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COMPOSITIONS AND METHODS FOR THE MODIFICATION OF GENE TRANSCRIPTION - Patent application
Patent application title: COMPOSITIONS AND METHODS FOR THE MODIFICATION OF GENE TRANSCRIPTION
Inventors: Marion Wood (Auckland, NZ) Michael A. Shenk (Amsterdam, NL) Annette Mcgrath (Victoria, AU) Matthew Glenn (Bethlehem, NZ) William H. Rottmann (Summerville, SC, US) Robert J. Kodrzycki (Summerville, SC, US)
Patent application number: 20110047644
COMPOSITIONS AND METHODS FOR THE MODIFICATION OF GENE TRANSCRIPTION - Patent application <?php require_once('/home/patents/php/mtc.config.php'); require_once('/home/patents/php/mtc.class.php'); $MTC = new MTC(); $MTC->init(); ?>
Inventors: Marion WOOD Michael A. SHENK Annette MCGRATH Matthew GLENN William H. ROTTMANN Robert J. KODRZYCKI
Novel isolated polynucleotides that encode plant transcription factors are provided, together with genetic constructs comprising such polynucleotides. Methods for using such constructs in modulating the expression of endogenous and/or heterologous genes are also disclosed, together with transgenic plants comprising such constructs.
1. An isolated polynucleotide comprising a sequence selected from the group consisting of: consisting of: SEQ ID NO: 1-591, 1183-1912, 1931-2106, 2371, 2374, 2377, 2385, 2387, 2389, 2391, 2393, 2395, 2397, 2399, 2401, 2403, 2405, 2407, 2409, 2411, 2413, 2415, 2417, 2419, and 2421.
2. An isolated polynucleotide comprising a sequence selected from the group consisting of:(a) complements of a sequence of SEQ ID NO: 1-591, 1183-1912, 1931-2106, 2371, 2374, 2377, 2385, 2387, 2389, 2391, 2393, 2395, 2397, 2399, 2401, 2403, 2405, 2407, 2409, 2411, 2413, 2415, 2417, 2419, and 2421;(b) reverse complements of a sequence of SEQ ID NO: 1-591, 1183-1912, 1931-2106, 2371, 2374, 2377, 2385, 2387, 2389, 2391, 2393, 2395, 2397, 2399, 2401, 2403, 2405, 2407, 2409, 2411, 2413, 2415, 2417, 2419, and 2421; and(c) reverse sequences a sequence of SEQ ID NO: 1-591, 1183-1912, 1931-2106, 2371, 2374, 2377, 2385, 2387, 2389, 2391, 2393, 2395, 2397, 2399, 2401, 2403, 2405, 2407, 2409, 2411, 2413, 2415, 2417, 2419, and 2421.
3. An isolated polynucleotide comprising a sequence selected from the group consisting of:(a) nucleotide sequences having at least 75% identity to a sequence of SEQ ID NO: 1-591, 1183-1912, 1931-2106, 2371, 2374, 2377, 2385, 2387, 2389, 2391, 2393, 2395, 2397, 2399, 2401, 2403, 2405, 2407, 2409, 2411, 2413, 2415, 2417, 2419, and 2421;(b) nucleotide sequences having at least 90% identity to a sequence of SEQ ID NO: 1-591, 1183-1912, 1931-2106, 2371, 2374, 2377, 2385, 2387, 2389, 2391, 2393, 2395, 2397, 2399, 2401, 2403, 2405, 2407, 2409, 2411, 2413, 2415, 2417, 2419, and 2421;(c) nucleotide sequence having at least 95% identity to a sequence of SEQ ID NO: 1-591, 1183-1912, 1931-2106, 2371, 2374, 2377, 2385, 2387, 2389, 2391, 2393, 2395, 2397, 2399, 2401, 2403, 2405, 2407, 2409, 2411, 2413, 2415, 2417, 2419, and 2421;(d) nucleotide sequences that hybridize to a sequence of SEQ ID NO: 1-591, 1183-1912, 1931-2106, 2371, 2374, 2377, 2385, 2387, 2389, 2391, 2393, 2395, 2397, 2399, 2401, 2403, 2405, 2407, 2409, 2411, 2413, 2415, 2417, 2419, and 2421 under stringent hybridization conditions;(e) nucleotide sequences that are 200-mers of a sequence of SEQ ID NO: 1-591, 1183-1912, 1931-2106, 2371, 2374, 2377, 2385, 2387, 2389, 2391, 2393, 2395, 2397, 2399, 2401, 2403, 2405, 2407, 2409, 2411, 2413, 2415, 2417, 2419, and 2421;(f) nucleotide sequences that are 100-mers of a sequence of SEQ ID NO: 1-591, 1183-1912, 1931-2106, 2371, 2374, 2377, 2385, 2387, 2389, 2391, 2393, 2395, 2397, 2399, 2401, 2403, 2405, 2407, 2409, 2411, 2413, 2415, 2417, 2419, and 2421;(g) nucleotide sequences that are 40-mers of a sequence of SEQ ID NO: 1-591, 1183-1912, 1931-2106, 2371, 2374, 2377, 2385, 2387, 2389, 2391, 2393, 2395, 2397, 2399, 2401, 2403, 2405, 2407, 2409, 2411, 2413, 2415, 2417, 2419, and 2421; and(h) nucleotide sequences that are 20-mers of a sequence of SEQ ID NO: 1-591, 1183-1912, 1931-2106, 2371, 2374, 2377, 2385, 2387, 2389, 2391, 2393, 2395, 2397, 2399, 2401, 2403, 2405, 2407, 2409, 2411, 2413, 2415, 2417, 2419, and 2421; and(i) nucleotide sequences that are degeneratively equivalent to a sequence of SEQ ID NO: 1-591, 1183-1912, 1931-2106, 2371, 2374, 2377, 2385, 2387, 2389, 2391, 2393, 2395, 2397, 2399, 2401, 2403, 2405, 2407, 2409, 2411, 2413, 2415, 2417, 2419, and 2421,wherein the isolated polynucleotide encodes a transcription factor.
4. An oligonucleotide probe or primer comprising at least 10 contiguous residues complementary to 10 contiguous residues of a sequence of SEQ ID NO: 1-591, 1183-1912 and 1931-2106.
5. An isolated polypeptide encoded by a polynucleotide according to any one of claims 1-3.
6. An isolated polypeptide comprising an amino acid sequence selected from the group consisting of: SEQ ID NO: 592, 594-850, 852-930, 932-951, 953-1046, 1048-1182, 1913-1930, 2107-2293, 2296-2368, 2372, 2375, 2378, 2386, 2388, 2390, 2392, 2394, 2396, 2398, 2400, 2402, 2404, 2406, 2408, 2410, 2412, 2414, 2416, 2418, 2420, and 2422.
7. An isolated polypeptide comprising an amino acid sequence selected from the group consisting of:(a) sequences having at least 75% identity to a sequence of SEQ ID NO: 592, 594-850, 852-930, 932-951, 953-1046, 1048-1182, 1913-1930, 2107-2293, 2296-2368, 2372, 2375, 2378, 2386, 2388, 2390, 2392, 2394, 2396, 2398, 2400, 2402, 2404, 2406, 2408, 2410, 2412, 2414, 2416, 2418, 2420, and 2422;(b) sequences having at least 90% identity to a sequence of SEQ ID NO: 592, 594-850, 852-930, 932-951, 953-1046, 1048-1182, 1913-1930, 2107-2293, 2296-2368, 2372, 2375, 2378, 2386, 2388, 2390, 2392, 2394, 2396, 2398, 2400, 2402, 2404, 2406, 2408, 2410, 2412, 2414, 2416, 2418, 2420, and 2422; and(c) sequences having at least 95% identity to a sequence of SEQ ID NO: 592, 594-850, 852-930, 932-951, 953-1046, 1048-1182, 1913-1930, 2107-2293, 2296-2368, 2372, 2375, 2378, 2386, 2388, 2390, 2392, 2394, 2396, 2398, 2400, 2402, 2404, 2406, 2408, 2410, 2412, 2414, 2416, 2418, 2420, and 2422,wherein the isolated polypeptide functions as a transcription factor.
8. An isolated polynucleotide that encodes a polypeptide according to any one of claims 6 and 7.
9. A genetic construct comprising a polynucleotide according to any one of claims 1-3.
10. A transgenic cell comprising a genetic construct according to claim 9.
11. A genetic construct comprising, in the 5'-3' direction:(a) a gene promoter sequence;(b) a polynucleotide sequence comprising at least one of the following: (1) a polynucleotide coding for a polypeptide comprising a sequence of 592, 594-850, 852-930, 932-951, 953-1046, 1048-1182, 1913-1930, 2107-2293, 2296-2368, 2372, 2375, 2378, 2386, 2388, 2390, 2392, 2394, 2396, 2398, 2400, 2402, 2404, 2406, 2408, 2410, 2412, 2414, 2416, 2418, 2420, and 2422; and (2) a polynucleotide comprising a non-coding region of a polynucleotide of any one of claims 1-3; and(c) a gene termination sequence.
12. The genetic construct of claim 11 wherein the open reading frame is in a sense orientation.
13. The genetic construct of claim 11 wherein the open reading frame is in an antisense orientation.
14. A transgenic plant cell comprising a genetic construct of any one of claims 9 and 11.
15. A plant comprising a transgenic plant cell according to claim 14, or fruit or seeds or propagules thereof.
16. The plant of claim 15 wherein the plant is a woody plant.
17. The plant of claim 16 wherein the plant is selected from the group consisting of eucalyptus, pine, acacia, poplar, sweetgum, teak and mahogany species
18. A method for modifying gene expression in a plant comprising stably incorporating into the genome of the plant a genetic construct according to any one of claims 9 and 11.
19. The method of claim 18, wherein the plant is a woody plant.
20. The method of claim 18, wherein the plant is selected from the group consisting of eucalyptus, pine, acacia, poplar, sweetgum, teak and mahogany species.
21. A method for producing a plant having modified gene expression comprising:(a) transforming a plant cell with a genetic construct according to any one of claims 9 and 11 to provide a transgenic cell; and(b) cultivating the transgenic cell under conditions conducive to regeneration and mature plant growth.
22. The method of claim 21 wherein the plant is a woody plant.
23. The method of claim 21 wherein the plant is selected from the group consisting of eucalyptus, pine, acacia, poplar, sweetgum, teak and mahogany species.
24. A method for modifying the activity of a polypeptide in a plant comprising stably incorporating into the genome of the plant a genetic construct according to any one of claims 9 and 11.
25. The method of claim 24 wherein the plant is a woody plant.
26. The method of claim 24 wherein the plant is selected from the group consisting of eucalyptus, pine, acacia, poplar, sweetgum, teak and mahogany species.
27. An isolated polypeptide comprising a DNA-binding domain, wherein the DNA-binding domain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 2279-2293 and 2296-2368.
[0001]This application is a continuation-in-part of U.S. patent application Ser. No. 10/856,499 filed May 24, 2004, which is a continuation-in-part of U.S. patent application Ser. No. 09/640,211 filed Aug. 16, 2000, now U.S. Pat. No. 6,833,446, which claims priority to International Patent Application No. PCT/US/00/06112, filed Mar. 9, 2000, and to U.S. Provisional Patent Application No. 60/149,485, filed Aug. 18, 1999, and is a continuation-in-part of U.S. patent application Ser. No. 09/266,513, filed Mar. 11, 1999, now abandoned, each of which is herein incorporated by reference in its entirety.
[0002]The contents of the text file submitted electronically herewith are incorporated herein by reference in their entirety: A computer readable format copy of the Sequence Listing (filename: ARBG--003--04US SeqList_ST25.txt, date recorded: Apr. 19, 2010, file size 2,329 kilobytes).
[0003]This invention relates to compositions isolated from plants and their use in the modification of gene transcription and/or expression. More specifically, this invention relates to plant polynucleotide sequences encoding transcription factors that are components of the cellular transcription apparatus and the use of such polynucleotide sequences in the modification of gene expression.
[0004]Eucaryotic gene expression is regulated, in part, by the cellular processes involved in transcription. During transcription, a single-stranded RNA complementary to the DNA sequence to be transcribed is formed by the action of RNA polymerases. Initiation of transcription in eucaryotic cells is regulated by complex interactions between cis-acting DNA motifs, located upstream of the gene to be transcribed, and trans-acting protein factors. Among the cis-acting regulatory regions are sequences of DNA, teinied promoters, which are located close to the transcription initiation site and to which RNA polymerase is first bound, either directly or indirectly. Promoters usually consist of proximal (e.g., TATA box) and more distant elements (e.g., CCAAT box). Enhancers are cis-acting DNA motifs which may be situated further up- and/or down-stream from the initiation site.
[0005]Both promoters and enhancers are generally composed of several discrete, often redundant, elements each of which may be recognized by one or more trans-acting regulatory proteins, known as transcription factors. Regulation of the complex patterns of gene expression observed both spatially and temporally, in all developing organisms, is thought to arise from the interaction of enhancer- and promoter-bound, general and tissue-specific transcription factors with DNA (Izawa T, Foster R and Chua N H, J. Mol. Biol. 230:1131-1144, 1993; Menkens A E, Schindler U and Cashmore A R, Trends in Biochem. Sci. 13:506-510, 1995). Developmental decisions in organisms as diverse as Drosophila melanogaster, Saccharomyces cerevisiae, Arabidopsis thaliana and Pinus radiata are regulated by transcription factors. These DNA-binding regulatory molecules have been shown to control the expression of genes responsible for the differentiation of different cell types, for example, the differentiation of leaf trichomes and xylem tissue in Arabidopsis thaliana, formation of endoderm from embryonic cells in Xenopus laevis and the initiation of gene expression in response to environmental and phytohoinional stress in plants (Yanagisawa S and Sheen J, The Plant Cell 10:75-89, 1998).
[0006]Transcription factors generally bind DNA in a sequence-specific manner and either activate or repress transcription initiation. The specific mechanisms of these interactions remain to be fully elucidated. At least three separate domains have been identified within transcription factors. One is essential for sequence-specific DNA recognition, one for the activation/repression of transcriptional initiation, and one for the formation of protein-protein interactions (such as dimerization). Four motifs, or domains, involved in DNA sequence recognition and/or transcription factor dimerization have been identified to date: zinc fingers; helix-turn-helix; leucine zipper; and helix-loop-helix. Both helix-loop-helix and leucine zipper protein motifs have been implicated in the binding of transcription factors to DNA via their ability to readily form homo- or hetero-dimers in vivo. "Activating" domains are rich in either proline, glutamine or acidic amino acids. It has been proposed that this net negative region of the transcription factor interacts with the TATA box-binding transcription factor TFIID, RNA polymerase, and/or another protein associated with the transcription apparatus.
[0007]Studies indicate that many plant transcription factors can be grouped into distinct classes based on their conserved DNA binding domains (Katagiri F and Chua N H, Trends Genet. 8:22-27, 1992; Menkens A E, Schindler U and Cashmore A R, Trends in Biochem. Sci. 13:506-510, 1995; Martin C and Paz-Ares J, Trends Genet. 13:67-73, 1997). Each member of these families interacts and binds with distinct DNA sequence motifs that are often found in multiple gene promoters controlled by different regulatory signals. Several classes of transcription factors that have been identified to date are described below.
[0008]The basic/leucine zipper (bZIP) is a conserved family of transcription factors defined by a basic/leucine zipper (bZIP) motif (Landschultz et al., Science 240:1759-1764, 1988; McKnight, Sci. Am. 264:54-64, 1991; Foster et al., FASEB J. 8[2]:192-200, 1994). Transcriptional regulation of gene expression is mediated by both the bZIPs and other families of transcription factors, through the concerted action of sequence-specific transcription factors that interact with regulatory elements residing in the promoter regions of the corresponding gene. The bZIP bipartite DNA binding structure consists of a region enriched in basic amino acids (basic region) adjacent to a leucine zipper that is characterized by several leucine residues regularly spaced at seven amino acid intervals (Vinson et al., Science 246:911-916, 1989). Whereas the basic region directly contacts the DNA, the leucine zipper mediates homodimerisation and heterodimerisation of protein monomers through a parallel interaction of the hydrophobic dimerization interfaces of two α-helices, resulting in a coiled-coil structure (O'Shea et al., Science 243:538-542, 1989; Science 254:539-544, 1991; Hu et al., Science 250:1400-1403, 1990; Rasmussen et al., Proc. Natl. Acad. Sci. USA 88:561-564, 1991).
[0009]Dof proteins are a relatively new class of transcription factor and are thought to mediate the regulation of some patterns of plant gene expression in part by combinatorial interactions between bZIP proteins and other types of transcription factors binding to closely linked sites. Such an example of this combinatorial interaction has been observed between bZIP and D of transcription factors (Singh, Plant Physiol. 118:1111-1120, 1998). These D of proteins possess a single zinc-finger DNA binding domain that is highly conserved in plants (Yanagisawa, Trends Plant Sci. 1:213, 1996). Specific binding of the D of protein to bZIP transcription factors has been demonstrated and it has been proposed that this specific interaction results in the stimulation of bZIP binding to DNA target sequences in plant promoters (Chen et al., Plant J. 10:955-966, 1996). Examples of such Do f/bZIP interactions have been reported in the literature, including for example, the Arabidopsis thaliana glutathionine S-transferase-6 gene (GST6) promoter which has been shown to contain several Dof-binding sites closely linked to the ocs element, a recognized bZIP binding site (Singh, Plant Physiol. 118:1111-1120, 1998).
[0010]The bZIP family of G-box binding factors from Arabidopsis (including GBF1, GBF2 and GBF3, for example) interact with the palindromic G-box motif (CCACGTGG). However, it has been demonstrated that the DNA binding specificity of such transcription factors, for example GBF1, may be influenced by the nature of the nucleotides flanking the ACGT core (Schindler et al., EMBO J. 11:1274-1289, 1992a). In vivo transient and transgenic plant expression studies have shown that these ACGT elements are necessary for maximal transcriptional activation and have been identified in a multitude of plant genes regulated by diverse environmental, physiological, and environmental cues. Classification of these transcription factors based upon their ability to bind to the ACGT core motif yielded a relatively diverse group of proteins, including, for example the CamV 35S promoter as-1-binding protein which exhibits DNA binding site requirements distinct from those proteins interacting with the G-box (Tabata et al., EMBO J. 10:1459-1467, 1991). Thus, in addition to defining the individual classes of bZIP proteins on the basis of their DNA binding specificity, such proteins can also be classified according to their heterodimerisation characteristics (Cao et al., Genes Dev. 5:1538-1552, 1991; Schindler et al., EMBO J. 11:1261-1273, 1992b).
[0011]Environmentally inducible promoters require the presence of two cis-acting elements, critical for promoter activity, one of which is the moderately conserved G-box (CCACGTGG) (deVetten et al., Plant Cell 4[10]:1295-1307, 1992). A mutation in one of the two elements abolishes or severely reduces the ability of the promoter to respond to environmental changes. The sequence of the second cis-acting element, positioned near the G-box, is not conserved among different environmentally-inducible promoters, but may be similar among promoters induced by the same signal. The spacing between the G-box and the second cis-acting element appears to be critical, suggesting a direct interaction between the respective binding factors (deVetten and Ferl, Int. J. Biochem. 26[9]:1055-1068, 1994; Ramachandran et al., Curr. Opin. Genet. Dev. 4[5]:642-646, 1994).
[0012]Basic helix-loop-helix zipper proteins represent an additional class of bZIP transcription factors described in the literature and include, for example, the Myc proteins. These proteins contain two regions characteristic of transcription factors: an N-terminal transactivation domain consisting of several phosphorylation sites, and a C-terminal basic helix-loop-helix (bHLH) leucine zipper motif known to mediate dimerization and sequence specific DNA binding via three distinct domains: the leucine zipper, helix-loop-helix, and basic regions.
[0013]The Myb family of transcription factors is a group of functionally diverse transcriptional activators found in both plants and animals that is characterized by a conserved amino-terminal DNA-binding domain containing either two (in plant species) or three (in animal species) imperfect tandem repeats of approximately 50 amino acids (Rosinski and Atchley, J. Mol. Evol. 46(1):74-83, 1998; Stober-Grasser et al., Oncogene 7[3]:589-596, 1992). Comparisons between the amino acid sequences of representative plant and mammalian MYB proteins indicate that there is a greater conservation between the same repeat from different proteins, than between the R2 and R3 repeats from the same protein (Martin and Paz-Ares, Trends Genet. 13[2]:67-73, 1997). More than 100 MYB genes have been reported from Arabidopsis thaliana (Romero et al., Plant J. 14[3]:273-284, 1998), representing the largest regulatory gene family currently known in plants. DNA-binding studies have demonstrated that there are differences, but also frequent overlaps, in binding specificity among plant MYB proteins, in line with the distinct but often related functions that are beginning to be recognized for these proteins. Studies involving the eight putative base-contacting residues in MYB DNA binding domains have revealed that at least six are fully conserved in all plant MYB proteins identified to date and the remaining two are conserved in at least 80% of these proteins (Martin and Paz-Ares, Trends Genet. 13[2]:67-73, 1997). Mutational analysis involving residues that do not contact bases have indicated that the sequence-specific binding capacity of MYBs is affected and this may account for some of the differences in the DNA-binding specificity between plant MYB proteins (Solan et al., J. Biol. Chem. 272[5]:2889-2895, 1997). This large-sized gene family may contribute to the regulatory flexibility underlying the developmental and metabolic plasticity displayed by plants.
[0014]Homeotic transcription factors have, in animals, been implicated in a number of developmental processes including, for example, the control of pattern formation in insects and vertebrate embryos and the specification of cell differentiation in many tissues (Ingham, Nature 335:25-34, 1988; McGinnis and Krumlauf, Cell 68:283-302, 1992). Homeodomain secondary structures are characterized by a distinctive helix-turn-helix motif initially identified in bacterial DNA binding domains. This helix-turn-helix sequence/structure motif spans approximately 20 amino acids and is characterized by two short helices separated by a sharp 90 degree bend or turn (Harrison and Aggarwal, Ann. Rev. Biochem. 59:933-969, 1990). This helix has been shown to bind in the major groove of the DNA helix.
[0015]Plant homeobox genes have been identified in a number of plant species including Arabidopsis thaliana, maize, parsley and soybean. Expression pattern analysis of maize homeobox gene family members suggests that these transcription factors may be involved in defining specific regions in the vegetative apical meristem, potentially involved in the initiation of leaf structures (Jackson et al., Development 120:405-413, 1994). Such observations imply that the plant homeobox genes, as for the animal homeobox genes, may be involved in the determination of cell fate.
[0016]Homeodomain-zipper (HD-zip) represents an additional family of homeodomain proteins. These homeodomain-zipper proteins (HD-zip) possess both the characteristic homeodomain linked to an additional leucine zipper dimerization motif. This family includes, for example, Athb-1 and Athb-2 (Sessa et al., EMBO J. 12:3507-3517, 1993) and Athb-4 (Carabelli et al., Plant J. 4:469-479, 1993).
[0017]The LIM domain is a specialized double-zinc finger motif found in a variety of proteins, in association with domains of divergent functions, such as the homeodomain (see the sunflower pollen-specific SF3 transcription factor: Baltz et al., Plant J. 2:713-721, 1992; or forming proteins composed primarily of LIM domains: Dawid et al., Trends Genet. 14[4]:156-162, 1998). LIM domains interact specifically with other LIM domains and with many different protein domains. LIM domains are thought to function as protein interaction modules, mediating specific contacts between members of functional complexes and modulating the activity of some of the constituent proteins. Nucleic acid binding by LIM domains, while suggested by structural considerations, remains an unproven possibility. However, it is possible that together with the homeodomain, the LIM domain could bind to the regulatory regions of developmentally controlled genes, as has been proposed for the paired box, a conserved sequence motif first identified in the paired (PRD) and gooseberry (GSB) homeodomain proteins from Drosophila (Triesman et al., Genes Dev. 5:594-604, 1991). The PRD box is also able to bind DNA in the absence of the homeodomain. LIM-domain proteins can be nuclear, cytoplasmic, or can shuttle between compartments. In the animal systems, several important LIM proteins have been shown to be associated with the cytoskeleton, having a role in adhesion-plaque and actin-microfilament organization. Among nuclear LIM proteins, the LIM homeodomain proteins form a major subfamily with important functions in cell lineage determination and pattern formation during animal development.
[0018]The AP2 (APETALA2) and EREBPs (ethylene-responsive element binding proteins) are the prototypic members of a family of transcription factors unique to plants, whose distinguishing characteristic is that they contain the so-called AP2 DNA-binding domain. AP2/EREBP genes form a large multigene family, and they play a variety of roles throughout the plant life cycle: from being key regulators of several developmental processes, like floral organ identity determination or control of leaf epidermal cell identity, to forming part of the mechanisms used by plants to respond to various types of biotic and environmental stress. In Arabidopsis thaliana, the homeotic gene APETALA2 (AP2) has been shown to control three salient processes during development: (1) the specification of flower organ identity and the regulation of floral organogenesis (Jofuku et al., Plant Cell 6:1211-1225, 1994); (2) establishment of flower meristem identity (Irish and Sussex, Plant Cell 2[8]:741-753, 1990); and (3) the temporal and spatial regulation of flower homeotic gene activity (Drews et al., Cell 65[6]:991-1002, 1991). DNA sequence analysis suggests that AP2 encodes a theoretical polypeptide of 432 aa, with a distinct 68 aa repeated motif termed the AP2 domain. This domain has been shown to be essential for AP2 functions and contains within the 68 aa, an eighteen amino acid core region that is predicted to form an amphipathic α-helix (Jofuku et al., Plant Cell 6:1211-1225, 1994). Ap2-like domain-containing transcription factors have been also been identified in both Arabidopsis thaliana (Okamuro et al., Proc. Natl. Acad. Sci. USA 94:7076-7081, 1997) and in tobacco with the identification of the ethylene responsive element binding proteins (EREBPs) (Ohme-Takagi and Shinshi, Plant Cell 7[2]:173-182, 1995). In Arabidopsis, these RAP2 (related to AP2) genes encode two distinct subfamilies of AP2 domain containing proteins designated AP2-like and EREBP-like (Okamuro et al., Proc. Natl. Acad. Sci. USA 94:7076-7081, 1997). In vitro DNA binding has not been shown to date using the RAP2 proteins; however, based upon the presence of two highly conserved motifs YRG and RAYD within the AP2 domain, it has been proposed that binding DNA binding occurs in a manner similar to that of AP2 proteins.
[0019]Zinc finger domains of the type Cys2H is2 appear to represent the most abundant DNA binding motif in eukaryotic transcription factors, with several thousand being identified to date (Berg and Shi, Science 271[5252]:1081-1085, 1996). A structural role for zinc in transcription factors was initially proposed in 1983 for the transcription factor IIIA (TFIIIA) (Hanas et al., J Biol. Chem. 258[23]:14120-14125, 1983). The Cys2H is2 Zinc finger domains are characterized by tandem arrays of sequences of C-x(2,4)-C-x(3)-[LIVMFYWC]-x(8)-H-x(3,5)-H (where X represents a variable amino acid). Structurally, the zinc finger consists of two antiparallel β strands followed by an α helix (Lee et al., Science 245[4918]:635-637, 1989). This structural arrangement allows for the cysteine and histidine side chains to coordinate the zinc with the three other conserved residues forming the hydrophobic core adjacent to the metal coordination unit (Berg and Shi, Science 271[5252]:1081-1085, 1996). Many proteins possessing a Cys2H is2 domain have been shown to interact with DNA in a sequence-specific manner. Crystal structure analysis of the mouse transcription factor Zif268 bound to a specific DNA target indicates that the zinc fingers in the protein/DNA complex reside in the major groove of the double helix and interacts with the DNA bases through amino acid side chains referred to as the contact residues (Pavletich and Pabo, Science 252[5007]:809-817, 1991). The orientations of the zinc finger domains with respect to the DNA are usually identical, with each domain contacting a contiguous 3-base pair subsite, the majority of which are directed to one strand. There are few interdomain interactions and the DNA recognition by each zinc finger appears to be largely independent of the other domains (Berg and Shi, Science 271[5252]:1081-1085, 1996).
[0020]The CCAAT-box element identified by Gelinas et al. (Nature 313[6000]:323-325, 1985) has been shown to occur between 80 bp and 300 bp from the transcription start site and may operate in either orientation, with possible cooperative interactions with multiple boxes (Tasanen et al., J Biol. Chem. 267[16]:11513-11519, 1992); or other conserved motifs (Muro et al., J. Biol. Chem. 267[18]:12767-12774, 1992; Rieping and Schoffl, Mol. Gen. Genet. 231[2]:226-232, 1992). CCAAT-box related motifs have been identified in a number of promoters in a variety of organisms including yeast (Hahn et al., Science 240[4850]:317-321, 1988), rat (Maity et al., Proc. Natl. Acad. Sci. USA 87[14]:5378-5382, 1990; Vuorio et al., J. Biol. Chem. 265[36]:22480-22486, 1990); and plants (Rieping and Schoffl, Mol. Gen. Genet. 231[2]:226-232, 1992; Kehoe et al., Plant Cell 6[8]:1123-1134, 1994). In both yeast and vertebrates, a protein complex has been shown to bind to the CCAAT-motif. In yeast the complex consists of three proteins, known as HAP2, HAP3 and HAPS (Pinkham and Guarente, Mol. Cell. Biol. 5[12]:3410-3416, 1985).
[0021]MADS box transcription factors interact with a conserved region of DNA known as the MADS box. All MADS box transcription factors contain a conserved DNA-binding/dimerization region, known as the MADS domain, which has been identified throughout the different kingdoms (Riechmann and Meyerowitz, Biol. Chem. 378[10]:1079-1101, 1997). Many of the MADS box genes isolated from plants are expressed primarily in floral meristems or floral organs, and are believed to play a role either in specifying inflorescence and floral meristem identity or in determining floral organ identity. One class of regulatory genes responsible for floral meristem identity and the pattern of meristem development includes the genes APETALA1 (AP1), APETALA2 (AP2), CAULIFLOWER (CAL), LEAFY (LFY) and AGAMOUS (AG) from Arabidopsis thaliana. Both LFY and AP1 have been shown to encode putative transcription factors (Weigel et al., Cell 69:843-859, 1992), with AP1 and AG each encoding putative transcription factors of the MADS box domain family (Yanofsky et al., Nature 346:35-39, 1990). Mutations in the Lfy gene have been shown to result in a partial conversion of flowers into infloresence shoots.
[0022]Briefly, the present invention provides polynucleotides isolated from plants that encode transcription factors, together with polypeptides encoded by such polynucleotides. The isolated polynucleotides and polypeptides of the present invention may be usefully employed in the modification of gene expression in plants, since both tissue- and temporal-specific gene expression patterns have been shown to be governed by transcription factors during the natural development of a plant. The inventive polynucleotides and polypeptides may thus be employed in the manipulation of plant phenotypes.
[0023]In a first aspect, the present invention provides polynucleotides isolated from eucalyptus and pine which encode transcription factors, including transcription factors from the following families of regulatory proteins: bZIP; bZIP family of G-box binding factors; basic helix-loop-helix zipper (bHLH); homeotic/homeodomain/homeobox/MADS; homeodomain zipper (ZIP); LIM domain; AP2 and EREBs; zinc finger domains of type Cys2H is2; CCAAT box elements; and MYB. In specific embodiments, the isolated polynucleotides of the present invention comprise a DNA sequence selected from the group consisting of: (a) sequences recited in SEQ ID NOS: 1-591, 1183-1912, 1931-2106, 2371, 2374, 2377, 2385, 2387, 2389, 2391, 2393, 2395, 2397, 2399, 2401, 2403, 2405, 2407, 2409, 2411, 2413, 2415, 2417, 2419, and 2421; (b) complements of the sequences identified as SEQ ID NOS: 1-591, 1183-1912, 1931-2106, 2371, 2374, 2377, 2385, 2387, 2389, 2391, 2393, 2395, 2397, 2399, 2401, 2403, 2405, 2407, 2409, 2411, 2413, 2415, 2417, 2419, and 2421; (c) reverse complements of the sequences identified as SEQ ID NOS: 1-591, 1183-1912, 1931-2106, 2371, 2374, 2377, 2385, 2387, 2389, 2391, 2393, 2395, 2397, 2399, 2401, 2403, 2405, 2407, 2409, 2411, 2413, 2415, 2417, 2419, and 2421; (d) reverse sequences of the sequences identified as SEQ ID NOS: 1-591, 1183-1912, 1931-2106, 2371, 2374, 2377, 2385, 2387, 2389, 2391, 2393, 2395, 2397, 2399, 2401, 2403, 2405, 2407, 2409, 2411, 2413, 2415, 2417, 2419, and 2421; and (e) sequences having either 60%, 75%, 80%, 90% or 95% identity, as defined herein, to a sequence of (a)-(d).
[0024]In a further aspect, isolated polypeptides encoded by the inventive polynucleotides are provided. In specific embodiments, such polypeptides comprise an amino acid sequence selected from the group consisting of: (a) sequences provided in SEQ ID NOS: SEQ ID NOS: 592-1182, 1913-1930, 2107-2278, 2372, 2375, 2378, 2386, 2388, 2390, 2392, 2394, 2396, 2398, 2400, 2402, 2404, 2406, 2408, 2410, 2412, 2414, 2416, 2418, 2420, and 2422; and (b) sequences having either 60%, 75%, 80%, 90% or 95% identity, as defined herein, to a sequence of (a).
[0025]In another aspect, the present invention provides polypeptides isolated from eucalyptus and pine which comprise transcription factor DNA-binding domains. In specific embodiments, such polypeptides comprise an amino acid sequence selected from the group consisting of: (a) sequences provided in SEQ ID NOS: 2279-2293 and 2296-2368; and (b) sequences having either 60%, 75%, 80%, 90% or 95% identity, as defined herein, to a sequence of (a).
[0026]In a further aspect, the invention provides genetic constructs comprising a polynucleotide of the present invention, either alone, in combination with one or more other polynucleotides disclosed herein, or in combination with one or more known DNA sequences, together with transformed cells comprising such constructs.
[0027]In specific embodiments, the inventive genetic constructs comprise, in the 5'-3' direction, a gene promoter sequence; an open reading frame coding for at least a functional portion of a polypeptide encoded by an inventive polynucleotide, or a variant thereof; and a gene termination sequence. The open reading frame may be orientated in either a sense or antisense direction. Genetic constructs comprising an untranslated, or non-coding, region of a polynucleotide coding for a transcription factor polypeptide of the present invention or a nucleotide sequence complementary to an untranslated region, together with a gene promoter sequence and a gene termination sequence, are also provided. Preferably, the gene promoter and termination sequences are functional in a host plant. Most preferably, the gene promoter and termination sequences are those of the original genes but others generally used in the art, such as the Cauliflower Mosaic Virus (CMV) promoter, with or without enhancers such as the Kozak sequence or Omega enhancer, and Agrobacterium tumefaciens nopalin synthase terminator may be usefully employed in the present invention. Tissue-specific promoters may be employed in order to target expression to one or more desired tissues. The genetic construct may further include a marker for the identification of transformed cells.
[0028]In yet a further aspect, transgenic cells comprising the genetic constructs of the present invention are provided, together with organisms, such as plants, comprising such transgenic cells. Fruits, seeds, derivatives, progeny, propagules and other products of such transgenic plants are also contemplated and encompassed by the present invention. As used herein, the term "propagule" means any part of a plant that may be used in reproduction or propagation, sexual or asexual, including cuttings.
[0029]In yet another aspect, methods for modifying gene expression in a target organism are provided, such methods including stably incorporating into the genome of the organism a genetic construct of the present invention. In a preferred embodiment, the target organism is a plant, preferably a woody plant, more preferably selected from the group consisting of eucalyptus and pine species, and most preferably from the group consisting of Eucalyptus grandis and Pinus radiata. In a related aspect, a method for producing a target organism, such as a plant, having modified gene expression is provided, the method comprising transforming a plant cell with a genetic construct of the present invention to provide a transgenic cell and cultivating the transgenic cell under conditions conducive to regeneration and mature plant growth.
[0030]The present invention further provides methods for modifying the activity of a transcription factor in a target organism, such as a plant, comprising stably incorporating into the genome of the plant a genetic construct of the present invention. In a preferred embodiment, the target plant is a woody plant, preferably selected from the group consisting of eucalyptus and pine species, and most preferably from the group consisting of Eucalyptus grandis and Pinus radiata.
[0031]The above-mentioned and additional features of the present invention and the manner of obtaining them will become apparent, and the invention will be best understood by reference to the following more detailed description. All references disclosed herein are hereby incorporated by reference in their entirety as if each was incorporated individually.
[0032]FIG. 1 depicts a map of the resulting plasmid, pWVK249 which is a binary vector used to transform plants and corresponds to SEQ ID: 2373.
[0033]FIG. 2 depicts a graph of basic gravity for wood from control and pWVK249-transformed cottonwood. The horizontal lines indicate the means for each set. The difference between the controls and the pWVK249 lines was significant at the 5% level.
[0034]The present invention provides isolated polynucleotides that encode plant transcription factors, together with isolated polypeptides encoded by such polynucleotides. As discussed above, transcription factors are components of the cellular "transcription apparatus" and are involved in the regulation of gene expression. Transcription factors are known to play a critical role in the growth and development of plants, and in cellular responses to external stimuli, such as environmental factors and disease pathogens. Transformation of plants with polynucleotides that encode proteins involved the cellular transcription process may thus be employed to modify properties such as lignin deposition, flower development, and male and female sterility. Transcription factors that regulate the formation of the vessel or fiber cells in secondary xylem or wood can be used to alter the characteristics of the wood. If the transcription factor up-regulates genes involved in lignin biosynthesis, overexpressing the transcription factor can increase the amount of lignin in the wood, while suppressing or knocking down the transcription factor can decrease the amount of lignin in wood. Certain transcription factors inhibit expression of genes involved in lignin formation and overexpression of one of these regulatory proteins would result in reduction of lignin. Overexpression of AmMYB308 from snapdragon led to reduction of lignin in transgenic tobacco (Tamagnone et al., Plant Cell 10: 135-154, 1998). Wood with high lignin is potentially useful as a fuel to be burned or converted to charcoal, while wood with low lignin is useful for pulping or conversion to ethanol. A transcription factor that up-regulates the full set of genes involved in cell wall biosynthesis can be used to alter the thickness of the cell walls and the density of the wood. Overexpressing the transcription factor can cause more production or prolonged production of the cell wall biosynthetic genes, resulting in cell walls that are thicker and wood that is denser and stronger. Goicoechea and co-workers showed that overexpression of a eucalyptus MYB gene in tobacco resulted in thicker cell walls and altered lignin composition (Plant J. 43: 553-567, 2005).
[0035]Using the methods and materials of the present invention, the amount of a specific transcription factor may be increased or reduced by incorporating additional copies of polynucleotides, or fragments of said polynucleotides, encoding the transcription factor into the genome of a target organism, such as a plant. Similarly, an increase or decrease in the amount of the transcription factor may be obtained by transfoiming the target plant with antisense copies of such genes.
[0036]In one embodiment, the present invention provides isolated polynucleotides encoding, or partially encoding, plant transcription factors that are involved in the regulation of gene expression. The polynucleotides of the present invention were isolated from forestry plant sources, namely from Eucalyptus grandis and Pinus radiata, but they may alternatively be synthesized using conventional synthesis techniques. In specific embodiments, isolated polynucleotides of the present invention comprise a sequence selected from the group consisting of sequences identified as SEQ ID NOS: 1-591, 1183-1912, 1931-2106, 2371, 2374, 2377, 2385, 2387, 2389, 2391, 2393, 2395, 2397, 2399, 2401, 2403, 2405, 2407, 2409, 2411, 2413, 2415, 2417, 2419, and 2421; complements of the sequences identified as SEQ ID NOS: 1-591, 1183-1912, 1931-2106, 2371, 2374, 2377, 2385, 2387, 2389, 2391, 2393, 2395, 2397, 2399, 2401, 2403, 2405, 2407, 2409, 2411, 2413, 2415, 2417, 2419, and 2421; reverse complements of the sequences identified as SEQ ID NOS: 1-591, 1183-1912, 1931-2106, 2371, 2374, 2377, 2385, 2387, 2389, 2391, 2393, 2395, 2397, 2399, 2401, 2403, 2405, 2407, 2409, 2411, 2413, 2415, 2417, 2419, and 2421; reverse sequences of the sequences identified as SEQ ID NOS: 1-591, 1183-1912, 1931-2106, 2371, 2374, 2377, 2385, 2387, 2389, 2391, 2393, 2395, 2397, 2399, 2401, 2403, 2405, 2407, 2409, 2411, 2413, 2415, 2417, 2419, and 2421; sequences comprising at least a specified number of contiguous residues (x-mers) of any of the above-mentioned polynucleotides; extended sequences corresponding to any of the above polynucleotides; antisense sequences corresponding to any of the above polynucleotides; and variants of any of the above polynucleotides, as that term is described in this specification.
[0037]In another embodiment, the present invention provides isolated polypeptides encoded by the polynucleotides of SEQ ID NOS: 1-591, 1183-1912, 1931-2106, 2371, 2374, 2377, 2385, 2387, 2389, 2391, 2393, 2395, 2397, 2399, 2401, 2403, 2405, 2407, 2409, 2411, 2413, 2415, 2417, 2419, and 2421. In certain specific embodiments, such isolated polypeptides comprise a sequence selected from the group consisting of SEQ ID NOS: 592-1182, 1913-1930, 2107-2278, 2372, 2375, 2378, 2386, 2388, 2390, 2392, 2394, 2396, 2398, 2400, 2402, 2404, 2406, 2408, 2410, 2412, 2414, 2416, 2418, 2420, and 2422.
[0038]The inventive polynucleotides and polypeptides have demonstrated similarity to transformation factors that are known to be involved in regulation of transcription and/or expression in plants as shown below in Table 1.
TABLE-US-00001 TABLE 1 Transcription factor family Polynucleotide SEQ ID NO: Basic leucine zipper (bZIP) 133, 148, 194, 206, 246, 258, 261, 265, 279, 284, 285, 286, 290, 294, 303, 318, 331, 455, 470, 473, 497, 501, 512, 533, 538, 554, 558, 575, 1896-1899, 1938, 1939, 1950, 1958, 1959, 1961, 1963, 1969, 1973, 1981, 1983, 1989, 1991, 1998, 2002, 2004, 2021, 2022, 2025, 2028, 2029, 2033- 2035, 2039, 2042, 2043, 2046, 2054, 2056, 2061, 2063, 2073, 2078, 2079, 2089, 2090, 2101, 2103, 2106 bZIP family of G-box binding 128, 136, 141, 142, 184, 202, 222, 244, factors 329, 541, 545 Basic helix-loop-helix zipper 157, 179, 223, 271, 274, 305, 317, 548, 563 Myb 138, 167, 214, 221, 232, 248, 252, 254, 255, 270, 276, 278, 280, 281, 282, 283, 292, 293, 315, 319, 328, 463, 483, 485, 486, 491, 492, 494, 502, 504, 507, 508, 510, 515, 518, 519, 520, 521, 527, 534, 536, 537, 540, 553, 559, 566, 572, 588, 1905, 1906, 1931, 1932, 1934-1936, 1940, 1948, 1949, 1951, 1953-1955, 1957, 1960, 1962, 1964-1968, 1974, 1975, 1977-1979, 1982, 1984-1988, 1992, 1994-1997, 2001, 2003, 2013-2015, 2024, 2026, 2027, 2030, 2032, 2036-2038, 2041, 2044, 2045, 2047- 2049, 2051, 2052, 2057-2060, 2065, 2067, 2071, 2072, 2074-2077, 2080-2088, 2104, 2105 Homeotic/homeodomain/homeobox/ 2, 3, 4, 7, 9, 10, 11, 12, 13, 17, 19, 25, 26, MADS 27, 28, 29, 31, 32, 34, 35, 36, 37, 39, 40, 44, 45, 49, 50, 51, 52, 54, 55, 57, 60, 62, 63, 64, 65, 66, 69, 72, 74, 76, 77, 79, 82, 84, 88, 89, 92, 94, 96, 97, 98, 100, 102, 103, 104, 105, 106, 107, 108, 11, 112, 114, 116, 117, 123, 125, 127, 168, 185, 249, 250, 332, 333, 334, 336, 337, 338, 340, 341, 343, 344, 345, 346, 347, 348, 349, 350, 351, 353, 354, 355, 356, 357, 359, 360, 361, 362, 364, 365, 366, 367, 368, 370, 371, 372, 373, 374, 375, 376, 379, 380, 383, 384, 385, 386, 387, 389, 392, 393, 394, 398, 399, 400, 401, 402, 403, 406, 408, 409, 410, 412, 414, 416, 417, 418, 420, 422, 424, 425, 426, 475, 526, 529, 580, 591, 1901, 1902, 1937, 1941- 1947, 1952, 1970-1972, 1976, 1980, 1990, 1993, 1999, 2000, 2006-2012, 2016-2020, 2023, 2031, 2040, 2050, 2053, 2055, 2062, 2064, 2066, 2068-2070, 2091-2100 Homeodomain zipper (HDZIP) 1, 5, 6, 14, 16,20, 21, 22, 23, 30, 33, 41, 42, 47, 58, 59, 61, 68, 70, 71, 73, 75, 80, 86, 87, 90, 91, 93, 115, 119, 121, 126, 335, 339, 342, 352, 358, 363, 369, 377, 381, 388, 390, 396, 397, 415, 419, 421, 423, 2005, 2102 LIM domain 15, 18, 24, 43, 78, 81, 83, 198, 210, 225, 273, 378, 391, 433, 437, 450, 452 AP2 and EREBs 120, 124, 170, 171, 219, 220, 224, 226, 229, 230, 238, 242, 243, 245, 247, 256, 301, 320, 330, 432, 434, 435, 436, 445, 447, 451, 453, 454, 459, 466, 469, 476, 481, 490, 524, 546, 549, 570, 1895 Zinc finger domains of type 132, 146, 154, 180, 181, 182, 183, 191, Cys2His2 207, 227, 234, 288, 323, 324, 325, 326, 404, 535, 567, 584, 585, 586, 587, 589, 590 CCAAT box elements 155, 174, 266, 309, 431, 460, 484, 499, 542, 551, 574, 583 Other transcription factors 8, 38, 46, 48, 53, 56, 67, 85, 95, 99, 101, 109, 110, 113, 118, 122, 129, 130, 131, 134, 135, 137, 139, 140, 143, 1444, 145, 147, 149, 150, 151, 152, 153, 156, 158, 159, 160, 161, 162, 163, 164, 165, 166, 169, 172, 173, 175, 176, 177, 178, 186, 187, 188, 189, 190, 192, 193, 195, 196, 197, 199, 200, 201, 203, 204, 205, 208, 209, 211, 212, 213, 215, 216, 217, 218, 228, 231, 233, 235, 236, 237, 239, 240, 241, 251, 253, 257, 259, 260, 262, 263, 264, 267, 268, 269, 272, 275, 277, 287, 289, 291, 295, 296, 297, 298, 299, 300, 302, 304, 306, 307, 308, 310, 311, 312, 313, 314, 316, 321, 322, 327, 382, 395, 405, 407, 411, 413, 427, 428, 429, 430, 438, 439, 440, 441, 442, 443, 444, 446, 449, 456, 457, 458, 461, 462, 464, 465, 467, 468, 471, 472, 474, 477, 478, 479, 480, 482, 487, 488, 489, 493, 495, 496, 498, 500, 505, 506, 509, 511, 513, 514, 516, 517, 522, 523, 525, 528, 530, 531, 532, 539, 543, 544, 547, 550, 552, 555, 556, 557, 560, 561, 562, 564, 565, 568, 569, 571, 573, 577, 578, 579, 581, 582, 448, 1183-1894, 1900, 1903, 1904, 1907, 1908-1912, 1933, 1956
[0039]The term "polynucleotide(s)," as used herein, means a single or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases and includes DNA and corresponding RNA molecules, including HnRNA and mRNA molecules, both sense and anti-sense strands, and comprehends cDNA, genomic DNA and recombinant DNA, as well as wholly or partially synthesized polynucleotides. An HnRNA molecule contains introns and corresponds to a DNA molecule in a generally one-to-one manner. An mRNA molecule corresponds to an HnRNA and DNA molecule from which the introns have been excised. A polynucleotide may consist of an entire gene, or any portion thereof. Operable anti-sense polynucleotides may comprise a fragment of the corresponding polynucleotide, and the definition of "polynucleotide" therefore includes all such operable anti-sense fragments. Anti-sense polynucleotides and techniques involving anti-sense polynucleotides are well known in the art and are described, for example, in Robinson-Benion et al., "Antisense techniques," Methods in Enzymol. 254[23]: 363-375, 1995; and Kawasaki et al., Artific. Organs 20[8]:836-848, 1996.
[0040]The definition of the terms "complement", "reverse complement" and "reverse sequence", as used herein, is best illustrated by the following example. For the sequence 5' AGGACC 3', the complement, reverse complement and reverse sequence are as follows:
TABLE-US-00002 complement 3' TCCTGG 5' reverse complement 3' GGTCCT 5' reverse sequence 5' CCAGGA 3'.
[0041]Preferably, sequences that are complements of a specifically recited polynucleotide sequence are complementary over the entire length of the specific polynucleotide sequence. The temi "polypeptide", as used herein, encompasses amino acid chains of any length including full length proteins, wherein amino acid residues are linked by covalent peptide bonds. Polypeptides of the present invention may be naturally purified products, or may be produced partially or wholly using recombinant techniques. The term "polypeptide encoded by a polynucleotide" as used herein, includes polypeptides encoded by a nucleotide sequence which includes the partial isolated DNA sequences of the present invention.
[0042]All of the polynucleotides and polypeptides described herein are isolated and purified, as those terms are commonly used in the art. Preferably, the polypeptides and polynucleotides are at least about 80% pure, more preferably at least about 90% pure, and most preferably at least about 99% pure.
[0043]Some of the polynucleotides of the present invention are "partial" sequences, in that they do not represent a full length gene encoding a full length polypeptide. Such partial sequences may be extended by analyzing and sequencing various DNA libraries using primers and/or probes and well known hybridization and/or PCR techniques. Partial sequences may be extended until an open reading frame encoding a polypeptide, a full length polynucleotide and/or gene capable of expressing a polypeptide, or another useful portion of the genome is identified. Such extended sequences, including full length polynucleotides and genes, are described as "corresponding to" a sequence identified as one of the sequences of SEQ ID NOS: 1-591, 1183-1912, 1931-2106, 2371, 2374, 2377, 2385, 2387, 2389, 2391, 2393, 2395, 2397, 2399, 2401, 2403, 2405, 2407, 2409, 2411, 2413, 2415, 2417, 2419, and 2421, or a variant thereof, or a portion of one of the sequences of SEQ ID NOS: 1-591, 1183-1912, 1931-2106, 2371, 2374, 2377, 2385, 2387, 2389, 2391, 2393, 2395, 2397, 2399, 2401, 2403, 2405, 2407, 2409, 2411, 2413, 2415, 2417, 2419, and 2421, or a variant thereof, when the extended polynucleotide comprises an identified sequence or its variant, or an identified contiguous portion (x-mer) of one of the sequences of SEQ ID NOS: 1-591, 1183-1912, 1931-2106, 2371, 2374, 2377, 2385, 2387, 2389, 2391, 2393, 2395, 2397, 2399, 2401, 2403, 2405, 2407, 2409, 2411, 2413, 2415, 2417, 2419, and 2421, or a variant thereof. Such extended polynucleotides may have a length of from about 50 to about 4,000 nucleic acids or base pairs, and preferably have a length of less than about 4,000 nucleic acids or base pairs, more preferably yet a length of less than about 3,000 nucleic acids or base pairs, more preferably yet a length of less than about 2,000 nucleic acids or base pairs. Under some circumstances, extended polynucleotides of the present invention may have a length of less than about 1,800 nucleic acids or base pairs, preferably less than about 1,600 nucleic acids or base pairs, more preferably less than about 1,400 nucleic acids or base pairs, more preferably yet less than about 1,200 nucleic acids or base pairs, and most preferably less than about 1,000 nucleic acids or base pairs.
[0044]Similarly, RNA sequences, reverse sequences, complementary sequences, antisense sequences, and the like, corresponding to the polynucleotides of the present invention, may be routinely ascertained and obtained using the cDNA sequences identified as SEQ ID NOS: 1-591, 1183-1912, 1931-2106, 2371, 2374, 2377, 2385, 2387, 2389, 2391, 2393, 2395, 2397, 2399, 2401, 2403, 2405, 2407, 2409, 2411, 2413, 2415, 2417, 2419, and 2421.
[0045]The polynucleotides identified as SEQ ID NOS: 1-591, 1183-1912, 1931-2106, 2371, 2374, 2377, 2385, 2387, 2389, 2391, 2393, 2395, 2397, 2399, 2401, 2403, 2405, 2407, 2409, 2411, 2413, 2415, 2417, 2419, and 2421 may contain open reading frames ("ORFs") or partial open reading frames encoding polypeptides. Open reading frames may be identified using techniques that are well known in the art. These techniques include, for example, analysis for the location of known start and stop codons, most likely reading frame identification based on codon frequencies, etc. Suitable tools and software for ORF analysis include, for example, GeneWise, available from The Sanger Center, Wellcome Trust Genome Campus, Hinxton, Cambridge, CB10 1SA, United Kingdom; Diogenes, available from Computational Biology Centers, University of Minnesota, Academic Health Center, UMHG Box 43 Minneapolis Minn. 55455; and GRAIL, available from the Informatics Group, Oak Ridge National Laboratories, Oak Ridge, Tenn. TN. Open reading frames and portions of open reading frames may be identified in the polynucleotides of the present invention. Once a partial open reading frame is identified, the polynucleotide may be extended in the area of the partial open reading frame using techniques that are well known in the art until the polynucleotide for the full open reading frame is identified. Thus, open reading frames encoding polypeptides may be identified using the polynucleotides of the present invention.
[0046]Once open reading frames are identified in the polynucleotides of the present invention, the open reading frames may be isolated and/or synthesized. Expressible genetic constructs comprising the open reading frames and suitable promoters, initiators, temiinators, etc., which are well known in the art, may then be constructed. Such genetic constructs may be introduced into a host cell to express the polypeptide encoded by the open reading frame. Suitable host cells may include various prokaryotic and eukaryotic cells, including plant cells, mammalian cells, bacterial cells, algae and the like.
[0047]Polypeptides encoded by the polynucleotides of the present invention may be expressed and used in various assays to determine their biological activity. Such polypeptides may be used to raise antibodies, to isolate corresponding interacting proteins or other compounds, and to quantitatively determine levels of interacting proteins or other compounds.
[0048]As used herein, the term "variant" comprehends nucleotide or amino acid sequences different from the specifically identified sequences, wherein one or more nucleotides or amino acid residues is deleted, substituted, or added. Variants may be naturally occurring allelic variants, or non-naturally occurring variants. Variant sequences (polynucleotide or polypeptide) preferably exhibit at least 50%, more preferably at least 75%, more preferably at least 85%, more preferably at least 90% and most preferably at least 95% identity to a sequence of the present invention. The percentage identity is determined by aligning the two sequences to be compared as described below, determining the number of identical residues in the aligned portion, dividing that number by the total number of residues in the inventive (queried) sequence, and multiplying the result by 100. By way of illustration only, assume an inventive polynucleotide having 220 nucleotides has a hit to a polynucleotide sequence in the EMBL database having 520 nucleotides over a stretch of 23 nucleotides in the alignment produced by the BLASTN algorithm using the parameters described above. The 23 nucleotide region includes 21 identical nucleotides, one gap and one different nucleotide. The percentage identity of the inventive polynucleotide to the hit in the EMBL library is thus 21/220 times 100, or 9.5%. The polynucleotide sequence in the EMBL database is thus not a variant of the inventive polynucleotide.
[0049]Polynucleotide and polypeptide sequences may be aligned, and percentage of identical residues in a specified region may be deteiinined against another polynucleotide or polypeptide sequence, using computer algorithms that are publicly available. Two exemplary algorithms for aligning and identifying the similarity of polynucleotide sequences are the BLASTN and FASTA algorithms. Polynucleotides may also be analyzed using the BLASTX algorithm, which compares the six-frame conceptual translation products of a nucleotide query sequence (both strands) against a protein sequence database. The similarity of polypeptide sequences may be examined using the BLASTP algorithm. The BLASTN, BLASTX and BLASTP programs are available on the NCBI anonymous FTP server, and from the National Center for Biotechnology Information (NCBI) National Library of Medicine, Building 38A, Room 8N8005, Bethesda, Md. 20894, USA. The BLASTN algorithm Version 2.0.4 [Feb. 24, 1998] and Version 2.0.6 [Sep. 16, 1998], set to the default parameters described in the documentation and distributed with the algorithm, are preferred for use in the determination of polynucleotide variants according to the present invention. The BLASTP algorithm is preferred for use in the determination of polypeptide variants according to the present invention. The use of the BLAST family of algorithms, including BLASTN, BLASTP, and BLASTX, is described at NCBI's Internet website and in the publication of Altschul et al., Nucleic Acids Res. 25:3389-3402, 1997.
[0050]The computer algorithm FASTA is available on the Internet, and from the University of Virginia by contacting David Hudson, Assistant Provost for Research, University of Virginia, PO Box 9025, Charlottesville, Va. Version 2.0u4 [Feb. 1996], set to the default parameters described in the documentation and distributed with the algorithm, may be used in the determination of variants according to the present invention. The use of the FASTA algorithm is described in Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444-2448, 1988; and Pearson, Methods in Enzymol. 183:63-98, 1990.
[0051]The following running parameters are preferred for determination of alignments and similarities using BLASTN that contribute to the E values and percentage identity for polynucleotide sequences: Unix running command: blastall -p blastn -d embldb -e 10 -G0 -E0-r 1 -v 30 -b 30 -i queryseq -o results; the parameters are: -p Program Name [String]; -d Database [String]; -e Expectation value (E) [Real]; -G Cost to open a gap (zero invokes default behavior) [Integer]; -E Cost to extend a gap (zero invokes default behavior) [Integer]; -r Reward for a nucleotide match (blastn only) [Integer]; -v Number of one-line descriptions (V) [Integer]; -b Number of alignments to show (B) [Integer]; -i Query File [File In]; and -o BLAST report Output File [File Out] Optional.
[0052]The following running parameters are preferred for determination of alignments and similarities using BLASTP that contribute to the E values and percentage identity of polypeptide sequences: blastall -p blastp -d swissprotdb -e 10 -G 0 -E 0 -v 30 -b 30 -i queryseq -o results; wherein the parameters are: -p Program Name [String]; -d Database [String]; -e Expectation value (E) [Real]; -G Cost to open a gap (zero invokes default behavior) [Integer]; -E Cost to extend a gap (zero invokes default behavior) [Integer]; -v Number of one-line descriptions (v) [Integer]; -b Number of alignments to show (b) [Integer]; -I Query File [File In]; -o BLAST report Output File [File Out] Optional.
[0053]The "hits" to one or more database sequences by a queried sequence produced by BLASTN, FASTA, BLASTP or a similar algorithm, align and identify similar portions of sequences. The hits are arranged in order of the degree of similarity and the length of sequence overlap. Hits to a database sequence generally represent an overlap over only a fraction of the sequence length of the queried sequence.
[0054]The BLASTN, FASTA and BLASTP algorithms also produce "Expect" values for alignments. The Expect value (E) indicates the number of hits one can "expect" to see over a certain number of contiguous sequences by chance when searching a database of a certain size. The Expect value is used as a significance threshold for determining whether the hit to a database, such as the preferred EMBL database, indicates true similarity. For example, an E value of 0.1 assigned to a polynucleotide hit is interpreted as meaning that in a database of the size of the EMBL database, one might expect to see 0.1 matches over the aligned portion of the sequence with a similar score simply by chance. By this criterion, the aligned and matched portions of the polynucleotide sequences then have a probability of 90% of being the same. For sequences having an E value of 0.01 or less over aligned and matched portions, the probability of finding a match by chance in the EMBL database is 1% or less using the BLASTN or FASTA algorithm.
[0055]According to one embodiment, "variant" polynucleotides and polypeptides, with reference to each of the polynucleotides and polypeptides of the present invention, preferably comprise sequences having the same number or fewer nucleic or amino acids than each of the polynucleotides or polypeptides of the present invention and producing an E value of 0.01 or less when compared to the polynucleotide or polypeptide of the present invention. That is, a variant polynucleotide or polypeptide is any sequence that has at least a 99% probability of being the same as the polynucleotide or polypeptide of the present invention, measured as having an E value of 0.01 or less using the BLASTN, FASTA, or BLASTP algorithms set at parameters described above.
[0056]Alternatively, variant polynucleotides of the present invention hybridize to the polynucleotide sequences recited in SEQ ID NOS: 1-591, 1183-1912, 1931-2106, 2371, 2374, 2377, 2385, 2387, 2389, 2391, 2393, 2395, 2397, 2399, 2401, 2403, 2405, 2407, 2409, 2411, 2413, 2415, 2417, 2419, and 2421, or complements, reverse sequences, or reverse complements of those sequences, under stringent conditions. As used herein, "stringent conditions" refers to prewashing in a solution of 6×SSC, 0.2% SDS; hybridizing at 65° C., 6×SSC, 0.2% SDS overnight; followed by two washes of 30 minutes each in 1×SSC, 0.1% SDS at 65° C. and two washes of 30 minutes each in 0.2×SSC, 0.1% SDS at 65° C.
[0057]The present invention also encompasses polynucleotides that differ from the disclosed sequences but that, as a consequence of the degeneracy of the genetic code, encode a polypeptide which is the same as that encoded by a polynucleotide of the present invention. Thus, polynucleotides comprising sequences that differ from the polynucleotide sequences recited in SEQ ID NOS: 1-591, 1183-1912, 1931-2106, 2371, 2374, 2377, 2385, 2387, 2389, 2391, 2393, 2395, 2397, 2399, 2401, 2403, 2405, 2407, 2409, 2411, 2413, 2415, 2417, 2419, and 2421; or complements, reverse sequences, or reverse complements thereof, as a result of conservative substitutions are contemplated by and encompassed within the present invention. Additionally, polynucleotides comprising sequences that differ from the polynucleotide sequences recited in SEQ ID NOS: 1-591, 1183-1912, 1931-2106, 2371, 2374, 2377, 2385, 2387, 2389, 2391, 2393, 2395, 2397, 2399, 2401, 2403, 2405, 2407, 2409, 2411, 2413, 2415, 2417, 2419, and 2421, or complements, reverse complements or reverse sequences thereof, as a result of deletions and/or insertions totaling less than 10% of the total sequence length are also contemplated by and encompassed within the present invention. Similarly, polypeptides comprising sequences that differ from the polypeptide sequences recited in SEQ ID NOS: 592-1182, 1913-1930, 2107-2278, 2372, 2375, 2378, 2386, 2388, 2390, 2392, 2394, 2396, 2398, 2400, 2402, 2404, 2406, 2408, 2410, 2412, 2414, 2416, 2418, 2420, and 2422 as a result of amino acid substitutions, insertions, and/or deletions totaling less than 10% of the total sequence length are contemplated by and encompassed within the present invention.
[0058]In addition to having a specified percentage identity to an inventive polynucleotide or polypeptide sequence, variant polynucleotides and polypeptides preferably have additional structure and/or functional features in common with the inventive polynucleotide or polypeptide. Polypeptides having a specified degree of identity to a polypeptide of the present invention share a high degree of similarity in their primary structure and have substantially similar functional properties. In addition to sharing a high degree of similarity in their primary structure to polynucleotides of the present invention, polynucleotides having a specified degree of identity to, or capable of hybridizing to an inventive polynucleotide preferably have at least one of the following features: (i) they contain an open reading frame, or partial open reading frame, encoding a polypeptide having substantially the same functional properties as the polypeptide encoded by the inventive polynucleotide; or (ii) they contain identifiable domains in common. Thus, in preferred embodiments, variants of the inventive polypeptides possess biological activities that are the same or similar to those of the inventive polypeptides. Such variant polypeptides function as transcription factors and are thus capable of modifying gene expression in a plant. Similarly, variant polynucleotides may encode polypeptides that function as transcription factors.
[0059]Polynucleotides of the present invention also comprehend polynucleotides comprising at least a specified number of contiguous residues (x-mers) of any of the polynucleotides identified as SEQ ID NOS: 1-591, 1183-1912, 1931-2106, 2371, 2374, 2377, 2385, 2387, 2389, 2391, 2393, 2395, 2397, 2399, 2401, 2403, 2405, 2407, 2409, 2411, 2413, 2415, 2417, 2419, and 2421, complements, reverse sequences, and reverse complements of such sequences, and their variants. Similarly, polypeptides of the present invention comprehend polypeptides comprising at least a specified number of contiguous residues (x-mers) of any of the polypeptides identified as SEQ ID NOS: 592-1182, 1913-1930, 2107-2278, 2372, 2375, 2378, 2386, 2388, 2390, 2392, 2394, 2396, 2398, 2400, 2402, 2404, 2406, 2408, 2410, 2412, 2414, 2416, 2418, 2420, and 2422, and their variants. As used herein, the term "x-mer," with reference to a specific value of "x," refers to a sequence comprising at least a specified number ("x") of contiguous residues of any of the polynucleotides identified as SEQ ID NOS: 1-591, 1183-1912, 1931-2106, 2371, 2374, 2377, 2385, 2387, 2389, 2391, 2393, 2395, 2397, 2399, 2401, 2403, 2405, 2407, 2409, 2411, 2413, 2415, 2417, 2419, and 2421, or the polypeptides identified as SEQ ID NOS: 592-1182, 1913-1930, 2107-2278, 2372, 2375, 2378, 2386, 2388, 2390, 2392, 2394, 2396, 2398, 2400, 2402, 2404, 2406, 2408, 2410, 2412, 2414, 2416, 2418, 2420, and 2422. According to preferred embodiments, the value of x is preferably at least 20, more preferably at least 40, more preferably yet at least 60, and most preferably at least 80. Thus, polynucleotides and polypeptides of the present invention comprise a 20-mer, a 40-mer, a 60-mer, an 80-mer, a 100-mer, a 120-mer, a 150-mer, a 180-mer, a 220-mer, a 250-mer, a 300-mer, a 400-mer, a 500-mer or a 600-mer of a polynucleotide or polypeptide identified as SEQ ID NOS: 1-2372, 2374, 2375, 2377, 2378, 2385, 2386, 2387, 2388, 2389, 2390, 2391, 2392, 2393, 2394, 2395, 2396, 2397, 2398, 2399, 2400, 2401, 2402, 2403, 2404, 2405, 2406, 2407, 2408, 2409, 2410, 2411, 2412, 2413, 2414, 2415, 2416, 2417, 2418, 2419, 2420, 2421, and 2422 and variants thereof.
[0060]The inventive polynucleotides may be isolated by high throughput sequencing of cDNA libraries prepared from Eucalyptus grandis and Pinus radiata as described below in Examples 1 and 2. Alternatively, oligonucleotide primer and probes based on the sequences provided in SEQ ID NOS: 1-591, 1183-1912, 1931-2106, 2371, 2374, 2377, 2385, 2387, 2389, 2391, 2393, 2395, 2397, 2399, 2401, 2403, 2405, 2407, 2409, 2411, 2413, 2415, 2417, 2419, and 2421 may be prepared as detailed below and used to identify positive clones in either cDNA or genomic DNA libraries from Eucalyptus grandis and Pinus radiata by means of hybridization or PCR techniques. Probes may be shorter than the sequences provided herein but should be at least about 10, preferably at least 15, and most preferably at least about 20 nucleotides in length. Hybridization and PCR techniques suitable for use with such oligonucleotides are well known in the art, and include those taught by Sambrook et al., Ibid. Positive clones may be analyzed by restriction enzyme digestion, DNA sequencing or the like.
[0061]The polynucleotides of the present invention may alternatively be synthesized using techniques that are well known in the art. The polynucleotides may be synthesized, for example, using automated oligonucleotide synthesizers (e.g., Beckman Oligo 1000M DNA Synthesizer) to obtain polynucleotide segments of up to 50 or more nucleic acids. A plurality of such polynucleotide segments may then be ligated using standard DNA manipulation techniques that are well known in the art of molecular biology. One conventional and exemplary polynucleotide synthesis technique involves synthesis of a single stranded polynucleotide segment having, for example, 80 nucleic acids, and hybridizing that segment to a synthesized complementary 85 nucleic acid segment to produce a 5 nucleotide overhang. The next segment may then be synthesized in a similar fashion, with a 5 nucleotide overhang on the opposite strand. The "sticky" ends ensure proper ligation when the two portions are hybridized. In this way, a complete polynucleotide of the present invention may be synthesized entirely in vitro.
[0062]In certain embodiments, the genetic constructs of the present invention include an open reading frame coding for at least a functional portion of a polypeptide of the present invention or a variant thereof. As used herein, the "functional portion" of a polypeptide is that portion which contains the active site essential for regulating gene expression, i.e., the portion of the molecule that is capable of binding to, or interacting with, the promoter of the gene to be expressed. The DNA-binding domain(s) for certain of the inventive polypeptides are identified below in Table 2. These DNA binding domains were identified using PROSITE 15.0 pattern or profile sequences as listed in the PROSITE database. PROSITE is available on the internet and its use is described in Hofman et al., Nucleic Acids Res. 27:215-219, 1999; and in Bairoch, Nucleic Acids Res. 20:Suppl. 2013-2018, 1992.
TABLE-US-00003 TABLE 2 Polynucleotide DNA-binding Domain(s) SEQ ID NO: SEQ ID NO: 1931 2283 1934 2284, 2285 1940 2288 1949 2293 1951 2279, 2280 1953 2296, 2297 1957 2298 1960 2301, 2302 1962 2307 1965 2308, 2309 1967 2281, 2282 1978 2320 1979 2321 1982 2322, 2323 1986 2324 1992 2335 1994 2336, 2337 1995 2338, 2339 1997 2340 2003 2286, 2287 2013 2289, 2290 2020 2291, 2292 2027 2299, 2300 2030 2303, 2304 2032 2305, 2306 2036 2310, 2311 2038 2312, 2313 2049 2314, 2315 2051 2316, 2317 2052 2318, 2319 2057 2325, 2326 2059 2327, 2328 2060 2329, 2330 2065 2331, 2332 2067 2333, 2334 2074 2342, 2343 2075 2344, 2345 2076 2346, 2347 2077 2348, 2349 2080 2352 2081 2353 2082 2354 2083 2355, 2356 2084 2357, 2358 2085 2359, 2360 2086 2361, 2362 2087 2365, 2366 2088 2367, 2368 2104 2350, 2351 2105 2363, 2364
[0063]The functional portion of a polypeptide may also be determined by targeted mutagenesis and screening of modified protein products with protocols well known in the art (Solano et al., J. Biol. Chem. 272:2889-95, 1997). The active site will generally exhibit high substrate specificity. Portions of the inventive polypeptides may be generated by synthetic or recombinant means. Synthetic polypeptides having fewer than about 100 amino acids, and generally fewer than about 50 amino acids, may be generated using techniques well known to those of ordinary skill in the art. For example, such polypeptides may be synthesized using any of the commercially available solid-phase techniques, such as the Merrifield solid-phase synthesis method, where amino acids are sequentially added to a growing amino acid chain. See Merrifield, J. Am. Chem. Soc. 85:2149-2154, 1963. Equipment for automated synthesis of polypeptides is commercially available from suppliers such as Perkin Elmer/Applied BioSystems, Inc. (Foster City, Calif.), and may be operated according to the manufacturer's instructions.
[0064]An open reading frame may be inserted in the genetic construct in a sense or antisense orientation, such that transfoimation of a target plant with the genetic construct will lead to a change in the amount of polypeptide compared to the wild-type plant. Transformation with a genetic construct comprising an open reading frame in a sense orientation will generally result in over-expression of the selected gene, while transformation with a genetic construct comprising an open reading frame in an antisense orientation will generally result in reduced expression of the selected gene. A population of plants transformed with a genetic construct comprising an open reading frame of the present invention in either a sense or antisense orientation may be screened for increased or reduced expression of the gene in question using techniques well known to those of skill in the art, and plants having the desired phenotypes may thus be isolated.
[0065]Alternatively, expression of a gene encoding a plant transcription factor may be inhibited by inserting a portion of an open reading frame of the present invention, in either sense or antisense orientation, in the genetic construct. Such portions need not be full-length but preferably comprise at least 25, and more preferably at least 50, residues of an inventive DNA sequence. A much longer portion or even the full length DNA corresponding to the complete open reading frame may be employed. The portion of the open reading frame does not need to be precisely the same as the endogenous sequence, provided that there is sufficient sequence similarity to achieve inhibition of the target gene. Thus a sequence derived from one species may be used to inhibit expression of a gene in a different species. A population of plants transformed with a genetic construct comprising an open reading frame of the present invention in either a sense or antisense orientation may be screened for increased or reduced expression of the gene in question using techniques well known to those of skill in the art, and plants having the desired phenotypes may thus be isolated.
[0066]In another embodiment, the inventive genetic constructs comprise a DNA sequence including an untranslated, or non-coding, region of a gene coding for a polypeptide of the present invention, or a DNA sequence complementary to such an untranslated region. Examples of untranslated regions which may be usefully employed in such constructs include introns and 5'-untranslated leader sequences. Transformation of a target plant with such a genetic construct may lead to a reduction in the amount of the polypeptide expressed in the plant by the process of cosuppression, in a manner similar to that discussed, for example, by Napoli et al. (Plant Cell 2:279-290, 1990), and de Carvalho Niebel et al. (Plant Cell 7:347-358, 1995).
[0067]Alternatively, regulation of polypeptide expression can be achieved by inserting appropriate sequences or subsequences (e.g. DNA or RNA) in ribozyme constructs (McIntyre and Manners, Transgenic Res. 5[4]:257-262, 1996). Ribozymes are synthetic RNA molecules that comprise a hybridizing region complementary to two regions, each of which comprises at least 5 contiguous nucleotides in a mRNA molecule encoded by one of the inventive polynucleotides. Ribozymes possess highly specific endonuclease activity, which autocatalytically cleaves the mRNA.
[0068]The genetic constructs of the present invention further comprise a gene promoter sequence and a gene termination sequence, operably linked to the DNA sequence to be transcribed, which control expression of the gene. The gene promoter sequence is generally positioned at the 5' end of the DNA sequence to be transcribed, and is employed to initiate transcription of the DNA sequence. Gene promoter sequences are generally found in the 5' untranslated region of a gene but they may exist downstream of the open reading frame, in introns (Luehrsen, Mol. Gen. Genet. 225:81-93, 1991) or in the coding region, as for example in a plant defence gene (Douglas et al., EMBO J. 10:1767-1775, 1991). When the construct includes an open reading frame in a sense orientation, the gene promoter sequence also initiates translation of the open reading frame. For genetic constructs comprising either an open reading frame in an antisense orientation or an untranslated region, the gene promoter sequence may consist only of a transcription initiation site having a RNA polymerase binding site.
[0069]A variety of gene promoter sequences which may be usefully employed in the genetic constructs of the present invention are well known in the art. The gene promoter sequence, and also the gene termination sequence, may be endogenous to the target plant host or may be exogenous, provided the promoter is functional in the target host. For example, the promoter and termination sequences may be from other plant species, plant viruses, bacterial plasmids and the like. Preferably, gene promoter and teimination sequences are from the inventive sequences themselves.
[0070]Factors influencing the choice of promoter include the desired tissue specificity of the construct, and the timing of transcription and translation. For example, constitutive promoters, such as the 35S Cauliflower Mosaic Virus (CaMV 35S) promoter, will affect the activity of the enzyme in all parts of the plant. Use of a tissue specific promoter will result in production of the desired sense or antisense RNA only in the tissue of interest. With genetic constructs employing inducible gene promoter sequences, the rate of RNA polymerase binding and initiation can be modulated by external stimuli, such as light, heat, anaerobic stress, alteration in nutrient conditions and the like. Temporally regulated promoters can be employed to effect modulation of the rate of RNA polymerase binding and initiation at a specific time during development of a transformed cell. Preferably, the original promoters from the enzyme gene in question, or promoters from a specific tissue-targeted gene in the organism to be transformed, such as eucalyptus or pine are used. Other examples of gene promoters which may be usefully employed in the present invention include mannopine synthase (mas), octopine synthase (ocs) and those reviewed by Chua et al. (Science 244:174-181, 1989).
[0071]The gene termination sequence, which is located 3' to the DNA sequence to be transcribed, may come from the same gene as the gene promoter sequence or may be from a different gene. Many gene termination sequences known in the art may be usefully employed in the present invention, such as the 3' end of the Agrobacterium tumefaciens nopaline synthase gene. However, preferred gene terminator sequences are those from the original gene or from the target species to be transformed.
[0072]The genetic constructs of the present invention may also contain a selection marker that is effective in cells of the target organism, such as a plant, to allow for the detection of transformed cells containing the inventive construct. Such markers, which are well known in the art, typically confer resistance to one or more toxins. One example of such a marker is the NPTII gene whose expression results in resistance to kanamycin or hygromycin, antibiotics which are usually toxic to plant cells at a moderate concentration (Rogers et al., in Weissbach, A and Weissbach H, eds., Methods for Plant Molecular Biology, Academic Press Inc.: San Diego, Calif., 1988). Transformed cells can thus be identified by their ability to grow in media containing the antibiotic in question. Alternatively, the presence of the desired construct in transformed cells can be determined by means of other techniques well known in the art, such as Southern and Western blots.
[0073]A transcription initiation site is additionally included in the genetic construct when the sequence to be transcribed lacks such a site.
[0074]Techniques for operatively linking the components of the inventive genetic constructs are well known in the art and include the use of synthetic linkers containing one or more restriction endonuclease sites as described, for example, by Sambrook et al., (Molecular cloning: a laboratory manual, CSHL Press: Cold Spring Harbor, N.Y., 1989). The genetic construct of the present invention may be linked to a vector having at least one replication system, for example E. coli, whereby after each manipulation, the resulting construct can be cloned and sequenced and the correctness of the manipulation determined.
[0075]The genetic constructs of the present invention may be used to transform a variety of target organisms including, but not limited to, plants. Plants which may be transformed using the inventive constructs include both monocotyledonous angiosperms (e.g., grasses, corn, grains, oat, wheat and barley); and dicotyledonous angiosperms (e.g., Arabidopsis, tobacco, legumes, alfalfa, oaks, eucalyptus, maple); and gymnosperms (e.g., Scots pine; Aronen, Finnish Forest Res. Papers, Vol. 595, 1996); white spruce (Ellis et al., Biotechnology 11:84-89, 1993); and larch (Huang et al., In Vitro Cell 27:201-207, 1991). In a preferred embodiment, the inventive genetic constructs are employed to transform woody plants, herein defined as a tree or shrub whose stem lives for a number of years and increases in diameter each year by the addition of woody tissue. Preferably the target plant is selected from the group consisting of eucalyptus and pine species, most preferably from the group consisting of Eucalyptus grandis and Pinus radiata. Other species which may be usefully transformed with the genetic constructs of the present invention include, but are not limited to: pines such as Pinus banksiana, Pinus brutia, Pinus caribaea, Pinus clausa, Pinus contorta, Pinus coulteri, Pinus echinata, Pinus eldarica, Pinus ellioti, Pinus jeffreyi, Pinus lambertiana, Pinus monticola, Pinus nigra, Pinus palustrus, Pinus pinaster, Pinus ponderosa, Pinus resinosa, Pinus rigida, Pinus serotina, Pinus strobus, Pinus sylvestris, Pinus taeda, Pinus virginiana; other gymnosperms, such as Abies amabilis, Abies balsamea, Abies concolor, Abies grandis, Abies lasiocarpa, Abies magnifica, Abies procera, Chamaecyparis lawsoniona, Chamaecyparis nootkatensis, Chamaecyparis thyoides, Huniperus virginiana, Larix decidua, Larix laricina, Larix leptolepis, Larix occidentalis, Larix siberica, Libocedrus decurrens, Picea abies, Picea engelmanni, Picea glauca, Picea mariana, Picea pungens, Picea rubens, Picea sitchensis, Pseudotsuga menziesii, Sequoia gigantea, Sequoia sempervirens, Taxodium distichum, Tsuga canadensis, Tsuga heterophylla, Tsuga mertensiana, Thuja occidentalis, Thuja plicata; and Eucalypts, such as Eucalyptus alba, Eucalyptus bancroftii, Eucalyptus botyroides, Eucalyptus bridgesiana, Eucalyptus calophylla, Eucalyptus camaldulensis, Eucalyptus citriodora, Eucalyptus cladocalyx, Eucalyptus coccifera, Eucalyptus curtisii, Eucalyptus dalrympleana, Eucalyptus deglupta, Eucalyptus delagatensis, Eucalyptus diversicolor, Eucalyptus dunnii, Eucalyptus ficifolia, Eucalyptus globulus, Eucalyptus gomphocephala, Eucalyptus gunnii, Eucalyptus henryi, Eucalyptus laevopinea, Eucalyptus macarthurii, Eucalyptus macrorhyncha, Eucalyptus maculata, Eucalyptus marginata, Eucalyptus megacarpa, Eucalyptus melliodora, Eucalyptus nicholii, Eucalyptus nitens, Eucalyptus nova-anglica, Eucalyptus obliqua, Eucalyptus obtusiflora, Eucalyptus oreades, Eucalyptus pauciflora, Eucalyptus polybractea, Eucalyptus regnans, Eucalyptus resinifera, Eucalyptus robusta, Eucalyptus rudis, Eucalyptus saligna, Eucalyptus sideroxylon, Eucalyptus stuartiana, Eucalyptus tereticornis, Eucalyptus torelliana, Eucalyptus urnigera, Eucalyptus urophylla, Eucalyptus viminalis, Eucalyptus viridis, Eucalyptus wandoo and Eucalyptus youmanni; and hybrids of any of these species.
[0076]Techniques for stably incorporating genetic constructs into the genome of target plants are well known in the art and include Agrobacterium tumefaciens mediated introduction, electroporation, protoplast fusion, injection into reproductive organs, injection into immature embryos, high velocity projectile introduction and the like. The choice of technique will depend upon the target plant to be transformed. For example, dicotyledonous plants and certain monocots and gymnospelins may be transformed by Agrobacterium Ti plasmid technology, as described, for example by Bevan (Nucleic Acids Res. 12:8711-8721, 1984). Targets for the introduction of the genetic constructs of the present invention include tissues, such as leaf tissue, dissociated cells, protoplasts, seeds, embryos, meristematic regions; cotyledons, hypocotyls, and the like. The preferred method for transforming eucalyptus and pine is a biolistic method using pollen (see, for example, Aronen, in Finnish Forest Res. Papers 595:53, 1996) or easily regenerable embryonic tissues.
[0077]Once the cells are transformed, cells having the inventive genetic construct incorporated in their genome may be selected by means of a marker, such as the kanamycin resistance marker discussed above. Transgenic cells may then be cultured in an appropriate medium to regenerate whole plants, using techniques well known in the art. In the case of protoplasts, the cell wall is allowed to reform under appropriate osmotic conditions. In the case of seeds or embryos, an appropriate germination or callus initiation medium is employed. For explants, an appropriate regeneration medium is used. Regeneration of plants is well established for many species. For a review of regeneration of forest trees see Dunstan et al., "Somatic embryogenesis in woody plants," in Thorpe T A, ed., In vitro embryogenesis of plants (Current Plant Science and Biotechnology in Agriculture, 20[12]:471-540, 1995. Specific protocols for the regeneration of spruce are discussed by Roberts et al. ("Somatic embryogenesis of spruce," in Redenbaugh K, ed., Synseed: applications of synthetic seed to crop improvement, CRC Press: 23:427-449, 1993). Transformed plants having the desired phenotype may be selected using techniques well known in the art. The resulting transformed plants may be reproduced sexually or asexually, using methods well known in the art, to give successive generations of transgenic plants.
[0078]As discussed above, the production of RNA in target cells can be controlled by choice of the promoter sequence, or by selecting the number of functional copies or the site of integration of the DNA sequences incorporated into the genome of the target host. A target organism may be transformed with more than one genetic construct of the present invention, thereby modulating the activity of more than one transcription factor, for example affecting gene expression in more than one tissue, or at more than one time in the development of the target organism. Similarly, a genetic construct may be assembled containing more than one open reading frame coding for a polypeptide of the present invention or more than one untranslated region of a gene coding for such a polypeptide. The polynucleotides of the present inventive may also be employed in combination with other known sequences encoding transcription factors.
[0079]Polynucleotides of the present invention may also be used to specifically suppress gene expression by methods that operate post-transcriptionally to block the synthesis of products of targeted genes, such as RNA interference (RNAi), and quelling. For a review of techniques of gene suppression see Science, 288:1370-1372, 2000. Exemplary gene silencing methods are also provided in WO 99/49029 and WO 99/53050. Posttranscriptional gene silencing is brought about by a sequence-specific RNA degradation process that results in the rapid degradation of transcripts of sequence-related genes. Studies have provided evidence that double-stranded RNA may act as a mediator of sequence-specific gene silencing (see, e.g., review by Montgomery and Fire, Trends in Genetics, 14:255-258, 1998). Gene constructs that produce transcripts with self-complementary regions are particularly efficient at gene silencing. A unique feature of this posttranscriptional gene silencing pathway is that silencing is not limited to the cells where it is initiated. The gene-silencing effects may be disseminated to other parts of an organism and even transmitted through the germ line to several generations.
[0080]The polynucleotides of the present invention may be employed to generate gene silencing constructs and or gene-specific self-complementary RNA sequences that can be delivered by conventional art-known methods to plant tissues, such as forestry tree tissues. Within genetic constructs, sense and antisense sequences can be placed in regions flanking an intron sequence in proper splicing orientation with donor and acceptor splicing sites, such that intron sequences are removed during processing of the transcript, and sense and antisense sequences, as well as splice junction sequences, bind together to form double-stranded RNA. Alternatively, spacer sequences of various lengths may be employed to separate self-complementary regions of sequence in the construct. During processing of the gene construct transcript, intron sequences are spliced-out, allowing sense and anti-sense sequences, as well as splice junction sequences, to bind forming double-stranded RNA. Select ribonucleases bind to and cleave the double-stranded RNA, thereby initiating the cascade of events leading to degradation of specific mRNA gene sequences, and silencing specific genes. Alternatively, rather than using a gene construct to express the self-complementary RNA sequences, the gene-specific double-stranded RNA segments are delivered to one or more targeted areas to be internalized into the cell cytoplasm to exert a gene silencing effect. Gene silencing RNA sequences comprising the polynucleotides of the present invention are useful for creating genetically modified plants with desired phenotypes as well as for characterizing genes (e.g., in high-throughput screening of sequences), and studying their functions in intact organisms.
[0081]Oligonucleotide probes and primers complementary to and/or corresponding to SEQ ID NOS: 1-591, 1183-1912, 1931-2106, 2371, 2374, 2377, 2385, 2387, 2389, 2391, 2393, 2395, 2397, 2399, 2401, 2403, 2405, 2407, 2409, 2411, 2413, 2415, 2417, 2419, and 2421, and variants of those sequences, are also comprehended by the present invention. Such oligonucleotide probes and primers are substantially complementary to the polynucleotide of interest. As used herein, the term "oligonucleotide" refers to a relatively short segment of a polynucleotide sequence, generally comprising between 6 and 60 nucleotides, and comprehends both probes for use in hybridization assays and primers for use in the amplification of DNA by polymerase chain reaction. An oligonucleotide probe or primer is described as "corresponding to" a polynucleotide of the present invention, including one of the sequences set out as SEQ ID NOS: 1-591, 1183-1912, 1931-2106, 2371, 2374, 2377, 2385, 2387, 2389, 2391, 2393, 2395, 2397, 2399, 2401, 2403, 2405, 2407, 2409, 2411, 2413, 2415, 2417, 2419, and 2421, or a variant, if the oligonucleotide probe or primer, or its complement, is contained within one of the sequences set out as SEQ ID NOS: 1-591, 1183-1912, 1931-2106, 2371, 2374, 2377, 2385, 2387, 2389, 2391, 2393, 2395, 2397, 2399, 2401, 2403, 2405, 2407, 2409, 2411, 2413, 2415, 2417, 2419, and 2421, or a variant of one of the specified sequences.
[0082]Two single stranded sequences are said to be substantially complementary when the nucleotides of one strand, optimally aligned and compared, with the appropriate nucleotide insertions and/or deletions, pair with at least 80%, preferably at least 90% to 95%, and more preferably at least 98% to 100%, of the nucleotides of the other strand. Alternatively, substantial complementarity exists when a first DNA strand will selectively hybridize to a second DNA strand under stringent hybridization conditions. Stringent hybridization conditions for determining complementarity include salt conditions of less than about 1 M, more usually less than about 500 mM, and preferably less than about 200 mM. Hybridization temperatures can be as low as 5° C., but are generally greater than about 22° C., more preferably greater than about 30° C., and most preferably greater than about 37° C. Longer DNA fragments may require higher hybridization temperatures for specific hybridization. Since the stringency of hybridization may be affected by other factors such as probe composition, presence of organic solvents and extent of base mismatching, the combination of parameters is more important than the absolute measure of any one alone. The DNA from plants or samples or products containing plant material can be either genomic DNA or DNA derived by preparing cDNA from the RNA present in the sample.
[0083]In addition to DNA-DNA hybridization, DNA-RNA or RNA-RNA hybridization assays are also possible. In the first case, the mRNA from expressed genes would then be detected instead of genomic DNA or cDNA derived from mRNA of the sample. In the second case, RNA probes could be used. In addition, artificial analogs of DNA hybridizing specifically to target sequences could also be used.
[0084]In specific embodiments, the oligonucleotide probes and/or primers comprise at least about 6 contiguous residues, more preferably at least about 10 contiguous residues, and most preferably at least about 20 contiguous residues complementary to a polynucleotide sequence of the present invention. Probes and primers of the present invention may be from about 8 to 100 base pairs in length or, preferably from about 10 to 50 base pairs in length or, more preferably from about 15 to 40 base pairs in length. The probes can be easily selected using procedures well known in the art, taking into account DNA-DNA hybridization stringencies, annealing and melting temperatures, and potential for formation of loops and other factors, which are well known in the art. Tools and software suitable for designing probes and PCR primers, are available on the Internet. An exemplary software program suitable for designing probes, and especially for designing PCR primers, is available from Premier Biosoft International, 3786 Corina Way, Palo Alto, Calif. 94303-4504. Preferred techniques for designing PCR primers are also disclosed in Dieffenbach and Dyksler, PCR primer: a laboratory manual, CSHL Press: Cold Spring Harbor, N.Y., 1995.
[0085]A plurality of oligonucleotide probes or primers corresponding to a polynucleotide of the present invention may be provided in a kit form. Such kits generally comprise multiple DNA or oligonucleotide probes, each probe being specific for a polynucleotide sequence. Kits of the present invention may comprise one or more probes or primers corresponding to a polynucleotide of the present invention, including a polynucleotide sequence identified in SEQ ID NOS: 1-591, 1183-1912, 1931-2106, 2371, 2374, 2377, 2385, 2387, 2389, 2391, 2393, 2395, 2397, 2399, 2401, 2403, 2405, 2407, 2409, 2411, 2413, 2415, 2417, 2419, and 2421.
[0086]In one embodiment useful for high-throughput assays, the oligonucleotide probe kits of the present invention comprise multiple probes in an array format, wherein each probe is immobilized in a predefined, spatially addressable location on the surface of a solid substrate. Array formats which may be usefully employed in the present invention are disclosed, for example, in U.S. Pat. Nos. 5,412,087, 5,545,531, and PCT Publication No. WO 95/00530, the disclosures of which are hereby incorporated by reference.
[0087]The significance of high-throughput screening systems is apparent for applications such as plant breeding and quality control operations in which there is a need to identify large numbers of seed lots and plant seedlings, to examine samples or products for unwanted plant materials, to identify plants or samples or products containing plant material for quarantine purposes etc. or to ascertain the true origin of plants or samples or products containing plant material. Screening for the presence or absence of polynucleotides of the present invention used as identifiers for tagging plants is valuable for later detecting the amount of gene flow in plant breeding, introgression of genes via dispersed pollen, etc.
[0088]In this manner, oligonucleotide probe kits of the present invention may be employed to examine the presence/absence (or relative amounts in case of mixtures) of polynucleotides of the present invention in different samples or products containing different materials rapidly and in a cost-effective manner. Examples of plant species that may be examined using the present invention include forestry species, such as pine and eucalyptus species, other tree species, agricultural plants including crop and forage plants, and horticultural plants.
[0089]Another aspect of the present invention involves collections of polynucleotides of the present invention. A collection of polynucleotides of the present invention, particularly the polynucleotides identified as SEQ ID NOS: 1-591, 1183-1912, 1931-2106, 2371, 2374, 2377, 2385, 2387, 2389, 2391, 2393, 2395, 2397, 2399, 2401, 2403, 2405, 2407, 2409, 2411, 2413, 2415, 2417, 2419, and 2421, and variants and x-mers thereof, may be recorded and/or stored on a storage medium and subsequently accessed for purposes of analysis, comparison, etc. Suitable storage media include magnetic media such as magnetic diskettes, magnetic tapes, CD-ROM storage media, optical storage media, and the like. Suitable storage media and methods for recording and storing information, as well as accessing information such as polynucleotide sequences recorded on such media, are well known in the art. The polynucleotide information stored on the storage medium is preferably computer-readable and may be used for analysis and comparison of the polynucleotide information.
[0090]Another aspect of the present invention thus involves storage medium on which are recorded a collection of the polynucleotides of the present invention, particularly a collection of the polynucleotides identified as SEQ ID NOS: 1-591, 1183-1912, 1931-2106, 2371, 2374, 2377, 2385, 2387, 2389, 2391, 2393, 2395, 2397, 2399, 2401, 2403, 2405, 2407, 2409, 2411, 2413, 2415, 2417, 2419, and 2421, and variants thereof, as well as x-mers of the polynucleotides of SEQ ID NOS: 1-591, 1183-1912, 1931-2106, 2371, 2374, 2377, 2385, 2387, 2389, 2391, 2393, 2395, 2397, 2399, 2401, 2403, 2405, 2407, 2409, 2411, 2413, 2415, 2417, 2419, and 2421, and extended sequences, probes and primers comprising or corresponding to a polynucleotide of SEQ ID NOS: 1-591, 1183-1912, 1931-2106, 2371, 2374, 2377, 2385, 2387, 2389, 2391, 2393, 2395, 2397, 2399, 2401, 2403, 2405, 2407, 2409, 2411, 2413, 2415, 2417, 2419, and 2421. According to one embodiment, the storage medium includes a collection of at least 20, preferably at least 50, more preferably at least 100, and most preferably at least 200 of the polynucleotides of the present invention, preferably the polynucleotides identified as SEQ ID NOS: 1-591, 1183-1912, 1931-2106, 2371, 2374, 2377, 2385, 2387, 2389, 2391, 2393, 2395, 2397, 2399, 2401, 2403, 2405, 2407, 2409, 2411, 2413, 2415, 2417, 2419, and 2421, or variants of such polynucleotides.
Isolation and Characterization of cDNA Clones from Eucalyptus grandis
[0092]Nine Eucalyptus grandis cDNA expression libraries (prepared from either mature shoot buds, early wood phloem, floral tissue, leaf tissue (two independent libraries), feeder roots, structural roots, xylem or early wood xylem) were constructed and screened as follows.
[0093]Total RNA was extracted from the plant tissue using the protocol of Chang et al. (Plant Molecular Biology Reporter 11:113-116, 1993). mRNA was isolated from the total RNA preparation using either a Poly(A) Quik mRNA Isolation Kit (Stratagene, La Jolla, Calif.) or Dynal Beads Oligo (dT)25 (Dynal, Skogen, Norway). A cDNA expression library was constructed from the purified mRNA by reverse transcriptase synthesis followed by insertion of the resulting cDNA clones in Lambda ZAP using a ZAP Express cDNA Synthesis Kit (Stratagene), according to the manufacturer's protocol. The resulting cDNAs were packaged using a Gigapack II Packaging Extract (Stratagene) using an aliquot (1-5 μl) from the 5 μl ligation reaction dependent upon the library. Mass excision of the library was done using XL1-Blue MRF' cells and XLOLR cells (Stratagene) with ExAssist helper phage (Stratagene). The excised phagemids were diluted with NZY broth (Gibco BRL, Gaithersburg, Md.) and plated out onto LB-kanamycin agar plates containing X-gal and isopropylthio-beta-galactoside (IPTG).
[0094]Of the colonies plated and picked for DNA miniprep, 99% contained an insert suitable for sequencing. Positive colonies were cultured in NZY broth with kanamycin and cDNA was purified by means of alkaline lysis and polyethylene glycol (PEG) precipitation. Agarose gel at 1% was used to screen sequencing templates for chromosomal contamination. Dye primer sequences were prepared using a Turbo Catalyst 800 machine (Perkin Elmer/Applied Biosystems Division, Foster City, Calif.) according to the manufacturer's protocol.
[0095]DNA sequence for positive clones was obtained using a Perkin Elmer/Applied Biosystems Division Prism 377 sequencer. cDNA clones were sequenced first from the 5' end and, in some cases, also from the 3' end. For some clones, internal sequence was obtained using either Exonuclease III deletion analysis, yielding a library of differentially sized subclones in pBK-CMV, or by direct sequencing using gene-specific primers designed to identified regions of the gene of interest.
[0096]The determined cDNA sequences were compared to known sequences in the EMBL database (up to mid-July 1999) using the computer algorithms FASTA and/or BLASTN. Multiple alignments of redundant sequences were used to build up reliable consensus sequences. The determined cDNA sequences are provided in SEQ ID NOS: 1-331, 1183-1536, 1896-1901, 1905, 1906, 1908-1910, 1932-1968, 2001-2036, 2074-2079 and 2104. Based on similarity to known sequences from other plant species, the isolated DNA sequences were identified as encoding transcription factors, as detailed in Table 1 above. The predicted amino acid sequences corresponding to the DNA sequences of SEQ ID NOS: 1-331, 1896-1901, 1905, 1906, 1908, 1909, 1910, 1932-1968, 2001-2036, 2074-2079 and 2104 are provided in SEQ ID NOS: 592-922, 1914-1919, 1923, 1924, 1926-1928, 2108-2142, 2175-2210, 2247-2252 and 2276, respectively.
Isolation and Characterization of cDNA Clones from Pinus radiata
[0097]Fourteen Pinus radiata cDNA expression libraries (prepared from either shoot bud tissue, suspension cultured cells, early wood phloem (two independent libraries), fascicle meristem tissue, male strobilus, root (unknown lineage), feeder roots, structural roots, female strobilus, cone primordia, female receptive cones and xylem (two independent libraries)) were constructed and screened as described above in Example 1.
[0098]DNA sequence for positive clones was obtained using forward and reverse primers on a Perkin Elmer/Applied Biosystems Division Prism 377 sequencer and the determined sequences were compared to known sequences in the database as described above.
[0099]Based on similarity to known sequences from other plant species, the isolated DNA sequences (SEQ ID NOS: 332-591, 1537-1894, 1895, 1902-1904, 1907, 1911, 1912, 1931, 1969-2000, 2037-2073, 2080-2103, 2105 and 2106) were identified as encoding transcription factors as detailed above in Table 1. The predicted amino acid sequences corresponding to the DNA sequences of SEQ ID NOS: 332-591, 1895, 1902-1904, 1907, 1911, 1912, 1931, 1969-2000, 2037-2073, 2080-2103, 2105 and 2106 are provided in SEQ ID NOS: 923-1182, 1913, 1920-1922, 1925, 1929-1930, 2107, 2143-2174, 2211-2246, 2253-2275, 2277 and 2278, respectively.
Use of a Myb Transcription Factor Gene to Modify Gene Expression in Plants
[0100]Transformation of tobacco plants with a Eucalyptus grandis Myb transcription factor gene is performed as follows. Genetic constructs comprising sense and anti-sense constructs containing a DNA sequence including the coding region of the Myb transcription factor of SEQ ID NO: 2076 are constructed and inserted into Agrobacterium tumefaciens by direct transformation using published methods (see An G, Ebert P R, Mitra A, Ha SB, "Binary vectors," in Gelvin SB and Schilperoort R A, eds., Plant Molecular Biology Manual, Kluwer Academic Publishers Dordrecht, 1988). The constructs of sense DNAs are made by direct cloning from PBK-CMV plasmid by cloning cDNA insert into pART7 plasmid, which is then cut by NotI enzyme and 35S-Insert-OCS 3'UTR put into pART27 plant expression vector (see Gleave, Plant Molecular Biology 20:1203-1207, 1992). The presence and integrity of the transgenic constructs are verified by restriction digestion and DNA sequencing.
[0101]Tobacco (Nicotiana tabacum cv. Samsun) leaf sections are transformed with the sense and anti-sense constructs using the method of Horsch et al. (Science 227:1229-1231, 1985). Arabidopsis thaliana (ecotype: Columbia) whole plants are transformed with the sense and anti-sense constructs using either the vacuum infiltration (Bechtold et al., C.R. Acad. 316:1194-1199, 1992), or floral dip (Clough and Bent, The Plant Journal 16:735-743, 1998) procedures. Transformed plants containing the appropriate construct are verified using Southern blot experiments. Expression of the Eucalyptus Myb transcription factor gene in transformed plants is confirmed by isolating total RNA from each independent transformed plant line created with the Myb transcription factor gene sense and anti-sense constructs. The RNA samples are analyzed in Northern blot experiments to determine the level of expression of the transgene in each transformed line. The expression level of the Myb transcription factor, encoded by the Eucalyptus Myb transcription factor gene and by the endogenous Myb transcription factor gene, for each transformed plant line created with the sense and anti-sense constructs is compared to that of wild-type control plants.
[0102]To circumvent potential lethal phenotypes commonly observed with the ectopic constitutive expression of transcription factors in planta, constructs expressing the 5' terminal DNA binding domains of MYB transcription factors (MYB TF) can be generated, under the control of either a constitutive or cell-specific promoter. It is hypothesized that ectopic expression of the truncated MYB TF creates an in planta competitive scenario for the endogenous MYB TFs and transgene product. Binding of this DNA binding domain product, with no associated activation domain, inhibits, or severely reduces, both the ability of the endogenous TF to bind to the appropriate cis-element and inhibits transcriptional activation of the downstream gene, resulting in a knock-out like phenotypic effect.
[0103]The Eucalyptus grandis transcription factor given in SEQ ID NO: 2076 shares amino acid sequence identity with MYB transcription factor genes from other plant species. This gene was identified from a cDNA library derived from Eucalyptus grandis floral buds. Analysis of the amino acid sequence using publicly available tools such as PROSITE and InterPro, readily identified putative DNA binding domains present in SEQ ID NO: 2076 (see Jin and Martin, Plant Mol. Biol. 41:577-885, 1999). The amino acid sequences of the identified DNA binding domains are given in SEQ ID NO: 2346 and 2347.
[0104]The adjacent putative DNA binding domains were amplified as a single fragment by polymerase chain reaction (PCR) (Forward primer 5' CGTCTGTCTAGAAACAAGCTGAACATGGACAAGAAGC 3' (SEQ ID NO: 2369) and Reverse primer 5' TGGCCTTCTAGACTAGCTCTGACCAGAGAAA 3' (SEQ ID NO: 2370)) with the aim of adding XbaI restriction sites on both the 5' and 3' termini to facilitate cloning using conventional restriction enzyme-based protocols, and a termination codon (TAG) to the 3' terminus only. These primers amplified nucleotides 21 to 424 of SEQ ID NO: 2076. The resulting DNA binding domains fragment was cloned into the pART7/pART27 plant binary vector system (Gleave, Plant Mol. Biol. 20:1203-1207, 1992) for expression in planta under the 35S constitutive promoter.
[0105]The binary vectors were introduced into Arabidopsis thaliana by Agrobacterium tumefaciens-mediated transformation using standard floral dip procedures, as described for Arabidopsis thaliana (Clough and Bent, Plant J. 16: 735-743, 1998).
[0106]Phenotypic analysis of the resulting T2 plant translines (Table 1 below) showed that down-regulation of MYB TF activity in planta, associated with the over-expression of the DNA binding domains (sense orientation), resulted in a dwarfed phenotype (approximately 40% decrease in height compared to wild type (WT) or pART27 empty vector control), with spindly inflorescence bolts. Floral development and maturation were unaffected at the temporal level. However, evidence of floral aberrations was observed in lines displaying severe growth retardation. Gross phenotypic analysis of the other translines yielded no significant phenotypes compared to wild type or pART27 empty vector control, indicating that this observed phenotype is due to the sense expression of the DNA binding domains as hypothesized. The T2 observations were made from 15 plants from 5 independent transformation lines per construct.
TABLE-US-00004 TABLE 3 Phenotypic analysis of 35S::E. grandis MYB TF DNA binding domain or entire ORF Plant Histological Construct Description Line Plant Phenotype Phenotype PCR data MW2 DNA Binding 2 2 Dwarf with Decreased Sense orientation of Domain (Sense) spindly number of DNA Binding inflorescence lignified cells Domains verified bolt. associated with cellular collapse MW2 DNA Binding 2 3 Dwarf with Collapsed Sense orientation of Domain (Sense) spindly lignified cells DNA Binding inflorescence Domains verified bolt. MW2 DNA Binding 2 4 Dwarf with Not tested Sense orientation of Domain (Sense) spindly DNA Binding inflorescence Domains verified bolt. MW2 DNA Binding 2 9 Dwarf with Decreased Sense orientation of Domain (Sense) spindly number of DNA Binding inflorescence lignified cells Domains verified bolt. associated with cellular collapse MW2 DNA Binding 2 11 Dwarf with Not tested Sense orientation of Domain (Sense) spindly DNA Binding inflorescence Domains not bolt. verified MW7 DNA Binding 1 7 Normal WT No vascular Antisense Domain perturbation orientation of DNA (Antisense) Binding Domains verified MW7 DNA Binding 1 8 Normal WT Putative Antisense Domain ectopic orientation of DNA (Antisense) lignification Binding Domains verified MW7 DNA Binding 1 13 Normal WT Not tested Antisense Domain orientation of DNA (Antisense) Binding Domains verified MW7 DNA Binding 1 14 Normal WT Putative Antisense Domain ectopic orientation of DNA (Antisense) lignification Binding Domains verified pART27 Empty Vector na 1 Normal WT No vascular Kan DNA verified control perturbation pART27 Empty Vector na 3 Normal WT No vascular Kan DNA verified control perturbation pART27 Empty Vector na 5 Normal WT No vascular Kan DNA verified control perturbation pART27 Empty Vector na 10 Normal WT No vascular Kan DNA verified control perturbation
[0107]Histological analysis of tissue from plants transformed with the 35S::MYB DNA binding domains (sense orientation) constructs stained with Phloroglucinol and Maules showed aberrations in the morphology and lignification of the vascular structures, with obvious xylem cell collapse when compared with the controls that were transformed with empty vector. Inflorescence bolt sections from plants transformed with the DNA binding domain of SEQ ID NO: 2076 were determined to have reduced cell population in the vascular bundle and an overall reduced gross size of the vascular bundle per se.
[0108]The histological data showed that introduction of the binding domains of SEQ ID NO: 2076 (in sense orientation) resulted in the pertubation of vascular structure development. Further analysis of the T3 generation of the MW2 line 2 (15 individual progeny) indicated that the aberrant phenotype was stably inherited, with 11 of the 15 plants analyzed exhibiting a spindly phenotype.
[0109]These studies demonstrate that the sequence of SEQ ID NO: 2076 encodes a transcription factor and that the introduction into a plant of genetic constructs containing the binding domains of SEQ ID NO: 2076 in a sense orientation may be successfully employed to modify the phenotype of the plant.
Expression of a SHAQKY MYB Transcription Factor in Transgenic Cottonwood to Alter Wood Quality
[0110]The eucalyptus-derived cDNA corresponding to SEQ ID NO: 1953 was analyzed to obtain the complete DNA sequence, SEQ ID NO: 2371. This cDNA encodes the full-length protein sequence, SEQ ID NO: 2372, which is a divergent member of the MYB family of transcription factors. Instead of the more usual R2R3 repeat in the MYB domain, this protein comprises a single MYB repeat, which includes a variant of the SHAQKY motif (Mercy et al., J. Exp. Bot. 54: 1117-1119, 2003). The cDNA was cloned into an expression cassette in a binary transformation vector using standard methods such as restriction digestion and ligation. The promoter used in the expression cassette was a 4CL promoter from Pinus taeda, which has been demonstrated to exhibit xylem-preferred expression (U.S. Pat. No. 6,252,135). A map of the resulting plasmid, pWVK249, is shown in FIG. 1. The full DNA sequence of pWVK249 is SEQ ID NO: 2373 and the cDNA corresponds to the region between positions 8537 and 9774 of the plasmid. The T-DNA region of the plasmid (between the left and right borders) also comprises an NPTII cassette for kanamycin selection of transformed plants.
[0111]The plasmid was transferred into Agrobacterium strain GV2260, which was then used to transform eastern cottonwood (Populus deltoides), similarly to Che et al. (Plant Biotech. J. 1: 311-319. (2003)). After generation of shoots, development of roots, transfer to soil and hardening off, the transformed cottonwood plants were put in a field test where they grew for three years. Control trees, transformed with a reporter gene, were included in the test. After harvest, the wood from the trees was characterized for density or, more strictly, basic gravity.
[0112]Basic gravity determination was performed on small chunks of wood approximately 10 to 25 cm3 in volume. Basic gravity was calculated as (weight of wood when completely dry).-+.(weight of water displaced by wood when saturated). It was found that half of the pWVK249 lines had basic gravity greater than any of the control samples. The results are shown in FIG. 2. The average of the pWVK249 set is approximately 4% greater than the control mean, and the best lines are 10% greater in density than the control mean.
[0113]Similar results can be found for other members of the genus Populus (aspens and cottonwoods), members of the genus Eucalyptus, and other hardwood trees. The increase of wood density may also be achieved in pine and other conifers using the present methods. The vascular-preferred promoter used here can be substituted by other known vascular-preferred promoters. Examples of vascular-preferred promoters are P. radiata cellulose synthase and E. grandis arabinogalactan protein, which can be found in U.S. Pat. No. 7,442,786.
Expression of a SHAQKY MYB Transcription Factor in Transgenic Pine to Alter Wood Quality
[0114]The construct provided in SEQ ID: 2373 is transformed into embryogenic callus culture of loblolly pine (Pinus taeda) as described by Connett-Porceddu et al. (U.S. Pat. No. 7,157,620, issued Jan. 2, 2007). Embryos are germinated and rooted in soil. When plantlets are approximately 6 inches in height, they are transferred outdoors to acclimate to unprotected conditions, and two to three months later they are planted in a field test at approximately 10-ft×8-ft spacing. After at least two years of growth, plants are harvested, the wood is collected and measurement of basic gravity is performed to determine the amount of increase.
Overexpression of Pine Transcription Factors in Transgenic Trees
[0115]Pine-derived cDNAs corresponding to SEQ ID NOs 341, 377, 388, 396, 413, 434, 454, 458, 462, 497, 504, 573, 1551, 1675, and 1910 were analyzed to obtain DNA sequences equivalent to full-length or near-full-length transcripts. The cDNAs were selected by searching for partially sequenced cDNAs (expressed sequence tags or ESTs) that overlapped the sequence of the original clone. By examining each set (or cluster or contig) of overlapping ESTs, it was possible to identify the cDNAs starting nearest to the 5'-end of the transcripts.
[0116]In order to demonstrate the effect of overexpression of these genes on the growth and development of trees, the full-length cDNAs were cloned into binary transformation vectors. A variety of base vectors was used in the construction of the overexpression cassettes. Vector pOX1 (SEQ ID NO: 2423) used a vascular-preferred pine 4CL promoter to drive the inserted sequence. Vector pOX28 (SEQ ID NO: 2424) used a constitutive pine polyubiquitin promoter to drive the inserted sequence. pAGSM49 (SEQ ID NO: 2425) was derived from pOX28 by addition of a cassette to prevent pollen formation. pAGSM50 was very similar to pAGSM49, differing only in the multiple cloning region. In pOX28 and pAGSM49, the multiple cloning region sequence was 5'-TTTCATTCAACCCGGGCTGCAGAACAATTGGCTAGCAAAGTA-CTTAAAGCTT-3'(SEQ ID NO: 2431) and in pAGSM50 the cloning region sequence was 5'-TTTCATTCAACCCGGGCTGCAGAAAGGCCTTTATCGATGGGCTAGCAAAAGTACT TAAAGCTT-3'(SEQ ID NO: 2432). All plasmids comprised an NPTII cassette in the T-DNA region for kanamycin selection of transformed plants.
[0117]SEQ ID NO: 2385 was cloned into three base vectors different from those provided above, yielding SEQ ID NOs: 2426, 2427 and 2428.
[0118]SEQ ID NO: 2387 was cloned into pOX28 (SEQ ID NO: 2424) and pAGSM49 (SEQ ID NO: 2425).
[0119]SEQ ID NO: 2389 was cloned into pAGSM50.
[0120]SEQ ID NO: 2391 was cloned into pOX28 (SEQ ID NO: 2424) and pAGSM49 (SEQ ID NO: 2425).
[0121]SEQ ID NO: 2393 was cloned into pAGSM50.
[0122]SEQ ID NO: 2395 was cloned into pOX1 (SEQ ID NO: 2423).
[0123]SEQ ID NO: 2401 was cloned into pOX28 (SEQ ID NO: 2424) and pAGSM49 (SEQ ID NO: 2425).
[0124]SEQ ID NO: 2403 was cloned into pOX1 (SEQ ID NO: 2423) and pOX28 (SEQ ID NO: 2424).
[0125]SEQ ID NO: 2409 was cloned into pOX1 (SEQ ID NO: 2423) and pOX28 (SEQ ID NO: 2424).
[0126]SEQ ID NO: 2411 was cloned into pOX1 (SEQ ID NO: 2423).
[0127]Table 4 below shows the correspondences between the partial and full-length sequences and the predicted translation products of the full-length sequences.
TABLE-US-00005 TABLE 4 Plasmid Original Full-length Protein SEQ ID SEQ ID NO Gene Family SEQ ID NO SEQ ID NO NO(s) 24 LIM 2382 2383 2384 1953 MYB 2371 2372 2373 1986 MYB 2374 2375 2376 2072 MYB 2377 2378 2379, 2380 2088 MYB same 2261 2381 158 AP2/EREBP same 749 341 Synaptobrevin 2385 2386 2426, 2427, 2428 377 HOMEObox 2387 2388 388 HOMEObox 2389 2390 396 HOMEObox 2391 2392 413 HOMEObox 2393 2394 434 AP2/EREBP 2395 2396 454 AP2/EREBP 2397 2398 458 HSF 2399 2400 462 CCAAT DR1 2401 2402 497 bZIP 2403 2404 504 MYB 2405 2406 573 Trihelix 2407 2408 1551 C2C2 GATA 2409 2410 1675 AP2/EREBP 2411 2412 1910 SBP 2413 2414 1985 MYB 2415 2416 2430 2042 bZIP 2417 2418 2046 HOMEObox 2419 2420 2085 MYB 2421 2422
Reduction of Expression of Pine Transcription Factors in Transgenic Trees
[0128]For each of the full-length cDNAs, SEQ ID NOs: 2393, 2395, 2397, 2399, 2405, 2407, and 2421, a fragment of the cDNA was cloned into a transitive RNAi cassette in a binary transformation vector using standard methods such as PCR, restriction digestion and ligation. In transitive RNAi, an inverted repeat of a nontarget sequence is linked 3' to a segment of the target gene, so that RNA-dependent RNA polymerase can extend into the target sequence and siRNAs with homology to the target sequence are generated (Filichkin et al., Plant Biotechnol. J. 5: 615-626, 2007). The sequence of the base transitive RNAi vector is provided as SEQ ID NO: 2429. Table 5 lists the full length SEQ ID NOs and provides the endpoints for the segment of each full-length sequence that was used in the constructs.
TABLE-US-00006 TABLE 5 Original Full-length SEQ ID NO SEQ ID NO Fragment ends 413 2393 1195-1948 434 2395 684-992 454 2397 334-835 458 2399 2038-2326 504 2405 574-1568 573 2407 376-1265 1985 2415 1068-1360 2085 2421 687-1656
[0129]The fragments were amplified by PCR, with primers designed to add BamHI restriction site at the 5'-end and an SpeI restriction site at the 3'-end. The purified fragments were inserted between the BamHI and SpeI sites of the vector (positions 9297 to 9644 of SEQ ID NO: 2429). When the restriction enzyme digestion of the vector was performed prior to insertion of the target gene fragment, a short "stuffer" fragment comprising positions 9320-9642 was deleted. The promoter used to drive the transitive RNAi cassette was a 4CL promoter from Pinus taeda. The T-DNA region of the plasmid (between the left and right borders) also comprised an NPTII cassette for kanamycin selection of transformed plants.
[0130]The suppression construct targeting SEQ ID NO: 2415 was constructed to comprise an inverted repeat of the target gene driven by a strong constitutive promoter. The inverted repeat of the cDNA fragment included a short spacer of noncoding DNA between the two repeats. It would also be possible to use an intron as the spacer, for example the intron from the YABBY gene of Arabidopsis thaliana, which was used in SEQ ID NO: 2384. The sequence of the plasmid bearing the inverted repeat of the fragment of SEQ ID NO: 2415 is provided as SEQ ID NO: 2430.
[0131]Each of the suppression plasmids described was introduced into Agrobacterium via electroporation and was then transformed into embryogenic pine callus cultures as described by Connett-Porceddu et al. (U.S. Pat. No. 7,157,620, issued Jan. 2, 2007). Transgenic plants were produced, grown in soil either in a greenhouse or in field tests, and characterized for the presence of novel phenotypes.
[0132]The effect of a suppression plasmid on expression of the target gene is characterized by determining the amount of target gene RNA present. Methods such as RNA hybridization, microarrays, or quantitative reverse transcription PCR (qPCR or RT-PCR) are commonly used to measure RNA levels for a specific gene. The TaqMan assay from Applied Biosystems is a version of real-time PCR that allows the level of the target gene to be compared to an internal standard.
[0133]SEQ ID NOS: 1-2430 are set out in the attached Sequence Listing. The codes for nucleotide and amino acid sequences used in the attached Sequence Listing, including the symbols "n" and "Xaa", conform to WIPO Standard ST.25 (1998), Appendix 2, Table 1.
[0134]Although the present invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, changes and modifications can be carried out without departing from the scope of the invention which is intended to be limited only by the scope of the claims.
Sequence CWU 0 SQTB SEQUENCE LISTING The patent application contains a lengthy "Sequence Listing" section. A copy of the "Sequence Listing" is available in electronic form from the USPTO web site (http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20110047644A1). An electronic copy of the "Sequence Listing" will also be available from the USPTO upon request and payment of the fee set forth in 37 CFR 1.19(b)(3).
Patent applications by William H. Rottmann, Summerville, SC US
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