicum, e.g. virgatum); trees such as poplar (Populus, e.g. tremula), pine (Pinus); shrubs, such as cotton (e.g., Gossypium hirsutum); and tubers, such as kohlrabi (Brassica, e.g. oleraceae), potato (Solanum, e.g. tuberosum), and the like.
Plant TransformationThe expression constructs described herein can be introduced into the plant cell in a number of art-recognized ways. The term “introducing” in the context of a polynucleotide, for example, a nucleotide construct of interest, is intended to mean presenting to the plant the polynucleotide in such a manner that the polynucleotide gains access to the interior of a cell of the plant. Where more than one polynucleotide is to be introduced, these polynucleotides can be assembled as part of a single nucleotide construct, or as separate nucleotide constructs, and can be located on the same or different transformation vectors. Accordingly, these polynucleotides can be introduced into the host cell of interest in a single transformation event, in separate transformation events, or, for example, in plants, as part of a breeding protocol. The methods of the invention do not depend on a particular method for introducing one or more polynucleotides into a plant, only that the polynucleotide(s) gains access to the interior of at least one cell of the plant. Methods for introducing polynucleotides into plants are known in the art including, but not limited to, transient transformation methods, stable transformation methods, and virus-mediated methods.
“Transient transformation” in the context of a polynucleotide is intended to mean that a polynucleotide is introduced into the plant and does not integrate into the genome of the plant.
By “stably introducing” or “stably introduced” in the context of a polynucleotide introduced into a plant is intended the introduced polynucleotide is stably incorporated into the plant genome, and thus the plant is stably transformed with the polynucleotide.
“Stable transformation” or “stably transformed” is intended to mean that a polynucleotide, for example, a nucleotide construct described herein, introduced into a plant integrates into the genome of the plant and is capable of being inherited by the progeny thereof, more particularly, by the progeny of multiple successive generations.
Numerous transformation vectors available for plant transformation are known to those of ordinary skill in the plant transformation arts, and the genes pertinent to this invention can be used in conjunction with any such vectors. The selection of vector will depend upon the preferred transformation technique and the target species for transformation. For certain target species, different antibiotic or herbicide selection markers may be preferred. Selection markers used routinely in transformation include the nptll gene, which confers resistance to kanamycin and related antibiotics (Messing & Vierra. Gene 19: 259-268 (1982); Bevan et al., Nature 304:184-187 (1983)), the bar gene, which confers resistance to the herbicide phosphinothricin (White et al., Nucl. Acids Res 18: 1062 (1990), Spencer et al. Theor. Appl. Genet 79: 625-631 (1990)), the hph gene, which confers resistance to the antibiotic hygromycin (Blochinger & Diggelmann, Mol Cell Biol 4: 2929-2931), and the dhfr gene, which confers resistance to methatrexate (Bourouis et al., EMBO J. 2(7): 1099-1104 (1983)), the EPSPS gene, which confers resistance to glyphosate (U.S. Pat. Nos. 4,940,935 and 5,188,642), and the mannose-6-phosphate isomerase gene, which provides the ability to metabolize mannose (U.S. Pat. Nos. 5,767,378 and 5,994,629).
Methods for regeneration of plants are also well known in the art. For example, Ti plasmid vectors have been utilized for the delivery of foreign DNA, as well as direct DNA uptake, liposomes, electroporation, microinjection, and microprojectiles. In addition, bacteria from the genus Agrobacterium can be utilized to transform plant cells. Below are descriptions of representative techniques for transforming both dicotyledonous and monocotyledonous plants, as well as a representative plastid transformation technique.
Many vectors are available for transformation using Agrobacterium tumefaciens. These typically carry at least one T-DNA border sequence and include vectors such as pBIN19 (Bevan, Nucl. Acids Res. (1984)). For the construction of vectors useful in Agrobacterium transformation, see, for example, US Patent Application Publication No. 2006/0260011, herein incorporated by reference.
Transformation without the use of Agrobacterium tumefaciens circumvents the requirement for T-DNA sequences in the chosen transformation vector and consequently vectors lacking these sequences can be utilized in addition to vectors such as the ones described above which contain T-DNA sequences. Transformation techniques that do not rely on Agrobacterium include transformation via particle bombardment, protoplast uptake (e.g. PEG and electroporation) and microinjection. The choice of vector depends largely on the preferred selection for the species being transformed. For the construction of such vectors, see, for example, US Application No. 20060260011, herein incorporated by reference.
For expression of a nucleotide sequence of the present invention in plant plastids, plastid transformation vector pPH143 (WO 97/32011, example 36) is used. The nucleotide sequence is inserted into pPH143 thereby replacing the PROTOX coding sequence. This vector is then used for plastid transformation and selection of transformants for spectinomycin resistance. Alternatively, the nucleotide sequence is inserted in pPH143 so that it replaces the aadH gene. In this case, transformants are selected for resistance to PROTOX inhibitors.
Transformation techniques for dicotyledons are well known in the art and include Agrobacterium-based techniques and techniques that do not require Agrobacterium. Non-Agrobacterium techniques involve the uptake of exogenous genetic material directly by protoplasts or cells. This can be accomplished by PEG or electroporation mediated uptake, particle bombardment-mediated delivery, or microinjection. Examples of these techniques are described by Paszkowski et al., EMBO J. 3: 2717-2722 (1984), Potrykus et al., Mol. Gen. Genet. 199: 169-177 (1985), Reich et al., Biotechnology 4: 1001-1004 (1986), and Klein et al., Nature 327: 70-73 (1987). In each case the transformed cells are regenerated to whole plants using standard techniques known in the art.
Agrobacterium-mediated transformation is a preferred technique for transformation of dicotyledons because of its high efficiency of transformation and its broad utility with many different species. Agrobacterium transformation typically involves the transfer of the binary vector carrying the foreign DNA of interest (e.g. pCIB200 or pCIB2001) to an appropriate Agrobacterium strain which may depend of the complement of vir genes carried by the host Agrobacterium strain either on a co-resident Ti plasmid or chromosomally (e.g. strain CIB542 for pCIB200 and pCIB2001 (Uknes et al. Plant Cell 5: 159-169 (1993)). The transfer of the recombinant binary vector to Agrobacterium is accomplished by a triparental mating procedure using E. coli carrying the recombinant binary vector, a helper E. coli strain which carries a plasmid such as pRK2013 and which is able to mobilize the recombinant binary vector to the target Agrobacterium strain. Alternatively, the recombinant binary vector can be transferred to Agrobacterium by DNA transformation (Hofgen & Willmitzer, Nucl. Acids Res. 16: 9877 (1988)).
Transformation of the target plant species by recombinant Agrobacterium usually involves co-cultivation of the Agrobacterium with explants from the plant and follows protocols well known in the art. Transformed tissue is regenerated on selectable medium carrying the antibiotic or herbicide resistance marker present between the binary plasmid T-DNA borders.
Another approach to transforming plant cells with a gene involves propelling inert or biologically active particles at plant tissues and cells. This technique is disclosed in U.S. Pat. Nos. 4,945,050, 5,036,006, and 5,100,792 all to Sanford et al. Generally, this procedure involves propelling inert or biologically active particles at the cells under conditions effective to penetrate the outer surface of the cell and afford incorporation within the interior thereof. When inert particles are utilized, the vector can be introduced into the cell by coating the particles with the vector containing the desired gene. Alternatively, the target cell can be surrounded by the vector so that the vector is carried into the cell by the wake of the particle. Biologically active particles (e.g., dried yeast cells, dried bacterium or a bacteriophage, each containing DNA sought to be introduced) can also be propelled into plant cell tissue.
Transformation of most monocotyledon species has now also become routine. Preferred techniques include direct gene transfer into protoplasts using PEG or electroporation techniques, and particle bombardment into callus tissue. Transformations can be undertaken with a single DNA species or multiple DNA species (i.e. co-transformation) and both of these techniques are suitable for use with this invention. Co-transformation may have the advantage of avoiding complete vector construction and of generating transgenic plants with unlinked loci for the gene of interest and the selectable marker, enabling the removal of the selectable marker in subsequent generations, should this be regarded desirable. However, a disadvantage of the use of co-transformation is the less than 100% frequency with which separate DNA species are integrated into the genome (Schocher et al. Biotechnology 4: 1093-1096 (1986)).
Patent Applications EP 0 292 435, EP 0 392 225, and WO 93/07278 describe techniques for the preparation of callus and protoplasts from an elite inbred line of maize, transformation of protoplasts using PEG or electroporation, and the regeneration of maize plants from transformed protoplasts. Gordon-Kamm et al. (Plant Cell 2: 603-618 (1990)) and Fromm et al. (Biotechnology 8: 833-839 (1990)) have published techniques for transformation of A188-derived maize line using particle bombardment. Furthermore, WO 93/07278 and Koziel et al. (Biotechnology 11: 194-200 (1993)) describe techniques for the transformation of elite inbred lines of maize by particle bombardment. This technique utilizes immature maize embryos of 1.5-2.5 mm length excised from a maize ear 14-15 days after pollination and a PDS-1000He Biolistics device for bombardment.
Transformation of rice can also be undertaken by direct gene transfer techniques utilizing protoplasts or particle bombardment. Protoplast-mediated transformation has been described for Japonica-types and Indica-types (Zhang et al. Plant Cell Rep 7: 379-384 (1988); Shimamoto et al. Nature 338: 274-277 (1989); Datta et al. Biotechnology 8: 736-740 (1990)). Both types are also routinely transformable using particle bombardment (Christou et al. Biotechnology 9: 957-962 (1991)). Furthermore, WO 93/21335 describes techniques for the transformation of rice via electroporation.
Patent Application EP 0 332 581 describes techniques for the generation, transformation and regeneration of Pooideae protoplasts. These techniques allow the transformation of Dactylis and wheat. Furthermore, wheat transformation has been described by Vasil et al. (Biotechnology 10: 667-674 (1992)) using particle bombardment into cells of type C long-term regenerable callus, and also by Vasil et al. (Biotechnology 11:1553-1558 (1993)) and Weeks et al. (Plant Physiol. 102: 1077-1084 (1993)) using particle bombardment of immature embryos and immature embryo-derived callus. A preferred technique for wheat transformation, however, involves the transformation of wheat by particle bombardment of immature embryos and includes either a high sucrose or a high maltose step prior to gene delivery. Prior to bombardment, any number of embryos (0.75-1 mm in length) are plated onto MS medium with 3% sucrose (Murashiga & Skoog, Physiologia Plantarum 15: 473-497 (1962)) and 3 mg/l 2,4-D for induction of somatic embryos, which is allowed to proceed in the dark. On the chosen day of bombardment, embryos are removed from the induction medium and placed onto the osmoticum (i.e. induction medium with sucrose or maltose added at the desired concentration, typically 15%). The embryos are allowed to plasmolyze for 2-3 hours and are then bombarded. Twenty embryos per target plate is typical, although not critical. An appropriate gene-carrying plasmid (such as pCIB3064 or pSOG35) is precipitated onto micrometer size gold particles using standard procedures. Each plate of embryos is shot with the DuPont BIOLISTICS® helium device using a burst pressure of about 1000 psi using a standard 80 mesh screen. After bombardment, the embryos are placed back into the dark to recover for about 24 hours (still on osmoticum). After 24 hrs, the embryos are removed from the osmoticum and placed back onto induction medium where they stay for about a month before regeneration. Approximately one month later the embryo explants with developing embryogenic callus are transferred to regeneration medium (MS+1 mg/liter NAA, 5 mg/liter GA), further containing the appropriate selection agent (10 mg/l basta in the case of pCIB3064 and 2 mg/l methotrexate in the case of pSOG35). After approximately one month, developed shoots are transferred to larger sterile containers known as “GA7s” which contain half-strength MS, 2% sucrose, and the same concentration of selection agent.
Transformation of monocotyledons using Agrobacterium has also been described. See, WO 94/00977 and U.S. Pat. No. 5,591,616, and, Negrotto et al., Plant Cell Reports 19: 798-803 (2000).
For example, rice (Oryza sativa) can be used for generating transgenic plants. Various rice cultivars can be used (Hiei et al., 1994, Plant Journal 6:271-282; Dong et al., 1996, Molecular Breeding 2:267-276; Hiei et al., 1997, Plant Molecular Biology, 35:205-218). Also, the various media constituents described below may be either varied in quantity or substituted. Embryogenic responses are initiated and/or cultures are established from mature embryos by culturing on MS-CIM medium (MS basal salts, 4.3 g/liter; B5 vitamins (200×), 5 ml/liter; Sucrose, 30 g/liter; proline, 500 mg/liter; glutamine, 500 mg/liter; casein hydrolysate, 300 mg/liter; 2,4-D (1 mg/ml), 2 ml/liter; adjust pH to 5.8 with 1 N KOH; Phytagel, 3 g/liter). Either mature embryos at the initial stages of culture response or established culture lines are inoculated and co-cultivated with the Agrobacterium tumefaciens strain LBA4404 (Agrobacterium) containing the desired vector construction. Agrobacterium is cultured from glycerol stocks on solid YPC medium (100 mg/L spectinomycin and any other appropriate antibiotic) for about 2 days at 28° C. Agrobacterium is re-suspended in liquid MS-CIM medium. The Agrobacterium culture is diluted to an OD600 of 0.2-0.3 and acetosyringone is added to a final concentration of 200 uM. Acetosyringone is added before mixing the solution with the rice cultures to induce Agrobacterium for DNA transfer to the plant cells. For inoculation, the plant cultures are immersed in the bacterial suspension. The liquid bacterial suspension is removed and the inoculated cultures are placed on co-cultivation medium and incubated at 22° C. for two days. The cultures are then transferred to MS-CIM medium with Ticarcillin (400 mg/liter) to inhibit the growth of Agrobacterium. For constructs utilizing the PMI selectable marker gene (Reed et al., In Vitro Cell. Dev. Biol.-Plant 37:127-132), cultures are transferred to selection medium containing Mannose as a carbohydrate source (MS with 2% Mannose, 300 mg/liter Ticarcillin) after 7 days, and cultured for 3-4 weeks in the dark. Resistant colonies are then transferred to regeneration induction medium (MS with no 2,4-D, 0.5 mg/liter IAA, 1 mg/liter zeatin, 200 mg/liter timentin 2% Mannose and 3% Sorbitol) and grown in the dark for 14 days. Proliferating colonies are then transferred to another round of regeneration induction media and moved to the light growth room. Regenerated shoots are transferred to GA7 containers with GA7-1 medium (MS with no hormones and 2% Sorbitol) for 2 weeks and then moved to the greenhouse when they are large enough and have adequate roots. Plants are transplanted to soil in the greenhouse (T0 generation) grown to maturity, and the T1 seed is harvested.
The plants obtained via transformation with a nucleic acid sequence of the present invention can be any of a wide variety of plant species, including those of monocots and dicots; however, the plants used in the method of the invention are preferably selected from the list of agronomically important target crops set forth supra. The expression of a gene of the present invention in combination with other characteristics important for production and quality can be incorporated into plant lines through breeding. Breeding approaches and techniques are known in the art. See, for example, Welsh J. R., Fundamentals of Plant Genetics and Breeding, John Wiley & Sons, NY (1981); Crop Breeding, Wood D. R. (Ed.) American Society of Agronomy Madison, Wis. (1983); Mayo O., The Theory of Plant Breeding, Second Edition, Clarendon Press, Oxford (1987); Singh, D. P., Breeding for Resistance to Diseases and Insect Pests, Springer-Verlag, NY (1986); and Wricke and Weber, Quantitative Genetics and Selection Plant Breeding, Walter de Gruyter and Co., Berlin (1986).
For the transformation of plastids, seeds of Nicotiana tabacum c.v. “Xanthiene” are germinated seven per plate in a 1″ circular array on T agar medium and bombarded 12-14 days after sowing with 1 um tungsten particles (M10, Biorad, Hercules, Calif.) coated with DNA from plasmids pPH143 and pPH145 essentially as described (Svab, Z. and Maliga, P. (1993) PNAS 90, 913-917). Bombarded seedlings are incubated on T medium for two days after which leaves are excised and placed abaxial side up in bright light (350-500 umol photons/m2/s) on plates of RMOP medium (Svab, Z., Hajdukiewicz, P. and Maliga, P. (1990) PNAS 87, 8526-8530) containing 500 μg/ml spectinomycin dihydrochloride (Sigma, St. Louis, Mo.). Resistant shoots appearing underneath the bleached leaves three to eight weeks after bombardment are subcloned onto the same selective medium, allowed to form callus, and secondary shoots isolated and subcloned. Complete segregation of transformed plastid genome copies (homoplasmicity) in independent subclones is assessed by standard techniques of Southern blotting (Sambrook et al., (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor). BamHI/EcoRI-digested total cellular DNA (Mettler, I. J. (1987) Plant Mol Biol Reporter 5, 346349) is separated on 1% Tris-borate (TBE) agarose gels, transferred to nylon membranes (Amersham) and probed with .sup.32P-labeled random primed DNA sequences corresponding to a 0.7 kb BamHI/HindIII DNA fragment from pC8 containing a portion of the rps 7/12plastid transit sequence. Homoplasmic shoots are rooted aseptically on spectinomycin-containing MS/IBA medium (McBride, K. E. et al. (1994) PNAS 91, 7301-7305) and transferred to the greenhouse.
The genetic properties engineered into the transgenic seeds and plants described above are passed on by sexual reproduction or vegetative growth and can thus be maintained and propagated in progeny plants. Generally, maintenance and propagation make use of known agricultural methods developed to fit specific purposes such as tilling, sowing or harvesting.
Use of the advantageous genetic properties of the transgenic plants and seeds according to the invention can further be made in plant breeding. Depending on the desired properties, different breeding measures are taken. The relevant techniques are well known in the art and include but are not limited to hybridization, inbreeding, backcross breeding, multi-line breeding, variety blend, interspecific hybridization, aneuploid techniques, etc. Thus, the transgenic seeds and plants according to the invention can be used for the breeding of improved plant lines that, for example, increase the effectiveness of conventional methods such as herbicide or pesticide treatment or allow one to dispense with said methods due to their modified genetic properties.
All publications, patents and patent applications are incorporated herein by reference. While in the foregoing specification this invention has been described in relation to certain preferred embodiments thereof, and many details have been set forth for purposes of illustration, it will be apparent to those skilled in the art that the invention is susceptible to additional embodiments and that certain of the details described herein may be varied considerably without departing from the basic principles of the invention.
The following examples are offered by way of illustration and not by way of limitation.
EXPERIMENTALStandard recombinant DNA and molecular cloning techniques used here are well known in the art and are described by J. Sambrook, E. F. Fritsch and T. Maniatis, Molecular Cloning: A Laboratory manual, Cold Spring Harbor laboratory, Cold Spring Harbor, N.Y. (1989) and by T. J. Silhavy, M. L. Berman, and L. W. Enquist, Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1984) and by Ausubel, F. M. et al., Current Protocols in Molecular Biology, pub. by Greene Publishing Assoc. and Wiley-Interscience (1987).
Example 1 Biochemical Method of Identifying a Chloroplast Transit Peptide Sequence from a GlaucocystophyteA Glaucocystophyte will be grown essentially as described by Bayer and Schenk, Curr Genet 16: 311-313 (1989). The culture will be grown with continuous light at 24 degrees C. and 4% carbon dioxide. The cyanelle organelle will be isolated essentially as described by Bayer and Schenk, Curr Genet 16: 311-313 (1989) by first harvesting the cells by centrifugation followed by organelle isolation by osmotic shock (Zook and Schenk, Endocyt Cell Res 3: 203-211 (1986)). The isolated cyanelles are further treated with DNasI and subsequently washed in phosphate buffered saline containing 1 mM magnesium chloride and 1 mM calcium chloride.
Cyanelle proteins will be recovered by lysing the purified cyanelles with lysozyme (0.5 micrograms per microliter of the above phosphate buffered saline solution). The cyanelle proteins will be purified from the lysate by desalting in water and concentrating the resulting lysate using standard techniques.
The cyanelle proteins will be end sequenced to derive an amino terminal partial sequence of the proteins. The protein sequence will be compared to a database containing genomic DNA sequence to identify candidate open reading frames in the nuclear DNA that encodes the cyanelle targeted protein. The cyanelle transit peptide sequence is derived by comparing the amino terminal sequence of isolated protein and the coding region of the nuclear gene to identify the amino terminal extension which serves as the cyanelle transit peptide sequence.
Example 2 Plant Transformation2.A. Maize Transformation:
Transformation of immature maize embryos is performed essentially as described in Negrotto et al., Plant Cell Reports 19:798-803 (2000). Various media constituents described therein can be substituted.
Agrobacterium strain LBA4404 (Invitrogen) containing the plant transformation plasmid is grown on YEP (yeast extract (5 g/L), peptone (10 g/L), NaCl (5 g/L), 15 g/l agar, pH 6.8) solid medium for 2 to 4 days at 28° C. Approximately 0.8×109 Agrobacteria are suspended in LS-inf media supplemented with 100 μM acetosyringone (As) (LSAs medium) (Negrotto et al., Plant Cell Rep 19:798-803 (2000)). Bacteria are pre-induced in this medium for 30-60 minutes.
Immature embryos from maize line, A188, or other suitable maize genotypes are excised from 8-12 day old ears into liquid LS-inf+100 μM As (LSAs). Embryos are vortexed for 5 seconds and rinsed once with fresh infection medium. Infection media is removed and Agrobacterium solution is then added and embryos are vortexed for 30 seconds and allowed to settle with the bacteria for 5 minutes. The embryos are then transferred scutellum side up to LSAs medium and cultured in the dark for two to three days. Subsequently, between 20 and 25 embryos per petri plate are transferred to LSDc medium supplemented with cefotaxime (250 mg/l) and silver nitrate (1.6 mg/l) (Negrotto et al., Plant Cell Rep 19:798-803 (2000)) and cultured in the dark for 28° C. for 10 days.
Immature embryos producing embryogenic callus are transferred to LSD1M0.5S medium (LSDc with 0.5 mg/l 2,4-D instead of Dicamba, 10 g/l mannose, 5 g/l sucrose and no silver nitrate). The cultures are selected on this medium for 6 weeks with a subculture step at 3 weeks. Surviving calli are transferred either to LSD1M0.5S medium to be bulked-up or to Reg1 medium (as described in Negrotto et al., Plant Cell Rep 19:798-803 (2000). Following culturing in the light (16 hour light/8 hour dark regiment), green tissues are then transferred to Reg2 medium without growth regulators (as described in Negrotto et al., Plant Cell Rep 19:798-803 (2000) and incubated for 1-2 weeks. Plantlets are transferred to Magenta GA-7 boxes (Magenta Corp, Chicago Ill.) containing Reg3 medium (as described in Negrotto et al. 2000) and grown in the light. Plants that are PCR positive for any component of the expression cassette are transferred to soil and grown in the greenhouse.
The presence of any component of the expression cassette is determined by +/−PCR assay or by a Taqman copy number assay.
2.B. Tobacco Transformation:
Agrobacterium tumefaciens strain LBA4404 containing a transformation vector containing an expression cassette are used to infect leaf explants of Nicotiana tabacum c.v. Petit Havana (SR1). Initially, the tobacco leaves are cut into 1-2 mm wide slices, exposed to the Agrobacterium for 5 minutes, and are placed on sterile paper to blot away excess liquid and then placed on co-cultivation medium for 3 days. The leaf slices are moved to selection/regeneration medium containing the appropriate selection agent. Selection agent resistant shoots are transplanted to soil. The plants are selfed or outcrossed with pollens from nontransgenic SR1 plants to produce seeds. The presence of any component of the expression cassette is determined by +/−PCR assay or by a Taqman copy number assay.
Example 3 Cyanophora paradoxa FNR Chloroplast Transit Peptide Sequence and cZsGreen in MaizeA cloning vector was created whereby the FNR-transit peptide was fused in-frame to a reporter gene (cZsGreen) with expression driven by the Cestrum Yellow Leaf Curl viral promoter and the NOS-gene terminator. This expression cassette was ligated into a plant transformation binary vector and subsequently used to generate Agrobacterium mediated transgenic maize events essentially as described in Example 2.
Western blot analysis of the T0-maize events found the reporter gene is expressed and that the reporter gene protein was cleaved to a mature size. Subsequently, chloroplasts were isolated from green leaf tissues from both transgenic and non-transgenic maize plants. Fluorescence microscopy (λ=496 nm excitation, λ=506 nm emission) revealed auto-fluorescence in chloroplasts isolated from the transgenic events but not from the negative control non-transgenic chloroplasts confirming the targeting of the expressed reporter gene protein to the chloroplast.
Example 4 Cyanophora paradoxa FNR Chloroplast Transit Peptide Sequence and Endoglucanase in Maize and Tobacco4.A: Expression Cassette Construction
Expression vectors capable of directing the expression of endoglucanase in transgenic plants were designed for both monocot and dicot optimized endoglucanase. Tobacco expression vectors used the constitutive promoter Cestrum yellow leaf curl virus (CYLCV) promoter to drive expression of the dicot optimized endoglucanase gene. Plastid targeting of the endoglucanase was via the transit peptide (SEQ ID NO:2) from ferredoxin-NADP+reductase (FNR) of Cyanophora paradoxa fused to the N-terminus (FEBS Letters 1996: 381, 153-155).
The maize PepC promoter (The Plant Journal 1994: 6(3), 311-319) was used to drive maize leaf specific expression of the monocot optimized endoglucanase. Plastid targeted constructs contained the FNR transit peptide as described in Example 3. All expression cassettes were subcloned into a binary vector for transformation into tobacco and maize using recombinant DNA techniques that are known in the art.
4.B: Transgenic Maize Plants Characterization
Protein extracts were obtained from approximately 100-500 mg of leaf tissue or 100 mg flour generated from maize seed from non-transgenic and transgenic plants. Leaf material was placed into 96 deep well blocks containing small steel balls and pre-cooled on dry ice. Samples were ground to a fine powder using a Geno/Grinder (SPEC/CertiPrep, Metuchen, N.J.). Flour samples were prepared by pooling approximately 10-20 seed and grinding to a fine powder using a KLECO Grinder (Gracia Machine Company, Visalia, Calif.).
Preparation of samples for SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analysis was carried out by extracting 50 to 100 mg leaf material in 250-500 μl of either Western Extraction Buffer (WEB=12.5 mM sodium borate, pH 10; 2% BME; and 1% SDS) or assay buffer, as described below, at room temperature for approximately 30 minutes followed by centrifugation for 5 minutes at 13,000 rpm.
SDS-PAGE was performed by transferring 100 μl of WEB samples to an eppendorf tube and add 25 μl 4XBioRad LDS or modified BioRad loading buffer (4×BioRad LDS:BME at a ratio of 2:1). Samples were heated for 10 minutes at 70° C. then immediately place on ice for 5 minutes. Spin samples briefly, and transfer back on to ice. Sample extracts (5-10 μl) were run on BioRad 4-12% Bis/Tris protein gel (18 well) using MOPS buffer.
Immunoblot analysis was performed by transferring SDS-PAGE gels onto a nitrocellulose membrane using chilled Nupage transfer buffer (Invitrogen) for 30 minutes at 100 volts. Total protein transferred to the blot was visualized using Ponceau stain (Sigma). Following Ponceau staining, the membrane was incubated in blocking buffer for 30 minutes in TBST wash buffer (30 mM Tris-HCL, pH 7.5, 100 mM NaCl, and 0.05% Tween 20) with 3% dry milk, then washed three times for 5 minutes in TBST. Polyclonal goat or rabbit primary antibody was added at 1 ug/ml in TBST wash buffer with 3% milk, and the blot incubated 2 hours to overnight. Following overnight incubation, the blot was washed three times for 5 minutes each in TBST wash buffer. Secondary antibody (Rabbit-AP or Goat-AP) was diluted 1:8000 (in TBST) and added to blot for 30 minutes. Following incubation in the secondary antibody, the blot was again washed three times for 5 minutes each. Visualization of immuno reactive bands was carried out by adding Moss BCIP/NBT-alkaline phosphatase substrate. Blots were rinsed thoroughly in water following incubation in the BCIP/NBT substrate and allowed to air dry.
Approximately 100-500 mg of fresh leaf tissue of approximately 15 day old T0 transgenic plant was extracted in 2 to 10 ml of one of the following buffers: (A) 100 mM Na acetate, 0.02% Tween, 0.02% Na azide pH 4.75, 1% PVP and Complete protease inhibitor cocktail tablets (Roche) or (B) 100 mM Na acetate, 0.02% Tween, and 0.02% Na azide pH 4.75 and Complete protease inhibitor cocktail tablets (Roche); or (C) 1 mM 0.02% Tween-20, 0.02% NaN3 Alternative buffers for extracting protein from leaf are well known in the art. Samples were placed on benchtop rotators for 30-60 minutes then centrifuged at 3000 rpm for 10 minutes. For fresh leaf samples, the amount of total protein extracted was measured by Pierce BCA protocol as outlined in product literature. Endoglucanase activity assays were carried out using one of the following substrates: pNP-lactoside, methylumbelliferyl-lactoside (MUL), carboxymethyl-cellulose (CMC), oat-μ glucan, phosphoric acid treated cellulose (PASC), Avicel, or other available substrates. Other substrates can be used for measuring cellulase activity following previously published protocols (Methods in Enzymology, Vol 160).
The following describes the method used for extracting and assaying, transgenically-expressed endoglucanase from leaf tissue. This method measures endoglucanase enzyme in μmol/min/mg of protein in terms of liberated glucose produced on CM-cellulose (0.5%) at 40° C., pH 4.75. Glucose oxidase/peroxidase (GOPOD) chemistry is used in this assay to measure glucose relative to a standard curve. The glucose-based assay method is a colorimetric assay in which GOPOD reacts with glucose at 40° C. to generate a light to dark pinkish chromophore. The assay consists of 4 basic steps: (1) grinding/milling transgenic tissue, (2) weighing out ground tissue samples, (3) extracting enzyme in Na Acetate buffer, and (4) assaying for enzymatic activity/protein quantification.
Enzymatic data and western blot data for transgenic maize plants expressing endoglucanase is outlined in Table 2. Table 3 outlines transgenic tobacco data generated as described above. The enzymatic data generated in table 2 and 3 was performed using CMC as the substrate as described above.
4.C: Chloroplast Targeting
The above described transgenic maize and tobacco plants will be used to isolate chloroplasts using the Chloroplast Isolation Kit (Sigma) following the manufacturers directions. The isolated chloroplasts will be treated with the peptidase, thermolysin, to remove proteins bound to the outer membrane of the isolated chloroplast. Total protein will be isolated from the treated chloroplasts using standard techniques and the endoglucanase activity measured essentially as described in Example 4.
Example 5 Additional Chloroplast Transit Peptide Sequences Derived from Cyanophora paradoxaAdditional cyanelle transit peptides will be evaluated in transgenic corn plants. The cyanelle transit peptides Rieske1 (transit peptide of SEQ ID NO:3), Rieske2 (maize optimized coding sequence of SEQ ID NO:4 and transit peptide sequence of SEQ ID NO:5) and cytochrome c6 (maize optimized coding sequence of SEQ ID NO:6 and transit peptide sequence of SEQ ID NO:7) from Cyanophora paradoxa will be cloned to the 5′ end of a reporter gene such that the cyanelle transit peptide is at the amino-terminus of the encoded polypeptide of a reporter gene. The reporter gene could be a selectable marker, GUS, CzsGreen or AmCyan (SEQ ID NO:8). The polynucleotide sequences of SEQ ID NOs: 6 and 8 will be cloned to the 5′ end of a selectable marker, GUS, CzsGreen or AmCyan such that the transit peptide is at the amino-terminus of the encoded polypeptide. The reporter gene could be a selectable marker, GUS, CzsGreen or AmCyan.
The coding sequence described above will be cloned into an expression cassette such that the coding sequence is operably linked to a cestrum promoter. The expression cassette will be cloned into a transformation vector containing the PPO gene as a selectable marker for plant transformation. Transgenic maize or tobacco plants will be generated using standard transformation techniques. Transgenic events will be confirmed by Taqman analysis designed to detect any of the components within the expression cassette.
The transit peptides will be evaluated using both western blots (immuno-blotting) and fluorescence. Whole plant extracts from transgenic plant leaf tissue will be prepared as described in the following paragraph and run on an 8-16% tris-glycine gel. The gel will be blotted and then probed with an antibody to the reporter gene (i.e. antibody to AmCyan). Two bands are expected corresponding to the processed (AmCyan without transit peptide) and unprocessed (transit peptide and AmCyan) form of the protein. From there, chloroplast preparations will be made by isolating chloroplasts from the transgenic leaf tissue using the Chloroplast Isolation Kit (Sigma). The purified chloroplasts will be lysed and the resulting lysate run on an 8-16% tris-glycine gel, blotted and probed with the antibody to the reporter gene (such as the anti-AmCyan antibody). We expect to find a band whose size corresponds to the processed form of the protein and the absence of a band that would correspond to an unprocessed form of the protein.
Western blots from whole extracts are performed by placing 3 hole punches of leaf tissue into a microcentrifuge tube and adding 100 μl of Laemmli buffer. The punches are then ground using a pestle and the preparation is then boiled at 100° C. for 10 min. The sample is then centrifuged at 13000 rpm (Eppendorf Centrifuge 5417R) and 10 ml of the supernatant are loaded onto a gel (8-16% tris-glycine, Invitrogen). The gel is run at 150V for 60 min. The separated proteins are then transferred to a PVDF membrane via electrophoresis (350 mA for 60 min.). The membrane is blocked for 1 hour at 25° C. in 5% milk. Blocked membranes were placed in a primary antibody solution of 1:5000 in PBST (PBS with 0.1% TWEEN 20) and placed on a rocker at 25° C. for 2 hours. The membrane is removed from the primary solution, washed in PBST (5, 10 min washes on a rocker at 25° C.) and placed in a secondary antibody solution (1:5000 dilution in PBST) for 2 hours at 25° C. on a rocker. The membrane is washed (5, 10 min washes on a rocker at 25° C.) and developed in BCIP. The BCIP reaction is stopped with tap water and the developed blot is allowed to dry at room temperature.
When using the AmCyan as a reporter gene, we can use confocal microscopy (Zeiss, LSM 710) to analyze the reporter protein (AmCyan) in relation to endogenous, auto-fluorescence within the cell. For our experiments we would expect the AmCyan fluorescence and the chlorophyll auto-fluorescence to co-localize within the cell when the transit peptide is performing as expected.
All publications and patent applications mentioned in the specification are indicative of the level of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.
Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended claims.
Claims
1. An isolated nucleic acid comprising a first nucleotide sequence encoding a plastid transit peptide from a Glaucocystophyte wherein said first nucleotide sequence is operably linked to a second nucleotide sequence encoding a heterologous polypeptide.
2. The isolated nucleic acid of claim 1, wherein the Glaucocystophyte is Cyanophora.
3. The isolated nucleic acid of claim 1, wherein the Glaucocystophyte is Cyanophora paradoxa.
4. The isolated nucleic acid of claim 1, wherein the plastid transit peptide is selected from the group consisting of SEQ ID NOs: 2, 3, 5 and 7.
5. A transgenic plant comprising a first nucleotide sequence encoding a plastid transit peptide from a Glaucocystophyte wherein said first nucleotide sequence is operably linked to a second nucleotide sequence encoding a heterologous polypeptide, wherein said heterologous polypeptide is targeted to a chloroplast.
6. The transgenic plant of claim 5, wherein the Glaucocystophyte is Cyanophora.
7. The transgenic plant of claim 5, wherein the Glaucocystophyte is Cyanophora paradoxa.
8. The transgenic plant of claim 5, wherein the plant is a monocot.
9. The transgenic plant of claim 5, wherein the plant is a dicot.
10. The transgenic plant of claim 5, wherein the plastid transit peptide encodes the polypeptide selected from the group consisting of SEQ ID NOs:2, 3, 5 and 7.
11. The transgenic plant of claim 8, wherein the monocot is a maize or a sugarcane plant.
12. The transgenic plant of claim 9, wherein the dicot is a soybean or a sugarbeet plant.
13. A method of stably expressing a heterologous polypeptide in a plant, said method comprising introducing into said plant a DNA construct comprising a first nucleotide sequence encoding a plastid transit peptide from a Glaucocystophyte, wherein said first nucleotide sequence is operably linked to a second nucleotide sequence encoding said heterologous polypeptide.
14. The method of claim 13, wherein the Glaucocystophyte is Cyanophora.
15. The method of claim 13, wherein the Glaucocystophyte is Cyanophora paradoxa.
16. The method of claim 13, wherein the plant is a monocot.
17. The method of claim 13, wherein the plant is a dicot.
18. The method of claim 13, wherein the plastid transit peptide encodes the polypeptide selected from the group consisting of SEQ ID NO:2, 3, 5 and 7.
19. The method of claim 16, wherein the monocot is a maize or a sugarcane plant.
20. The method of claim 17, wherein the dicot is a soybean or a sugarbeet plant.
21. An isolated polypeptide comprising a plastid transit peptide from a Glaucocystophyte operably linked to a heterologous polypeptide.
22. The isolated polypeptide of claim 21, wherein the Glaucocystophyte is Cyanophora.
23. The isolated polypeptide of claim 21, wherein the Glaucocystophyte is Cyanophora paradoxa.
24. The isolated polypeptide of claim 21, wherein the plant is a monocot.
25. The isolated polypeptide of claim 21, wherein the plant is a dicot.
26. The isolated polypeptide of claim 21, wherein the plastid transit sequence encodes the polypeptide selected from the group consisting of SEQ ID NOs:2, 3, 5, and 7.
27. The isolated polypeptide of claim 24, wherein the monocot is a maize or a sugarcane plant.
28. The isolated polypeptide of claim 25, wherein the dicot is a soybean or a sugarbeet plant.
Type: Application
Filed: Jan 15, 2009
Publication Date: Aug 13, 2009
Inventors: Stacy Miles (Durham, NC), John Hipskind (Durham, NC), Gregory W. Warren (Durham, NC)
Application Number: 12/354,216
International Classification: C12N 15/82 (20060101); C12N 15/11 (20060101); A01H 5/00 (20060101); C07K 14/00 (20060101);
