Plant promoter derived from luminal binding protein gene and methods for its use
The invention provides a luminal binding protein promoter (PmBiPPro1), deletions thereof, and variants thereof. The promoter is useful for, among other things, directing the expression of transgenes.
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[0001] This invention relates to an isolated luminal binding protein promoter sequence and methods for its use.
BACKGROUND[0002] Molecular Chaperone Proteins
[0003] Luminal binding proteins (BiP) have been identified as a type of molecular chaperone localized within the endoplasmic reticulum (ER) and nuclear envelope of eukaryotic cells. BiP is a member of the heat-shock protein 70 (HSP70) family of proteins (Haas, Experimentia 50:1012-1020, 1994). BiP has been found to assist in the co-translational translocation of newly synthesized polypeptides across the ER membrane in yeast (Vogel et al., J. Cell Biol. 110:1885-1895, 1990; and Nguyen et al., Proc. Natl. Acad. Sci. USA 88:1565-1569, 1991). BiP remains associated with polypeptides until they attain their properly folded conformation and/or subunit assembly. For polypeptides that are unable to attain their mature conformation due to misfolding (Schmitz et al., EMBO J. 14:1091-1098, 1995) or lack of a subunit component (Knittler et al., Proc. Natl. Acad. Sci. USA 92:1764-1768, 1995), BiP remains associated with the polypeptide until the polypeptide is degraded.
[0004] In angiosperms, the expression of BiP is subject to developmental, hormonal, stress-induced, and diurnal regulation (Denecke et al., Plant Cell 3:1025-1035, 1991; Jones et al., Plant Physiol. 97:456-459, 1991; Anderson et al., Plant Physiol. 104:1359-1370, 1994; Kalinski et al., Planta 195:611-621, 1995; and Figueiredo et al., Braz. J. Plant Physiol. 9:103-110, 1997). BiP associates with the bean storage protein phaseolin (D'Amico et al., Plant J. 2:443-455, 1992; Pedrazzini et al., Plant J. 5:103-110, 1994) and with rice prolamines (Li et al., Science 262:1054-1056, 1993). High levels of BiP expression are associated with the accumulation of protein intermediates that are unable to attain their proper folded conformation because of mutations such as those seen in the maize zein regulatory mutants “floury-2,” “defective endosperm-B30,” and “mucronate” (Boston et al., Plant Cell 3:497-505, 1991; Fontes et al., Plant Cell 3:483-496, 1991).
[0005] Treatment with tunicamycin, which inhibits N-linked glycosylation and proper protein folding, also results in increased levels of BiP expression (Denecke et al., Plant Cell 3:1025-1035, 1991; and D'Amico et al., Plant J. 2:443-455, 1992). However, the increased expression resulting from unfolded proteins and from increased levels of secretory protein traffic may be mediated through different signals (Pahl et al., EMBO J. 14:2580-2588, 1995).
SUMMARY[0006] The invention provides a Douglas-fir (Pseudotsuga menziesii) lumninal binding protein promoter (PmBiPPro1; SEQ ID NO: 31). Expression of the PmBiP protein (SEQ IDNO: 36) is shown herein to be developmentally-regulated and inducible by environmental changes. The promoter (PmBiPPro1; SEQ ID NO: 31), fragments thereof, and variants thereof are useful for expressing heterologous proteins either transiently in host cells or transgenically in stably transformed cells and plants.
[0007] One aspect of the invention provides the PmBiP promoter (SEQ ID NO: 31), fragments/deletions of the PmBiP promoter (SEQ ID NO: 31) and variants thereof The variant promoters are characterized by their retention of at least 50% sequence identity with the disclosed promoter sequences (SEQ ID NOS: 16, 17, 18, and 31, respectively), or by their retention of at least 20, 30, 40, 50, or 60 consecutive nucleic acid residues of the disclosed promoter sequences (SEQ ID NOS: 16, 17, 18, and 31). In each case these promoters, at a minimum, retain promoter activity. In some cases these promoters retain native PmBiP promoter activity.
[0008] It is also contemplated that promoters such as the CaMV35S promoter may be altered through the introduction of sequences found in the PmBiP promoter (SEQ ID NO: 31). The resulting promoter also will be characterized by its retention of at least 20, 30, 40, 50, or 60 consecutive nucleic acid residues of the disclosed promoter sequences (SEQ ID NOS: 16, 17, 18, and 31).
[0009] Another aspect of the invention provides vectors containing the above-described promoters and variants thereof. The vectors can be transformed into host cells. If the host cell is a plant cell, the transformed host cell can give rise to a transgenic plant.
[0010] The invention also provides transgenes. These transgenes include one of the above-described promoter sequences operably linked to one or more open reading frames (ORFs). The transgenes can be cloned into vectors and subsequently used to transform host cells such as bacterial, insect, mammalian, fungal, yeast, or plant cells.
[0011] Accordingly, the invention provides transgenic plants such as maize, wheat, rice, millet, tobacco, sorghum, rye, barley, brassica, sunflower, seaweeds, lemna, oat, soybean, cotton, legumes, rape/canola, alfalfa, flax, sunflower, safflower, brassica, cotton, flax, peanut, and clover; lettuce, tomato, cucurbits, cassava, potato, carrot, radish, pea, lentil, cabbage, cauliflower, broccoli, Brussel sprouts, peppers and other vegetables; citrus, apples, pears, peaches, apricots, walnuts, and other fruit trees; orchids, carnations, roses, and other flowers; cacao; poplar, elms, and other deciduous trees; pine, Douglas-fir, spruce, and other conifers; turf grasses; cacao; and rubber trees and other members of the genus Hevea.
[0012] In yet another embodiment, the invention provides methods for expressing proteins in host cells, such as plant host cells. Such methods involve operably linking a promoter, such as those described above, to at least one ORF to produce a transgene, and introducing the transgene into a plant. Accordingly, the invention also provides proteins that are produced by these methods.
[0013] The PmBiP promoter (SEQ ID NO: 31) is shown herein to be inducible (at least via wounding and probably via cold temperatures), and the amount of mRNA encoding PmBiP protein as well as BiP protein itself has been shown to be increased at cold temperatures, thus making the PmBiP promoter ideal for use in the expression of proteins. This is because cold temperatures serve to stabilize the protein during translation. Accordingly, another aspect of the invention provides inducible promoters derived through the use of fragments of the promoter described herein.
[0014] An alternative method of characterizing promoters is by analyzing the various promoter elements found within a promoter sequence. Hence, the invention also provides promoters that maintain promoter activity and include at least 8 promoter elements selected from the group consisting of the E-box motif (SEQ ID NO: 1), the MNF1 element (SEQ ID NO: 28), the POLLEN1LELAT52 element (SEQ ID NO: 29), the ROOTMOTIF element (SEQ ID NO: 30), the 2SSEEDPROTBANAP element (SEQ ID NO: 32), the BOXIIPCCHS element (SEQ ID NO: 33), the ASF1MOTIF element (SEQ ID NO: 34), ACGT-core elements (SEQ ID NO: 4), the CAAT-box (SEQ ID NO: 9), the CANABNNAPA element (SEQ ID NO: 12), the HEXMOTIF element (SEQ ID NO: 27), and duplicates thereof, wherein the promoter displays promoter activity. The invention also provides promoters that contain the following promoter elements in the following orientation: 3′-ACGT-core element (SEQ ID NO: 4), E-box motif (SEQ ID NO: 1), CAAT-box (SEQ ID NO: 9), 2SSEEDPROTBANAP (SEQ ID NO: 32), CANABNNAPA element (SEQ ID NO: 12), HEXMOTIF element (SEQ ID NO: 27), CAAT-box (SEQ ID NO: 9), BOXIIPCCHS element (SEQ ID NO: 33), E-box motif (SEQ ID NO: 1), ASF1MOTIF (SEQ ID NO: 34), POLLEN1LELAT52 element (SEQ ID NO: 29), and MNF1 element (SEQ ID NO: 28)-5′.
[0015] Finally, the invention also provides vectors, host cells, and transgenic plants that include the promoters described above by their inclusion of various promoter elements.
[0016] These and other aspects of the invention will become readily apparent from the following detailed description.
BRIEF DESCRIPTION OF THE FIGURES[0017] FIG. 1 is a graph depicting the expression of PmBiP protein during Douglas-fir seed development. Total RNA was isolated from whole developing seeds collected at the indicated time points and subjected to northern blot analysis (20 &mgr;g per lane) using the PmBiP cDNA as probe. The indicated time points correspond to the following developmental stages, based on morphological characteristics of embryos established by Allen and Owens (Allen and Owens, The Life History of Douglas-fir, Environment Canada, Canadian Forestry Service, Ottawa, 1972): May 31—pre-fertilization, June 19—proembryo, July 12—early to mid-cotyledonary embryo, August 1—mid to late embryo, August 15—late to mature embryo. The same membrane was stripped and re-probed with a genomic DNA probe encoding the Douglas-fir 18S rRNA subunit to account for differences in the amount of RNA loaded per lane. Differences in the amount of PmBiP transcript were adjusted for differences in the amount of RNA loaded per lane (given in units of integrated optical density).
[0018] FIG. 2 is a graph depicting the expression of PmBiP RNA during germination and early seedling development. Total RNA was isolated from tissue collected at the indicated time points and subjected to northern blot analysis (20 &mgr;g per lane) using the PmBiP cDNA as probe. The same membrane was stripped and re-probed with a genomic DNA probe encoding the Douglas-fir 18S rRNA subunit to account for differences in the amount of RNA loaded per lane. Differences in the amount of PmBiP transcript were adjusted for differences in the amount of RNA loaded per lane (given in units of integrated optical density).
[0019] FIG. 3 is a graph showing changes in PmBiP RNA levels in response to cold treatment. The RNA was isolated from 14-day-old seedlings and analyzed by northern blot procedures.
[0020] FIG. 4 is a graph showing the seasonal variation of PmBiP protein in needles from 1-year-old seedlings. Total protein was isolated from the needles of 1-year-old Douglas-fir seedlings at the indicated times and subjected to western blot analysis (15 &mgr;g per lane). Following blot development, immunoreactive bands were quantified using scanning densitometry and displayed graphically (given in units of arbitrary density). (A) shows PmBiP protein levels, and (B) shows the monthly average temperatures (given in degrees Celsius) to allow direct comparison at each time point.
[0021] FIGS. 5A-5I are a table of cis-acting elements located in the PmBiP promoter (SEQ ID NO: 31) using the plant cis-acting regulatory database (PLACE; Higo et al, Plant Mol. Biol. Rep. 5:387-405. 1987; and Prestridge, CABIOS 7:203-206, 1991). Cis elements are grouped according to type. Elements deleted from PmBiPPro1-1 construct to form PmBiPPro1-3 are darkly shaded, elements deleted from PmBiPPro1-3 construct to form PmBiPPro1-5 are lightly shaded, and elements remaining in the PmBiPPro1-5 construct are not shaded.
[0022] FIG. 6 is a schematic diagram showing a comparison of the full-length PmBiPPro1 sequence (SEQ ID NO: 31; PmBiPPro1) to the deletion constructs PmBiPPro1-1 (SEQ ID NO: 16), PmBiPPro1-3 (SEQ ID NO: 17), and PmBiPPro1-5 (SEQ ID NO: 18).
[0023] FIG. 7 is a graph of the results from the transient expression of GUS in Douglas-fir zygotic embyros under the control of the CaMV35S, PmBiPPro1-1 (SEQ ID NO: 16), PmBiPPro1-3 (SEQ ID NO: 17), and PmBiPPro1-5 (SEQ ID NO: 18) promoter sequences. The results represent the average of two trials, each trial involving ten embryos, and the data is reported as an average number of expression foci per embyro.
[0024] FIG. 8 is a graph of data from in vitro GUS activity of 19-day-old transgenic Arabidopsis plants containing various PmBiPPro1 constructs. GUS activity was measured in 6 &mgr;g of total protein extracted from whole seedlings as described herein. Two transformants were examined for each construct. Results represent the average and standard deviation of three trials for each plant extract.
[0025] FIG. 9 is a graph of the results from PmBiPPro1-1 (SEQ ID NO: 16) expression in response to wounding in 21-day-old Arabidopsis cotyledons. One cotyledon was wounded from each plant, while the other served as control. GUS activity was measured in 6 &mgr;g of total protein extracted, 18 hours after wounding (pinching with forceps). GUS activity is measured as change in absorbance (&Dgr;OD) after 18 hours. GUS assays also were performed on wounded untransformed and CaMV35S:GUS plant cotyledons. Results represent the average and standard deviation of three trials for each plant extract induced via wounding.
[0026] FIG. 10 is the nucleotide and deduced amino acid sequence of PmBiP cDNA. The nucleotide sequence is numbered on the left, and the amino acid sequence is numbered on the right. Untranslated regions are in lower-case letters and the open reading frame is capitalized. The three potential start codons are underlined, with the amino acid sequence beginning at the 3rd codon. The predicted signal-peptide cleavage site and beginning of the mature PmBiP amino acid sequence is indicated by an asterisk (Nielsen et al., Protein Eng. 10: 1-6, 1997). The ER-retention signal sequence is boxed. The 13 carboxy-terminal amino acids used to generate an antiserum to the peptide are indicated in bold italics.
SEQUENCE LISTING[0027] The nucleic and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, and three-letter code for amino acids. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood to be included by any reference to the displayed strand.
[0028] SEQ ID NO: 1 is the nucleic acid sequence of an E-box motif.
[0029] SEQ ID NO: 2 is the nucleic acid sequence of a RY-repeated element.
[0030] SEQ ID NO: 3 is the nucleic acid sequence of an AT-rich region.
[0031] SEQ ID NO: 4 is the nucleic acid sequence of an ACGT-core element.
[0032] SEQ ID NO: 5 is the nucleic acid sequence of an opaque-2-like binding site.
[0033] SEQ ID NOs: 6 and 7 are the nucleic acid sequences of respective conserved gymnosperm-like regions.
[0034] SEQ ID NO: 8 is the nucleic acid sequence of a TATA box.
[0035] SEQ ID NO: 9 is the nucleic acid sequence of a CAAT box.
[0036] SEQ ID NO: 10 is the nucleic acid sequence of a MYBPZM element.
[0037] SEQ ID NO: 11 is the nucleic acid sequence of a GT1 consensus sequence.
[0038] SEQ ID NO: 12 is the is the nucleic acid sequence of a CANBNAPA element.
[0039] SEQ ID NO: 13 is the nucleic acid sequence of a MARARS element.
[0040] SEQ ID NOS: 14 and 15 are specific examples of opaque-2-like binding sites.
[0041] SEQ ID NO: 16 is the nucleic acid sequence of the PmBiPPro1-1 promoter construct.
[0042] SEQ ID NO: 17 is the nucleic acid sequence of the PmBiPPro1-3 construct.
[0043] SEQ ID NO: 18 is the nucleic acid sequence of the PmBiPPro1-5 construct.
[0044] SEQ ID NOS: 19-22 are respective PCR primers used in inverse-PCR reactions.
[0045] SEQ ID NOS: 23-26 are respective PCR primers used to clone the PmBiP promoter.
[0046] SEQ ID NO: 27 is the nucleic acid sequence of a HEXMOTIF element.
[0047] SEQ ID NO: 28 is the nucleic acid sequence of a MNF1 element.
[0048] SEQ ID NO: 29 is the nucleic acid sequence of a POLLEN1LELAT52 element.
[0049] SEQ ID NO: 30 is the nucleic acid sequence of a ROOTMOTIF element.
[0050] SEQ ID NO: 31 is the complete PmBiP promoter sequence.
[0051] SEQ ID NO: 32 is the nucleic acid sequence of a 2SSEEDPROTBANAP element.
[0052] SEQ ID NO: 33 is the nucleic acid sequence of a BOXIIPCCHS element.
[0053] SEQ ID NO: 34 is the nucleic acid sequence of an ASF1MOTIF.
[0054] SEQ ID NO: 35 is the cDNA sequence encoding the luminal binding protein BiP.
[0055] SEQ ID NO: 36 is the amino acid sequence of the luminal binding protein.
[0056] SEQ ID NO: 37 is the amino acid sequence of the endoplasmic reticulum (ER) retention sequence, HEEL.
DETAILED DESCRIPTION[0057] I. Definitions
[0058] Unless otherwise noted, technical terms are used according to conventional usage. Definitions of common terms in molecular biology may be found in Lewin, Genes VII, Oxford University Press, 1999 (ISBN 0-19-879276-X); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, Blackwell Science, Ltd., 1994 (ISBN 0-632-02182-9); and Meyers (ed.), Molecular Biology and Biotechnology: A Comprehensive Desk Reference, VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8).
[0059] In order to facilitate review of the various embodiments of the invention, the following definitions are provided:
[0060] “cDNA (complementary DNA).” A “cDNA” is a piece of DNA lacking internal, non-coding segments (introns) and transcriptional regulatory sequences. cDNA also may contain untranslated regions (UTRs) that are responsible for translational control in the corresponding RNA molecule. cDNA usually is synthesized in the laboratory by reverse transcription from messenger RNA extracted from cells.
[0061] “Cationic Peptides.” “Cationic peptides” are endogenous antimicrobial peptides produced by plants and animals typically consisting of 12-45 amino acids. Additionally, they are amphipathic molecules having a net positive charge (cationic) at physiological pH. Although cationic antimicrobial peptides (CAPs) are structurally diverse, they fall into two general classes of structures: &agr;-helical peptides, such as the cecropins and magainans, and &bgr;-sheet peptides stabilized by intramolecular disulphide bonds, such as the defensins, protegrins, and tachyplesins. Hancock and Lehrer, Trends Biotechnol. 16:82-88, 1998; Zasloff, Curr. Opin. Immunol. 4:3-7, 1992; Cociancich et al., Biochem. J. 300:567-575 1994; and Piers and Hancock, Mol. Microbiol. 12:951-958, 1994. Natural CAPs vary greatly in their respective spectra of biological activities, including killing bacteria (Gram-positive and -negative), fungi, protozoa, and viruses. CAPs normally kill susceptible microorganisms in vitro at concentrations from 0.25 &mgr;g/ml to 4 &mgr;g/ml (Hancock and Lehrer, Trends Biotechnol. 16:82-88, 1998), providing exciting possibilities in the face of the declining efficacy of conventional antibiotics. Furthermore, the expression of CAP in plants may introduce broad-spectrum resistance to phytopathogenic microorganisms. Jaynes, Plant Science 89:43-53, 1993; and Misra and Zhang, Plant Physiol. 106:977-981, 1994.
[0062] Cationic peptides are one type of protein that might be expressed under the control of the disclosed PmBiP promoter (SEQ ID NO: 31). Other proteins that confer disease resistance, resistance to environmental stress, resistance to insect infestation, or herbicide resistance, or alter consumer-related characteristics such as shelf-life, color, or nutritional value, also may be expressed under the control of the PmBiP promoter (SEQ ID NO: 31) described herein.
[0063] “Deletion.” A “deletion” is the removal of one or more nucleic acid residues from a DNA sequence, the regions on either side of the removed sequence being joined together.
[0064] “Douglas-fir luminal binding protein promoter (PmBiPPro1).” The nucleic acid sequence of the PmBiP promoter is provided in SEQ ID NO: 31. However, the invention also encompasses variants and fragments of the PmBiP promoter (SEQ ID NO: 31) that are characterized by their ability to maintain promoter activity, at a minimum, and in some cases maintain native PmBiP promoter activity. These variants have at least 50%, 60%, 70%, 80%, or 90% sequence identity when compared to the nucleic acid sequences shown in SEQ ID NOS: 16, 17, 18, and 31. These variants can be isolated from nature using the hybridization or PCR techniques described below, or they can be made by manipulating the nucleic acid sequences shown in SEQ ID NOS: 16, 17, 18, and 31.
[0065] The PmBiP promoter shown in SEQ ID NO: 31 contains several distinct promoter elements and inter-element spaces that are arranged in series in the DNA fragment. One or more of these elements or inter-element spaces can be altered, deleted, and/or duplicated without loss of promoter activity. Also, one of ordinary skill in the art will appreciate other promoter elements may be added to the promoter shown in SEQ ID NO: 31 without loss of promoter activity and/or native PmBiP promoter activity. Hence, the invention provides promoters that maintain native promoter activity and/or promoter activity and include at least 10, 12, 14, 16, 18, 20, 22, 30, or 35 of the promoter elements contained within the PmBiP promoter (SEQ ID NO: 31).
[0066] Variants of the PmBiP promoter also can be characterized by the number of contiguous nucleic acid residues they share with the PmBiP promoter (SEQ ID NO: 31). For example, a variant of the PmBiP promoter can share at least 20, 25, 30, 40, 50, or 60 contiguous nucleic acid residues with the PmBiP promoter shown in SEQ ID NO: 31. Such variants additionally will be characterized by their ability to drive the expression of a transgene operably linked to it.
[0067] “Insertion.” An “insertion” is the addition of one or more nucleotide or amino acid residues into a nucleic acid sequence or an amino acid sequence, respectively.
[0068] “Isolated.” An “isolated” biological component (such as a nucleic acid, protein, or organelle) has been substantially separated or purified from other biological components in the cell of the organism in which the component naturally occurs, i.e., other chromosomal and extra-chromosomal DNA and RNA, proteins, and organelles. Nucleic acids and proteins that have been “isolated” include nucleic acids and proteins purified by standard purification methods. The term also embraces nucleic acids and proteins prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acids.
[0069] “Native PmBiP Promoter Activity.” “Native PmBiP promoter activity” is characterized by developmental-specific transcription. mRNA encoding the PmBiP protein has been shown to be expressed in seeds, following stratification and exposure to germination conditions, to a greater extent than in mature seeds. Hence, it is believed that the PmBiP promoter (SEQ ID NO: 31) will drive the expression of transgenes in a similar pattern. Developmental-specific activity is defined as the ability of a promoter to drive transcription at a higher level during one stage in development compared to another stage of development.
[0070] Furthermore, developmental-specific expression can be determined by creating transgenic plants and assaying the resulting transgenic tissues (e.g., leaves, flowers, seeds, roots) for transgene mRNA or by assaying for a reporter gene such as GUS. Developmental-specific expression is quantified by comparing the level of mRNA expressed in a tissue during one stage in development compared to the level expressed in the same tissue at another stage of development. The degree of developmental-specific expression is expressed in terms of a percentage of expression, i.e., the percentage of mRNA in one developmental stage compared to another. For example 100% (1×) expression denotes that an equal amount of expression is observed during two distinct stages of development, 200% (2×) denotes that twice as much mRNA is expressed in one tissue compared to another tissue. Native PmBiP promoter activity is, therefore, defined by the ability of the PmBiP promoter to drive the expression of mRNA to a greater degree during one stage in plant development compared to another stage in development (i.e., at least 101%). Of course, the PmBiP promoter (SEQ ID NO: 31) can show an even stronger bias for developmental-specific expression, such as at least 125%, 150%, 200%, 250%, or 300% developmental-specific expression in seeds.
[0071] “Oligonucleotide (“oligo”). An “oligonucleotide” refers to a linear polynucleotide sequence of up to about 100 nucleotide bases in length.
[0072] “Open reading frame (ORF).” An “open reading frame” is a series of nucleotide triplets (codons) coding for amino acids without any internal termination codons. These sequences are usually translatable into a peptide.
[0073] “Operably linked.” A first nucleic acid sequence is “operably linked” with a second nucleic acid sequence whenever the first nucleic acid sequence is situated in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, where necessary to join two protein-coding regions, are in the same reading frame.
[0074] “Orthologs.” “Orthologs” are nucleic acid or amino acid sequences that share a common ancestral sequence, but that diverged when a species carrying that ancestral sequence split into two species. Orthologous sequences are usually also homologous sequences.
[0075] “Probes and primers.” Nucleic acid “probes and primers” readily may be prepared based on the nucleic acid sequences provided by this invention. A “probe” comprises an isolated nucleic acid sequence attached to a detectable label or reporter molecule. These labeled nucleic acid sequences are useful for identifying other promoters and seed-storage proteins. Typical labels include radioactive isotopes, ligands, chemiluminescent agents, and enzymes. Methods for labeling and guidance in the choice of labels appropriate for various purposes are discussed, e.g., in Sambrook et al. (eds.), Molecular Cloning: A Laboratory Manual, 2nd ed., vol. 1-3, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989; and Ausubel et al. (ed.), Current Protocols in Molecular Biology, Greene Publishing and Wiley-Interscience, New York (with periodic updates), 1987.
[0076] “Primers” are short nucleic acids, preferably DNA oligonucleotides 15 nucleotides or more in length, that can be annealed to a complementary target DNA strand by nucleic acid hybridization to form a hybrid between the primer and the target DNA strand. The annealed primers can be extended along the target DNA strand by a DNA polymerase enzyme. Primer pairs can be used for amplification of a nucleic acid sequence, e.g., by the polymerase chain reaction (PCR) or other nucleic-acid amplification methods known in the art.
[0077] As noted, probes and primers are preferably 15 nucleotides or more in length, but, to enhance specificity, probes and primers of 20 or more nucleotides may be preferred.
[0078] Methods for preparing and using probes and primers are described, for example, in Sambrook et al. (eds.), Molecular Cloning: A Laboratory Manual, 2nd ed., vol. 1-3, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989; Ausubel et al. (eds.), Current Protocols in Molecular Biology, Greene Publishing and Wiley-Interscience, New York (with periodic updates), 1987; and Innis et al., PCR Protocols: A Guide to Methods and Applications, Academic Press: San Diego, 1990. PCR primer pairs can be derived from a known sequence, for example, by using computer programs intended for that purpose such as Primer™ (Version 0.5, 1991, Whitehead Institute for Biomedical Research, Cambridge, Mass.). One of skill in the art will appreciate that the specificity of a particular probe or primer increases with the length of the probe or primer. For example, a primer comprising 20 consecutive nucleotides will anneal to a target with a higher specificity than a corresponding primer of only 15 nucleotides. Thus, in order to obtain greater specificity, probes and primers may be selected that comprise, by way of example, 10, 20, 25, 30, 35, 40, 50 or more consecutive nucleotides.
[0079] “Promoter Activity.” “Promoter activity” is defined as the ability of a DNA sequence to drive transcription. Promoter activity varies with the number and position of the promoter elements. For example, the PmBiP promoter (SEQ ID NO: 31) can be altered to remove its developmental-specific activity (native activity) without loss of its ability to drive transcription.
[0080] “Promoter elements.” “Promoter elements” as used herein refers to sub-domains within the promoter that confer tissue-specific expression, enhance expression, or inhibit expression. A promoter can contain multiple promoter elements. Furthermore, some elements can appear more than once within a single promoter. Examples of such elements are E-box motifs (SEQ ID NO: 1), RY-repeat elements (SEQ ID NO: 2), AT-rich regions (SEQ ID NO: 3), ACGT-core elements (SEQ ID NO: 4), Opaque-2-like elements (SEQ ID NO: 5), and conserved gymnosperm-like regions (SEQ ID NOS: 6 and 7). Additional examples of promoter elements can be found in U.S. Pat. Nos.: 5,723,751 to Chua; 5,608,149 to Barry et al.; 5,589,615 to De Clercq et al.; 5,589,583 to Klee et al.; 5,677,474 to Rogers; 5,487,991 to Vandekerckhove et al.; and 5,530,194 to Knauf et al. Typically, a TATA box is found on the 3′-end of the series of promoter elements.
[0081] Examples of specific promoter elements are provided above and in the sequence listing. However, one of skill in the art will appreciate that the specific examples shown in the sequence listing can be modified while still maintaining activity. For example a base in an RY-repeat element can be altered by the substitution of one or more acid residues without the RY-repeat element losing its functionality within the overall promoter sequence.
[0082] After a promoter has been identified, the promoter elements can be characterized, such as is described below for the PmBiP promoter (SEQ ID NO: 31; FIG. 5). This promoter contains a series of identifiable promoter elements. These elements appear in series in the genomic DNA as is shown schematically in FIG. 6. The space between the elements is hereinafter referred to as “inter-element space.” An inter-element space can be modified through the addition, deletion, and/or substitution of nucleotides without loss of promoter activity.
[0083] The PmBiP promoter (SEQ ID NO: 31) also can be modified by deleting elements from the promoter and/or duplicating elements within the promoter. One of ordinary skill in the art will appreciate that such modifications to the promoter can enhance promoter activity, inhibit promoter activity, or alter the level of tissue-specific expression of the promoter.
[0084] One of skill in the art will appreciate that, by modifying the order of the promoter elements, the number of the promoter elements, and/or the length of the inter-element space(s), one can modify promoter activity and/or native PmBiP promoter activity. However, in each case, the PmBiP promoter (SEQ ID NO: 31) will be capable of driving the expression of the gene operably linked to it. Assays for quantifying PmBiP activity as well as native PmBiP activity are provided below.
[0085] “Protein,” A biological molecule expressed by a gene and comprised of amino acids.
[0086] “Purified.” The term “purified” does not require absolute purity; rather, it is intended as a relative term. Thus, for example, a purified protein preparation is one in which the protein referred to is purer than the protein in its natural environment within a cell or within a production reaction chamber (as appropriate).
[0087] “Recombinant.” A “recombinant” nucleic acid is one having a sequence that does not occur naturally or having a sequence made by an artificial combination of two otherwise separated sequences. This artificial combination is often accomplished by chemical synthesis or, more commonly, by the manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques.
[0088] “Sequence identity.” The term “sequence identity” is used to describe the similarity between two nucleic acid sequences or between two amino acid sequences. Sequence identity is typically expressed in terms of percentage identity; the higher the percentage, the more similar the two sequences.
[0089] Methods for aligning sequences for comparison purposes are well-known in the art. Various programs and alignment algorithms are described in: Smith and Waterman, Adv. Appl. Math. 2:482, 1981; Needleman and Wunsch, J. Mol. Biol. 48:443, 1970; Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444-2448, 1988; Higgins and Sharp, Gene 73:237-244, 1988; Higgins and Sharp, CABIOS 5:151-153, 1989; Corpet et al., Nucl. Acids Res. 16:10881-10890, 1988; Huang et al., Comput. Applic. Biosciences 8:155-165, 1992; and Pearson et al., Meth. Mol. Biol. 24:307-331, 1994. Altschul et al., J. Mol. Biol. 215:403-410, 1990, presents a detailed discussion of sequence-alignment methods and homology calculations.
[0090] The NCBI Basic Local Alignment Search Tool (BLAST™, Altschul et al. J. Mol. Biol. 215:403-410, 1990) is available from several sources, including the National Center for Biotechnology Information (NCBI, Bethesda, Md.) and on the Internet, for use in connection with the sequence-analysis programs blastp, blastn, blastx, tblastn and tblastx. BLAST™ can be accessed at the web site maintained by the NCBI. A description of how to determine sequence identity using this program is also available at the same web site.
[0091] For comparisons of amino acid sequences of greater than about 30 amino acids, the “Blast 2 sequences” function in the BLAST™ program can be employed using the default BLOSUM62 matrix set to default parameters, (gap existence cost of 11, and a per-residue gap cost of 1). When aligning short peptides (fewer than about 30 amino acids), the alignment should be performed using the Blast 2 sequences function, employing the PAM30 matrix set to default parameters (open gap 9, extension gap 1 penalties). Proteins having even greater similarity to the reference sequences will show increasing percentage identities when assessed by this method, such as at least 45%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity.
[0092] A first nucleic acid is “substantially similar” to a second nucleic acid if, when optimally aligned (with appropriate nucleotide insertions or deletions) with the other nucleic acid (or its complementary strand), nucleotide-sequence identity occurs in at least about 60%, 75%, 80%, 85%, 90% or 95% of the nucleotide bases. (As used herein, “optimally aligned” sequences exhibit a maximal possible sequence identity). Sequence similarity can be determined by comparing the nucleotide sequences of two nucleic acids using the BLAST™ sequence-analysis software (blastn) available from The National Center for Biotechnology Information. Such comparisons may be made using the software set to default settings (expect=10, filter=default, descriptions=500 pairwise, alignments=500, alignment view=standard, gap existence cost=11, per residue existence=1, per residue gap cost=0.85). Similarly, a first polypeptide is substantially similar to a second polypeptide if it shows sequence identity of at least about 75%-90% or greater when optimally aligned and compared using BLAST™ software (blastp) using default settings.
[0093] “Transformed.” A “transformed” cell is a cell into which a nucleic acid molecule has been introduced by molecular biology techniques. As used herein, the term “transformation” encompasses all techniques by which a nucleic acid molecule might be introduced into such a cell, including transfection with a viral vector, transformation with a plasmid vector, and introduction of naked DNA by electroporation, lipofection, and particle gun acceleration.
[0094] “Transgenic plant.” As used herein a “transgenic plant” refers to a plant that contains recombinant genetic material (“transgene”) normally not found in a wild-type plant of the same species. Thus, a plant that is grown from a plant cell into which recombinant DNA is introduced by transformation is a transgenic plant, as are all offspring of that plant containing the introduced transgene (whether produced sexually or asexually).
[0095] “Vector.” A “vector” is a nucleic acid molecule as introduced into a host cell, thereby producing a transformed host cell. A vector may include one or more nucleic acid sequences, such as an origin of replication, that permit the vector to replicate in a host cell. A vector also may include one or more selectable marker genes and other genetic elements known in the art.
[0096] Unless otherwise defined, all technical and scientific terms used herein have the same respective meanings as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
[0097] II. Assessment of Promoter Activity
[0098] A. Experimental Overview
[0099] A cDNA encoding the Douglas-fir luminal binding protein (PmBiP) was isolated by screening a Douglas-fir cDNA library with a previously isolated partial PmBiP cDNA clone. Four potential clones were isolated. The nucleotide and deduced amino acid sequence of the largest cDNA clone (PmBiP3) were used in subsequent experiments showing that PmBiP protein expression is developmentally and environmentally regulated. More specifically, PmBiP RNA levels to increased upon exposure to cold temperature, before and after fertilization and during germination.
[0100] The promoter responsible for the expression of the PmBiP protein was isolated by extracting genomic DNA from Douglas-fir spring-flush needles. The genomic DNA was then digested with either XbaI or SacI, re-circularized, subjected to PCR amplification, and identified via Southern blotting with a probe generated from a PmBiP cDNA clone.
[0101] Constructs containing the full-length PmBiP promoter (SEQ ID NO: 31) and deletions thereof then were cloned upstream from the uidA(&bgr;-glucuronidase (GUS)) gene, and experiments were designed to determine the activity of the promoter. The PmBiP promoter (SEQ ID NO: 31) as well as the various deletions thereof (SEQ ID NOS: 16, 17, and 18) were highly active. In fact, in some cases, the PmBiP promoter (SEQ ID NO: 31) and fragments thereof were significantly more active than the commonly used 35S CaMV promoter.
[0102] B. Materials and Methods Relating to the Expression Pattern of PmBiP
[0103] 1. Plant Material.
[0104] Coastal Douglas-fir (Pseudotsuga menziesii [Mirb] Franco) seeds (seed-lot #952) were grown as previously described (Tranbarger et al., Gene 172:221-226, 1995). Germinating and young seedlings were collected at midday at the times indicated, frozen in liquid nitrogen, and stored at −80° C. until further use. Growth of young Douglas-fir seedlings (high elevation seed-lot #6485) used for seasonal expression analysis was as described in Ekramoddoullah et al., Can. J. For Res. 25:1137-1147, 1995. One needle from each of 112 trees was collected. The needles were pooled on the morning of the dates indicated, frozen in liquid nitrogen, freeze dried, ground to a powder, and stored at −20° C. until further use. Developing seeds were collected from an open-pollinated seed orchard during midday on the dates indicated at Pacific Forest Products Ltd., Saanichton, B.C., Canada. Developing seeds were promptly dissected from cones, frozen on dry ice, and stored at −80° C. until further use.
[0105] 2. Isolation of Full Length BiP cDNA's.
[0106] A partial length BiP cDNA clone from a Douglas-fir cDNA library prepared from poly A+ RNA isolated from 4-6-day old seedlings was used as a probe (Tranbarger et al., Gene 172:221-226, 1995). The cDNA was 32P-labelled with a random primers DNA labelling kit (GIBCO BRL, Burlington, Ontario, Canada) and used to re-screen the cDNA library, according to the manufacturer's instructions (Strategene, La Jolla, Calif., USA), to obtain a full-length cDNA. Plasmid DNA from each positive clone was digested with EcoR1 and electrophoresed on a 1% agarose gel. The DNA was transferred to a Zeta Probe™ membrane (BioRad, Mississauga, Ontario, Canada) for Southern blotting. Clones containing a BiP cDNA insert of an appropriate size were selected for DNA sequencing.
[0107] 3. DNA Sequencing and Analysis.
[0108] The largest cDNA clone was selected for double-stranded DNA sequencing using Sequenase™ (United States Biochemical, Cleveland, Ohio, USA) and oligo primers synthesized on a PCR MATE™ 391 DNA synthesizer (Applied Biosystems, Mississauga, Ontario, Canada). Prediction of the signal sequence and signal-peptide cleavage site from deduced amino acid sequences was performed using the “SignalP” V1.1 World Wide Web Server (Nielsen et al., Protein Eng. 10:1-6, 1997). Amino-acid-sequence alignments were constructed using “CLUSTAL W” v1.7 (Thompson et al., Nucl. Acids Res. 22:4673-4680, 1994). The phylogenetic tree was constructed using the PHYLIP package (Felsenstein, Cladistics 5:164-166, 1989). The amino acid sequences (and database accession numbers) used for this analysis were: Aspergillus awamorii (EMBL: Y12504), Aplysia californica (PIR: S24782), Arabidopsis thaliana 1 (DDBJ: D89341), Arabidopsis thaliana 2 (DDBJ: D89342), Caenorhabditis elegans (GENBANK: U56965), Drosophila melanogaster (PIR:JN0666), Echinococcus granulosus (GENBANK: M63605), Echinococcus multilocularis (GENBANK: M63604), Eimeria tenella (EMBL: Z66492), Gallus gallus (PIR: 150242), Glycine max A (GENBANK: U08384), Glycine max B (GENBANK: U08383), Homo sapiens (SWISS-PROT: P11021), Lycopersicon esculentum (SWISSPROT: P49118), Mesocricetus auratus (SWISS-PROT: P07823), Mus musculus (SWISS-PROT: P20029), Neurospora crassa (EMBL: Y09011), Nicotiana tabacum 4 (SWISS-PROT: Q03684), Nicotiana tabacum 5 (PIR: JQ1361), Oryza sativa (GENBANK: AF006825), Phytophthora cinnamomi (PIR: S38890), Plasmodium falciparum (EMBL: X69121), Phaeodactylum tricornutum (GENBANK: U29675), Rattus norvegicus (SWISS-PROT: P06761), Saccharomyces cerevisiae (SWISS-PROT: P16474), Spinacia oleracea (GENBANK: L23551), Trypanosoma brucei (GENBANK: L14477), Xenopus laevis (GENBANK: U62807), Zea mays E2 (GENBANK: U58208), and Zea mays E3 (GENBANK: U58209).
[0109] 4. Genomic DNA Extraction and Restriction Analysis.
[0110] Douglas-fir genomic DNA was extracted from spring-flush needles by a modification of the “CTAB” method (De Vemo et al., Constructing Conifer Genomic Libraries: A Basic Guide, Information Report, Petawawa National Forestry Institute, Canadian Forest Service, PI-X-88, 1989). Aliquots of 10 &mgr;g of DNA were digested for 26 hours with restriction enzymes, then separated on a 0.7% agarose gel. Hybridization methods were based on those described in Lueders and Fewell, Biotechniques 16:66-67, 1994, as follows: The gel was incubated at room temperature with shaking in denaturing solution (0.5 N NaOH, 150 mM NaCl) for 30 minutes, rinsed in distilled water, and incubated in neutralizing solution (500 mM Tris-HCl pH 8, 150 mM NaCl) for 30 minutes. The gel was dried on a vacuum gel drier for 30 minutes with vacuum only, followed by 1 hour at 60° C. The dried gel (unblot) was probed with 32P-labelled, random primed, PmBiP cDNA in hybridization solution (0.5 M Na2HPO4 pH 7.2, 7% SDS, 100 &mgr;g/ml denatured salmon sperm DNA) at 65° C. overnight, then washed at low stringency twice in hybridization solution for 45 minutes each at 65° C. The unblot was exposed for 7 days under a phosphor-imaging screen and developed using the STORM 820™ Phosphorimager (Molecular Dynamics, Sunnyvale, Calif., USA). Following development, the unblot was washed at high stringency twice in wash buffer (20 mM Na2HPO4 pH 7.2, 1% SDS) for 45 minutes each at 65° C. and exposed for 8 days and developed as above. Quantification was performed using the Image Quant NT™ software (Molecular Dynamics). Calculation of gene copy number was as described in Pasternak, Glick, and Thompson (eds.), Methods in Plant Molecular Biology and Biotechnology,CRC Press, Inc., Boca Raton, Fla., USA, pp. 29-36, 1993, using a Douglas-fir genome size of 25 pg per haploid nucleus (Ingle et al., Plant Physiol. 55:496-501, 1975).
[0111] 5. Northern Blotting.
[0112] Total RNA was isolated as described in Kaukinen et al., Plant Mol. Biol. 30:1115-1128, 1996, separated on a 1% agarose/formaldehyde gel, and transferred to a Zeta-Probe GT membrane (BioRad, Mississauga, Ontario, Canada). Blots were then probed with 32P-labelled, random primed, PmBiP cDNA following the basic hybridization conditions described in the Zeta-Probe manual. Blots were stripped and re-probed with a PCR-amplified genomic fragment representing the Douglas-fir 18S rRNA gene to account for differences in the amount of RNA loaded per lane. Densitometry and adjustment for differences in the amount of RNA loaded per lane (calculation of integrated optical density) were performed as described in Tranbarger and Misra, Physiol. Plant 95:456-464, 1996. Densitometry for these figures was performed using the Chemilmager™ 4000 system (Alpha Innotech Corporation, San Leandro, Calif., U.S.A.).
[0113] 6. Antibody Production.
[0114] A synthetic peptide corresponding to the 13 C-terminal amino acids of the PmBiP deduced amino acid sequence was synthesized at the University of Victoria Protein Micro-Chemistry Centre using a Model 430A peptide synthesizer (Applied Biosystems, Foster City, Calif., USA) with the “FastMoc” chemistry software. The peptide, with an additional cysteine residue added to the N-terminal end, was then conjugated to a KLH carrier protein using the Imject™ kit and following the manufacturer's instructions (Pierce, Rockford, Ill., USA). The conjugated peptide was mixed with Freund's complete adjuvant and injected into New Zealand white rabbits. Subsequent booster injections were given at 2-week intervals using the conjugated peptide prepared in Freund's incomplete adjuvant.
[0115] 7. Protein Extraction and Western Blotting.
[0116] Protein extractions from whole developing seeds, mature seeds, germinating seeds, and young seedlings were performed by grinding approximately 100 mg of tissue in liquid nitrogen with a mortar and pestle. The powders were suspended individually in extraction buffer containing 65 mM Tris-HCl, pH 6.8, 1% SDS, 5% glycerol, and 2.5% &bgr;-mercaptoethanol, boiled for 5 minutes, frozen at −80° C. for 1 hour, boiled for 5 minutes, then centrifuged at 16,000× g for 25 minutes. The supernatants were collected and saved for further analysis. For subcellular fractionations (FIG. 1B), approximately 5 g of tissue were frozen in liquid nitrogen and ground to a fine powder using a mortar and pestle. The individual powders were suspended and vortexed in buffer A (100 mM Tris-HCl pH 7.5, 250 mM sucrose, 2 mM MgCl2, 10 mM KCl, 1 mM phenylmethylsulfonylflouride (PMSF), and 2.8 mM &bgr;-mercaptoethanol), then filtered through two layers of Miracloth™ (Calbiochem, La Jolla, Calif., USA). The filtrates were centrifuged at 25,000× g for 30 minutes. The supernatants were collected and centrifuged at 140,000× g for 1 hour. The supernatants (soluble fractions) were saved, and the respective pellets (microsomal fraction) were suspended in buffer B (50 mM phosphate buffer pH 7.5, 20% glycerol, and 10 mM &bgr;-mercaptoethanol).
[0117] Microsomes were separated into soluble and membrane fractions according to Fujiki et al., J. Cell Biol. 93:97-102, 1982.
[0118] To purify nuclei (nuclear fraction), the pellets from the 25,000× g centrifugation were resuspended in buffer A, layered on a 25%/75% Percoll™ (Sigma, Oakville, Ontario, Canada) step gradient, and centrifuged at 1000× g for 20 minutes. Nuclei were collected from the 25%/75% interface, washed 2× in buffer A, and suspended in buffer B.
[0119] Protein concentrations were determined by the BioRad Reagent protein assay (BioRad). Extraction and quantification of needle proteins from seasonal samples and densitometry of western blots were performed as described in Ekramoddoullah et al., Can. J. For. Res. 25:1137-1147, 1995. Protein samples were suspended in protein sample buffer (12.5 mM Tris-HCl pH 6.8, 2 SDS, 10% glycerol, 5%,&bgr;-mercaptoethanol, and 0.1 bromophenol blue), boiled for 3 minutes, and separated by SDS-PAGE using the Mini-PROTEAN II™ gel electrophoresis system (BioRad) with a 4% (w/v) acrylamide stacking gel (80 volts; constant voltage) and an 11 (w/v) acrylamide separating gel (200 volts; constant voltage). The proteins were stained with Coomassie brilliant blue R250 or transferred to nitrocellulose membranes (Schleicher & Schuell, Keene, N.H., USA) using a Mini-Trans-Blot™ cell (BioRad) at 100 volts for 1 hour in transfer buffer (25 mM Tris, 190 mM glycine, 20% methanol, and 0.1% SDS). The membranes were blocked overnight at 4° C. in Tris-buffered saline (“TBS”; 20 mM Tris, 500 mM NaCl; pH 7.5) containing 0.05% Tween-20 (“TTBS”), incubated with primary antibody (diluted 1:3000 in TTBS) for 90 minutes at room temperature, then washed two times with TTBS (5 minutes each). The membranes were then incubated with an alkaline phosphatase-conjugated goat anti-rabbit antibody (1:3000 dilution in TTBS) (Cedar Lane Laboratories Ltd., Hornby, Ontario, Canada) for 45 minutes at room temperature, followed by washing in TTBS (5 minutes) and TBS (5 minutes). Immunoreactive bands were visualized by incubating the membrane with 5-bromo-4-chloro-3-indolyl-phosphate (0.165 mg/ml) and nitroblue tetrazolium (0.33 mg/ml) as substrate in buffer containing 100 mM NaHCO3 pH 9.8 and 1 mM MgCl2.
[0120] C. Expression Pattern of PmBiP mRNA and Protein
[0121] To understand better the developmental regulation of PmBiP, the pattern of PmBiP mRNA and protein expression during seed and seedling development was examined using northern and western blotting. Western blotting also was used to determine whether seasonal variations exist in PmBiP protein levels in the needles of one-year-old seedlings.
[0122] Both before and soon after fertilization (FIG. 1; May 31 and June 19, respectively), the amount of PmBiP mRNA observed in developing seeds was approximately 50-to 100-fold higher than the amount observed during embryogenesis (FIG. 1; July 12-August 15). During embryogenesis, northern blotting of dissected material showed similar amounts of PmBiP mRNA in both the megagametophyte and developing embryo. Western blot analysis of seeds collected at various stages of seed development showed that the amount of PmBiP protein also was high before and soon after fertilization, and decreased thereafter.
[0123] Following imbibition and stratification, PmBiP mRNA increased slightly (FIG. 2). Upon exposure of the seeds to germination conditions, the amount of PmBiP mRNA increased to levels greater than the level observed during early stages of seed development. Levels increased 150-to 200-fold over levels observed in mature or imbibed seeds after only a 2-day exposure to germination conditions. PmBiP mRNA amounts were highest after 8 days, approximately 250-fold higher than observed in mature seeds. The amount of PmBiP protein did not show an increase until 8 days after exposure of the stratified seeds to germination conditions, with the highest amounts appearing after 14 days.
[0124] Temperature regulation of PmBiP protein expression was observed at both the mRNA level and the protein level. 14-day-old seedlings were subjected to cold treatment, and mRNA and protein levels were assessed. After the cold treatment both protein levels and mRNA levels (FIG. 3) were found to be increased.
[0125] In an additional set of experiments, the abundance of PmBiP protein was followed over a one-year period in needles collected from 1-year-old Douglas-fir seedlings kept under natural day-length and temperature in an outdoor shelter house (FIG. 4). PmBiP protein levels showed seasonal variation, with the highest amounts occurring in needles taken from seedlings during the winter when the monthly average temperature was below 10° C.
[0126] D. Materials and Methods Relating to the Characterization of the PmBiP Promoter
[0127] 1. Plant Material
[0128] Tobacco plants (Xanthi) and 4-6 week old potato plants (Desiree) for use in transformation were grown in Majenta jars on hormone free MS medium (Murashige and Skoog, Physiol. Plant 115:473-497, 1962 under a 16 hours light/8 hours dark photoperiod at a constant temperature of 23° C.
[0129] Growth of Arabidopsis plants used for transformation was as follows:
[0130] Approximately 10-20 Arabidopsis thaliana (L.) Heynh. seeds of ecotype Columbia were placed on a nylon screen covering moistened Sunshine™ mix #3 soil (Sun Gro Horticulture, Bellevue, Wash., USA) on 10-cm-diameter pots, and then covered with Saran Wrap™ secured with an elastic band. Pots were then placed at 4° C. for 2 days to promote uniform germination. Pots were placed in a growth chamber with an 18 hours 24° C. day/ 6 hours 22° C. night cycle with 150 &mgr;Em−2s−1 of light. The Saran Wrap™ was removed when plants began to push against its surface (approximately 2 days).
[0131] Growth of Douglas-fir embryos used for transient expression was as follows:
[0132] Interior Douglas-fir (Pseudotsuga menziesii [Mirb.] Franco) seeds (seed-lot 8912) were imbibed for 2 days at 4° C., then surface-sterilized in 50% industrial bleach (6% sodium hypochlorite) for 20 minutes at room temperature. Embryos were aseptically dissected from seeds and placed on woody plant medium (WPM; Table 1; Lloyd and McCown, Proc. Int. Plant Prop. Soc. 30:421-427, 1980) at 22° C. in the dark for 16 hours before particle bombardment.
[0133] 2. Inverse PCR and Cloning
[0134] Inverse PCR was conducted based on the method described in Ochman et al. et al., Amplification of Flanking Sequences by Inverse PCR, 1980, and Innis et al. (eds.) PCR Protocols; A Guide to Methods and Applications, Academic Press Inc., San Diego, 1990, as follows. Douglas-fir genomic DNA was extracted from spring-flush needles by a modification of the CTAB method of De Verno et al., Constructing Conifer Genomic Libraries; A Basic Guide, Petawawa National Forestry Institute, Canadian Forest Service, 1989. Approximately 18 &mgr;g of DNA was digested with XbaI or SacI overnight at 37° C. Each reaction was heat-inactivated at 65° C. for 20 minutes, then suspended in 10 ml of ligation buffer (50 mM Tris-HCl pH 7.4, 10 mM MgCl2, 10 mM dithiothreitol, and 1 mM ATP) with 0.02 Weiss units/&mgr;L T4 DNA ligase for 16 hours at 15° C. Circularized DNA was precipitated by the addition of {fraction (1/10)}volume of 2.5 M ammonium acetate, followed by 2 volumes of −20° C. 100% ethanol on ice, then centrifuged at 25,000× g for 10 minutes at 4° C. Precipitated DNA was suspended in 60 &mgr;L of sterile distilled H2O of which 5 &mgr;L was subjected to PCR using Taq PCR MasterMix™ (QIAGEN, Mississauga, Ontario, Canada) and 250 pmol of the following primers in a 100-&mgr;L reaction: XbaI used primer combinations p5-3z8 (5′-AAT GAA AGC GAA GTG ACA CC-3′; SEQ ID No: 19) and p14-5a4 (5′-CAG AAC CAT TAA CAA GAG CAA GAT 3′; SEQ ID NO: 20) or p14-5z1.1 (5′-AAC CAG CAG TGA TAA ACG CC-3′; SEQ ID NO: 21) and p14-5a4; SacI used primer combinations p5-3z8 and p14-5a3 (5′-TAT GGT TTG GAT AAA AAG GGA G-3′; SEQ ID NO: 22) or p14 5z1.1 and 14-5a3. Conditions for PCR consisted of 1 cycle of denaturing at 95° C. for 5 minutes and 1 minute at 75° C., 30 cycles of denaturing at 94° C. for 1 minute, primer annealing at 56° C. for 1 minute and an extension of 72° C. for 2 minutes, followed by a final elongation step at 72° C. for 5 minutes. Aliquots of each reaction (20 &mgr;L) were separated on an agarose gel and subjected to Southern blotting to identify potential promoter fragments. PCR reactions containing positive fragments were cloned into the pC®2.1-TOPO vector using the TOPO TA™ Cloning Kit (Invitrogen, Carlsbad, Calif., U.S.A.) according to the manufacturer's instructions. Colonies containing an appropriately sized insert were screened using PCR with the appropriate primers followed by Southern blotting. Plasmid DNA for colony screening using PCR was obtained by suspending colonies in 200 &mgr;L of distilled H2O, followed by incubation at 85° C. for 5 minutes. Samples were centrifuged at 16,000× g for 5 minutes, and 36 &mgr;L were removed and used as a template for PCR.
[0135] 3. DNA Sequencing
[0136] PmBiP promoter (SEQ ID NO: 31) and expression constructs were sequenced using the Big Dye™ Superscript Terminator Cycle sequencing Ready Reaction (Perkin Elmer) and oligo primers with the ABI Prism automated 377 DNA Sequencer (Perkin Elmer). Plasmid DNA for sequencing was isolated using the Wizard™ 373 DNA Purification System (Promega, Madison, Wis., U.S.A.). DNA sequence trace files were assembled using the DNASTAR™ program SeqMan (DNASTAR Inc, Madison, Wis., U.S.A.).
[0137] 4. Sequence Analysis
[0138] Analysis of the PmBiPPro1 DNA sequence and identification of putative regulatory elements were performed done by searching the plant cis-acting DNA regulatory database (PLACE; Higo et al., Nucl. Acids Res. 27:297-300, 1999).
[0139] 5. Southern Blotting
[0140] DNA was electrophoresed on a 1% agarose gel and transferred to a Zeta Probe™ membrane (BioRad, Mississauga, Ontario, Canada) according to the manufacturer's instructions. The PmBiP3 cDNA was 32P-labelled with a random-primers DNA labeling kit (GIBCO BRL, Burlington, Ontario, Canada). Hybridization and washing were performed according to the standard protocol in the Zeta Probe manufacturer's instructions. Blots were exposed using Kodak X-OMAT AR film (Eastman Kodak Company, Rochester, N.Y., U.S.A.) overnight at −80° C.
[0141] 6. Construction of Vectors Containing PmBiP Promoter Sequences
[0142] The following promoter-gene fusions were constructed for expression in the cytosol: Plasmids were constructed from parent plasmids pBI121 and pBI221 for stable and transient expressions, respectively. PmBiP promoter constructs were generated using PCR with either Taq polymerase (PmBiPpro1-1; Quiagen) or DeepVent™ polymerase (PmBippro1-3 and PmBiPpro1 5; NEB) and the PmBiPpro1 clone as template. The primers, containing HindIII and XbaI sites (bold), used for amplification of the various promoter constructs, employed the same 3′-primer (5′-TCG AAG CGC AAA TCT AGA GTT TAA ACT TCC-3′; SEQ ID NO: 23) and the following 5′-primers: PmBiPPro1-1 (5′-AAG AAG GCA AGC TTT CAA CTA A-3′; SEQ ID NO: 24), PmBiPPro1-3 (5′-GCA TAA GAA AGC TTC TAC CCT G-3′; SEQ ID NO: 25), and PmBiPPro1-5 (5′-GCA CTA GGA AGC TTG GGA ACT C-3′; SEQ ID NO: 26). Following restriction digestion, the resulting products (PmBiPpro1-1, 2263 bp, SEQ ID NO:16; PmBiPpro1-3, 1259 bp, SEQ ID NO: 17; PmBiPpro1-5, 263 bp, SEQ ID NO: 18) were cloned into the HindIII and XbaI sites of pBI221, replacing the CaMV 35S promoter (˜0.8 kb). The resulting plasmids, containing PmBiP promoter sequences, were labeled pPRO1-1221, pPRO1-3221, and pPRO1-5221. Replacing the HindIII-XbaI fragment in pBI121 (containing the CaMV 35S promoter) by HindIII-XbaI fragments from pPRO1-1221, pPRO1-3221, and pPRO1-5221 respectively, created the plasmids pPRO1-1121, pPRO1-3121, and pPRO1-5121.
[0143] 7. Transient Expression
[0144] Germinated Douglas-fir zygotic embryos were bombarded using the model PDS-1000/He Biolistic® Particle Delivery System (BioRad). DNA (pBI221 plasmid derivatives) was coated onto gold particles (1-3 &mgr;m diameter; Sigma-Aldrich Canada Ltd, Oakville, Ontario, Canada) as described by Jefferson, Plant Mol. Biol. Rep. 5:387-405, 1987, as follows. A gold suspension (60 mg/ml) was prepared in 50% glycerol of which 15 &mgr;L was placed in 1.5-ml microfuge tubes with 4.2×1011 copies of either a CaMV 35S:GUS plasmid (pBI221, 5700 bp; Clontech Laboratories Inc, Palo Alto, Calif., U.S.A.), a plasmid containing PmBiPpro1-1 (SEQ ID NO: 16) (7188 bp), a plasmid containing PmBiPpro1-3 (SEQ ID NO: 17) (6184 bp), or a plasmid containing PmBiPpro1-5 (SEQ ID NO: 18) (5189 bp), 15 &mgr;L of 2.5 M CaCl2, and 6 &mgr;L of 0.1M spermidine with continuous vortexing. The particles were allowed to settle on ice, then pelleted by a brief centrifugation. The supernatant was discarded, and 70 &mgr;L cold 70% ethanol was added without disturbing the pellet. The 70% ethanol was then removed, and an additional 70 &mgr;L cold 100% ethanol was added without disturbing the pellet. This too was removed and the particles suspended in 30 &mgr;L cold 100% ethanol with slow vortexing. Aliquots of 10 &mgr;L were placed on a macrocarrier disk and allowed to dry in the presence of silica gel desiccant. Each bombardment delivered 1.4×1011 constructs and was conducted using the following parameters. The gap distance between the rupture disk and macrocarrier was 0.6 cm, the macrocarrier travel distance was 0.6 cm, the target tissue distance was 8 cm from the microcarrier launch assembly platform, the sample chamber vacuum was 25 inches of mercury, and rupture pressure was 1550 psi. Tissue then was incubated on WPM at 22° C. in the dark for 48 hours prior to histochemical GUS staining. Following GUS staining (see below), the number of blue spots were counted under a stereo dissecting microscope.
[0145] 8. Arabidopsis Transformation
[0146] Arabidopsis thaliana plants were transformed according to the method of Clough and Bent, Plant J. 16: 735-743, 1998. Agrobacterium tumefaciens strain MP90 carrying the plasmid CaMV 35S:GUS or PmBiPPro1 (SEQ ID NO:16):GUS was grown to stationary phase in liquid culture at 28° C., 250 rpm, in sterile LB broth (10 g tryptone, 5 g yeast extract, 5 g NaCl per liter of water) containing 50 &mgr;g/ml kanamycin and 10 &mgr;g/ml gentamycin. Cells were harvested by centrifugation for 20 minutes at room temperature at S500× g, then suspended in infiltration medium (5.0 % sucrose and 0.05 Silwet L-77 (Lehle Seeds, Round Rock, Tex., U.S.A.)) to a final OD600 of approximately 0.8 prior to use. The above-ground portions of plants were dipped in infiltration medium containing Agrobacterium for 3-5 sec with gentle swirling 2 days after removal of the primary bolt. One subsequent dip was made 7 days later. Following each dip, the plants were covered with a plastic bag for 24 hours to retain moisture. Plants were grown normally and fed with HI•SOL™ 18-24-12 soluble plant food (1 g/L; Green Valley Fertilizer, Abbotsford, B.C, Canada) once a week via sub-irrigation. Plants were no longer watered after seed pods began to turn brown. When plants were fully dried, they were placed in a brown paper bag for 1 week prior to collecting seed. Seeds were collected by manually rubbing plants and pods, then filtering the debris through a 0.707-mm mesh sieve (W. S. Tyler Company of Canada Ltd., St. Catherines, Ont., Canada) several times until the seeds were reasonably free of other matter.
[0147] Seeds were sterilized using vapor-phase sterilization as follows (Clough and Bent, Plant J. 16:735-743, 1998). Collected seeds were placed in 15-ml conical tubes (2-3 ml seeds per tube) with lids attached loosely. Tubes were placed in a rack inside a plastic vacuum dessicator (Bel-Art #42025, 240-mm internal diameter) containing a 250-ml glass beaker with 150 ml bleach (5.25% sodium hypochlorite). Five ml of concentrated HCl were placed in a 10-ml glass beaker and floated on top of the bleach solution. The lid was placed on the dessicator and a slight vacuum applied. The dessicator was shaken slightly to spill the concentrated HCl into the bleach to liberate chlorine gas for overnight sterilization. Sterile seeds were sprinkled on 150×15 mm2 selection plates (½MS media/0.8% agar/1% sucrose, 50 &mgr;g/ml kanamycin, 100 &mgr;g/ml ampicillin) and placed in dark at 4° C. for 2 days. Plates were removed and placed in a growth chamber with 16 hours/8-hours light/dark at 22° C. for 2 weeks. Healthy green transformants were selected and placed in moist soil in a growth chamber with an 18-hour 24° C. day/6-hour 22° C. night cycle with 150 &mgr;Em−2s−1 of light. The plants were covered with Saran Wrap™ for the first 2 days.
[0148] 9. Tobacco and Potato Transformation
[0149] Leaf strips from tobacco and stem segments (5-10 mm pieces) and leaves (cut at the base) from potato were pre-cultured upside down for 3-5 days on MS 104 medium (MS medium supplemented with 1 &mgr;g/ml BAP, 0.1 &mgr;g/ml NAA, pH=5.7). Explants were incubated in S2 medium (MS medium without agar but supplemented with 0.5 g/L MES and 20 g/L mannitol) inoculated with a 1:200 (v:v) dilution of an overnight culture of Agrobacterium tumefaciens strain MP90 for 2-3 days under low light intensity. An overnight culture of Agrobacterium tumefaciens was grown at 28° C. in LB media supplemented with 50 &mgr;g/ml kanamycin and 10 &mgr;g/ml gentamycin. Explants were incubated at low light intensity on Stage I medium (MS medium supplemented with 6 g/L agarose (instead of 8 gL agar), 200 mg/L glutamine, 600 mg/L MES, 500 mg/L PVP, 20 g/L mannitol, 20 g/L glucose, 40 mg/L adenine-SO4, 2.5 mg/L zeatine-riboside, 0.1 mg/L NAA, and 0.02 mg/L GA3) for 3-5 days followed by transfer to Stage II medium (Stage I medium supplemented with 100 &mgr;/ml kanamycin and 500 &mgr;g/ml cefotaxime) for 7-12 days to initiate growth of callus. To initiate growth of shoots, explants containing callus were transferred to Stage III medium (Stage II medium containing no NAA) for 5-7 weeks. Following shoot formation, plantlets were transferred to rooting medium (MS medium supplemented with 50 &mgr;g/ml kanamycin and 250 &mgr;g/ml cefotaxim) and grown as described for parent plants.
[0150] 10. Histochemical GUS Staining
[0151] GUS staining was performed based on the method described by Jefferson, Plant Mol. Biol. Rep. 5:387-405, 1987. Tissue from particle bombardment or transgenic plants was immersed in solution containing 1 mM X-Gluc, 100 mM sodium phosphate buffer pH 7.0, 10 mM EDTA, 0.5 mM potassium ferricyanide, 0.5 mM potassium ferrocyanide, 0.1% triton X-100, and incubated overnight at 37° C.
[0152] 11. In Vitro GUS Assay
[0153] Fresh plant tissue was placed in a 1.5-ml Eppendorf tube containing ice-cold lysis buffer (50 mM sodium phosphate pH 7.0, 10 mM EDTA, 0.1% Triton X-100, 0.1% sarkosyl, 10 mM &bgr;-mercaptoethanol, and 0.02 g/ml insoluble polyvinylpyrrolidone (PVP)), and homogenized using a glass pestle connected to a Barnant series 10 mixer (Barnant Company, Barrington, Ill., USA). Homogenates were centrifuged at 16,000× g for 15 minutes at 4° C. Supernatants were collected and assayed for protein using the method of Bradford, Anal. Biochem. 72:248-254, 1976. GUS activity was measured in 100 &mgr;L extraction buffer (without PVP) containing 6 &mgr;g of total protein and 1 mM p-nitrophenyl-&bgr;-D-glucuronide as substrate at 37° C. using a Thermomax™ microplate reader and Softmax™ Pro v3.1 software (Molecular Dynamics Corporation). Absorbance was measured at 405 nm every 5 minutes or after 18 hours.
[0154] D. Expression Pattern of GUS Transgene Under the Control of PmBiP Constructs
[0155] Results from sequencing the PmBiP promoter (SEQ ID NO: 31) revealed that it contains several possible cis-acting elements (FIG. 5). Furthermore, several of the identified cis-acting elements are believed to be responsible for promoter activity in response to environmental changes (i.e., inducible), such as light, temperature, wounding, and water stress. Other identified promoter elements indicate that the endogenous PmBiP promoter (SEQ ID NO: 31) contains negative regulatory regions. One of skill in the art will appreciate that the identification of these regions, and the other elements shown in FIG. 6, facilitates the subsequent modification of the PmBiP promoter (SEQ ID NO: 31). Thus, the PmBiP promoter (SEQ ID NO: 31) can be modified via deletion of negative regulatory elements to increase transcription, or to alter the induciblity of the promoter via the addition or deletion of inducible elements.
[0156] The isolation and sequencing of the PmBiP promoter (SEQ ID NO: 31) also facilitated the creation of the deletion constructs PmBiPPro1-1, PmBiPPro1-3, and PmBiPPro1-5 (FIG. 6). These deletion constructs were used to stably transform Arabidopsis, potato, and tobacco, as well as to transiently transform Douglas-fir zygotic embryos.
[0157] Douglas-fir zygotic embryos were transiently transformed with construct containing the GUS open reading frame under the control of PmBiPpro1-1, PmBiPpro1-3, PmBiPpro1-5, or CaMV35S. The results (FIG. 7) showed that all of the PmBiP promoter constructs were capable driving the expression of GUS. Additionally, even the relatively weak PmBiP promoter construct (PmBiPpro1-3) was capable of driving expression of the transgene at a rate 7-fold higher than the rate exhibited by the control CaMV35S promoter construct (average expression levels taken over two trials, ten embyros per trial).
[0158] Potato and tobacco plantlets that were stably transformed with either the GUS open reading frame under the control of PmBiPpro1-1, PmBiPpro1-3, PmBiPpro1-5, or CaMV35S showed that even PmBiPpro1-5, the smallest deletion, was capable of driving the expression of GUS. Moreover, the PmBiPpro1-5 construct showed promoter activity that was comparable to the activity of the control CaMV35S construct.
[0159] Stably transformed 19-day-old Arabidopsis plants also were tested for GUS expression levels. The results showed that the highest level of GUS expression was in plants transformed with the PmBiPpro1-3 construct. However, plants transformed with PmBiPpro1-1 showed very similar levels of expression (FIG. 8).
[0160] The ability of the PmBiP promoter to be induced via wounding was tested in stably tranformed 21-day-old Arabidopsis cotyledons (FIG. 9). One cotyledon was wounded by pinching with forceps, and the second cotyledon was used as a control. The results showed that the PmBiPpro1-1 (SEQ ID NO: 16) construct is wound-inducible. This data, coupled with the PmBiP protein expression data described above, showed that the PmBiP promoter (SEQ ID NO: 31) is inducible at least by wounding and upregulated by temperature alterations. These attributes make this promoter particularly useful in situations where it is desirable to produce a protein at cold temperatures (i.e., where increased protein stability is desired). “Cold” implies that the plant is being grown at a colder temperature than otherwise would be optimal for host growth. For example, a plant or a plant part (i.e., a leaf, or stem) can be wounded and placed in a cold temperature, such as at less than 20° C., less than 15° C., or less than 10° C. Additionally, the table of cis-acting elements provided in FIG. 5 shows that it is likely that the PmBiP promoter (SEQ ID NO: 31) is inducible by many other environmental factors.
[0161] V. Alteration of Promoter Structure
[0162] A. Modifications of the Douglas-Fir Luminal Binding Protein (PmBiP) Promoter
[0163] The structure of a given promoter determines the level of mRNA expression as well as specificity of the promoter. However, expression levels and/or specificity can be maintained when deletions, substitutions, and/or additions are made to the promoter sequence. Hence, the scope of the invention encompasses PmBiP promoters that have been modified through the incorporation of deletions, substitutions, and/or additions. However, regardless of the number of mutations that are incorporated into the PmBiP promoter (SEQ ID NO: 31), the promoter continues to exhibit promoter activity, or native PmBiP promoter activity, as described above.
[0164] One possible method of modifying the PmBiP promoter (SEQ ID NO: 31) is by inserting additional promoter elements into the promoter sequence. For example, the promoter can be modified such that an E-box motif, RY-repeated element, AT-rich region, ACGT-core element, opaque-2-like binding site, and/or a conserved gymnosperm-like region is added. One of skill in the art will appreciate that standard molecular biology techniques can be used to insert one or more of these elements into the promoter sequence. The modified promoter then can be transiently transfected into gymnosperm, monocot, or dicot tissue and the tissue can be tested for transgene expression.
[0165] Similarly, one or more of the existing promoter elements can be deleted from the promoter sequence. The modified promoter can be tested for transcriptional activity and specificity. Given the disclosure of the PmBiP promoter (SEQ ID NO: 31) and the above-described materials and methods, it also is possible to make both additions and deletions and test for promoter activity.
[0166] Finally, the PmBiP promoter (SEQ ID NO: 31) also can be modified such that the inter-element spaces contain deletions, insertions, and/or substitutions. One of ordinary skill in the art can use standard molecular biology techniques to insert additional nucleic acid residues into the inter-element spaces, delete nucleic acid residues from the inter-element spaces, and/or substitute other sequences into the inter-element spaces. However, regardless of the number and combination of insertions, deletions, and substitutions, the PmBiP promoter (SEQ ID NO: 31) will maintain promoter activity. In some cases, the promoter will maintain native PmBiP promoter activity.
[0167] B. Methods for Producing the Douglas-Fir PmBiP Promoter, and Variants and Deletion Mutants Thereof
[0168] 1. Cloning Nucleic Acid Sequences Encoding PmBiP
[0169] Provided with the nucleic acid sequence of the Douglas-fir PmBiP promoter (SEQ ID NO: 31), one of ordinary skill in the art will appreciate that several different methods can be used to isolate the Douglas-fir PmBiP promoter (SEQ ID NO: 31). One example of such a method is the polymerase chain reaction (PCR) (U.S. Pat. No. 4,683,202 to Mullis; and Saiki et al., Science 239:487-491, 1988). After isolation, the PmBiP promoter (SEQ ID NO: 31) sequence is useful for driving the expression of transgenes.
[0170] When using PCR to isolate a sequence encoding a gene, a first primer can be designed that targets the extreme 5′-end of the sequence, and a second primer can be designed that targets the extreme 3′-end of the sequence. These primers can be used to generate multiple copies of the promoter sequence. The copies are isolated by separation on an agarose gel. The fragment of interest is then removed from the gel and ligated into an appropriate vector.
[0171] Alternatively, a promoter can be created by engineering synthetic strands of DNA that partially overlap each other (Beaucage and Caruthers, Tetrahedron Letters 22:1859-1869, 1981;and Matthes et al., EMBO J. 3:801-805, 1984). The synthetic strands are annealed, and a DNA polymerase is used to fill in the single-stranded regions. The resulting synthetic double-stranded DNA molecule can be cloned into a vector.
[0172] For use as primers and probes, nucleic acid sequences can contain at least 15 contiguous nucleic acid molecules of the sequences shown in SEQ ID NOS: 16, 17, 18, and 31, or the complementary strand of the molecule shown in SEQ ID NO: 31. The nucleic acid sequences are useful for performing hybridization protocols, such as northern blots or Southern blots as described in Sambrook et al. (eds.), Molecular Cloning, A Laboratory Manual, 2d ed., vol. 1-3, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.
[0173] These hybridization protocols can be used to identify nucleic acid sequences that are substantially similar to the sequences shown in SEQ ID NOS: 16, 17, 18, and 31. A successful hybridization to such sequences indicates that the analogous nucleic acid sequence hybridizes to the oligonucleotide probe that comprises at least a fragment of the sequences shown in SEQ ID NOS: 16, 17, 18, and 31. Generally, hybridization conditions are classified into categories, for example very high stringency, high stringency, and low stringency. The conditions corresponding to these categories for probes of approximately 600 bp are provided below. 1 Very High Stringency (detects sequences that share 90% sequence identity) Hybridization in 5 x SSC at 65° C. 16 hours Wash twice in 2 x SSC at room temp. 15 minutes each Wash twice in 0.2 x SSC at 65° C. 20 minutes each High Stringency (detects sequences that share 80% sequence identity or greater) Hybridization in 3 x SSC at 65° C. 16 hours Wash twice in 2 x SSC at room temp. 15 minutes each Wash twice in 0.5 x SSC at 55° C. 20 minutes each Low Stringency (detects sequences that share greater than 50% sequence identity) Hybridization in 3 x SSC at 65° C. 16 hours Wash twice in 2 x SSC at room temp. 20 minutes each
[0174] Variant PmBiP promoter (SEQ ID NO: 31) sequences may be produced by standard DNA-mutagenesis techniques, for example, M13 primer mutagenesis. Details of these techniques are provided in Sambrook et al. (eds.), Molecular Cloning: A Laboratory Manual, 2nd ed., vol. 1-3, Ch. 15, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989; and Ausubel et al. (eds.) Current Protocols in Molecular Biology, Greene Publishing and Wiley-Interscience, New York (with periodic updates), 1987. By the use of such techniques, variants can be created that differ slightly from the PmBiP promoter sequences specifically disclosed, yet that still encode a promoter having promoter activity. DNA molecules and nucleotide sequences that are derivatives of those specifically disclosed herein and that differ from those disclosed by the deletion, addition, or substitution of nucleotides while still maintaining promoter activity and/or native PmBiP promoter activity are comprehended by this invention.
[0175] 2. Transformation
[0176] The DNA constructs of the invention, containing the PmBiP promoter (SEQ ID NO: 31) operably linked to one or more transgenes may be either homologous or heterologous to the host in question. If homologous to the host cell, i.e., if the transgene is produced by the host cell in nature, then the construct may be connected operably to a different secretory signal sequence and/or terminator sequence than in the natural environment. In this context, the term “homologous” is intended to include a cDNA sequence encoding a transgene that is native to the host cell. The term “heterologous” is intended to include a transgene not expressed by the host cell in nature. Thus, the DNA sequence may be from another organism, or it may be a synthetic sequence.
[0177] The host cell of the invention, into which the DNA construct or the recombinant expression vector of the invention is to be introduced, may be any cell capable of driving expression of the PmBiP promoter (SEQ ID NO: 31). Such cells include bacteria cells, yeast cells, fungal cells, insect cells, plant cells, and other higher eukaryotic cells.
[0178] Various methods of introducing the DNA construct into host cells are well known in the art. For example, in some species, the Ti plasmid of A. tumefaciens can be used to transform host cells (Gouka et al., Nature Biotech. 6:598-602, 1999). The host cell also can be transformed using gene blasting techniques (described above) and standard chemical treatments.
[0179] Having illustrated and described the principles of the invention in multiple embodiments and examples, it should be apparent to those skilled in the art that the invention can be modified in arrangement and detail without departing from such principles. Therefore, the invention includes all modifications coming within the spirit and scope of the following claims.
Claims
1. A recombinant promoter, comprising a nucleic acid sequence selected from the group consisting of:
- (a) a nucleic acid sequences selected from the group consisting of SEQ ID NOS: 16,17, 18; and 31;
- (b) a nucleic acid sequence that shares at least 50% sequence identity with the nucleic acid sequences shown in (a); and
- (c) a nucleic acid sequence that comprises at least 20 consecutive nucleic acid residues of the nucleic acid sequences shown in (a), wherein the promoter is capable of driving the expression of an ORF that is operably linked to the promoter.
2. The promoter of claim 1, comprising the nucleic acid sequence shown in SEQ ID NO: 16.
3. The promoter of claim 1, comprising the nucleic acid sequence shown in SEQ ID NO: 17.
4. The promoter of claim 1, comprising the nucleic acid sequence shown in SEQ ID NO: 18.
5. The promoter of claim 1, comprising the nucleic acid sequence shown in SEQ ID NO: 31.
6. A vector, comprising a promoter as recited in claim 1.
7. A host cell, comprising a vector as recited in claim 6.
8. A transgenic plant, comprising a host cell as recited in claim 7.
9. A transgene, comprising the promoter of claim 1 and at least one ORF operably linked to the promoter.
10. A vector, comprising a transgene as recited in claim 9.
11. A plant cell, comprising a vector as recited in claim 10.
12. A transgene according to claim 9, wherein the ORF encodes a cationic peptide.
13. The plant cell of claim 11, wherein the plant cell is obtained from a plant selected from the group consisting of: maize, wheat, rice, millet, tobacco, sorghum, rye, barley, brassica, sunflower, seaweeds, lemna, oat, soybean, cotton, legumes, rape/canola, alfalfa, flax, sunflower, safflower, brassica, cotton, flax, peanut, and clover; lettuce, tomato, cucurbits, cassava, potato, carrot, radish, pea, lentil, cabbage, cauliflower, broccoli, Brussel sprouts, peppers and other vegetables; citrus, apples, pears, peaches, apricots, walnuts, and other fruit trees; orchids, carnations, roses, and other flowers; cacao; poplar, elms, and other deciduous trees; pine, Douglas-fir, spruce, and other conifers; turf grasses; cacao; and rubber trees and other members of the genus Hevea.
14. A method for expressing at least one protein in a host cell, comprising:
- provide a recombinant promoter as recited in claim 1;
- operably linking an ORF to the promoter to produce a transgene;
- introducing the transgene into a vector; and
- introducing the vector into a host cell, wherein the host cell produces a protein from the ORF.
15. The method according to claim 14, wherein the host cell is a plant host cell.
16. A protein produced according to the method of claim 14.
17. The protein according to claim 14, wherein the protein is a cationic peptide.
18. A promoter according to claim 1, wherein the promoter is inducible.
19. The promoter of claim 1, wherein the promoter is inducible at low temperatures.
20. The promoter of claim 1, wherein the promoter is developmentally specific.
21. The promoter of claim 20, wherein the promoter is expressed in actively dividing cells.
22. The promoter of claim 1, wherein the promoter is wound-inducible.
23. An isolated promoter, comprising at least 8 promoter elements selected from the group consisting of E-box motifs (SEQ ID NO: 1), MNF1 elements (SEQ ID NO: 28), POLLEN1LELAT52 elements (SEQ ID NO: 29) ROOTMOTIF elements (SEQ ID NO: 30), 2SSEEDPROTBANAP elements (SEQ ID NO: 32), BOXIIPCCHS elements (SEQ ID NO: 33), ASF1MOTIF elements (SEQ ID NO: 34), ACGT-core elements (SEQ ID NO: 4), CAAT-boxes (SEQ ID NO: 9), CANABNNAPA elements (SEQ ID NO: 12), HEXMOTIF elements (SEQ ID NO: 27), and duplicates thereof, wherein the promoter displays promoter activity.
24. The isolated promoter of claim 23, wherein the promoter comprises at least 10 promoter elements.
25. The isolated promoter of claim 23, comprising promoter elements in the following order: 3′-ACGT-core element (SEQ ID NO: 4), E-box motif (SEQ ID NO: 1), CAAT-box (SEQ ID NO: 9), 2SSEEDPROTBANAP element(SEQ ID NO: 32), CANABNNAPA element (SEQ ID NO: 12), HEXMOTIF element (SEQ ID NO: 27), CAAT-box (SEQ ID NO: 9), BOXIIPCCHS element (SEQ ID NO: 33), E-box motif (SEQ ID NO: 1), ASF1MOTIF element (SEQ ID NO: 34), POLLEN1LELAT52 element (SEQ ID NO: 29), and MNF1 element (SEQ ID NO: 28)-5′.
26. A transgene, comprising the promoter of claim 25 operably linked to an ORF.
27. A host cell, comprising the transgene of claim 26.
28. A transgenic plant, comprising the host cell of claim 27.
29. The transgenic plant of claim 28, wherein the plant is selected from the group consisting of maize, wheat, rice, millet, tobacco, sorghum, rye, barley, brassica, sunflower, seaweeds, lemna, oat, soybean, cotton, legumes, rape/canola, alfalfa, flax, sunflower, safflower, brassica, cotton, flax, peanut, and clover; lettuce, tomato, cucurbits, cassava, potato, carrot, radish, pea, lentil, cabbage, cauliflower, broccoli, Brussel sprouts, peppers and other vegetables; citrus, apples, pears, peaches, apricots, walnuts, and other fruit trees; orchids, carnations, roses, and other flowers; cacao; poplar, elms, and other deciduous trees; pine, Douglas-fir, spruce, and other conifers; turf grasses; cacao; and rubber trees and other members of the genus Hevea.
30. The transgene according to claim 26, wherein the ORF encodes a cationic peptide.
31. The transgene according to claim 26, wherein the ORF encodes the proteins shown in SEQ ID NO: 36.
32. A method for expressing a protein in a plant, the method comprising:
- providing a transgene plant according to claim 8; and
- growing the plant under conditions in which to the plant produces the protein.
33. The method of claim 32, further comprising wounding the plant.
34. The method of claim 32, further comprising growing the plant at a cold temperature.
35. A method for expressing a protein in a plant, the method comprising:
- growing a plant according to claim 28; wherein the plant produces the protein.
36. The method of claim 35, further comprising wounding the plant.
37. The method of claim 35, further comprising growing the plant in cold temperatures.
38. The method of claim 36, further comprising wounding a plant part and placing the plant part in a cold temperature.
Type: Application
Filed: Apr 3, 2002
Publication Date: Dec 19, 2002
Applicant: University of Victoria Innovation and Development Corporation
Inventors: Santosh Misra (Victoria), Benjamin S. Forward (Victoria)
Application Number: 10117641
International Classification: A01H005/00; C07H021/04; C12N005/04;
