Patent application title: Novel Genes Involved In Biosynthesis
Inventors:
Kerry Ruth Hancock (Palmerston North, NZ)
Margaret Greig (Palmerston North, NZ)
Assignees:
Grasslanz Technology Limited
IPC8 Class: AC12N1582FI
USPC Class:
800278
Class name: Multicellular living organisms and unmodified parts thereof and related processes method of introducing a polynucleotide molecule into or rearrangement of genetic material within a plant or plant part
Publication date: 2016-05-19
Patent application number: 20160138034
Abstract:
The invention provides a novel MYB class transcription factor gene
(nucleic acid sequences, protein sequences, and variants and fragments
thereof) designated MYB14 by the applicants, that is useful for
manipulating the production of flavonoids, specifically condensed
tannins, in plants. The invention provides the isolated nucleic acid
molecules encoding proteins with at least 70% identity to any one of
MYB14 polypeptide sequences of SEQ ID NO: 14 and 46 to 54. The invention
also provides, constructs, vectors, host cells, plant cells and plants
genetically modified to contain the polynucleotide. The invention also
provides methods for producing plants with altered flavonoid,
specifically condensed tannin production, making use of the MYB14 nucleic
acid molecules of the invention.Claims:
1-65. (canceled)
66. An isolated nucleic acid molecule encoding a MYB14 polypeptide comprising a sequence with at least 70% identity to SEQ ID NO: 14, or a functional fragment thereof, wherein % identity is calculated over the entire length of SEQ ID NO: 14.
67. The isolated polynucleotide of claim 66, wherein the MYB14 polypeptide comprises the sequence of SEQ ID NO: 14.
68. The isolated nucleic molecule of claim 66, wherein the MYB14 polypeptide regulates at least one of: (a) the production of flavonoids in a plant, (b) the production of condensed tannins in plants, (c) at least one gene in the flavonoid biosynthetic pathway in a plant, and (d) at least one gene in the condensed tannin biosynthetic pathway in a plant.
69. The isolated nucleic molecule of claim 66, wherein the MYB14 polypeptide, or functional fragment thereof, comprises an amino acid sequence with at least 70% identity to SEQ ID NO: 17.
70. The isolated nucleic molecule of claim 66, wherein the MYB14 polypeptide, or functional fragment thereof, comprises the amino acid sequence of SEQ ID NO: 17.
71. The isolated nucleic acid molecule of claim 66, wherein the nucleotide sequence is selected from the group consisting of: a) SEQ ID NO: 1, 2 or 55; b) a complement of the sequence(s) in a); c) a functional fragment or variant of the sequence(s) in a) or b); d) a homolog or an ortholog of the sequence(s) in a), b), or c); e) an antisense sequence to a RNA sequence obtained from a sequence in a), b), c) or d).
72. The isolated nucleic acid molecule of claim 71, wherein the variant has at least 70% identity to the specified sequence.
73. An isolated MYB14 polypeptide comprising a sequence with at least 70% identity to SEQ ID NO: 14, or a functional fragment thereof, wherein % identity is calculated over the entire length of SEQ ID NO: 14.
74. The MYB14 polypeptide of claim 73 that comprises the sequence of SEQ ID NO: 15 and SEQ ID NO: 17, but lacks the sequence of SEQ ID NO: 16.
75. The isolated polypeptide of claim 73, wherein the MYB14 polypeptide comprises the sequence of any one of SEQ ID NO: 14 and 46 to 54.
76. The isolated polypeptide of claim 73, wherein the MYB14 polypeptide comprises the sequence of SEQ ID NO: 14.
77. An isolated polypeptide encoded by a nucleic acid molecule of claim 66.
78. An isolated nucleic acid molecule comprising a sequence encoding a polypeptide of claim 73.
79. A construct including a nucleotide sequence substantially as described in claim 66.
80. The construct of claim 79 which includes: at least one promoter; and the nucleic acid molecule; wherein the promoter is operatively linked to the nucleic acid molecule to control the expression of the nucleic acid molecule.
81. A host cell which has been altered from the wild type to include a nucleic acid molecule substantially as described in claim 66.
82. A host cell comprising a genetic construct of claim 79.
83. The host cell of claim 82, wherein the host cell is a plant cell.
84. A plant cell or plant transformed with a nucleic acid molecule substantially as described in claim 66.
85. A plant cell comprising a genetic construct of claim 79.
86. The seed of a plant of claim 84.
87. A composition which includes an ingredient which is, or is obtained from, a plant of claim 84, or a part thereof.
88. Use of a nucleic acid molecule substantially as described in claim 66 to alter a plant or plant cell.
89. A method for producing an altered plant or plant cell using a nucleic acid molecule substantially as described in of claim 66 to alter the plant or plant cell, wherein the plant cell or plant is altered in at least one of: (a) the production of flavonoids, or an intermediate in the production of flavonoids, (b) the production of at least one condensed tannin, or monomer thereof, (c) the production of a condensed tannin selected from catechin, epicatechin, epigallocatechin and gallocatechin, (d) expression of at least one enzyme in a flavonoid biosynthetic pathway, (e) expression of at least one enzyme in the condensed tannin biosynthetic pathway, (f) altered expression of LAR and/or ANR.
90. The use or method of claim 89, wherein the altered production or expression, is increased production or expression.
91. The method of claim 89, wherein the altered production or expression, is in substantially all tissues of the plant.
92. The method of claim 89, wherein the altered production or expression is in the foliar tissue of the plant.
93. The method of claim 89, wherein the altered production or expression is in the vegetative portions of the plant.
94. The method of claim 89, wherein the altered production or expression is in the epidermal tissues of the plant.
95. The method of claim 89, wherein the altered production of flavonoids or condensed tannins, is in a tissue of the plant that is substantially devoid of the flavonoids or condensed tannins.
96. The method of claim 89, wherein the levels of flavonoids and/or condensed tannins altered by the present invention are sufficient to provide a therapeutic or agronomic benefit.
97. A plant produced by a method of claim 89.
98. A part, seed, fruit, harvested material, propagule or progeny of a plant of claim 84.
99. A part, seed, fruit, harvested material, propagule or progeny of a plant, wherein the plant, seed, fruit, harvested material, propagate or progeny is genetically modified to comprise at least one nucleic acid molecule of claim 66.
100. A part, seed, fruit, harvested material, propagule or progeny of a plant, wherein the plant, seed, fruit, harvested material, propagate or progeny is genetically modified to comprise at least one construct of claim 79.
Description:
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of pending U.S. application Ser. No. 13/224,720, filed Sep. 2, 2011, which is a continuation-in-part of pending U.S. application Ser. No. 12/996,117, which has a 371(c) date of Apr. 6, 2011, which is a National Stage Application filed under 35 U.S.C. ยง371 of PCT Application No. PCT/NZ2009/000099, filed on Jun. 5, 2009 and published in English on Dec. 10, 2009 as WO 2009/148336, which claims priority to U.S. Provisional Application 61/059,691, filed on Jun. 6, 2008, and New Zealand Application 568928, filed on Jun. 6, 2008, all of which are incorporated by reference in their entireties to the extent there is no inconsistency with the present disclosure.
TECHNICAL FIELD
[0002] The invention relates to a novel gene(s) involved in biosynthesis. In particular, the present invention relates to gene(s) encoding a regulatory factor controlling the expression of key genes involved in the production of flavonoids including condensed tannins in plants.
BACKGROUND ART
The Molecular Phenylpropanoid Pathway
[0003] The phenylpropanoid pathway (shown in FIG. 1) produces an array of secondary metabolites including flavones, anthocyanins, flavonoids, condensed tannins and isoflavonoids (Dixon et al., 1996; 2005). In particular, the condensed tannin (CT) biosynthetic pathway shares its early steps with the anthocyanin pathway before diverging to proanthocyanindin biosynthesis.
[0004] Anthocyanidins are precursors of flavan-3-ols (e.g. (-)-epicatechin), which are important building blocks for CTs. These cis-flavan-3-ols are formed from anthocyanidins by anthocyanidin reductase (ANR), which has been cloned from many species including A. thaliana and M. truncatula (Xie et al., 2003; 2004). In A. thaliana (-)-epicatechin is the exclusive CT monomer (Abrahams et al., 2002), but in many other species, including legumes, both (+)- and (-)-flavan-3-ols are polymerized to CTs. The biosynthesis of these alternate (+)-flavan-3-ols (catechins) is catalysed by leucoanthocyanidin reductase (LAR). This enzyme has been cloned and characterized from legumes including the CT-rich legume tree Desmodium uncinatum (Tanner et al., 2003), as well as from other species such as grapes and apples (Pfeiffer et al., 2006). The enzyme catalyses the reduction of leucopelargonidin, leucocyanidin, and leucodelphinidin to afzelechin, catechin, and gallocatechin, respectively. No homologues of LAR have been found in A. thaliana, consistent with the exclusive presence of (-)-epicatechin derived CT building blocks in this plant.
[0005] Whereas information on TF regulation of this pathway in Arabidopsis seeds is well defined, TFs that control leaf CT biosynthesis within the tribe of Trifolieae have yet to be identified. An important family of TF proteins, the MYB family, controls a diverse range of functions including the regulation of secondary metabolism such as the anthocyanin and CT pathways in plants. The expression of the MYB TF AtTT2 coordinately turns on or off the late structural genes in Arabidopsis thaliana, ultimately controlling the expression of the CT pathway.
[0006] An array of Arabidopsis thaliana transparent testa (TT) mutants (Winkel-Shirley, 2002; Debeaujon et al., 2001) and tannin deficient seed (TDS) mutants (Abrahams et al. 2002; 2003) have been made--all being deficient in CT accumulation in the seed coat. Molecular genetic studies of these mutants has allowed for the identification of a number of structural genes and transcription factors (TFs) that regulate the expression and tissue specificity of both anthocyanin and CT synthesis in A. thaliana (Walker et al., 1999; Nesi et al., 2000; 2002).
[0007] Although most of the structural genes within the CT pathway have been identified in a range of legumes, attempts to manipulate CT biosynthesis in leaves by engineering the expression of these individual genes has failed so far. The major reason for this is that not one (or a few) enzyme(s) are rate-limiting, but that activity of virtually all enzymes in a pathway has to be increased to achieve an overall increased flux into specific end-products such as condensed tannins.
[0008] Transcription factors (TFs) are regulatory proteins that act as repressors or activators of metabolic pathways. TFs can therefore be used as a powerful tool for the manipulation of entire metabolic pathways in plants. Many MYB TFs are important regulators of the phenylpropanoid pathway including both the anthocyanin and condensed tannin biosynthesis (Debaujon et al; 2003; Davies and Schwinn, 2003). For example, the A. thaliana TT2 (AtTT2) gene encodes an R2R3-MYB TF factor which is solely expressed in the seed coat during early stages of embryogenesis, when condensed tannin biosynthesis occurs (Nesi et al., 2001). TT2 has been shown to regulate the expression of the flavonoid late biosynthetic structural genes TT3 (DFR), TT18, TT12 (MATE protein) and ANR during the biosynthesis and storage of CTs. AtTT2 partially determines the stringent spatial and temporal expression of genes, in combination with two other TFs; namely TT8 (bHLH protein) and TTG1 (WD-40 repeat protein; Baudry et al., 2004).
[0009] Other MYB TFs in Vitis vinifera; grape (VvMYBPA1) Birdsfoot trefoil and Brassica napus (BnTT2) that are involved in the regulation of CT biosynthesis have also recently been reported (Wei et al., 2007; Bogs et al., 2007; Yoshida et al., 2008).
[0010] The AtTT2 gene has also been shown to share a degree of similarity to the rice (Oryza sativa) OsMYB3, the maize (Zea mays) ZmC1, AmMYBROSEA from Antirrhinum majus and PhMYBAN2 from Petunia hybrida, genes which have been shown to regulate anthocyanin biosynthesis (Stracke et al., 2001; Mehrtens et al., 2005).
Condensed Tannins
[0011] Condensed tannins (CTs) also called proanthocyanidins (PAs) are colourless polymers, one of several secondary plant metabolites. CTs are polymers of 2 to 50 (or more) flavonoid units (see compound (I) below) that are joined by carbon-carbon bonds which are not susceptible to being cleaved by hydrolysis. The base flavonoid structure is:
##STR00001##
[0012] Condensed tannins are located in a range of plant parts, for example; the leaves, stem, flowers, roots, wood products, bark, buds. CTs are generally found in vacuoles or on the surface epidermis of the plant
Condensed Tannins in Forage Plants
[0013] Forage plants, such as forage legumes, are beneficial in pasture-based livestock systems because they improve both the intake and quality of the animal diet. Also, their value to the nitrogen (N) economy of pastures and to ruminant production are considerable (Caradus et al., 2000). However, while producing a cost-effective source of feed for grazing ruminants, pasture is often sub-optimal when it comes to meeting the nutritional requirements of both the rumen microflora and the animal itself. Thus the genetic potential of grazing ruminants for meat, wool or milk production is rarely achieved on a forage diet.
[0014] New Zealand pastures contain up to 20% white clover, while increasing the levels of white clover in pastures helps address this shortfall, it also exacerbates the incidence of bloat. White clover (Trifolium repens), red clover (Trifolium pretense) and lucerne (Medicago sativa) are well documented causes of bloat, due to the deficiency of plant polyphenolic compounds, such as CT, in these species. Therefore the development of forage cultivars producing higher levels of tannins in plant tissue would be a important development in the farming Industry to reduce the incidence of bloat (Burggraaf et al., 2006).
[0015] In particular, condensed tannins, if present in sufficient amounts, not only helps eliminate bloat, but also strongly influences plant quality, palatability and nutritive value of forage legumes and can therefore help improve animal performance. The animal health and productivity benefits reported from increased levels of CTs include increased ovulation rates in sheep, increased liveweight gain, wool growth and milk production, changed milk composition and improved anthelmintic effects on gastrointestinal parasites (Rumbaugh, 1985; Marten et al., 1987; Niezen et al., 1993; 1995; Tanner et al., 1994; McKenna, 1994; Douglas et al., 1995; Waghorn et al., 1998; Aerts et al., 1999; McMahon et al., 2000; Molan et al., 2001; Sykes and Coop, 2001).
[0016] A higher level of condensed tannin also represents a viable solution to reducing greenhouse gases (methane, nitrous oxide) released into the environment by grazing ruminants (Kingston-Smith and Thomas, 2003). Ruminant livestock produce at least 88% of New Zealand's total methane emissions and are a major contributor of greenhouse gas emissions (Clark, 2001). The principle source of livestock methane is enteric fermentation in the digestive tract of ruminants. Methane production, which represents an energy loss to ruminants of around 3 to 9% of gross energy intake (Blaxter and Clapperton, 1965), can be reduced by as much as 5% by improving forage quality. Forage high in CT has been shown to reduce methane emission from grazing animals (Woodward, et al 2001; Puchala, et al., 2005). Increasing the CT content of pasture plants can therefore contribute directly to reduced levels of methane emission from livestock.
[0017] Therefore, the environmental and agronomical benefits that could be derived from triggering the accumulation of even a moderate amount of condensed tannins in forage plants including white clover are of considerable importance in the protection and nutrition of ruminants (Damiani et al., 1999).
Legumes
[0018] It is the inventors understanding that the regulation of CT foliar-specific pathway in Trifolium legumes, involving the interaction of regulatory transcription factors (TFs) with the pathway, remains unknown. Modification or manipulation of this pathway to influence the amount CT has been explored but, as the process is not straightforward, there has been little firm success in understanding this pathway.
[0019] The clover genus, Trifolium, for example, is one of the largest genera in the family Leguminosae (D Fabaceae), with ca. 255 species (Ellison et al., 2006). Only two Trifolium species; T. affine (also known as Trifolium preslianum Boiss. Is) and T. arvense (also known as hare-foot clover) are known to accumulate high levels of foliar CTs (Fay and Dale, 1993). Although significant levels of CTs are present in white clover flower heads (Jones et al., 1976), only trace amounts can be detected in leaf trichomes (Woodfield et al., 1998). Several approaches including gene pool screening and random mutagenesis have failed to provide white or red clover plants with increased levels of foliar CTs (Woodfield et al., 1998).
Genetic Manipulation of Condensed Tannins
[0020] The inventors in relation to US2006/012508 created a transgenic alfalfa plant using the TT2 MYB regulatory gene and managed to surprisingly produce CTs constitutively throughout the root tissues. However, importantly, the inventors were unable to achieve CT accumulation in the leaves of this forage legume. It has been previously reported no known circumstances exist that can induce proanthocyanidins (CTs) in alfalfa forage (Ray et al., 2003). The authors of this paper assessed amongst other things whether the LC myc-like regulatory gene (TF) from maize or the C1 myb regulatory gene (TF) from maize could stimulate the flavonoid pathway in alfalfa forage and seed coat. The authors of this paper found that only the LC gene, and not C1 could stimulate anthocyanin and proanthocyanidin biosynthesis in alfalfa forage, but stimulation only occurred in the presence of an unknown stress-responsive alfalfa factor.
[0021] Studies assessing condensed tannin production in Lotus plants using a maize bHLH regulatory gene (TF) found that transformation of this TF into Lotus plants resulted in CT's only a very small (1%) increase in levels of condensed tannins in leaves (Robbins et al., 2003).
[0022] Previous attempts to alter and enhance agriculturally important compounds in white clover involved altering anthocyanin biosynthesis-derived from the phenylpropanoid pathway. Despite attermpts to activate this pathway using several heterologous myc and MYB TFs only one success has been reported, using the maize myc TF B-Peru (de Majnik et al., 2000). All other TFs investigated resulted in poor or no regenerants, implying a deleterious effect from their over-expression.
[0023] More recently, TT2 homologs derived from the high-CT legume, Lotus japonicus, have been reported (Yoshida et al., 2008). Bombardment of these genes into A. thaliana leaf cells has shown transient expression resulting in detectable expression of ANR and limited CT accumulation as detected by DMACA. However, these genes have not been transformed and analysed in any legume species.
[0024] The expression of the maize Lc gene resulted in the accumulation of PA-like compounds in alfalfa only if the plants were under abiotic stress (Ray et al., 2003). The co-expression of three transcription factors, TT2, PAP1 and Lc in Arabidopsis was required to overcome cell-type-specific expression of PAs, but this constitutive accumulation of PAs was accompanied by death of the plants (Sharma and Dixon, 2005).
[0025] Introduction of PAs into plants by combined expression of a MYB family transcription factor and anthocyanidin reductase for conversion of anthocyanidin into (epi)-flavan-3-ol has been attempted by Xie et al. (2006).
[0026] This attempt to increase the levels of proanthocyanidins (PAs) in the leaves of tobacco by co-expressing PAP1 (a MYB TF) and ANR were reported as having levels of PAs in tobacco that if translated to alfalfa may potentially provide bloat protection (Xie et al., 2006). Anthocyanin-containign leaves of transgenic M. truncatula constitutively expressing MtANR contained up to three times more PAs than those of wild-type plants at the same stage of development, and these compounds were of a specific subset of PA oligomers. Additionally, these levels of PA produced in M. truncatula fell well short of those necessary for an improved agronomic benefit. The authors state that it remained unclear which additional biosynthetic and non-biosynthetic genes will be needed for engineering of PAs in any specific plant tissue that does naturally accumulate the compounds.
[0027] Similar difficulties in expressing CTs or PAs in leaves were also encountered when the TT2 and/or BAN genes were transformed into alfalfa--refer US 2004/0093632 and US 2006/0123508.
Condensed Tannins Useful in Natural Health Products
[0028] The use of any flavonoid including proanthocyanidins to form food supplements, compositions or medicaments is also widely known. For example;
[0029] US patent application NO: 2003/0180406 describes a method using polyphenol compositions specifically derived from cocoa to improve cognitive function.
[0030] Patent publication WO 2005/044291 describes use of grape seed (Vitus genus) to prevent degenerative brain diseases including; stroke, cerebral concussion, Huntington's disease, CJD, Alzheimer's, Parkinsons, and senile dementia.
[0031] Patent publication WO 2005/067915 discloses a synergistic combination of flavonoids and hydroxystilbenes (synthetic or from green tea) combined with flavones, flavonoids, proanthocyanidins and anthocyanidins (synthetic or from bark extract) to reduce neuronal degeneration associated with disease states such as dementia, Alzheimer's, cerebrovascular disease, age-related cognitive impairment and depression.
[0032] U.S. Pat. No. 5,719,178 describes use of proanthocyanidin extract to treat ADHD.
[0033] PCT publication number 06/126895 describes a composition containing bark extract from the genus Pinus to improve, or prevent a decline in, human cognitive abilities or improve, or prevent symptoms of, neurological disorders in a human.
[0034] None of the above considers use of legumes as a raw material source of CT.
[0035] It would therefore be useful if there could be provided nucleic acid molecules and polypeptides useful in studying the metabolic pathways involved in flavonoids and/or condensed tannin biosynthesis.
[0036] It would also be useful if there could be provided nucleic acid molecules and polypeptides which are capable of altering levels of flavonoids and/or condensed tannins in plants or parts thereof.
[0037] In particular, it would be useful if there could be provided nucleic acid molecules which can be used to produce flavonoids and/or condensed tannins in plants or parts thereof de novo.
[0038] It is therefore one object of the invention to provide a method to increase CT levels in the leaves of forage legume species. The identification of the gene also provides a method to prevent CT accumulation in legume species which produce detrimental high levels of CT in leaves or seeds.
[0039] It would also be useful if there could be provided nucleic acid molecules which can be used alone or together with other nucleic acid molecules to produce plants, particularly forages and legumes, with enhanced levels of flavonoids and/or condensed tannins.
[0040] It is an object of the present invention to address the foregoing problems or at least to provide the public with a useful choice.
SUMMARY OF THE INVENTION
[0041] The present invention is concerned with the identification and uses of a novel MYB gene and associated polypeptide which has been termed by the inventors `MYB14` which has been isolated by the applicants and shown to be involved in the production of flavonoid compounds including condensed tannins.
[0042] Throughout this specification the nucleic acid molecules and polypeptides of the present invention may be designated by the descriptor MYB14.
[0043] The present invention contemplates the use of MYB14 independently or together with other nucleic acid molecules to manipulate the flavonoid/condensed tannin biosynthetic pathway in plants.
Polynucleotides Encoding Polypeptides
[0044] In the one aspect the invention provides an isolated nucleic acid molecule encoding a MYB14 polypeptide as herein defined, or a functional variant or fragment thereof.
[0045] In one embodiment the MYB14 polypeptide comprises the sequence of SEQ ID NO: 15.
[0046] In one embodiment the MYB14 polypeptide comprises the sequence of SEQ ID NO: 17.
[0047] In one embodiment the MYB14 polypeptide comprises the sequence of SEQ ID NO: 15 and SEQ ID NO: 17, but lacks the sequence of SEQ ID NO: 16.
[0048] In a further embodiment the MYB14 polypeptide comprises a sequence with at least 70% identity to any one of SEQ ID NO: 14 and 46 to 54.
[0049] In a further embodiment the MYB14 polypeptide comprises a sequence with at least 70% identity to SEQ ID NO: 14.
[0050] In a further embodiment the MYB14 polypeptide comprises the sequence of any one of SEQ ID NO: 14 and 46 to 54.
[0051] In a further embodiment the MYB14 polypeptide comprises the sequence of SEQ ID NO: 14.
[0052] In a further embodiment the MYB14 polypeptide regulates the production of flavonoids in a plant.
[0053] In a further embodiment the flavonoids are condensed tannins.
[0054] In a further embodiment the MYB14 polypeptide regulates at least one gene in the flavonoid biosynthetic pathway in a plant.
[0055] In a further embodiment the MYB14 polypeptide regulates at least one gene in the condensed tannin biosynthetic pathway in a plant.
[0056] In a further embodiment the functional fragment has substantially the same activity as the MYB14 polypeptide.
[0057] In a further embodiment the functional fragment comprises an amino acid sequence with at least 70% identity to SEQ ID NO: 17.
[0058] In a further embodiment the functional fragment comprises the amino acid sequence of SEQ ID NO: 17.
[0059] In a further aspect invention provides a nucleic acid molecule encoding a polypeptide comprising an amino acid sequence substantially as shown in SEQ ID NO: 17.
[0060] In a further aspect invention provides a nucleic acid molecule encoding a polypeptide having an amino acid sequence substantially as shown in SEQ ID NO: 17.
[0061] In a further aspect invention provides a nucleic acid molecule encoding a polypeptide comprising an amino acid sequence substantially as shown in SEQ ID NO: 14.
[0062] In a further aspect invention provides a nucleic acid molecule encoding a polypeptide having an amino acid sequence substantially as shown in SEQ ID NO: 14.
[0063] In a further aspect invention provides an isolated nucleic acid molecule encoding a polypeptide comprising 3' amino acid sequence motif as set forth in SEQ ID NO: 17
Polynucleotides
[0064] In a further aspect invention provides an isolated nucleic acid molecule having a nucleotide sequence selected from the group consisting of:
[0065] a) at least one of SEQ ID NO: 1 to 13 and 55 to 64, or a combination thereof;
[0066] b) a complement of the sequence(s) in a);
[0067] c) a functional fragment or variant of the sequence(s) in a) or b);
[0068] d) a homolog or an ortholog of the sequence(s) in a), b), or c);
[0069] e) an antisense sequence to a RNA sequence obtained from a sequence in a), b), c) or d).
[0070] In one embodiment the variant has at least 70% identity to the coding sequence of the specified sequence.
[0071] In a further embodiment the variant has at least 70% identity to the specified sequence.
[0072] In a further embodiment the fragment comprises the coding sequence of the specified sequence.
[0073] In a further aspect invention provides an isolated nucleic acid molecule having a nucleotide sequence selected from the group consisting of:
[0074] a) SEQ ID NO: 1, 2 or 55;
[0075] b) a complement of the sequence(s) in a);
[0076] c) a functional fragment or variant of the sequence(s) in a) or b);
[0077] d) a homolog or an ortholog of the sequence(s) in a), b), or c);
[0078] e) an antisense sequence to a RNA sequence obtained from a sequence in a), b), c) or d).
[0079] In one embodiment the variant has at least 70% identity to the coding sequence of the specified sequence.
[0080] In a further embodiment the variant has at least 70% identity to the specified sequence.
[0081] In a further embodiment the fragment comprises the coding sequence of the specified sequence.
[0082] In a further embodiment isolated nucleic acid molecule comprises the sequence of SEQ ID NO: 2.
[0083] In a further embodiment isolated nucleic acid molecule comprises the sequence of SEQ ID NO: 1.
[0084] In a further embodiment isolated nucleic acid molecule comprises the sequence of SEQ ID NO:55.
Probes
[0085] In a further aspect the invention provides a probe capable of binding to a nucleic acid of the invention
[0086] According to another aspect of the present invention there is a probe capable of binding to a 3' domain of the MYB14 nucleic acid molecule substantially as described above.
[0087] In one embodiment the probe is capable of binding to a nucleic acid molecule that encodes the amino acid sequence of SEQ ID NO: 17, or to a complement of the nucleic acid molecule.
[0088] In one embodiment the probe is capable of binding to the nucleic acid molecule, or complement thereof under stringent hybridisation conditions.
[0089] According to a further aspect of the present invention there is provided a probe to a 3' sequence encoding the motif as set forth in SEQ ID NO: 17.
Primers
[0090] In a further aspect the invention provides a primer capable of binding to a nucleic acid of the invention
[0091] According to another aspect of the present invention there is a primer capable of binding to a 3' domain of the MYB14 nucleic acid molecule substantially as described above.
[0092] In one embodiment the probe is capable of binding to a nucleic acid molecule that encodes the amino acid sequence of SEQ ID NO: 15, or to a complement of the nucleic acid molecule.
[0093] In one embodiment the probe is capable of binding to the nucleic acid molecule, or complement thereof under PCR conditions.
[0094] According to a further aspect of the present invention there is provided a primer to a nucleic acid encoding a 3' sequence encoding the motif as set forth in SEQ ID NO: 17.
Polypeptides
[0095] In the one aspect the invention provides a MYB14 polypeptide as herein defined, or a functional fragment thereof.
[0096] In one embodiment the MYB14 polypeptide comprises the sequence of SEQ ID NO: 15 and SEQ ID NO: 17, but lacks the sequence of SEQ ID NO: 16.
[0097] In a further aspect the invention provides an isolated polypeptide having an amino acid sequence selected from the group consisting of:
[0098] a) any one of SEQ ID NO: 14 and 46 to 54;
[0099] b) a functional fragment or variant of the sequence listed in a).
[0100] In a further embodiment the variant comprises a sequence with at least 70% identity to any one of SEQ ID NO: 14 and 46 to 54.
[0101] In a further embodiment the variant comprises a sequence with at least 70% identity to SEQ ID NO: 14.
[0102] In a further embodiment the MYB14 polypeptide comprises the sequence of any one of SEQ ID NO: 14 and 46 to 54.
[0103] In a further embodiment the MYB14 polypeptide comprises the sequence of SEQ ID NO: 14.
[0104] In a further embodiment the MYB14 polypeptide regulates the production of flavonoids in a plant.
[0105] In a further embodiment the flavonoids are condensed tannins.
[0106] In a further embodiment the MYB14 polypeptide regulates at least one gene in the flavonoid biosynthetic pathway in a plant.
[0107] In a further embodiment the MYB14 polypeptide regulates the condensed tannin biosynthetic pathway in a plant.
[0108] In a further embodiment the MYB14 polypeptide regulates at least one gene in the condensed tannin biosynthetic pathway in a plant.
[0109] In a further embodiment the functional fragment has substantially the same activity as the MYB14 polypeptide.
[0110] According to another aspect of the present invention there is provided an isolated polypeptide having an amino acid sequence selected from the group consisting of:
[0111] a) SEQ ID NO: 14;
[0112] b) a functional fragment or variant of the sequence listed in a).
[0113] According to another aspect of the present invention there is provided an isolated polypeptide comprising a 3' amino acid sequence motif as set forth in SEQ ID NO: 17.
[0114] According to another aspect of the present invention there is provided an isolated polypeptide having a 3' amino acid sequence motif as set forth in SEQ ID NO: 17.
[0115] According to a further aspect of the present invention there is provided an isolated MYB14 polypeptide or a functional fragment thereof wherein said MYB14 polypeptide includes an amino acid sequence motif of subgroup 5 as shown in SEQ ID NO: 15 as well as an amino acid sequence 3' motif as shown in SEQ ID NO: 17 but which lacks an amino acid sequence motif of subgroup 6 as shown in SEQ ID NO: 16.
[0116] According to another aspect of the present invention there is provided an isolated polypeptide encoded by a nucleic acid molecule having a nucleotide sequence selected from those set forth in any one of SEQ ID NO:1 to 13 and 55 to 64.
[0117] According to another aspect of the present invention there is provided an isolated polypeptide encoded by a nucleic acid molecule having a nucleotide sequence as set forth in either SEQ ID NO: 1, 2 or 55.
[0118] In a further aspect the invention provides a nucleic acid molecule comprising a sequence encoding a polypeptide of the invention.
Constructs
[0119] According to a further aspect of the present invention there is provided a construct including a nucleotide sequence substantially as described above.
[0120] According to a further aspect of the present invention, there is provided a construct which includes:
[0121] at least one promoter; and
[0122] a nucleic acid molecule substantially as described above; wherein the promoter is operably linked to the nucleic acid molecule to control the expression of the nucleic acid molecule.
[0123] Preferably, the construct may include one or more other nucleic acid molecules of interest and/or one or more further regulatory sequences, such as inter alia terminator sequences.
[0124] Most preferably, the nucleic acid molecule in the construct may have a nucleotide sequence selected from SEQ ID NO: 1, 2 or 55.
Host Cells
[0125] According to a further aspect of the present invention there is provided a host cell which has been altered from the wild type to include a nucleic acid molecule substantially as described above.
[0126] In one embodiment the nucleic acid is part of a genetic construct of the invention.
[0127] In one embodiment the host cell does not form part of a human being.
[0128] In a further embodiment the host cell is a plant cell.
Plant Cells and Plants
[0129] According to a further aspect of the present invention there is provided a plant or plant cell transformed with a construct substantially as described above.
[0130] According to a further aspect of the present invention there is provided a plant transformed with a construct substantially as described above.
[0131] According to a further aspect of the present invention there is provided a plant or part thereof which has been altered from the wild type to include a nucleic acid molecule substantially as described above.
[0132] According to a further aspect of the present invention, there is provided a plant cell, plant or part thereof which has been manipulated via altered expression of a MYB14 gene to have increased or decreased levels of flavonoids and/or condensed tannins than a corresponding wild-type plant or part thereof.
[0133] According to a further aspect of the present invention, there is provided a plant cell, plant cell which has been manipulated via altered expression of a MYB14 gene to have increased or decreased levels of flavonoids and/or condensed tannins than a corresponding wild-type plant cell.
[0134] According to a further aspect of the present invention, there is provided a leaf of a plant which via altered expression of a MYB14 gene to have increased levels of flavonoids and/or condensed tannins than a corresponding wild-type plant or part thereof.
[0135] According to a further aspect of the present invention, there is provided the progeny of a plant cell or a plant substantially as described above which via altered expression of a MYB14 gene has increased or decreased to levels of flavonoids and/or condensed tannins than a corresponding wild-type plant cell or plant.
[0136] According to a further aspect of the present invention there is provided the seed of a transgenic plant substantially as described above.
Compositions
[0137] According to a further aspect of the present invention, there is provided a composition which includes an ingredient which is, or is obtained from, a plant and/or part thereof, wherein said plant or part thereof has been manipulated via altered expression of a MYB14 gene to have increased or decreased levels of flavonoids and/or condensed tannins compared to those of a corresponding wild type plant or part thereof.
Methods Using Polynucleotides
[0138] According to a further aspect of the present invention there is provided the use of a nucleic acid molecule substantially as described above to alter a plant or plant cell.
[0139] According to a further aspect of the present invention there is provided a method for producing an altered plant or plant cell using a nucleic acid molecule substantially as described above.
[0140] In one embodiment the plant or plant cell is altered in the production of flavonoids, or an intermediate in the production of flavonoids.
[0141] In a further embodiment the flavonoids include at least one condensed tannin.
[0142] In a further embodiment the condensed tannin is selected from catechin, epicatechin, epigallocatechin and gallocatechin.
[0143] In a preferred embodiment the alteration is an increase.
[0144] In a further embodiment the plant or plant cell is altered in expression of at least one enzyme in a flavonoid biosynthetic pathway.
[0145] In one embodiment the flavonoid biosynthetic pathway is the condensed tannin biosynthetic pathway.
[0146] In a preferred embodiment the altered expression is increased expression.
[0147] In a further embodiment the enzyme is LAR or ANR.
[0148] In a further embodiment the plant is altered in the expression of both LAR and ANR.
[0149] The plant may be any plant, and the plant cell may be from any plant.
[0150] In one embodiment the plant is a forage crop plant.
[0151] In a further embodiment the plant is a legumionous plant.
[0152] In one embodiment the altered production or expression, described above, is in substantially all tissues of the plant.
[0153] In one embodiment the altered production or expression, described above, is in the foliar tissue of the plant.
[0154] In one embodiment the altered production or expression, described above, is in the vegetative portions of the plant.
[0155] In one embodiment the altered production or expression, described above, is in the epidermal tissues of the plant.
[0156] For the purposes of this specification, the epidermal tissue refers to the outer single-layered group of cells, including the leaf, stems, and roots and young tissues of a vascular plant.
[0157] In one embodiment the altered production flavonoids, described above, is in a tissue of the plant that is substantially devoid of the flavonoids.
[0158] In one embodiment the altered production condensed tannins described above is in a tissue of the plant that is substantially devoid of the condensed tannins.
[0159] Therefore, in some embodiments of the invention, the production of flavonoids or condnesed tannins is de novo production.
[0160] In one embodiment the nucleic acid encodes a MYB14 protein as herein defined.
[0161] In a further embodiment the nucleic acid encodes a protein comprising an amino acid sequence as set forth in any one of SEQ ID NOs 1-13 and 55 to 64, or fragment or variant thereof.
[0162] In a further embodiment the nucleic acid comprises a sequence substantially as set forth in any one of SEQ ID NOs 1-13 and 55 to 64, or fragment or variant thereof.
[0163] In a further embodiment the nucleic acid comprises a sequence substantially as set forth in SEQ ID NOs 1, 2 or 55, or fragment or variant thereof.
[0164] In a further embodiment the nucleic acid is part of a construct substantially as described above.
[0165] In one embodiment the plant is altered by transforming the plant with the nucleic acid or construct.
[0166] In a further embodiment the plant is altered by manipulating the genome of a plant so as to express increase or decrease levels of the nucleic acid, or fragment or variant thereof, in the plant compared to that produced in a corresponding wild-type plant or plant thereof.
[0167] According to a further aspect of the present invention there is provided the use of a nucleic acid molecule or polypeptide of the present invention to identify other related flavonoid and/or condensed tannin regulatory genes/polypeptides.
[0168] According to a further aspect of the present invention there is provided the use of a nucleic acid molecule substantially as described above to alter a plant or plant cell wherein said plant is, or plant cell is from, a forage crop.
[0169] In one embodiment the plant is altered in production of condensed tannins.
[0170] In one embodiment the plant has increased production of condensed tannins.
[0171] Preferably, the forage crop may be a forage legume.
[0172] According to a further aspect of the present invention there is provided the use of a nucleic acid molecule substantially as described above to alter the levels of flavonoids or condensed tannins in leguminous plants or leguminous plant cells.
[0173] Preferably, the levels of condensed tannins are altered.
[0174] Preferably, the levels of condensed tannins are altered in foliar tissue.
[0175] According to a further aspect of the present invention there is provided the use of nucleic acid sequence information substantially as set forth in any one of SEQ ID NO: 1-13 and 55 to 64 to alter the flavonoid or condensed tannin biosynthetic pathway in planta.
[0176] According to a further aspect of the present invention there is provided the use of nucleic acid sequence information substantially as set forth in any one of SEQ ID NO:1, 2 and 55 to alter the flavonoid or condensed tannin biosynthetic pathway in planta.
[0177] According to a further aspect of the present invention there is provided use of a construct substantially as described above to transform a leguminous plant or plant cell to alter the levels of flavonoids and/or condensed tannins in the vegetative portions of the leguminous plant or plant cell.
[0178] According to a further aspect of the present invention, there is provided a method of altering flavonoids and/or condensed tannins production within a leguminous plant or part thereof, including the step of manipulating the genome of a plant so as to express increased or decreased levels a of leguminous MYB14 gene, or fragment or variant thereof, in the plant compared to that produced in a corresponding wild-type plant or plant thereof.
[0179] According to a further aspect of the present invention, there is provided a method of altering flavonoids and/or condensed tannins production within a leguminous plant or part thereof, including the step of manipulating the genome of a plant so as to express increased or decreased levels a of leguminous MYB14 gene, or fragment or variant thereof, in the plant compared to that produced in a corresponding wild-type plant or plant thereof.
[0180] According to a further aspect of the present invention, there is provided the use of a nucleic acid molecule to produce flavonoids or condensed tannins in planta in a leguminous plant or part thereof de novo.
[0181] According to a further aspect of the present invention, there is provided the use of a nucleic acid molecule substantially as described above to manipulate in a leguminous plant or part thereof the flavonoids and/or condensed tannin biosynthetic pathway in planta.
[0182] According to a further aspect of the present invention, there is provided the use of a construct substantially as described above, to manipulate the flavonoids and/or condensed tannin biosynthetic pathway in planta.
[0183] According to a further aspect of the present invention, there is provided the use of a MYB14 gene having a nucleic acid sequence substantially corresponding to a nucleic acid molecule of the present invention to manipulate the biosynthetic pathway in planta.
[0184] According to a further aspect of the present invention, there is provided the use of a nucleic acid molecule substantially as described above to produce a flavonoid and/or condensed tannin, enzyme, intermediate or other chemical compound associated with the flavonoid and/or condensed tannin biosynthetic pathway.
[0185] According to a further aspect of the present invention, there is provided a method of manipulating the flavonoid and/or condensed tannin biosynthetic pathway characterized by the step of altering a nucleic acid substantially as described above to produce a gene encoding a non-functional polypeptide.
[0186] According another aspect there is provided the use of an isolated nucleic acid molecule of the present invention in planta to manipulate the levels of LAR and/or ANR within a leguminous plant or plant cell.
[0187] According another aspect there is provided the use of an isolated nucleic acid molecule of the present invention in planta to manipulate the levels of catechin and/or epicatechin or other tannin monomer (epigallocatechin or gallocatechin) within a leguminous plant or plant cell.
[0188] According to a further aspect of the present invention there is provided the use of a nucleic acid molecule or polypeptide to identify other related flavonoid and/or condensed tannin regulatory genes/polypeptides.
[0189] In one embodiment, the whole of the plant tissue may be manipulated. In an alternative embodiment, the epidermal tissue of the plant may be manipulated. For the purposes of this specification, the epidermal tissue refers to the outer single-layered group of cells, the leaf, stems, and roots and young tissues of a vascular plant.
[0190] Most preferably, the levels of flavonoids and/or condensed tannins altered by the present invention are sufficient to provide a therapeutic or agronomic benefit to a subject consuming the plant with altered levels of flavonoids and/or condensed tannins.
Plants Produced Via the Methods
[0191] In a further embodiment the invention provides a plant produced by a method of the invention.
[0192] In a further embodiment the invention provides a part, seed, fruit, harvested material, propagule or progeny of a plant of any the invention.
[0193] In a further embodiment the part, seed, fruit, harvested material, propagule or progeny of the plant is genetically modified to comprise at least one nucleic acid molecule of the invention, or a construct of the invention.
[0194] In one embodiment, the transformed plant cells, plants or ancestors thereof, are transformed by any transformation method.
[0195] In a further embodiment, the transformed plant cells, plants or ancestors thereof, are transformed by agrobacterium-mediated transformation. Source of nucleic acids and proteins of the invention
[0196] The nucleic acids and proteins of the invention may derived from any plant, as described below, or may be synthetically or recombinantly produced.
Plants
[0197] The plant cells and plants of the invention, or those transformed or manipulated in methods and uses of the inventions, may be from any species.
[0198] In one embodiment the plant cell or plant, is derived from a gymnosperm plant species
[0199] In a further embodiment the plant cell or plant, is derived from an angiosperm plant species.
[0200] In a further embodiment the plant cell or plant, is derived from a from dicotyledonous plant species.
[0201] In a further embodiment the plant cell or plant, is derived from a monocotyledonous plant species.
[0202] Preferably the plants are from dicotyledonous species.
[0203] Other preferred plants are forage plant species from a group comprising but not limited to the following genera: Lolium, Festuca, Dactylis, Bromus, Thinopyrum, Trifolium, Medicago, Pheleum, Phalaris, Holcus, Lotus, Plantago and Cichorium.
[0204] Other preferred plants are leguminous plants. The leguminous plant or part thereof may encompass any plant in the plant family Leguminosae or Fabaceae. For example, the plants may be selected from forage legumes including, alfalfa, clover; leucaena; grain legumes including, beans, lentils, lupins, peas, peanuts, soy bean; bloom legumes including lupin, pharmaceutical or industrial legumes; and fallow or green manure legume species.
[0205] A particularly preferred genus is Trifolium.
[0206] Preferred Trifolium species include Trifolium repens; Trifolium arvense; Trifolium affine; and Trifolium occidentale.
[0207] A particularly preferred Trifolium species is Trifolium repens.
[0208] Another preferred genus is Medicago.
[0209] Preferred Medicago species include Medicago sativa and Medicago truncatula.
[0210] A particularly preferred Medicago species is Medicago sativa, commonly known as alfalfa.
[0211] Another preferred genus is Glycine.
[0212] Preferred Glycine species include Glycine max and Glycine wightii (also known as Neonotonia wightii)
[0213] A particularly preferred Glycine species is Glycine max, commonly known as soy bean
[0214] A particularly preferred Glycine species is Glycine wightii, commonly known as perennial soybean.
[0215] Another preferred genus is Vigna.
[0216] Preferred Vigna species include Vigna unguiculata
[0217] A particularly preferred Vigna species is Vigna unguiculata commonly known as cowpea.
[0218] Another preferred genus is Mucana.
[0219] Preferred Mucana species include Mucana pruniens
[0220] A particularly preferred Mucana species is Mucana pruniens commonly known as velvetbean.
[0221] Another preferred genus is Arachis
[0222] Preferred Mucana species include Arachis glabrata
[0223] A particularly preferred Arachis species is Arachis glabrata commonly known as perennial peanut.
[0224] Another preferred genus is Pisum
[0225] Preferred Pisum species include Pisum sativum
[0226] A particularly preferred Pisum species is Pisum sativum commonly known as pea.
[0227] Another preferred genus is Lotus
[0228] Preferred Lotus species include Lotus corniculatus, Lotus pedunculatus, Lotus glabar, Lotus tenuis and Lotus uliginosus.
[0229] A particularly preferred Lotus species is Lotus comiculatus commonly known as Birdsfoot Trefoil.
[0230] A particularly preferred Lotus species is Lotus glabar commonly known as Narrow-leaf Birdsfoot Trefoil
[0231] A particularly preferred Lotus species is Lotus pedunculatus commonly known as Big trefoil.
[0232] A particularly preferred Lotus species is Lotus tenuis commonly known as Slender trefoil.
[0233] Another preferred genus is Brassica.
[0234] Preferred Brassica species include Brassica oleracea
[0235] A particularly preferred Brassica species is Brassica oleracea, commonly known as forage kale and cabbage.
[0236] The term `plant` as used herein refers to the plant in its entirety, and any part thereof, may include but is not limited to: selected portions of the plant during the plant life cycle, such as plant seeds, shoots, leaves, bark, pods, roots, flowers, fruit, stems and the like. A preferred `part thereof` is leaves.
DETAILED DESCRIPTION OF THE INVENTION
[0237] In this specification where reference has been made to patent specifications, other external documents, or other sources of information, this is generally for the purpose of providing a context for discussing the features of the invention. Unless specifically stated otherwise, reference to such external documents is not to be construed as an admission that such documents, or such sources of information, in any jurisdiction, are prior art, or form part of the common general knowledge in the art.
[0238] The term "comprising" as used in this specification and claims means "consisting at least in part of"; that is to say when interpreting statements in this specification and claims which include "comprising", the features prefaced by this term in each statement all need to be present but other features can also be present. Related terms such as "comprise" and "comprised" are to be interpreted in similar manner. However, in preferred embodiments comprising can be replaced with consisting.
[0239] The term "MYB14 polypeptide" refers to an R2R3 class MYB transcription factor.
[0240] Preferably the MYB14 polypeptide comprises a sequence with at least 70% identity to any one of SEQ ID NO: 14 and 46 to 54.
[0241] Preferably the MYB14 polypeptide comprises the sequence motif of SEQ ID NO:15
[0242] Preferably the MYB14 polypeptide comprises the sequence motif of SEQ ID NO:17
[0243] More preferably the MYB14 polypeptide comprises the sequence of SEQ ID NO: 15 and SEQ ID NO: 17, but lacks the sequence of SEQ ID NO: 16.
[0244] Preferably MYB14 polypeptide comprises a sequence with at least 70% identity to SEQ ID NO: 14.
[0245] A "MYB14 gene" is a gene, by the standard definition of gene, that encodes a MYB14 polypeptide.
[0246] The term "MYB transcription factor" is a term well understood by those skilled in the art to refer to a class of transcription factors characterised by a structurally conserved DNA binding domain consisting of single or multiple imperfect repeats.
[0247] The term "R2R3 transcription factor" or "MYB transcription with an R2R3 DNA binding domain" is a term well understood by those skilled in the art to refer to MYB transcription factors of the two-repeat class.
[0248] The terms `proanthocyanidins` and `condensed tannins` may be used interchangeably throughout the specification
[0249] The term "sequence motif" as used herein means a stretch of amino acids or nucleotides. Preferably the stretch of amino acids or nucleotides is contiguous.
[0250] The term "altered" with respect to a plant with "altered production" or "altered expression", means altereded relative to the same plant, or plant of the same type, in the non-transformed state.
[0251] The term "altered" may mean increased or decreased. Preferably altered is increased
Polynucleotides and Fragments
[0252] The term "polynucleotide(s)," as used herein, means a single or double-stranded deoxyribonucleotide or ribonucleotide polymer of any length but preferably at least 15 nucleotides, and include as non-limiting examples, coding and non-coding sequences of a gene, sense and antisense sequences complements, exons, introns, genomic DNA, cDNA, pre-mRNA, mRNA, rRNA, siRNA, miRNA, tRNA, ribozymes, recombinant polypeptides, isolated and purified naturally occurring DNA or RNA sequences, synthetic RNA and DNA sequences, nucleic acid probes, primers and fragments.
[0253] The term "polynucleotide" can be used interchangably with "nucleic acid molecule".
[0254] A "fragment" of a polynucleotide sequence provided herein is a subsequence of contiguous nucleotides that is preferably at least 15 nucleotides in length. The fragments of the invention preferably comprises at least 20 nucleotides, more preferably at least 30 nucleotides, more preferably at least 40 nucleotides, more preferably at least 50 nucleotides and most preferably at least 60 contiguous nucleotides of a polynucleotide of the invention. A fragment of a polynucleotide sequence can be used in antisense, gene silencing, triple helix or ribozyme technology, or as a primer, a probe, included in a microarray, or used in polynucleotide-based selection methods.
[0255] Preferably fragments of polynucleotide sequences of the invention comprise at least 25, more preferably at least 50, more preferably at least 75, more preferably at least 100, more preferably at least 150, more preferably at least 200, more preferably at least 300, more preferably at least 400, more preferably at least 500, more preferably at least 600, more preferably at least 700, more preferably at least 800, more preferably at least 900, more preferably at least 1000 contiguous nucleotides of the specified polynucleotide.
[0256] The term "primer" refers to a short polynucleotide, usually having a free 3'OH group, that is hybridized to a template and used for priming polymerization of a polynucleotide complementary to the template. Such a primer is preferably at least 5, more preferably at least 6, more preferably at least 7, more preferably at least 9, more preferably at least 10, more preferably at least 11, more preferably at least 12, more preferably at least 13, more preferably at least 14, more preferably at least 15, more preferably at least 16, more preferably at least 17, more preferably at least 18, more preferably at least 19, more preferably at least 20 nucleotides in length.
[0257] The term "probe" refers to a short polynucleotide that is used to detect a polynucleotide sequence, that is complementary to the probe, in a hybridization-based assay. The probe may consist of a "fragment" of a polynucleotide as defined herein. Preferably such a probe is at least 5, more preferably at least 10, more preferably at least 20, more preferably at least 30, more preferably at least 40, more preferably at least 50, more preferably at least 100, more preferably at least 200, more preferably at least 300, more preferably at least 400 and most preferably at least 500 nucleotides in length.
Polypeptides and Fragments
[0258] The term "polypeptide", as used herein, encompasses amino acid chains of any length but preferably at least 5 amino acids, including full-length proteins, in which amino acid residues are linked by covalent peptide bonds. The polypeptides may be purified natural products, or may be produced partially or wholly using recombinant or synthetic techniques. The term may refer to a polypeptide, an aggregate of a polypeptide such as a dimer or other multimer, a fusion polypeptide, a polypeptide fragment, a polypeptide variant, or derivative thereof.
[0259] A "fragment" of a polypeptide is a subsequence of the polypeptide that performs a function that is required for the biological activity and/or provides three dimensional structure of the polypeptide. The term may refer to a polypeptide, an aggregate of a polypeptide such as a dimer or other multimer, a fusion polypeptide, a polypeptide fragment, a polypeptide variant, or derivative thereof capable of performing the above activity.
[0260] The term "isolated" as applied to the polynucleotide or polypeptide sequences disclosed herein is used to refer to sequences that are removed from their natural cellular environment. An isolated molecule may be obtained by any method or combination of methods including biochemical, recombinant, and synthetic techniques.
[0261] The term "derived from" with respect to a polynucleotide or polypeptide sequence being derived from a particular genera or species, means that the sequence has the same sequence as a polynucleotide or polypeptide sequence found naturally in that genera or species. The sequence, derived from a particular genera or species, may therefore be produced synthetically or recombinantly.
Variants
[0262] As used herein, the term "variant" refers to polynucleotide or polypeptide 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. Variants may be from the same or from other species and may encompass homologues, paralogues and orthologues. In certain embodiments, variants of the inventive polynucleotides and polypeptides possess biological activities that are the same or similar to those of the inventive polynucleotides or polypeptides. The term "variant" with reference to polynucleotides and polypeptides encompasses all forms of polynucleotides and polypeptides as defined herein.
Polynucleotide Variants
[0263] Variant polynucleotide sequences preferably exhibit at least 50%, more preferably at least 51%, more preferably at least 52%, more preferably at least 53%, more preferably at least 54%, more preferably at least 55%, more preferably at least 56%, more preferably at least 57%, more preferably at least 58%, more preferably at least 59%, more preferably at least 60%, more preferably at least 61%, more preferably at least 62%, more preferably at least 63%, more preferably at least 64%, more preferably at least 65%, more preferably at least 66%, more preferably at least 67%, more preferably at least 68%, more preferably at least 69%, more preferably at least 70%, more preferably at least 71%, more preferably at least 72%, more preferably at least 73%, more preferably at least 74%, more preferably at least 75%, more preferably at least 76%, more preferably at least 77%, more preferably at least 78%, more preferably at least 79%, more preferably at least 80%, more preferably at least 81%, more preferably at least 82%, more preferably at least 83%, more preferably at least 84%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, and most preferably at least 99% identity to a specified polynucleotide sequence. Identity is found over a comparison window of at least 20 nucleotide positions, more preferably at least 50 nucleotide positions, more preferably at least 100 nucleotide positions, more preferably at least 200 nucleotide positions, more preferably at least 300 nucleotide positions, more preferably at least 400 nucleotide positions, more preferably at least 500 nucleotide positions, more preferably at least 600 nucleotide positions, more preferably at least 700 nucleotide positions, more preferably at least 800 nucleotide positions, more preferably at least 900 nucleotide positions, more preferably at least 1000 nucleotide positions and most preferably over the entire length of the specified polynucleotide sequence.
[0264] Polynucleotide sequence identity can be determined in the following manner. The subject polynucleotide sequence is compared to a candidate polynucleotide sequence using BLASTN (from the BLAST suite of programs, version 2.2.5 [November 2002]) in bl2seq (Tatiana A. Tatusova, Thomas L. Madden (1999), "Blast 2 sequences--a new tool for comparing protein and nucleotide sequences", FEMS Microbiol Lett. 174:247-250), which is publicly available from NCBI (ftp<dot>ncbi<dot>nih<dot>gov/blast/). The default parameters of bl2seq are utilized except that filtering of low complexity parts should be turned off.
[0265] The identity of polynucleotide sequences may be examined using the following unix command line parameters:
bl2seq-i nucleotideseq1-j nucleotideseq2-FF-p blastn
[0266] The parameter -F F turns off filtering of low complexity sections. The parameter -p selects the appropriate algorithm for the pair of sequences. The bl2seq program reports sequence identity as both the number and percentage of identical nucleotides in a line "Identities=".
[0267] Polynucleotide sequence identity may also be calculated over the entire length of the overlap between a candidate and subject polynucleotide sequences using global sequence alignment programs (e.g. Needleman, S. B. and Wunsch, C. D. (1970) J. Mol. Biol. 48, 443-453). A full implementation of the Needleman-Wunsch global alignment algorithm is found in the needle program in the EMBOSS package (Rice, P. Longden, I. and Bleasby, A. EMBOSS: The European Molecular Biology Open Software Suite, Trends in Genetics June 2000, vol 16, No 6. pp. 276-277) which can be obtained from www<dot>hgmp<dot>mrc<dot>ac<dot>uk/Software/EMBOS- S/. The European Bioinformatics Institute server also provides the facility to perform EMBOSS-needle global alignments between two sequences on line at www<dot>ebi<dot>ac<dot>uk/emboss/align/.
[0268] Alternatively the GAP program, which computes an optimal global alignment of two sequences without penalizing terminal gaps, may be used to calculate sequence identity. GAP is described in the following paper: Huang, X. (1994) On Global Sequence Alignment. Computer Applications in the Biosciences 10, 227-235.
[0269] Sequence identity may also be calculated by aligning sequences to be compared using Vector NTI version 9.0, which uses a Clustal W algorithm (Thompson et al., 1994, Nucleic Acids Research 24, 4876-4882), then calculating the percentage sequence identity between the aligned sequences using Vector NTI version 9.0 (Sep. 2, 2003 ยฉ1994-2003 InforMax, licenced to Invitrogen).
[0270] Polynucleotide variants of the present invention also encompass those which exhibit a similarity to one or more of the specifically identified sequences that is likely to preserve the functional equivalence of those sequences and which could not reasonably be expected to have occurred by random chance. Such sequence similarity with respect to polynucleotides may be determined using the publicly available bl2seq program from the BLAST suite of programs (version 2.2.5 [November 2002]) from NCBI (ftp<dot>ncbi<dot>nih<dot>gov/blast/).
[0271] The similarity of polynucleotide sequences may be examined using the following unix command line parameters:
bl2seq-i nucleotideseq1-j nucleotideseq2-FF-p tblastx
[0272] The parameter -F F turns off filtering of low complexity sections. The parameter -p selects the appropriate algorithm for the pair of sequences. This program finds regions of similarity between the sequences and for each such region reports an "E value" which is the expected number of times one could expect to see such a match by chance in a database of a fixed reference size containing random sequences. The size of this database is set by default in the bl2seq program. For small E values, much less than one, the E value is approximately the probability of such a random match.
[0273] Variant polynucleotide sequences preferably exhibit an E value of less than 1ร10-10 more preferably less than 1ร10-20, more preferably less than 1ร10-30, more preferably less than 1ร10-40, more preferably less than 1ร10-50 more preferably less than 1ร10-60 more preferably less than 1ร10-70 more preferably less than 1ร10-80 more preferably less than 1ร10-90 and most preferably less than 1ร10-100 when compared with any one of the specifically identified sequences.
[0274] Alternatively, variant polynucleotides of the present invention hybridize to a specified polynucleotide sequence, or complements thereof under stringent conditions.
[0275] The term "hybridize under stringent conditions", and grammatical equivalents thereof, refers to the ability of a polynucleotide molecule to hybridize to a target polynucleotide molecule (such as a target polynucleotide molecule immobilized on a DNA or RNA blot, such as a Southern blot or Northern blot) under defined conditions of temperature and salt concentration. The ability to hybridize under stringent hybridization conditions can be determined by initially hybridizing under less stringent conditions then increasing the stringency to the desired stringency.
[0276] With respect to polynucleotide molecules greater than about 100 bases in length, typical stringent hybridization conditions are no more than 25 to 30ยฐ C. (for example, 10ยฐ C.) below the melting temperature (Tm) of the native duplex (see generally, Sambrook et al., Eds, 1987, Molecular Cloning, A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press; Ausubel et al., 1987, Current Protocols in Molecular Biology, Greene Publishing,). Tm for polynucleotide molecules greater than about 100 bases can be calculated by the formula Tm=81.5+0.41% (G+C-log (Na+). (Sambrook et al., Eds, 1987, Molecular Cloning, A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press; Bolton and McCarthy, 1962, PNAS 84:1390). Typical stringent conditions for polynucleotide of greater than 100 bases in length would be hybridization conditions such as prewashing in a solution of 6รSSC, 0.2% SDS; hybridizing at 65ยฐ C., 6รSSC, 0.2% SDS overnight; followed by two washes of 30 minutes each in 1รSSC, 0.1% SDS at 65ยฐ C. and two washes of 30 minutes each in 0.2รSSC, 0.1% SDS at 65ยฐ C.
[0277] With respect to polynucleotide molecules having a length less than 100 bases, exemplary stringent hybridization conditions are 5 to 10ยฐ C. below Tm. On average, the Tm of a polynucleotide molecule of length less than 100 bp is reduced by approximately (500/oligonucleotide length)ยฐ C.
[0278] With respect to the DNA mimics known as peptide nucleic acids (PNAs) (Nielsen et al., Science. 1991 Dec. 6; 254(5037):1497-500) Tm values are higher than those for DNA-DNA or DNA-RNA hybrids, and can be calculated using the formula described in Giesen et al., Nucleic Acids Res. 1998 Nov. 1; 26(21):5004-6. Exemplary stringent hybridization conditions for a DNA-PNA hybrid having a length less than 100 bases are 5 to 10ยฐ C. below the Tm.
[0279] Variant polynucleotides such as those in constructs of the invention encoding proteins to be expressed, also encompasses polynucleotides that differ from the specified sequences but that, as a consequence of the degeneracy of the genetic code, encode a polypeptide having similar activity to a polypeptide encoded by a polynucleotide of the present invention. A sequence alteration that does not change the amino acid sequence of the polypeptide is a "silent variation". Except for ATG (methionine) and TGG (tryptophan), other codons for the same amino acid may be changed by art recognized techniques, e.g., to optimize codon expression in a particular host organism.
[0280] Polynucleotide sequence alterations resulting in conservative substitutions of one or several amino acids in the encoded polypeptide sequence without significantly altering its biological activity are also contemplated. A skilled artisan will be aware of methods for making phenotypically silent amino acid substitutions (see, e.g., Bowie et al., 1990, Science 247, 1306).
[0281] Variant polynucleotides due to silent variations and conservative substitutions in the encoded polypeptide sequence may be determined using the publicly available bl2seq program from the BLAST suite of programs (version 2.2.5 [November 2002]) from NCBI (ftp<dot>ncbi<dot>nih<dot>gov/blast/) via the tblastx algorithm as previously described.
Polypeptide Variants
[0282] The term "variant" with reference to polypeptides encompasses naturally occurring, recombinantly and synthetically produced polypeptides. Variant polypeptide sequences preferably exhibit at least 50%, more preferably at least 51%, more preferably at least 52%, more preferably at least 53%, more preferably at least 54%, more preferably at least 55%, more preferably at least 56%, more preferably at least 57%, more preferably at least 58%, more preferably at least 59%, more preferably at least 60%, more preferably at least 61%, more preferably at least 62%, more preferably at least 63%, more preferably at least 64%, more preferably at least 65%, more preferably at least 66%, more preferably at least 67%, more preferably at least 68%, more preferably at least 69%, more preferably at least 70%, more preferably at least 71%, more preferably at least 72%, more preferably at least 73%, more preferably at least 74%, more preferably at least 75%, more preferably at least 76%, more preferably at least 77%, more preferably at least 78%, more preferably at least 79%, more preferably at least 80%, more preferably at least 81%, more preferably at least 82%, more preferably at least 83%, more preferably at least 84%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, and most preferably at least 99% identity to a sequences of the present invention. Identity is found over a comparison window of at least 20 amino acid positions, preferably at least 50 amino acid positions, more preferably at least 100 amino acid positions, and most preferably over the entire length of a polypeptide of the invention.
[0283] Polypeptide sequence identity can be determined in the following manner. The subject polypeptide sequence is compared to a candidate polypeptide sequence using BLASTP (from the BLAST suite of programs, version 2.2.5 [November 2002]) in bl2seq, which is publicly available from NCBI (ftp<dot>ncbi<dot>nih<dot>gov/blast/). The default parameters of bl2seq are utilized except that filtering of low complexity regions should be turned off.
[0284] Polypeptide sequence identity may also be calculated over the entire length of the overlap between a candidate and subject polynucleotide sequences using global sequence alignment programs. EMBOSS-needle (available at www<dot>ebi<dot>ac<dot>uk/emboss/align/) and GAP (Huang, X. (1994) On Global Sequence Alignment. Computer Applications in the Biosciences 10, 227-235) as discussed above are also suitable global sequence alignment programs for calculating polypeptide sequence identity.
[0285] Sequence identity may also be calculated by aligning sequences to be compared using Vector NTI version 9.0, which uses a Clustal W algorithm (Thompson et al., 1994, Nucleic Acids Research 24, 4876-4882), then calculating the percentage sequence identity between the aligned polypeptide sequences using Vector NTI version 9.0 (Sep. 2, 2003 ยฉ1994-2003 InforMax, licenced to Invitrogen).
[0286] Polypeptide variants of the present invention also encompass those which exhibit a similarity to one or more of the specifically identified sequences that is likely to preserve the functional equivalence of those sequences and which could not reasonably be expected to have occurred by random chance. Such sequence similarity with respect to polypeptides may be determined using the publicly available bl2seq program from the BLAST suite of programs (version 2.2.5 [November 2002]) from NCBI (ftp<dot>ncbi<dot>nih<dot>gov/blast/). The similarity of polypeptide sequences may be examined using the following unix command line parameters:
bl2seq-i peptideseq1-j peptideseq2-FF-p blastp
[0287] Variant polypeptide sequences preferably exhibit an E value of less than 1ร10-6 more preferably less than 1ร10-9, more preferably less than 1ร10-12, more preferably less than 1ร10-15, more preferably less than 1ร10-18, more preferably less than 1ร10-21, more preferably less than 1ร10-30, more preferably less than 1ร10-40, more preferably less than 1ร10-50, more preferably less than 1ร10-60, more preferably less than 1ร10-70, more preferably less than 1ร10-80, more preferably less than 1ร10-90 and most preferably 1ร10-100 when compared with any one of the specifically identified sequences.
[0288] The parameter -F F turns off filtering of low complexity sections. The parameter -p selects the appropriate algorithm for the pair of sequences. This program finds regions of similarity between the sequences and for each such region reports an "E value" which is the expected number of times one could expect to see such a match by chance in a database of a fixed reference size containing random sequences. For small E values, much less than one, this is approximately the probability of such a random match.
[0289] Conservative substitutions of one or several amino acids of a described polypeptide sequence without significantly altering its biological activity are also included in the invention. A skilled artisan will be aware of methods for making phenotypically silent amino acid substitutions (see, e.g., Bowie et al., 1990, Science 247, 1306).
Constructs, Vectors and Components Thereof
[0290] The term "genetic construct" refers to a polynucleotide molecule, usually double-stranded DNA, which may have inserted into it another polynucleotide molecule (the insert polynucleotide molecule) such as, but not limited to, a cDNA molecule. A genetic construct may contain a promoter polynucleotide including the necessary elements that permit transcribing the insert polynucleotide molecule, and, optionally, translating the transcript into a polypeptide. The insert polynucleotide molecule may be derived from the host cell, or may be derived from a different cell or organism and/or may be a synthetic or recombinant polynucleotide. Once inside the host cell the genetic construct may become integrated in the host chromosomal DNA. The genetic construct may be linked to a vector.
[0291] The term "vector" refers to a polynucleotide molecule, usually double stranded DNA, which is used to transport the genetic construct into a host cell. The vector may be capable of replication in at least one additional host system, such as E. coli.
[0292] The term "expression construct" refers to a genetic construct that includes the necessary elements that permit transcribing the insert polynucleotide molecule, and, optionally, translating the transcript into a polypeptide.
[0293] An expression construct typically comprises in a 5' to 3' direction:
[0294] a) a promoter functional in the host cell into which the construct will be transformed,
[0295] b) the polynucleotide to be expressed, and
[0296] c) a terminator functional in the host cell into which the construct will be transformed.
[0297] The term "coding region" or "open reading frame" (ORF) refers to the sense strand of a genomic DNA sequence or a cDNA sequence that is capable of producing a transcription product and/or a polypeptide under the control of appropriate regulatory sequences. The coding sequence is identified by the presence of a 5' translation start codon and a 3' translation stop codon. When inserted into a genetic construct, a "coding sequence" is capable of being expressed when it is operably linked to promoter and terminator sequences.
[0298] The term "operably-linked" means that the sequenced to be expressed is placed under the control of regulatory elements that include promoters, tissue-specific regulatory elements, temporal regulatory elements, enhancers, repressors and terminators.
[0299] The term "noncoding region" includes to untranslated sequences that are upstream of the translational start site and downstream of the translational stop site. These sequences are also referred to respectively as the 5' UTR and the 3' UTR. These sequences may include elements required for transcription initiation and termination and for regulation of translation efficiency. The term "noncoding" also includes intronic sequences within genomic clones.
[0300] Terminators are sequences, which terminate transcription, and are found in the 3' untranslated ends of genes downstream of the translated sequence. Terminators are important determinants of mRNA stability and in some cases have been found to have spatial regulatory functions.
[0301] The term "promoter" refers to a polynucleotide sequence capable of regulating or driving the expression of a polynucleotide sequence to which the promoter is operably linked in a cell, or cell free transcription system. Promoters may comprise cis-initiator elements which specify the transcription initiation site and conserved boxes such as the TATA box, and motifs that are bound by transcription factors.
Methods for Isolating or Producing Polynucleotides
[0302] The polynucleotide molecules of the invention can be isolated by using a variety of techniques known to those of ordinary skill in the art. By way of example, such polynucleotides can be isolated through use of the polymerase chain reaction (PCR) described in Mullis et al., Eds. 1994 The Polymerase Chain Reaction, Birkhauser, incorporated herein by reference. The polynucleotides of the invention can be amplified using primers, as defined herein, derived from the polynucleotide sequences of the invention.
[0303] Further methods for isolating polynucleotides of the invention, or useful in the methods of the invention, include use of all or portions, of the polynucleotides set forth herein as hybridization probes. The technique of hybridizing labeled polynucleotide probes to polynucleotides immobilized on solid supports such as nitrocellulose filters or nylon membranes, can be used to screen the genomic. Exemplary hybridization and wash conditions are: hybridization for 20 hours at 65ยฐ C. in 5.0รSSC, 0.5% sodium dodecyl sulfate, 1รDenhardt's solution; washing (three washes of twenty minutes each at 55ยฐ C.) in 1.0รSSC, 1% (w/v) sodium dodecyl sulfate, and optionally one wash (for twenty minutes) in 0.5รSSC, 1% (w/v) sodium dodecyl sulfate, at 60ยฐ C. An optional further wash (for twenty minutes) can be conducted under conditions of 0.1รSSC, 1% (w/v) sodium dodecyl sulfate, at 60ยฐ C.
[0304] The polynucleotide fragments of the invention may be produced by techniques well-known in the art such as restriction endonuclease digestion, oligonucleotide synthesis and PCR amplification.
[0305] A partial polynucleotide sequence may be used, in methods well-known in the art to identify the corresponding full length polynucleotide sequence and/or the whole gene/and/or the promoter. Such methods include PCR-based methods, 5'RACE (Frohman M A, 1993, Methods Enzymol. 218: 340-56) and hybridization-based method, computer/database--based methods. Further, by way of example, inverse PCR permits acquisition of unknown sequences, flanking the polynucleotide sequences disclosed herein, starting with primers based on a known region (Triglia et al., 1998, Nucleic Acids Res 16, 8186, incorporated herein by reference). The method uses several restriction enzymes to generate a suitable fragment in the known region of a polynucleotide. The fragment is then circularized by intramolecular ligation and used as a PCR template. Divergent primers are designed from the known region. Promoter and flanking sequences may also be isolated by PCR genome walking using a GenomeWalkerยฎ kit (Clontech, Mountain View, Calif.), following the manufacturers instructions. In order to physically assemble full-length clones, standard molecular biology approaches can be utilized (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press, 1987).
[0306] It may be beneficial, when producing a transgenic plant from a particular species, to transform such a plant with a sequence or sequences derived from that species. The benefit may be to alleviate public concerns regarding cross-species transformation in generating transgenic organisms. Additionally when down-regulation of a gene is the desired result, it may be necessary to utilise a sequence identical (or at least highly similar) to that in the plant, for which reduced expression is desired. For these reasons among others, it is desirable to be able to identify and isolate orthologues of a particular gene in several different plant species. Variants (including orthologues) may be identified by the methods described.
Methods for Identifying Variants
Physical Methods
[0307] Variant polynucleotides may be identified using PCR-based methods (Mullis et al., Eds. 1994 The Polymerase Chain Reaction, Birkhauser).
[0308] Alternatively library screening methods, well known to those skilled in the art, may be employed (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press, 1987). When identifying variants of the probe sequence, hybridization and/or wash stringency will typically be reduced relatively to when exact sequence matches are sought.
Computer-Based Methods
[0309] Polynucleotide and polypeptide variants may also be identified by computer-based methods well-known to those skilled in the art, using public domain sequence alignment algorithms and sequence similarity search tools to search sequence databases (public domain databases include Genbank, EMBL, Swiss-Prot, PIR and others). See, e.g., Nucleic Acids Res. 29: 1-10 and 11-16, 2001 for examples of online resources. Similarity searches retrieve and align target sequences for comparison with a sequence to be analyzed (i.e., a query sequence). Sequence comparison algorithms use scoring matrices to assign an overall score to each of the alignments.
[0310] An exemplary family of programs useful for identifying variants in sequence databases is the BLAST suite of programs (version 2.2.5 [November 2002]) including BLASTN, BLASTP, BLASTX, tBLASTN and tBLASTX, which are publicly available from (ftp<dot>ncbi<dot>nih<dot>gov/blast/) or from the National Center for Biotechnology Information (NCBI), National Library of Medicine, Building 38A, Room 8N805, Bethesda, Md. 20894 USA. The NCBI server also provides the facility to use the programs to screen a number of publicly available sequence databases. BLASTN compares a nucleotide query sequence against a nucleotide sequence database. BLASTP compares an amino acid query sequence against a protein sequence database. BLASTX compares a nucleotide query sequence translated in all reading frames against a protein sequence database. tBLASTN compares a protein query sequence against a nucleotide sequence database dynamically translated in all reading frames. tBLASTX compares the six-frame translations of a nucleotide query sequence against the six-frame translations of a nucleotide sequence database. The BLAST programs may be used with default parameters or the parameters may be altered as required to refine the screen.
[0311] The use of the BLAST family of algorithms, including BLASTN, BLASTP, and BLASTX, is described in the publication of Altschul et al., Nucleic Acids Res. 25: 3389-3402, 1997.
[0312] The "hits" to one or more database sequences by a queried sequence produced by BLASTN, BLASTP, BLASTX, tBLASTN, tBLASTX, 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.
[0313] The BLASTN, BLASTP, BLASTX, tBLASTN and tBLASTX algorithms also produce "Expect" values for alignments. The Expect value (E) indicates the number of hits one can "expect" to see by chance when searching a database of the same size containing random contiguous sequences. The Expect value is used as a significance threshold for determining whether the hit to a 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 database screened, one might expect to see 0.1 matches over the aligned portion of the sequence with a similar score simply by chance. 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 that database is 1% or less using the BLASTN, BLASTP, BLASTX, tBLASTN or tBLASTX algorithm.
[0314] Multiple sequence alignments of a group of related sequences can be carried out with CLUSTALW (Thompson, J. D., Higgins, D. G. and Gibson, T. J. (1994) CLUSTALW: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions-specific gap penalties and weight matrix choice. Nucleic Acids Research, 22:4673-4680, www-igbmc<dot>u-strasbg<dot>fr/Biolnfo/ClustalW/Top <dot>html) or T-COFFEE (Cedric Notredame, Desmond G. Higgins, Jaap Heringa, T-Coffee: A novel method for fast and accurate multiple sequence alignment, J. Mol. Biol. (2000) 302: 205-217)) or PILEUP, which uses progressive, pairwise alignments. (Feng and Doolittle, 1987, J. Mol. Evol. 25, 351).
[0315] Pattern recognition software applications are available for finding motifs or signature sequences. For example, MEME (Multiple Em for Motif Elicitation) finds motifs and signature sequences in a set of sequences, and MAST (Motif Alignment and Search Tool) uses these motifs to identify similar or the same motifs in query sequences. The MAST results are provided as a series of alignments with appropriate statistical data and a visual overview of the motifs found. MEME and MAST were developed at the University of California, San Diego.
[0316] PROSITE (Bairoch and Bucher, 1994, Nucleic Acids Res. 22, 3583; Hofmann et al., 1999, Nucleic Acids Res. 27, 215) is a method of identifying the functions of uncharacterized proteins translated from genomic or cDNA sequences. The PROSITE database (www<dot>expasy<dot>org/prosite) contains biologically significant patterns and profiles and is designed so that it can be used with appropriate computational tools to assign a new sequence to a known family of proteins or to determine which known domain(s) are present in the sequence (Falquet et al., 2002, Nucleic Acids Res. 30, 235). Prosearch is a tool that can search SWISS-PROT and EMBL databases with a given sequence pattern or signature.
Function of Variants
[0317] The function of the polynucleotides/polypeptides of the invention can be tested using methods provided herein. In particular, see Example 7.
Methods for Producing Constructs and Vectors
[0318] The genetic constructs of the present invention comprise one or more polynucleotide sequences of the invention and/or polynucleotides encoding polypeptides disclosed, and may be useful for transforming, for example, bacterial, fungal, insect, mammalian or particularly plant organisms. The genetic constructs of the invention are intended to include expression constructs as herein defined.
[0319] Methods for producing and using genetic constructs and vectors are well known in the art and are described generally in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press, 1987; Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing, 1987).
Methods for Producing Host Cells Comprising Constructs and Vectors
[0320] The invention provides a host cell which comprises a genetic construct or vector of the invention. Host cells may be derived from, for example, bacterial, fungal, insect, mammalian or plant organisms.
[0321] Host cells comprising genetic constructs, such as expression constructs, of the invention are useful in methods well known in the art (e.g. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press, 1987; Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing, 1987) for recombinant production of polypeptides. Such methods may involve the culture of host cells in an appropriate medium in conditions suitable for or conducive to expression of a polypeptide of the invention. The expressed recombinant polypeptide, which may optionally be secreted into the culture, may then be separated from the medium, host cells or culture medium by methods well known in the art (e.g. Deutscher, Ed, 1990, Methods in Enzymology, Vol 182, Guide to Protein Purification).
Methods for Producing Plant Cells and Plants Comprising Constructs and Vectors
[0322] The invention further provides plant cells which comprise a genetic construct of the invention, and plant cells modified to alter expression of a polynucleotide or polypeptide. Plants comprising such cells also form an aspect of the invention.
[0323] Methods for transforming plant cells, plants and portions thereof with polynucleotides are described in Draper et al., 1988, Plant Genetic Transformation and Gene Expression. A Laboratory Manual, Blackwell Sci. Pub. Oxford, p. 365; Potrykus and Spangenburg, 1995, Gene Transfer to Plants. Springer-Verlag, Berlin; and Gelvin et al., 1993, Plant Molecular Biol. Manual. Kluwer Acad. Pub. Dordrecht. A review of transgenic plants, including transformation techniques, is provided in Galun and Breiman, 1997, Transgenic Plants. Imperial College Press, London.
[0324] The following are representative publications disclosing genetic transformation protocols that can be used to genetically transform the following plant species: Rice (Alam et al., 1999, Plant Cell Rep. 18, 572); apple (Yao et al., 1995, Plant Cell Reports 14, 407-412); maize (U.S. Pat. Nos. 5,177,010 and 5,981,840); wheat (Ortiz et al., 1996, Plant Cell Rep. 15, 1996, 877); tomato (U.S. Pat. No. 5,159,135); potato (Kumar et al., 1996 Plant J. 9: 821); cassava (Li et al., 1996 Nat. Biotechnology 14, 736); lettuce (Michelmore et al., 1987, Plant Cell Rep. 6, 439); tobacco (Horsch et al, 1985, Science 227, 1229); cotton (U.S. Pat. Nos. 5,846,797 and 5,004,863); perennial ryegrass (Bajaj et al., 2006, Plant Cell Rep. 25, 651); grasses (U.S. Pat. Nos. 5,187,073, 6,020,539); peppermint (Niu et al., 1998, Plant Cell Rep. 17, 165); citrus plants (Pena et al., 1995, Plant Sci. 104, 183); caraway (Krens et al, 1997, Plant Cell Rep, 17, 39); banana (U.S. Pat. No. 5,792,935); soybean (U.S. Pat. Nos. 5,416,011; 5,569,834; 5,824,877; 5,563,04455 and 5,968,830); pineapple (U.S. Pat. No. 5,952,543); poplar (U.S. Pat. No. 4,795,855); monocots in general (U.S. Pat. Nos. 5,591,616 and 6,037,522); brassica (U.S. Pat. Nos. 5,188,958; 5,463,174 and 5,750,871); and cereals (U.S. Pat. No. 6,074,877); pear (Matsuda et al., 2005, Plant Cell Rep. 24(1):45-51); Prunus (Ramesh et al., 2006, Plant Cell Rep. 25(8):821-8; Song and Sink 2005, Plant Cell Rep. 2006; 25(2):117-23; Gonzalez Padilla et al., 2003, Plant Cell Rep. 22(1):38-45); strawberry (Oosumi et al., 2006, Planta; 223(6):1219-30; Folta et al., 2006, Planta. 2006 Apr. 14; PMID: 16614818), rose (Li et al., 2003, Planta. 218(2):226-32), Rubus (Graham et al., 1995, Methods Mol Biol. 1995; 44:129-33). Clover (Voisey et al., 1994, Plant Cell Reports 13: 309-314, and Medicago (Bingham, 1991, Crop Science 31: 1098). Transformation of other species is also contemplated by the invention. Suitable methods and protocols for transformation of other species are available in the scientific literature.
Methods for Genetic Manipulation of Plants
[0325] A number of strategies for genetically manipulating plants are available (e.g. Birch, 1997, Ann Rev Plant Phys Plant Mol Biol, 48, 297). For example, strategies may be designed to increase expression of a polynucleotide/polypeptide in a plant cell, organ and/or at a particular developmental stage where/when it is normally expressed or to ectopically express a polynucleotide/polypeptide in a cell, tissue, organ and/or at a particular developmental stage which/when it is not normally expressed. Strategies may also be designed to increase expression of a polynucleotide/polypeptide in response to external stimuli, such as environmental stimuli. Environmental stimuli may include environmental stresses such as mechanical (such as herbivore activity), dehydration, salinity and temperature stresses. The expressed polynucleotide/polypeptide may be derived from the plant species to be transformed or may be derived from a different plant species.
[0326] Transformation strategies may be designed to reduce expression of a polynucleotide/polypeptide in a plant cell, tissue, organ or at a particular developmental stage which/when it is normally expressed or to reduce expression of a polynucleotide/polypeptide in response to an external stimuli. Such strategies are known as gene silencing strategies.
[0327] Genetic constructs for expression of genes in transgenic plants typically include promoters, such as promoter polynucleotides of the invention, for driving the expression of one or more cloned polynucleotide, terminators and selectable marker sequences to detect presence of the genetic construct in the transformed plant.
[0328] Exemplary terminators that are commonly used in plant transformation genetic construct include, e.g., the cauliflower mosaic virus (CaMV) 35S terminator, the Agrobacterium tumefaciens nopaline synthase or octopine synthase terminators, the Zea mays zin gene terminator, the Oryza sativa ADP-glucose pyrophosphorylase terminator and the Solanum tuberosum PI-II terminator.
[0329] Selectable markers commonly used in plant transformation include the neomycin phophotransferase II gene (NPT II) which confers kanamycin resistance, the aadA gene, which confers spectinomycin and streptomycin resistance, the phosphinothricin acetyl transferase (bar gene) for Ignite (AgrEvo) and Basta (Hoechst) resistance, and the hygromycin phosphotransferase gene (hpt) for hygromycin resistance.
[0330] Use of genetic constructs comprising reporter genes (coding sequences which express an activity that is foreign to the host, usually an enzymatic activity and/or a visible signal (e.g., luciferase, GUS, GFP) which may be used for promoter expression analysis in plants and plant tissues are also contemplated. The reporter gene literature is reviewed in Herrera-Estrella et al., 1993, Nature 303, 209, and Schrott, 1995, In: Gene Transfer to Plants (Potrykus, T., Spangenberg. Eds) Springer Verlag. Berline, pp. 325-336.
[0331] Gene silencing strategies may be focused on the gene itself or regulatory elements which effect expression of the encoded polypeptide. "Regulatory elements" is used here in the widest possible sense and includes other genes which interact with the gene of interest.
[0332] Genetic constructs designed to decrease or silence the expression of a polynucleotide/polypeptide may include an antisense copy of a polynucleotide. In such constructs the polynucleotide is placed in an antisense orientation with respect to the promoter and terminator.
[0333] An "antisense" polynucleotide is obtained by inverting a polynucleotide or a segment of the polynucleotide so that the transcript produced will be complementary to the mRNA transcript of the gene, e.g.,
TABLE-US-00001 5'GATCTA 3' (coding strand) 3'CTAGAT 5' (antisense strand) 3'CUAGAU 5' mRNA 5'GAUCUCG 3' antisense RNA
[0334] Genetic constructs designed for gene silencing may also include an inverted repeat. An `inverted repeat` is a sequence that is repeated where the second half of the repeat is in the complementary strand, e.g.,
TABLE-US-00002 5'-GATCTA...TAGATC-3' 3'-CTAGAT...ATCTAG-5'
[0335] The transcript formed may undergo complementary base pairing to form a hairpin structure. Usually a spacer of at least 3-5 bp between the repeated region is required to allow hairpin formation.
[0336] Another silencing approach involves the use of a small antisense RNA targeted to the transcript equivalent to an miRNA (Llave et al., 2002, Science 297, 2053). Use of such small antisense RNA corresponding to polynucleotide of the invention is expressly contemplated.
[0337] The term genetic construct as used herein also includes small antisense RNAs and other such polynucleotides useful for effecting gene silencing.
[0338] Transformation with an expression construct, as herein defined, may also result in gene silencing through a process known as sense suppression (e.g. Napoli et al., 1990, Plant Cell 2, 279; de Carvalho Niebel et al., 1995, Plant Cell, 7, 347). In some cases sense suppression may involve over-expression of the whole or a partial coding sequence but may also involve expression of non-coding region of the gene, such as an intron or a 5' or 3' untranslated region (UTR). Chimeric partial sense constructs can be used to coordinately silence multiple genes (Abbott et al., 2002, Plant Physiol. 128(3): 844-53; Jones et al., 1998, Planta 204: 499-505). The use of such sense suppression strategies to silence the expression of a sequence operably-linked to promoter of the invention is also contemplated.
[0339] The polynucleotide inserts in genetic constructs designed for gene silencing may correspond to coding sequence and/or non-coding sequence, such as promoter and/or intron and/or 5' or 3' UTR sequence, or the corresponding gene.
[0340] Other gene silencing strategies include dominant negative approaches and the use of ribozyme constructs (McIntyre, 1996, Transgenic Res, 5, 257)
[0341] Pre-transcriptional silencing may be brought about through mutation of the gene itself or its regulatory elements. Such mutations may include point mutations, frameshifts, insertions, deletions and substitutions.
Plants
[0342] The term "plant" is intended to include a whole plant or any part of a plant, propagules and progeny of a plant.
[0343] The term `progeny` as used herein refers to any cell, plant or part thereof which has been obtained or derived from a cell or transgenic plant of the present invention. Thus, the term progeny includes but is not limited to seeds, plants obtained from seeds, plants or parts thereof, or derived from plant tissue culture, or cloning, techniques.
[0344] The term `propagule` means any part of a plant that may be used in reproduction or propagation, either sexual or asexual, including seeds and cuttings.
[0345] A "transgenic" or transformed" plant refers to a plant which contains new genetic material as a result of genetic manipulation or transformation. The new genetic material may be derived from a plant of the same species as the resulting transgenic of transformed plant or from a different species. A transformed plant includes a plant which is either stably or transiently transformed with new genetic material.
[0346] The plants of the invention may be grown and either self-ed or crossed with a different plant strain and the resulting hybrids, with the desired phenotypic characteristics, may be identified. Two or more generations may be grown. Plants resulting from such standard breeding approaches also form part of the present invention.
BRIEF DESCRIPTION OF DRAWINGS
[0347] Further aspects of the present invention will become apparent from the following description which is given by way of example only and with reference to the accompanying drawings in which:
[0348] FIG. 1 shows the general condensed tannin pathway;
[0349] FIG. 2(A) illustrates the cDNA sequence representing the full length cDNA sequence of TaMYB14, cloned from mature T. arvense leaf tissue.
[0350] FIG. 2(B) illustrates the amino acid translation of TaMYB14.
[0351] FIG. 3 shows the transcript levels of TaMYB14 in varying tissues from Trifolium species and cultivars grown in identical glasshouse conditions. Lane 1, (ladder); Lane 2, T. repens mature leaf cDNA library (Cultivar Huia); Lane 3, T. repens mature root cDNA library (Cultivar Huia); Lane 4, T. repens mature stolon cDNA library (Cultivar Huia); Lane 5, T. repens mature floral cDNA library (Cultivar DC111); Lane 6, T. repens emerging leaf cDNA (Cultivar Huia); Lane 7, T. repens mature leaf cDNA (High anthocyanin Cultivar Isabelle); Lane 8, T. arvense immature leaf cDNA (Cultivar AZ2925); Lane 9, T. arvense mature leaf cDNA (Cultivar AZ2925); Lane 10, T. repens meristem floral cDNA (Cultivar Huia); Lane 11, T. repens meristem leaf cDNA (Cultivar Huia); Lane 12, T. repens meristem trichome only cDNA (Cultivar Huia); Lane 13, T. occidentale mature plant (leaf, root and stolon cDNA library (Cultivar Huia); Lane 14, T. repens mature nodal cDNA library (Cultivar Huia); Lane 15, cloned T. arvense MYB14cDNA clone in TOPO, Lane 16, cloned T. arvense MYB14 genomic clone in TOPO, lane 17, T. occidentale genomic DNA; lane 17, T. repens genomic DNA; lane 17, T. arvense genomic DNA; Lane 20, (ladder).
[0352] FIG. 4 shows the transcript levels of BANYULS (A) and LAR (B) in varying tissues from Trifolium species and cultivars grown in identical glasshouse conditions. Lane 1, (ladder); Lane 2, T. repens mature leaf cDNA library (Cultivar Huia); Lane 3, T. repens mature root cDNA library (Cultivar Huia); Lane 4, T. repens mature stolon cDNA library (Cultivar Huia); Lane 5, T. repens mature floral cDNA library (Cultivar DC111); Lane 6, T. repens emerging leaf cDNA (Cultivar Huia); Lane 7, T. repens mature leaf cDNA (High anthocyanin Cultivar Isabelle); Lane 8, T. arvense immature leaf cDNA (Cultivar AZ2925); Lane 9, T. arvense mature leaf cDNA (Cultivar AZ2925); Lane 10, T. repens meristem floral cDNA (Cultivar Huia); Lane 11, T. repens meristem leaf cDNA (Cultivar Huia); Lane 12, T. repens meristem trichome only cDNA (Cultivar Huia); Lane 13, T. occidentale mature plant (leaf, root and stolon cDNA library (Cultivar Huia); Lane 14, T. repens mature nodal cDNA library (Cultivar Huia); Lane 15, cloned T. arvense cDNA BAN or LAR clone in TOPO, Lane 16, cloned T. arvense BAN or LAR genomic clone in TOPO, lane 17, T. occidentale genomic DNA; lane 17, T. repens genomic DNA; lane 17, T. arvense genomic DNA; Lane 20, (ladder).
[0353] FIG. 5 shows the results of DMACA staining of transformed white clover mature leaf tissue. DMACA staining (light/dark grey colour) of mature white clover leaf tissue identifying Condensed Tannins in (A) Wild Type and (B) transformed with TaMYB14 gene.
[0354] FIG. 6 shows the plasmid vector M14ApHZBarP, used for plant transformation. E1, E2 and E3 indicate the 3 exons of the genomic allele TaMYB14-1.
[0355] FIG. 7 shows the alignment of the full-length cDNA sequences of Trifolium MYB14, top BLASTN hits and AtTT2 with similarities highlighted in light grey.
[0356] FIG. 8 shows the alignment of the translated open reading frames of Trifolium arvense TaMYB14, top BLASTP hits and AtTT2 with similarities highlighted in light grey and motifs boxed.
[0357] FIG. 9 shows the alignment of the full-length protein sequences of TaMYB14 (expressed TaMYB14FTa and silent TaMYB14-2S), ToMYB14 allele, and TrMYB14 alleles with differences highlighted in dark grey/white regions and deletion/insertion areas highlight in boxes.
[0358] FIG. 10 shows the alignment of the full-length genomic DNA sequences of Trifolium repens TrMYB14 allelles (TRM*) aligned with Trifolium arvense TaMYB14 alleles (TaM3, TaM4), with differences in exons (light grey) and introns (dark grey) highlighted.
[0359] FIG. 11 shows the alignment of the full-length genomic DNA sequences of Trifolium occidentale ToMYB14 allelles (To1, To6) aligned with Trifolium arvense TaMYB14 alleles (TaM3, TaM4), with differences in exons (light grey) and introns (dark grey) highlighted.
[0360] FIG. 12 shows the alignment of the full-length genomic DNA sequences of Trifolium arvense TaMYB14 allelles (Ta*) and Trifolium affine TafMYB14 allelles (Tar) with exons (light grey) and introns (dark grey) showing differences.
[0361] FIG. 13 shows the Vector NTI map of the construct pHZbarSMYB containing the NotI fragment from MYB14pHANNIBAL, which contains a segment of TaMYB14 cDNA from T. arvense in sense (SMYB14F) and antisense (SMYB14R) orientation flanking the pdk intron.
[0362] FIG. 14 shows the PCR reaction for the presence of M14ApHZBAR from genomic DNA isolated from putatively transformed white clover. Lanes; A1, B1 Ladder; A2-18 and B2-B15 transformed clovers, B16 non-transformed white clover, B17 plasmid control, B18 water control. Primers were 35S (promoter) and PMYBR (to 3'end of gene) amplifying a 1,244 bp fragment.
[0363] FIG. 15 shows the results of DMACA screening of wild type (A) and transgenic (B to D) T. repens leaves, transformed with TaMYB14 construct.
[0364] FIG. 16 shows oil microscopy of trichomes (E-G), epidermal cells (H) and mesophyll cell (I-K) of DMACA stained transgenic leaflets expressing the TaMyb14A gene (SEQ ID NO:2).
[0365] FIG. 17 shows Grape Seed Extract Monomers--The SRM chromatograms of the monomers in a grape seed extract are shown below. Trace A is a sum of the product ions 123, 139 and 165 m/z of the SRM of 291.3 m/z (catechin (C) and epicatechin (EC)). Trace B is a sum of the product ions 139 and 151 m/z of the SRM of 307.3 m/z (gallocatechin (GC) and epigallocatechin (EGC)).
[0366] FIG. 18 shows Grape Seed Extract Dimers and Trimers. The SRM chromatograms of the dimers and trimers in a grape seed extract are shown below. Trace A is a sum of the product ions 291, 409 and 427 m/z of the SRM of 579.3 m/z (PC:PC dimer). Trace B is a sum of the product ions 291, 307, 427 and 443 m/z of the SRM of 595.3 m/z (PC:PD dimer). Trace C is a sum of the product ions 291, 577 and 579 m/z of the SRM of 867.3 m/z (3PC trimer). The MS2 spectra of a PC:PC dimer, a PC:PD dimer, and two 3PC trimers are provided as evidence of identification of these metabolites.
[0367] FIG. 19 shows the SRM chromatograms of monomers for the control (White Clover -ve) and transgenic (White Clover +ve) plants expressing MYB14 are shown below. Trace A is a sum of the product ions 123, 139 and 165 m/z of the SRM of 291.3 m/z (PC; catechin and epicatechin). Trace B is a sum of the product ions 139 and 151 m/z of the SRM of 307.3 m/z (PD; gallocatechin and epigallocatechin). The chromatogram scales are fixed to show the appearance of monomers in the modified plant. No monomers were detected in the control plant. The MS2 spectra of epicatechin (EC) and epigallocatechin (EGC) are provided from the modified plant as evidence of identification of these metabolites.
[0368] FIG. 20 shows the SRM chromatograms of dimers for the control (White Clover -ve) and transgenic (White Clover +ve) plants expressing MYB14 are shown below. Trace A is a sum of the product ions 291, 409 and 427 m/z of the SRM of 579.3 m/z (PC:PC dimer). Trace B is a sum of the product ions 291, 307, 427 and 443 m/z of the SRM of 595.3 m/z (PC:PD dimer). Trace C is a sum of the product ions 307 and 443 m/z of the SRM of 611.3 m/z (PD:PD dimer). The chromatogram scales are fixed to show the appearance of dimers in the modified plant. No dimers were detected in the control plant. The MS2 spectra of three PD:PD dimers (1-3) and one PC:PD mixed dimer (4) are provided from the modified plant as evidence of identification of these metabolites.
[0369] FIG. 21 shows the SRM chromatograms of trimers for the control (White Clover -ve) and transgenic (White Clover +ve) plants expressing MYB14 are shown below. Trace A is a sum of the product ions 291, 577 and 579 m/z of the SRM of 867.3 m/z (3PC trimer). Trace B is a sum of the product ions 291, 307, 427, 443, 577, 579, 593, 595 and 757 m/z of the SRM of 883.3 m/z (PC:PD dimer). Trace C is a sum of the product ions 291, 307, 443, 593, 595, 611, 731, 757 and 773 m/z of the SRM of 899.3 m/z (1 PC:2PD trimer). Trace D is a sum of the product ions 307, 443, 609, 611, 747, 773 and 789 m/z of the SRM of 915.3 m/z (3PD trimer). The chromatogram scales are fixed to show the appearance of trimers in the modified plant. No trimers were detected in the control plant. The MS2 spectra of a 3PD trimer and a 1 PC:2PD mixed trimer are provided from the modified plant as evidence of identification of these metabolites.
[0370] FIG. 22 shows the PCR reaction for the presence of M14ApHZBAR from genomic DNA isolated from putatively transformed tobacco plantlets. Lanes; A1, Ladder; A2-10 transformed tobacco, A13, 14, tobacco controls, A15 plasmid control, Primers were 35S (promoter) and PMYBR (to 3'end of gene) amplifying a 1,244 bp fragment.
[0371] FIG. 23 shows the results of DMACA screening of transgenic (A to G) tobacco (Nicotiana tabacum) leaves, transformed with M14ApHZBAR construct.
[0372] FIG. 24 shows the SRM chromatograms for the control (wild type) and modified (transgenic) plants expressing MYB14 are shown below. Trace A is a sum of the product ions 123, 139 and 165 m/z of the SRM of 291.3 m/z (PC; catechin and epicatechin). Trace B is a sum of the product ions 139 and 151 m/z of the SRM of 307.3 m/z (PD; gallocatechin and epigallocatechin). Trace C is a sum of the product ions 291, 409 and 427 m/z of the SRM of 579.3 m/z (PC:PC dimer). Trace D is a sum of the product ions 291, 577 and 579 m/z of the SRM of 867.3 m/z (PC:PC:PC timer). The chromatogram scales are fixed to show the appearance of monomers, dimers and trimers in the modified plant. Note, no mixed PC:PD or 100% PD dimers or trimers were detected.
[0373] FIG. 25 shows the MS2 spectra of epicatechin (EC), gallocatechin (GC), epigallocatechin (EGC), PC:PC dimer 1 and 2, and the PC:PC:PC trimer are provided from the modified (transgenic) plants expressing MYB14, as evidence of identification of these metabolites.
[0374] FIG. 26 shows the PCR reaction for the presence of M14pHANNIBAL in genomic DNA isolated from putatively transformed T. arvense. Lanes; A1 pHANNIBAL negative control vector, A2 M14ApHZBAR containing 35S and genomic gene construct-control amplifying a 1,244 bp fragment; A3 M14pHANNIBAL positive plasmid control containing hpRNA construct, A4 pHANNIBAL containing MYB fragment in antisense orientation upstream of ocs terminator (negative control), A5 pHZBARSMYB positive plasmid control, A6 Ladder, A7-18 transformed T. arvense, A19 genomic DNA wild type T. arvense, A20 water control.
[0375] B: B1 Ladder, B2-B11 transformed T. arvense, B12 M14pHANNIBAL positive plasmid control. Primers were 35S (promoter) and PHMYBR (to 3'end of gene) amplifying a 393 bp fragment.
[0376] FIG. 27 shows the results of DMACA screening of wild type T. arvense callus (A) and plantlets (B to D) regenerated on tissue culture media. No DMACA staining occurs in callus and DMACA screening of transgenic (E to L) T. arvense plantlets regenerated on tissue culture media. Staining is greatly diminished compared to wild type plants.
[0377] FIG. 28 shows the four monomer SRM chromatograms for T. arvense control and knockout plants: Trace A is a sum of the product ions 123, 139 and 165 m/z of the SRM of 291.3 m/z (PC; catechin and epicatechin) for a control plant. B is a sum of the product ions 123, 139 and 165 m/z of the SRM of 291.3 m/z (PC; catechin and epicatechin) for a knockout plant. C is a sum of the product ions 139 and 151 m/z of the SRM of 307.3 m/z (PD; gallocatechin and epigallocatechin) for a control plant. D is a sum of the product ions 139 and 151 m/z of the SRM of 307.3 m/z (PD; gallocatechin and epigallocatechin) for a knockout plant. The MS2 spectra are provided from the control plant as evidence of catechin and gallocatechin in the control plant. The chromatogram scales for traces A, B, C and D have been fixed to show the disappearance of catechin and gallocatechin in the knockout plant.
[0378] FIG. 29 shows the dimer SRM chromatograms for the control and knockout T. arvense plants. Trace A is a sum of the product ions 291 and 427 m/z of the SRM of 579.3 m/z (PC:PC dimer). Trace B is a sum of the product ions 307, 427 and 443 m/z of the SRM of 595.3 m/z (PC:PD dimer). Trace C is a sum of the product ions 307 and 443 m/z of the SRM of 611.3 m/z (PD:PD dimer). The chromatogram scales are fixed to show the disappearance of dimers in the knockout plant. The MS2 spectra are provided from the control plant as evidence of all three types of dimers in the control.
[0379] FIG. 30 shows the PCR analysis for the presence of pTaMyb14A from genomic DNA (SEQ ID NO:2) isolated from putatively transformed alfalfa. Lanes L; ladder; 1-3, non-transformed, 4-10 transformed, 11 wild type, 12 water control, 13 plasmid control. Primers were 35S and PMYBR (to 3'end of gene).
[0380] FIG. 31 shows the PCR analysis for the presence of M14ApHZBAR from genomic DNA isolated from putatively transformed brassica plantlets. Lane 8, brassica control; Lane 18 Ladder; Lane 1-7 and 9-17 transformed brassica. Primers were 35S (promoter) and PMYBR (to 3'end of gene) amplifying a 1,244 bp fragment.
[0381] FIG. 32 shows the results of DMACA screening of wild type brassica (Brassica oleracea) (A) and transgenic (B to D) leaves, transformed with M14ApHZBARP construct.
[0382] FIG. 33 shows the SRM chromatograms of the product ions 123, 139 and 165 m/z of the SRM of 291.3 m/z (catechin (C) and epicatechin (EC)) in two controls and a transgenic brassica expressing MYB14. The MS2 spectra of the epicatechin detected in the green control and the transgenic +ve sample are provided as evidence of identification of these metabolites. No epicatechin was detected in the red control sample.
[0383] FIG. 34 shows an alignment of all the Trifolium MYB14 protein sequences identified by the applicant.
[0384] FIG. 35 shows the percent identity between the sequences aligned in FIG. 34.
[0385] FIG. 36 shows DMACA staining of leaves from wild type (A) and transgenic (B) Medicao plants transformed with a CaMV35S::TaMYB14 construct (B)
[0386] FIG. 37 shows LC-MS/MS composite extracted ion chromatograms of ions 123+139+151+165 m/z for catechin (peak #1) and epicatechin (peak #2) (traces A1-B1) from MS2 product ion scans of 291 m/z and ions 139+151 m/z for gallocatechin (not detected) and epigallocatechin (not detected) (traces A2-B2) from MS2 product ion scans of 307 m/z in A)--M. sativa wild type and B)--M. sativa transformed with CaMV35S::TaMYB14.
[0387] FIG. 38 shows LC-MS/MS composite extracted ion chromatograms of ions 291+409+427 m/z from MS2 product ion scans of 579 m/z of PC:PC dimers in leaf extracts of A)--M. sativa wild type and B)--M. sativa transformed with CaMV35S::TaMYB14.
[0388] FIG. 39 shows LC-MS/MS composite extracted ion chromatograms of ions 291+579 m/z from MS2 product ion scans of 867 m/z for PC:PC:PC trimers (traces A1-B1); ions 291+307+443+579+595+757 m/z from the MS2 product ion scans of 883 m/z for PC:PC:PD trimers (traces A2-B2); ions 291+307+443+579+595+757+773 m/z from the MS2 product ion scans of 899 m/z for PC:PD:PD trimers (traces A3-B3); ions 307+611+773+789 m/z from the MS2 product ion scans of 915 m/z for PD:PD:PD trimers (traces A4-B4) in A)--M. sativa wild type and B)--M. sativa transformed with CaMV35S::TaMYB14.
TABLE-US-00003
[0389] BRIEF DESCRIPTION OF SEQUENCE LISTING SEQ ID NO: Description Corresponding sequence 1 Polynucleotide, Trifolium arvense, TaMYB14-1 cDNA Sequence of Ta MYB14 cDNA of expressed gene 2 Polynucleotide, Trifolium arvense, TaMYB14-1 gDNA Sequence genomic of Ta MYB14 1 from allele 1 from Trifolium arvense. 3 Polynucleotide, Trifolium arvense, TaMYB14-2 gDNA Sequence genomic of Ta MYB14 2 from allele 2 from Trifolium arvense. 4 Polynucleotide, Trifolium affine, TafMYB14-1 gDNA Sequence genomic of Taf MYB14 1 from allele 1 from Trifolium affine. 5 Polynucleotide, Trifolium affine, TafMYB14-1 cDNA Sequence of Taf MYB14 cDNA of expressed gene 6 Polynucleotide, Trifolium affine, TafMYB14-2 gDNA Sequence genomic of Taf MYB14 2 from allele 2 from Trifolium affine. 7 Polynucleotide, Trifolium occidentale, ToMYB14-1 Sequence genomic of ToMYB14 1 gDNA from allele 1 from Trifolium occidentale. 8 Polynucleotide, Trifolium occidentale, ToMYB14-2 Sequence genomic of ToMYB14 2 gDNA from allele 2 from Trifolium occidentale. 9 Polynucleotide, Trifolium repens, TrMYB14-1 gDNA Sequence genomic of TrMYB14 1 from allele 1 from Trifolium repens. 10 Polynucleotide, Trifolium repens, TrMYB14-2 gDNA Sequence genomic of TrMYB14 2 from allele 2 from Trifolium repens. 11 Polynucleotide, Trifolium repens, TrMYB14-3 gDNA Sequence genomic of TrMYB14 3 from allele 3 from Trifolium repens. 12 Polynucleotide, Trifolium repens, TrMYB14-4 gDNA Sequence genomic of TrMYB14 4 from allele 4 from Trifolium repens. 13 Polynucleotide, Trifolium arvense, TaMYB14-1 cDNA cDNA sequence representing the full length cDNA sequence of TaMYB14 14 Polypeptide, Trifolium arvense, TaMYB14-1 amino acid translation of TaMYB14 15 Polypeptide, artificial, consensus motif similar to Motif of subgroup 5 (Stracke et al., 2001) common to known CT MYB activators 16 Polypeptide, artificial, consensus motif common to known anthocyanin MYB activators (Motif of subgroup 6, Stracke et al., 2001) 17 Polypeptide, artificial, consensus novel MYB motif of MYB14 TFs 18 Polynucleotide, artificial, primer MYB domain hunt-MYBFX 19 Polynucleotide, artificial, primer MYB domain hunt-MYBFY 20 Polynucleotide, artificial, primer MYB domain hunt-MYBFZ 21 Polynucleotide, artificial, primer Isolation of full length-M14ATG 22 Polynucleotide, artificial, primer Isolation of full length-M14TGA 23 Polynucleotide, artificial, primer Gene walking-M14TSP1 24 Polynucleotide, artificial, primer Gene walking-M14TSP2 25 Polynucleotide, artificial, primer Gene walking-M14TSP3 26 Polynucleotide, artificial, primer Cloning into vector-M14FATG 27 Polynucleotide, artificial, primer Lotus corniculatus-MYBLF 28 Polynucleotide, artificial, primer Lotus corniculatus-MYBLR 29 Polynucleotide, artificial, primer 5' UTR end of MYB14-MYB148N 30 Polynucleotide, artificial, primer 3' UTR end of MYB14-MYB14RR 31 Polynucleotide, artificial, primer Primer for intron 1-I5 32 Polynucleotide, artificial, primer Primer for intron 1-I3 33 Polynucleotide, artificial, primer Gene walking-TSP4 34 Polynucleotide, artificial, primer Gene walking-TSP5 35 Polynucleotide, artificial, primer 5'start site Forward-MYB148F 36 Polynucleotide, artificial, primer 5'start site Reverse-MYB14RR 37 Polynucleotide, artificial, primer Expression analysis/ Silencing vector-MYB14F 38 Polynucleotide, artificial, primer Expression analysis/ Silencing vector-MYB14R 39 Polynucleotide, artificial, primer Gene walking-MYB14R2 40 Polynucleotide, artificial, primer Gene walking-MYB14R3 41 Polynucleotide, artificial, primer Sequencing-M13 Forward 42 Polynucleotide, artificial, primer Sequencing-M13 Reverse 43 Polynucleotide, artificial, primer cDNA production-BD SMART II ยฎ A Oligonucleotide 44 Polynucleotide, artificial, primer cDNA production-3' BD SMART ยฎ CDS Primer II A 45 Polynucleotide, artificial, primer Amplification of mRNA-5' PCR Primer II A 46 Polypeptide, Trifolium arvense, TaMYB14-2 47 Polypeptide, Trifolium affine, TafMYB14-1 48 Polypeptide, Trifolium affine, TafMYB14-2 49 Polypeptide, Trifolium occidentale, ToMYB14-1 50 Polynucleotide, Trifolium occidentale, ToMYB14-2 51 Polypeptide, Trifolium repens, TrMYB14-1 52 Polypeptide, Trifolium repens, TrMYB14-2 53 Polypeptide, Trifolium repens, TrMYB14-3 54 Polypeptide, Trifolium repens, TrMYB14-4 55 Polynucleotide, Trifolium arvense, TaMYB14-1 cDNA/ORF 56 Polynucleotide, Trifolium arvense, TaMYB14-2 cDNA/ORF 57 Polynucleotide, Trifolium affine, TafMYB14-1 cDNA/ORF 58 Polynucleotide, Trifolium affine, TafMYB14-2 cDNA/ORF 59 Polynucleotide, Trifolium occidentale, ToMYB14-1 cDNA/ORF 60 Polynucleotide, Trifolium occidentale, ToMYB14-2 cDNA/ORF 61 Polynucleotide, Trifolium repens, TrMYB14-1 cDNA/ORF 62 Polynucleotide, Trifolium repens, TrMYB14-2 cDNA/ORF 63 Polynucleotide, Trifolium repens, TrMYB14-3 cDNA/ORF 64 Polynucleotide, Trifolium repens, TrMYB14-4 cDNA/ORF 65 Polynucleotide, Trifolium arvense, silencing sequence 66 Polynucleotide, artifical, primer, MYB F1 67 Polynucleotide, artifical, primer, MYB R 68 Polynucleotide, artifical, primer, MYB F 69 Polynucleotide, artifical, primer, MYB R1 70 Polynucleotide, Lotus japonicus LjTT2a from FIG. 7 71 Polynucleotide, Trifolium affine MYB14 Taf from FIG. 7 72 Polynucleotide, Glycine max MYB92Gmax from FIG. 7 73 Polynucleotide, Daucus carota MYB3 from FIG. 7 74 Polynucleotide, Gossypium hirsutum GHMYB10 from FIG. 7 75 Polynucleotide, Brassica napus BnTT2-3 from FIG. 7 76 Polynucleotide, Gossypium hirsutum GHMYB36 from FIG. 7 77 Polypeptide, Arabidopsis thaliana AtTT2 from FIG. 8 78 Polypeptide, Brassica napus BnTT2-1 from FIG. 8 79 Polypeptide, Zea mays ZMP1 from FIG. 8 80 Polypeptide, Gossypium hirsutum GHMYB10 from FIG. 8 81 Polypeptide, Vitis vinifera VvMYBPA1 from FIG. 8 82 Polypeptide, Lotus japonicus LjTT2a from FIG. 8 83 Polypeptide, Glycine max MYB185Gmax from FIG. 8 84 Polypeptide, Malus domestica MYB11 Malus from FIG. 8 85 Polypeptide, Trifolium arvense TaMYB14-25 from FIG. 9 86 Polypeptide, Trifolium repens TrMYB14f from FIG. 9 87 Polypeptide, Trifolium occidentale ToMYB14 from FIG. 9 88 Polypeptide, Artificial Consensus sequence from FIG. 9 89 Polynucleotide, Trifolium repens TRM6 from FIG. 10 90 Polynucleotide, Trifolium repens TRM14 from FIG. 10 91 Polynucleotide, Trifolium occidentale To1 from FIG. 11 92 Polynucleotide, Trifolium occidentale To6 from FIG. 11 93 Polynucleotide, Trifolium affine Taf11 from FIG. 12 94 Polynucleotide, Trifolium affine Taf2 r#2 from FIG. 12 95 Polynucleotide, Trifolium affine Taf3 from FIG. 12 96 Polynucleotide, Trifolium affine Taf7 from FIG. 12 97 Polynucleotide, Trifolium affine Taf4 from FIG. 12 98 Polynucleotide, Trifolium affine Taf10 from FIG. 12 99 Polypeptide, Trifolium occidentale ToMYB14-2 from FIG. 12 100 Polypeptide, Artificial Consensus sequence from FIG. 34 101 Polypeptide, Artificial Motif associated with MYB TFs that regulate CT pathways 102 Polypeptide, Artificial Motif of subgroup 5 common to previously known CT MYB activators
[0390] The invention will now be illustrated with reference to the following non-limiting examples.
Example 1
Identification of the MYB14 Genes/Nucleic Acids/Proteins of the Invention, and Analysis of Expression Profiles
Introduction
[0391] Using primers designed to the MYB domain of legume species, the applicant has amplified sequences encoding novel MYB transcription factors (TFs) by PCR of cDNA and genomic DNA (gDNA) isolated from a range of Trifolium species. These species differ in their capacity to accumulate CTs in mature leaf tissue. Because white clover does not express CT genes in leaf tissue the applicants used an alternative strategy that allowed isolation of the expressed MYB TF from closely related Trifolium species (T. arvense; T. affine) which do accumulate CTs in all cells of foliar tissue throughout the life of the leaf. This was achieved by investigating the differential expression patterns of MYB TFs in various Trifolium leaf types; namely (a) within white clover (T. repens) leaf tissue, where CT gene expression is restricted to the leaf trichomes during meristematic development prior to leaf emergence; (b) within the closely related species (T. arvense), where CT gene expression is found within most cells of the leaf during its entire life span (except the trichome hairs); (c) with white clover mature leaf tissue where CT biosynthesis has already ceased. Such specific temporal and spatial expression requires the differential regulation by different MYB TFs specific to the CT branch pathway. Comparison of the MYB TFs from each leaf type eliminated common MYB factors that have functions other than in CT biosynthesis. Analysis of the remaining isolated MYB TFs allowed identification of those that are unique to CT accumulating tissues.
[0392] Sequencing of PCR products resulted in the identification of a previously unidentified MYB TFs from a number of Trifolium species. Full-length sequencing of these MYB genes revealed a highly dissimilar protein code when compared to the published AtTT2 sequence (NP_198405), including the presence of several deletions and insertions of bases in the genes from the different Trifolium species (FIGS. 7 and 8). Translation of the cDNA sequence revealed that the protein encoded by this MYB TF also has substantial number of amino acid deletions, insertions, and exchanges (FIG. 9). The applicants have designated this gene TaMYB14. Analysis of full-length gDNA sequences from 2 different Trifolium species revealed the presence of three exons and two introns of varying sizes in all TaMYB14 isoforms/alleles (FIGS. 10-12).
[0393] Seeds from a number of accessions representing various genotypes from four Trifolium species, respectively, were grown in a glasshouse and the presence or absence of CTs was determined in leaves using DMACA staining. Primers specific for TaMYB14 were designed and transcript levels in various tissues were determined by PCR. Expression of TaMYB14 was correlated with CT accumulation in leaf tissues. Its expression was undetectable in CT free tissues. TaMyb14 was very highly expressed in tissues actively accumulating CTs and coincided with the detectable expression of the two enzymes specifically involved in CT biosynthesis; namely ANR and LAR.
[0394] Transformation and over-expression of TaMYB14 in white clover (see Example 2) resulted in increased levels of CTs in tissues usually devoid of CTs. This shows that expression of TaMYB14 is critical for the accumulation of CTs. Overexpression of TaMYB14 in T. repens by means of transgenesis will therefore allow accumulation of significant levels of CTs in foliar tissues of various plant species, thereby providing the means to improve pasture quality for livestock.
Materials and Methods
Plant Material and Analysis of Condensed Tannin Levels
[0395] Seeds from several cultivars of four legume species differing in their levels of foliar CT were grown in glasshouses. Trifolium repens (Huia); T. arvense (AZ2925; AZ4755; AZ1353); T. affine (AZ925), and T. occidentale (AZ4270). Plant material of various ages and types were harvested and the material immediately frozen in liquid nitrogen and subsequently ground and used for isolation of DNA or RNA
DMACA Staining of Plant Material.
[0396] CTs were histochemically analysed using the acidified DMACA (4-dimethylamino-cinnamaldehyde) method essentially as described by Li et al. (1996). This method uses the DMACA (p-dimethylaminocinnamaldehyde) reagent as a rapid histochemical stain that allows specific screening of plant material for very low CT accumulation. The DMACA-HCl protocol is highly specific for proanthocyanidins. This method was preferentially used over the vanillin test as anthocyanins seriously interfere with the vanillin assay. Tissues of various ages were sampled and tested.
Selection Methods of MYB R2R3 Candidates
[0397] Two methods were used to identify legume sequences containing a MYB R2R3 DNA-binding domain: hidden Markov models (HMMs) and profiles. Both methods depend on first creating a "model" of the domain from known MYB R2R3 DNA-binding domain protein sequences, which is then used as the basis of the search. The HMM and profile models were created using known plant MYB R2R3 domains as indicated in Table 1 below. These were taken from FIG. 2 in Miyake et. al. (2003) and FIG. 4C in Nesi et. al. (2001; the human MYB sequence in this figure was excluded). The species distribution of the sequences used in constructing the model as follows:
TABLE-US-00004 TABLE 1 Plant MYB R2R3 domains taken from Miyake et. al. (2003) and Nesi et. al. (2001) Source Species Domain count Miyake et. al. (2003) Lotus japonicus 3 Glycine max 1 Nesi et. al. (2001) Arabidopsis thaliana 10 Zea mays 3 Hordeum vulgare subsp. vulgare 2 Oryza sativa 1 Petunia x hybrida 1 Picea mariana 1
[0398] The legume sequence sets searched are listed in Table 2 below. Prior to searching, all EST and EST contig sets were translated in six frames to generate protein sequences suitable for the HMM/profile analyses. The M. truncatula protein sequences were used as-is (these are FGENESH gene predictions obtained from TIGR).
[0399] The HMMER program hmmbuild was used to create an HMM from the model DNA-binding domains, and this was searched against the legume sequence sets using the HMMER program hmmsearch (E-value cut-off=0.01). The EMBOSS program prophecy was used to create a profile from the same domains, and this was also searched against the legume sequences using the EMBOSS program profit (score cut-off=50). The numbers of hits identified by each method in each set of sequences are listed in Table 2 below:
TABLE-US-00005 TABLE 2 Legume sequence sets searched Number of Number Number hits passed Total of hits- of hits- to number of Profile HMM phylogenetic Sequence set sequences method method analysis White clover EST 17,758 18 24 17 contigs (CS35) White clover PG NR 159,017 0 9 3 Red clover EST contigs 38,099 1 2 0 Lotus EST contigs 28,460 5 9 4 Soybean EST contigs 63,676 15 40 15 Medicago truncatula 41,315 60 80 69 predicted proteins Medicago sativa 5,647 1 2 1 glandular trichome ESTs Total 353,972 100 166 109
[0400] The HMM method appeared to be more sensitive than the profile method, identifying all profile hits as well as many additional hits. For this reason the HMM method was selected as the method of choice--the HMM hit proteins were used to generate the alignments and were passed to the phylogenetic analysis. The profile hits are still quite useful: the profile method is more stringent and therefore there is a higher likelihood that the profile candidates represent true hits.
Generation of Alignments
[0401] DNA-binding domain sequences were extracted from the 166 legume MYB R2R3 candidates identified above. The protein domains were aligned using the HMMER alignment program hmmalign, which aligns the domains using information in the original HMM model. Nucleotide alignments were generated by overlaying the corresponding nucleotide sequences onto the protein alignments, thereby preserving the structure of the alignments at the protein level. This was done to obtain a more accurate alignment that better represents the domain structure.
Phylogenetic Analysis
[0402] A phylogenetic analysis was performed on plant MYB R2R3 DNA-binding domains, to see whether the resulting tree nodes could be used to identify MYB R2R3 subtypes, related to TT2 transcription factors. 109 Full length DNA-binding domains were extracted from the 166 legume MYB R2R3 candidates identified in this study, and these were combined with the known MYB R2R3 genes from Nesi et. al. (2001) and Miyake et. al. (2003), giving 130 DNA-binding domains in total. A protein alignment of these 130 domains was generated using hmmalign, and corresponding nucleotide domain sequences were aligned based on this. The nucleotide alignment was submitted to a maximum likelihood analysis to generate a phylogenetic tree based on 100 bootstrap replicates, using the programs fastDNAml and the Phylip program consensus to generate the consensus tree. This information was used to design three primers to legume MYBR2R3 domain.
Isolation of DNA and RNA, and cDNA Synthesis
[0403] Genomic DNA was isolated from fresh or frozen plant tissues (100 mg) using DNeasyยฎ Plant Mini kit (Qiagen) following the manufacturer's instructions. DNA preparations were treated with RNAse H (Sigma) to remove RNA from the samples. Total RNA was isolated from fresh or frozen tissues using RNeasyยฎ Plant Mini kit (Qiagen). Isolated total RNA (100 ฮผg) was treated with RNAse free DNAse I to remove DNA from the samples during the isolation, following the manufacturer's instructions. Concentration and purity of DNA and RNA samples was assessed by determining the ratio of absorbance at 260 and 280 nm using a NanoDrop ND-100 spectrophotometer. Total RNA (1 ฮผg) was reverse-transcribed into cDNA using SMARTยฎ cDNA Synthesis Kit (Clontech) using the SMARTยฎ CDS primer IIA and SMART IIยฎ A oligonucleotides following manufacturer's instructions.
Polymerase Chain Reaction (PCR) and TOPO Cloning of PCR Products
[0404] Standard PCR reactions were carried out in a Thermal Cycler (Applied Biosystems), a quantity of approximately 5 ng DNA or 1 ฮผl cDNA was used as template. The thermal cycle conditions were as follows: Initial reaction at 94ยฐ C. for 30 sec, 35 cycles at 94ยฐ C. for 30 sec, 50-64ยฐ C. for 30 sec (depending on the Tm of the primers), and at 72ยฐ C. for 1-2 min (1 min/kb), respectively, and a final reaction at 72ยฐ C. for 10 min.
[0405] PCR products were separated by agarose gel electrophoresis and visualised by ethidium bromide staining. Bands of interest were cut out and DNA subsequently extracted from the gel slice using the QIAquick Gel Extraction Kit (Qiagen) following the manufacturer's instructions. Extracted PCR products were cloned into TOPO 2.1 vectors (Invitrogen) and transformed into OneShotยฎ Escherichia. coli cells by chemical transformation following the manufacturer's instructions. Bacteria were subsequently plated onto pre-warmed Luria-Bertani (LB; Invitrogen) agar plates (1% tryptone, 0.5% yeast extract, 1.0% NaCl, and 1.5% agar) containing 50 ฮผg ml-1 kanamycin and 40 ฮผl of 40 ฮผg ml-1 X-gal (5-bromo-4-chloro-3-indolyl-X-D- galactopyranoside; Invitrogen) and incubated at 37ยฐ C. overnight. Positive colonies were selected using white-blue selection in combination with antibiotic selection. Colonies were picked and inoculated into 6 ml LB broth (1% tryptone, 0.5% yeast extract, 1.0% NaCl) containing 50 ฮผg ml-1 kanamycin and incubated at 37ยฐ C. in a shaking incubator at 200 rpm.
[0406] Bacterial cultures were extracted and purified from LB broth culture using the Qiagen Prep Plasmid Miniprep Kit (Qiagen) following the manufacturer's instructions.
DNA Sequencing
[0407] Isolated plasmid DNA was sequenced using the dideoxynucleotide chain termination method (Sanger et al., 1977), using Big-Dye (Version 3.1) chemistry (Applied Biosystems). Either M13 forward and reverse primers or specific gene primers were used. The products were separated on an ABI Prism 3100 Genetic Analyser (Applied Biosystems) and sequence data were compared with sequence information published in GenBank (NCBI) using AlignX (Invitrogen).
Results
Identification and Sequencing of TaMYB14
[0408] Total RNA and genomic DNA (gDNA) were isolated from developing and mature T. arvense leaf tissue and total RNA was reverse transcribed into cDNA. Initially, primers were designed to the generic MYB region of the coding sequence and PCR performed. PCR products were separated on agarose gels and visualised by ethidium bromide staining. Bands ranging in size were cut out, DNA extracted, purified, cloned into TOPO vectors, and transformed into E. coli cells. Two hundred transformants from the cloning event were randomly chosen, plasmid DNA isolated and subsequently sequenced. Additional primers were designed to sequence the N-terminal regions where required (Table 4).
[0409] An array of partial MYBs were identified by sequencing of the isolated cDNA; >50% were unknowns, yielding no substantial hit to known MYB proteins. The remaining were identified as orthologues for MYBs expressed during abiotic stress, response to water deprivation, light stimulus, salt stress, ethylene stimulus, auxin stimulus, abscisic acid stimulus, gibberellic acid stimulus, salicylic acid stimulus, jasmonic acid stimulus, cadmium, light, stomatal movement and control, regulation, mixta-like (epidermal cell growth), down-regulation of caffeic acid O-methyl-transferase, and meristem control.
[0410] Two partial MYB cDNAs coded for a protein that fell within the correct MYB clades (NO8 and NO9) whose members include those known to activate anthocyanin or CT biosynthesis. Primers were designed to the 3' end of the gene to isolate the remaining 5' end and hence the entire cDNA clone. The full-length TaMYB14 contains a 942 bp coding region coding for a 314 amino acid protein. In comparison, AtTT2 codes for a 258 amino acid protein.
Blast Results for TaMYB14
[0411] The cDNA sequence of TaMYB14 from T. arvense genotype AZ2925 was blasted against the public databases. BlastN returned the following top 5 hits:
TABLE-US-00006 AB300033.1 "Lotus japonicus LjTT2-1 mRNA for R2R3-MYB transcription factor", (e-value 3e-69) AB300035.1 Lotus japonicus LjTT2-3 mRNA for R2R3-MYB transcription factor",. (e-value 4e-62) AB300034.1 Lotus japonicus LjTT2-2 mRNA for R2R3-MYB transcription factor", (e-value 4e-59) AF336284.1 Gossypium hirsutum GhMYB36 mRNA, (e-value 1e-40) AB298506.1 Daucus carota DcMYB3-1 mRNA for transcription factor, (e-value 7e-39)
[0412] While BlastX of the translated sequence of TaMYB14 from T. arvense genotype AZ2925 returned the following 5 top hits:
TABLE-US-00007 BAG12893.1 "Lotus japonicus R2R3-MYB transcription factor LjTT2-1", (e-value 2e-81) AAK19615.1AF336282_1 "Gossypium hirsutum GhMYB10", (e-value 3e-76); BAG12895.1 "Lotus japonicus R2R3-MYB transcription factor LjTT2-3", (e-value 8e-74); BAG12894.1 "Lotus japonicus R2R3-MYB transcription factor LjTT2-2", (e-value 2e-72); AAZ20431 .1 "MYB11" [Malus x domestica], (e-value 2e-66)
[0413] Alignment of TaMYB14 cDNA to AtTT2 and other BLAST hits are shown in FIG. 7 with highest similarities shown in yellow. Translation of the open reading frame also showed substantial differences in the amino acid composition, sharing 52% homology to A. thaliana TT2 (FIG. 8). Moreover TaMYB14 shares the motifs common to known CT MYB activators (N09).
[0414] Alignment of TaMYB14 cDNA to AtTT2 and other BLAST hits are shown in FIG. 7. with similarities highlighted in yellow and blue. Translation of the open reading frame (FIG. 8) also showed substantial differences in the amino acid composition, sharing 52% homology to A. thaliana TT2, primarily within the MYB domain region.
[0415] TaMYB14 includes a motif similar to the motif of subgroup 5 (DExWRLxxT (SEQ ID NO:102)) according to Stracke et al., 2001, that is common to previously known CT MYB activators.
[0416] TaMYB14 lacks the motif of subgroup 6 (KPRPR[S/T, shown in SEQ ID NO:16) according to Stracke et al., 2001, that is common to previously known anthocyanin MYB activators.
[0417] Moreover this alignment has identified a novel MYB motif (VI/VRTKAxR/KxSK (SEQ ID NO:101)). This new motif (highlighted in FIG. 8) appears associated with a number of novel MYB14 TFs that regulate CT pathways.
TaMYB14 Transcript Levels
[0418] CT accumulation occurred in the species T. arvense and T. affine, where they were detectable throughout the entire leaf lamina in the abaxial and adaxial epidermal layer, and the petiole; except for the petiolule region. CTs are only detectable in T. repens and T. occidentale in the leaf trichomes on the abaxial epidermal surface, Transcript analysis using primers specific to TaMYB14 revealed that this gene was expressed only in tissues actively accumulating CTs. TaMYB14 was expressed in T. arvense mature and immature leaf tissue, but not in callus (which does not synthesise CTs). Primers designed to TaMYB14 also amplified a MYB14 in T. repens, which was expressed in meristem leaf and early meristematic trichomes, where CTs are actively accumulating, but were not detected in mature or emergent leaf tissue, stolons, internodes, roots, and petioles. MYB14 was also not detected in mature T. occidentale tissues where CTs are only present in leaf trichomes. Results of the analysis are shown in Table 3 below:
TABLE-US-00008 TABLE 3 The expression of MYB14 also coincides with expression of anthocyanidin reductase (ANR; BAN) and LAR, two key enzymes specific to CT biosynthesis in legumes. Species Library Result Expect Pathway T. repens Huia Mature Leaf - - CT? T. repens Huia young leaf - - T. repens Huia meristem leaf + + T. repens Huia early trichome + + T. repens Huia stolon nodes and - - internodes T. repens Huia Roots - - T. repens Huia floral -+ + T. repens Huia petioles - - T. occidentale mature plant - - T. repens Isabelle Mature leaf - - Anthocyanin T. arvense callus - - CT-ve T. arvense mature leaf + + CT T. arvense immature leaf + +
[0419] FIGS. 3 and 4 also showed the comparison of transcript levels in various tissues in the Trifolium species; FIG. 3 shows transcript levels of TaMYB14 in varying tissues from Trifolium species and cultivars grown in identical glasshouse conditions; Lane 1, (ladder); Lane 2, T. repens mature leaf cDNA library (Cultivar Huia); Lane 3, T. repens mature root cDNA library (Cultivar Huia); Lane 4, T. repens mature stolon cDNA library (Cultivar Huia); Lane 5, T. repens mature floral cDNA library (Cultivar DC111); Lane 6, T. repens emerging leaf cDNA (Cultivar Huia); Lane 7, T. repens mature leaf cDNA (High anthocyanin Cultivar Isabelle); Lane 8, T. arvense immature leaf cDNA (Cultivar AZ2925); Lane 9, T. arvense mature leaf cDNA (Cultivar AZ2925); Lane 10, T. repens meristem floral cDNA (Cultivar Huia); Lane 11, T. repens meristem leaf cDNA (Cultivar Huia); Lane 12, T. repens meristem trichome onlycDNA (Cultivar Huia); Lane 13, T. occidentale mature plant (leaf, root and stolon cDNA library (Cultivar Huia); Lane 14, T. repens mature nodal cDNA library (Cultivar Huia); Lane 15, cloned T. arvense MYB14cDNA clone in TOPO, Lane 16, cloned T. arvense MYB14 genomic clone in TOPO, lane 17, T. occidentale genomic DNA; lane 17, T. repens genomic DNA; lane 17, T. arvense genomic DNA; Lane 20, (ladder).
[0420] While FIG. 4 shows transcript levels of BANYULS(A) and LAR (B) in varying tissues from Trifolium species and cultivars grown in identical glasshouse conditions. Lane 1, (ladder); Lane 2, T. repens mature leaf cDNA library (Cultivar Huia); Lane 3, T. repens mature root cDNA library (Cultivar Huia); Lane 4, T. repens mature stolon cDNA library (Cultivar Huia); Lane 5, T. repens mature floral cDNA library (Cultivar DC111); Lane 6, T. repens emerging leaf cDNA (Cultivar Huia); Lane 7, T. repens mature leaf cDNA (High anthocyanin Cultivar Isabelle); Lane 8, T. arvense immature leaf cDNA (Cultivar AZ2925); Lane 9, T. arvense mature leaf cDNA (Cultivar AZ2925); Lane 10, T. repens meristem floral cDNA (Cultivar Huia); Lane 11, T. repens meristem leaf cDNA (Cultivar Huia); Lane 12, T. repens meristem trichome onlycDNA (Cultivar Huia); Lane 13, T. occidentale mature plant (leaf, root and stolon cDNA library (Cultivar Huia); Lane 14, T. repens mature nodal cDNA library (Cultivar Huia); Lane 15, cloned T. arvense cDNA BAN or LAR clone in TOPO, Lane 16, cloned T. arvense BAN or LAR genomic clone in TOPO, lane 17, T. occidentale genomic DNA; lane 17, T. repens genomic DNA; lane 17, T. arvense genomic DNA; Lane 20, (ladder).
Identification and Sequencing of MYB14 from gDNA of T. arvense, T. Affine, T. occidentale and T. repens
[0421] Using primers designed to the start and stop region of TaMYB14 (see Table 4) the inventors amplified homologues of TaMYB14 by PCR of cDNA and gDNA isolated from a range of several Trifolium species; namely T. arvense, T. affine, T. repens and T. occidentale. Isolation of the genomic DNA sequence and full-length sequencing of the cloned PCR products showed T. arvense has two isoforms or alleles of this gene, one of which corresponds to the expressed cDNA sequence, the other corresponding to a previously unidentified isoform/allelic variant of TaMYB14.
[0422] Alignment of these isoform or allelic variant revealed the presence of several deletions and insertions of bases compared to the cDNA sequence of TaMYB14 (see FIG. 10). Translation of the putative cDNA sequence revealed that the protein encoded by this isoform or allelic variant also has amino acid deletions, insertions, and exchanges (see FIG. 9). The inventors designated the allelic variant as TaMYB14-2.
[0423] The corresponding full-length gDNA sequences for this gene were also isolated from three other Trifolium species; T. affine, T. repens and T. occidentale. All MYB14 alleles had three exons and two introns of varying sizes (see FIGS. 10-12). T. affine and T. occidentale both have one allele, while T. repens has two alleles. The translated sequences of MYB14 from the various species were 95% homologous to TaMYB14 with changes in amino acid composition. The majority of amino acid differences are located in the 3' unique region downstream of the MYB domain.
TABLE-US-00009 TABLE 4 Primer sequences for PCR, cloning and sequencing of MYB14 from various Trifolium species (T. arvense; T. repens; T. affine; T. occidentale). Primer usage Code Primer sequence SEQ ID NO: MYB domain hunt MYBFX GACAATGAGATAAAGAA 18 TTACTTG MYB domain hunt MYBFY AAGAGTTGTAGACTTAG 19 MTGG MYB domain hunt MYBFZ YTKGGSAACAGGTTGTC 20 Isolation of M14ATG ATGGGGAGAAGCCCTTG 21 full length TTGTGC Isolation of M14TGA TCATTCTCCTAGTACTTC 22 full length CTCACTGG Gene walking M14TSP1 CTCTTTTTGGAAGGTTTC 23 TCC Gene walking M14TSP2 TTCTCCATTTTCCTTCAC 24 CATGG Gene walking M14TSP3 TCCAAGCACCTCTATTCA 25 AGCC Cloning into vector M14FATG CTCGAGATGCAATGCTG 26 GTTGATGGTGTGGC Lotus corniculatus MYBLF CATTGCCTGTAGATTCT 27 GTAGCC Lotus corniculatus MYBLR TGAAGATTGTTGGACAC 28 ATTGG 5' UTR end of MYB14 MYB148N AGGTTGGAATACAAGAC 29 AGAC 3' UTR end of MYB14 MYB14RR TCTCCTAGTACTTCCTCA 30 CTGG Primer for intron 1 15 ATAATCATACTAATTAAC 31 ATCAC Primer for intron 1 13 GTGTGATAGATCATGTCATT 32 Gene walking TSP4 GCCTTCCTTTGCACAAC 33 AAGGGC Gene walking TSP5 GCACAACAAGGGCTTCT 34 CCCC 5'start site Forward MYB148F ATGGGGAGAAGCCCTTG 35 TTGTGC 5'start site Reverse MYB14RR TCTCCTAGTACTTCCTCA 36 CTGG Expression analysis/ MYB14F CTCGAGCAATGCTGGTT 37 Silencing vector GATGGTGTGGC Expression analysis/ MYB14R TCTAGAGGACACATTTG 38 Silencing vector TCTCATCAGC Gene walking MYB14R2 TCTAGATTGAGTTTGGT 39 CCGAACAAGG Gene walking MYB14R3 TCTAGAAATCTTCTAGCA 40 AATCTGCGG Sequencing M13 GTAAAACGACGGCCAG 41 Forward M13 CAGGAAACAGCTATGAC 42 Reverse cDNA production BD AAGCAGTGGTATCAACG 43 SMART CAGAGTACGCGGG II ยฎ A Oligo- nucleotide cDNA production 3' BD AAGCAGTGGTATCAACG 44 SMART ยฎ CAGAGTACT(30)V N-3' CDS Primer II A Amplification of 5' PCR AAGCAGTGGTATCAACG 45 mRNA Primer II CAGAGT A
[0424] In summery the applicants have identified and isolated ten novel MYB14 proteins/genes, as summarized in Table 5 below, which also shows the SEQ ID NO: associated with each sequence in the sequence listing:
TABLE-US-00010 TABLE 5 Summary of MYB14 sequences of the invention. SEQ ID NO: Full-length Species, and sequence reference cDNA gDNA Protein ORF Trifolium arvense, TaMYB14-1 1, 13 2 14 55 Trifolium arvense, TaMYB14-2 -- 3 46 56 Trifolium affine, TafMYB14-1 5 4 47 57 Trifolium affine, TafMYB14-2 -- 6 48 58 Trifolium occidentale, ToMYB14-1 -- 7 49 59 Trifolium occidentale, ToMYB14-2 -- 8 50 60 Trifolium repens, TrMYB14-1 -- 9 51 61 Trifolium repens, TrMYB14-2 -- 10 52 62 Trifolium repens, TrMYB14-3 -- 11 53 63 Trifolium repens, TrMYB14-4 -- 12 54 64
[0425] An alignment of all of these MYB14 sequences is shown in FIG. 34. The applicants identified two sequence motifs common to all of the MYB14 protein sequences.
[0426] The first motif is DDEILKN (SEQ ID NO:15)
[0427] The second motif is X1VVRTX2AX3KCSK (SEQ ID NO:17), where X1=N, Y or H, X2=K or R, and X3=T or I.
[0428] The presence of either or both of these mofits appears to be diagnostic for MYB14 proteins, particulary when associated with a lack of motif of SEQ ID NO:16.
[0429] FIG. 35 shows the percent identity between each of the MYB14 proteins aligned in FIG. 34.
[0430] The applicants have also shown that spatial and temperal expression pattern of TaMYB14 is consistently correlated with production of CT in plants in vivo.
Example 2
Use of the MYB14 Nucleic Acid Sequence of the Invention to Produce Condensed Tannins in White Clover (Trifolium repens)
Materials and Methods
Genetic Constructs Used in the Transformation Protocol
[0431] The plant transformation vector, pHZBar is derived from pART27 (Gleave 1992). The pnos-nptII-nos3' selection cassette has been replaced by the CaMV35S-BAR-OCS3' selection cassette with the bar gene (which confers resistance to the herbicide ammonium glufosinate) expressed from the CaMV 35S promoter. Cloning of expression cassettes into this binary vector is facilitated by a unique NotI restriction site and selection of recombinants by blue/white screening for ฮฒ-galactosidase. White clover was transformed using M14ApHZBarP which contains the expressed allele from Trifolium arvense. Over-expression cassettes for M14ApHZBarP were firstly cloned in pART7. The construct were then shuttled to pHZBar as a NotI fragment. T-DNAs of the genetic constructs, showing orientation of cloned genes, are represented graphically in FIG. 6.
[0432] Genetic constructs in pHZBar were transferred into Agrobacterium tumefaciens strain GV3101 as plasmid DNA using freeze-thaw transformation method (Ditta et al 1980). The structure of the constructs maintained in Agrobacterium was confirmed by restriction digest of plasmid DNA's prepared from bacterial culture. Agrobacterium cultures were prepared in glycerol and transferred to -80ยฐ C. for long term storage. Genetic constructs maintained in Agrobacterium strain GV3101 are inoculated into 25 mL of MGL broth containing spectinomycin at a concentration of 100 mg/L. Cultures are grown overnight (16 hours) on a rotary shaker (200 rpm) at 28ยฐ C. Bacterial cultures are harvested by centrifugation (3000รg, 10 minutes). The supernatant is removed and the cells resuspended in a 5 mL solution of 10 mM MgSO4.
Transformation of Cotyledonary Explants.
[0433] Clover was transformed using a modified method of Voisey et al. (1994). Seeds are weighed to provide approximately 400-500 cotyledons (ie. 200-250 seeds) for dissection (0.06 gm=100 seeds). In a centrifuge tube, seeds are rinsed with 70% ethanol for 1 minute. Seeds are surface sterilised in bleach (5% available chlorine) by shaking on a circular mixer for 15 minutes followed by four washes in sterile water. Seeds are imbibed overnight at 4ยฐ C. Cotyledons are dissected from seeds using a dissecting microscope. Initially, the seed coat and endosperm are removed. Cotyledons are separated from the radical with the scalpel by placing the blade between the cotyledons and slicing through the remaining stalk. Cotyledonary explants are harvested onto a sterile filter disk on CR7 media.
[0434] For transformation, a 3 ul aliquot of Agrobacterium suspension is dispensed on to each dissected cotyledon. Plates are sealed and cultured at 25ยฐ C. under a 16 hour photoperiod. Following a 72 hour period of co-cultivation, transformed cotyledons are transferred to plates containing CR7 medium supplemented with ammonium glufosinate (2.5 mg/L) and timentin (300 mg/L) and returned to the culture room. Following the regeneration of shoots, explants are transferred to CR5 medium supplemented with ammonium glufosinate (2.5 mg/L) and timentin (300 mg/L). Regenerating shoots are subcultured three weekly to fresh CR5 media containing selection. As root formation occurs, plantlets are transferred into tubs containing CR0 medium containing ammonium glufosinate selection. Large clumps of regenerants are divided to individual plantlets at this stage. Whole, rooted plants growing under selection are then potted into sterile peat plugs.
LCMSMS Methodology for HPLC Analysis
[0435] To extract flavonoids for HPLC analysis, leaf tissue (0.5 g fresh weight) was frozen in liquid N2, ground to a fine powder and extracted with acetic acid: methanol (80:20 v/v) for 30 mins at 4ยฐ C. The plant debris was pelleted in a microcentrifuge at 13K rpm for 10 mins. The supernatant was removed and placed at -20ยฐ C. for 30 mins. An aliquot was used for HPLC analysis. An aliquot was analysed by HPLC using both UV-PDA and MS/MS detection on a Thermo LTQ Ion Trap Mass Spectrometer System. The extracts were resolved on a Phenomonex Luna C18 reversed phase column by gradient elution with water and acetonitrile with 0.1% formic acid as the mobile phase system. Detection of the anthocyanins were by UV absorption at 550 nm, and the other metabolites were estimated by either MS1 or MS2 detection by the mass spectrometer.
[0436] The instrument used was a linear ion trap mass spectrometer (Thermo LTQ) coupled to a Thermo Finnigan Surveyor HPLC system (both San Jose, Calif., USA) equipped with a Thermo photo diode array (PDA) detector. Thermo Finnigan Xcalibur software (version 2.0) was used for data acquisition and processing.
[0437] A 5 ฮผL aliquot of sample was injected onto a 150ร2.1 mm Luna C18(2) column (Phenomenex, Torrance, Calif.) held at a constant 25ยฐ C. The HPLC solvents used were: solvent A=0.1% formic acid in H2O; solvent B=0.1% formic acid in Acetonitrile. The flow rate was 200 ฮผL min-1 and the solvent gradient used is shown in Table 6 below. PDA data was collected across the range of 220 nm-600 nm for the entire chromatogram.
TABLE-US-00011 TABLE 6 HPLC gradient Time (min) Solvent A % Solvent B % 0 95 5 6 95 5 11 90 10 26 83 17 31 77 23 41 70 30 45 50 50 52 50 50 52 3 97 59 3 97 62 95 5 70 95 5
[0438] The mass spectrometer was set for electrospray ionisation in positive mode. The spray voltage was 4.5 kV and the capillary temperature 275ยฐ C., and flow rates of sheath gas, auxiliary gas, and sweep gas were set (in arbitrary units/min) to 20, 10, and 5, respectively. The first 4 and last 11 minutes of flow from the HPLC were diverted to waste. The MS was programmed to scan from 150-2000 m/z (MS1scan), then perform data dependant MS3 on the most intense MS1 ion. The isolation windows for the data dependant MS3 method was 2 mu (nominal mass units) and fragmentation (35% CE (relative collision energy)) of the most intense ion from the MS1 spectrum was followed by the isolation (2 mu) and fragmentation (35% CE) of the most intense ion from the MS2 spectrum. The mass spectrometer then sequentially performed selected reaction monitoring (SRM) on the masses in Table 7 below, with isolation windows for each SRM of 2.5 mu and fragmentation CE of 35%. These masses listed cover the different combinations of procyanidin (catechin and/or epicatechin) and prodelphinidin (gallocatechin or epigallocatechin) masses up to trimer.
TABLE-US-00012 TABLE 7 SRM masses for monomers, dimers and trimers: SRM mass (m/z) MS2 scan range (m/z) Target compound 291.3 80-700 PC monomers 307.3 80-700 PD monomers 579.3 155-2000 PC:PC dimers 595.3 160-2000 PC:PD dimers 611.3 165-2000 PD:PD dimers 867.3 235-2000 PC:PC:PC timers 883.3 240-2000 PC:PC:PD trimers 899.3 245-2000 PC:PD:PD trimers 915.3 250-2000 PD:PD:PD trimers
Results
[0439] DMACA Analysis of White Clover with MYB14 from gDNA of T. arvense
[0440] White clover cotyledons were transformed with the T. arvense allele corresponding to the expressed cDNA sequence, under the control of the CaMV 35S promoter, and regenerated as described in the methods. Leaves from all regenerated plantlets were screened for CT production with DMACA staining, as described in Example 1. A number of these transformed plants were positive for CT production, resulting in blue staining when stained with DMACA. Such staining occurred in most epidermal cells of leaf tissues, including the six middle cells of leaf trichomes. In comparison, non-transformed wild type white clover plants were negative for CT, apart from the trichomes on the abaxial leaf side (FIG. 5). CTs were also present within some root and petiolar cells of some plants. This indicates that constitutive expression of TaMYB14 alters the temporal and spatial patterning of CT accumulation in white clover plants.
Molecular Analysis, DMACA Screen and Biochemistry of Transgenic White Clover
White Clover Molecular Analysis
[0441] DNA extracted from transgenic white clover plants was tested for integration of the M14ApHZBAR vector. PCR reactions were performed using primer sets designed to amplify a product including a portion of the 35S promoter and the majority of the TaMYB14 gene. Results of this analysis indicated integration of the binary vector containing the TaMyb14A gene (SEQ ID NO:2) into the white clover genome (FIG. 14).
White Clover DMACA Analysis
[0442] The results achieved from DMACA staining of white clover leaf tissues are shown (FIG. 15). The CT specific stain, DMACA, has heavily stained the leaf blade and petiole of the transgenic clover leaves (B, C, D, G, H), compared to wild type white clover leaf (A, E, F).
[0443] In addition (FIG. 16), the trichome tier cells and apical cells were much more strongly stained (F, G) than normally seen in wild type leaves (E). The guard cells of the stomata had also strongly stained (H). There was definite staining in the nucleus of the epidermal cells as in the stalk trichome cell. Epidermal cells were more uniformly stained than normal and the basal cell of the rosette were also strongly stained (G). Leaf tears were carried out to help establish what specific cells have DMACA staining (I to K). This instance the lower epidermis (outside surface topmost) has been separated from the mesophyll layer. The epidermal cells (apart from specialised cells such as stomata and trichomes) had little activity compared to the mesophyll cell layer. The mesophyll cells showed definite strong staining throughout the cell with definite sub localization into specific vacuole-like organelles, which are obviously multiple per cell. There is therefore compartmentalization of the DMACA staining within the mesophyll cells.
White Clover HPLC/LCMS Analysis
[0444] The applicant's biochemical analysis of the transgenic tissue transformed with M14ApHZBAR provided indisputable evidence that over expression of TaMYB14 leads to the accumulation of condensed tannin monomers, dimers and trimers in foliar tissue in white clover and tobacco. It is also possible that longer chain tannins are present but resolving these are beyond the scope of our equipment.
[0445] Purified grape seed extract was used as the standard for all LCMSMS HPLC measurements because its tannin profile has been well characterised and is shown in FIGS. 17 and 18. This extract allows definite identification of catechin (C), epicatechin (EC), gallocatechin (GC) and epigallocatechin (EGC) as well as detection of PC:PC dimers, a PC:PD dimers and two 3PC trimers.
[0446] The MS2 spectra of all four monomers are provided as evidence of identication of these metabolites.
[0447] Flavonoids were extracted from transgenic and wild type control white clover plants, and processed via HPLC/LCMS. Results of these analyses confirmed the presence of CT in leaf extracts from the transgenic clover samples. The majority of monomers detected were epicatechin and epigallocatechin with traces of gallocatechin. This is consistent as clover tannins are delphinidin derived. No monomers were detected in wild type white clover leaf tissue (FIG. 19). Dimers and trimers were also detected (FIGS. 20, 21).
Example 3
Use of the MYB14 Nucleic Acid Sequence of the Invention to Produce Condensed Tannins in Tobacco (Nicotiana tabacum)
Materials and Methods
Genetic Construct Used in Transformation Protocols.
[0448] The NotI fragment from the plasmid M14ApHZBAR (FIG. 6) was isolated and cloned into pART27 (Gleave, 1992) for transformation of tobacco. This binary vector contains the nptll selection gene for kanamycin resistance under the control of the CaMV 35S promoter.
Tobacco Transformation
[0449] Tobacco was transformed via the leaf disk transformation-regeneration method (Horsch et al. 1985). Leaf disks from sterile wild type W38 tobacco plants were inoculated with an Agrobacterium tumefaciens strain containing the binary vector, and were cultured for 3 days. The leaf disks were then transferred to MS selective medium containing 100 mg/L of kanamycin and 300 mg/L of cefotaxime. Shoot regeneration occurred over a month, and the leaf explants were placed on hormone free medium containing kanamycin for root formation.
Results
Molecular Analysis, DMACA Screen and Biochemistry of Transgenic Tobacco
Tobacco Molecular Analysis
[0450] DNA extracted from transgenic tobacco plants was tested for integration of the M14ApHZBAR binary vector. PCR reactions were performed using primer sets designed to amplify a portion of the 35S promoter and the majority of the gene. Results of this analysis indicated integration of the binary vector containing the TaMyb14A gene (SEQ ID NO:2) into the white clover genome (FIG. 22).
Tobacco DMACA Analysis
[0451] DMACA analysis was performed on the tobacco plants, as described for clover in Example 1. Transgenic tobacco plantlets expressing TaMYB14A (under the control of the cauliflower mosaic virus 35S promoter) showed no significant differences in growth compared to wild-type plants. Moreover, CT was detected in leaf tissue of transgenic tobacco plantlets derived from cells of either the wild type or the transgenic tobacco (already accumulating anthocyanin) compared to wild type untransformed tobacco that does not accumulate CT in vegetative tissues. This indicates that the T. arvense MYB14 gene is able to activate all the genes of the CT pathway in tobacco, on its own. Examples of the DMACA staining of transgenic tobacco leaves are shown (FIG. 23). The CT specific stain, DMACA, heavily stained the leaf blade of the transgenic tobacco leaves (A to G) compared to wild type leaves, which are always devoid of CT.
Tobacco HPLC/LCMS Analysis
[0452] HPLC/LCMS analysis was performed for tobacco as described for clover in Example 2. Flavonoids were extracted from transgenic and wild type control tobacco plants, and processed via HPLC. Results of these analyses confirmed the presence of CT in leaf extracts from the transgenic tobacco samples. The tobacco control samples were devoid of CT units. The majority of monomers detected were epicatechin, with small amounts of epigallocatechin and gallocatechin monomers (FIG. 24). Dimers and trimers were also detected (FIG. 25).
Example 4
Use of the MYB14 Nucleic Acid Sequence of the Invention to Reduce Production Condensed Tannins in Trifolium arvense
Materials and Methods
Genetic Construct Used in Silencing Protocol
[0453] pHANNIBAL (Helliwell and Waterhouse, 2003), a hairpin RNAi plant vector, was used to transform T. arvense cotyledons with a construct expressing self-complementary portions of a sequence homologous to a portion of the cDNA of TaMYB14. The entire cDNA for the MYB14 (previously isolated from a leaf library) was used to amplify a 299 bp long fragment of the cDNA from the 3' end of the gene (caatgctggttgatggtgtggctagtgattcaatgagtaacaacgaaatggaacacggttatggatttttgt- cattttgcgatgaa gagaaagaactatccgcagatttgctagaagattttaacatcgcggatgatatttgcttatctgaacttttga- actctgatttctcaaa tgcgtgcaatttcgattacaatgatctattgtcaccttgttcggaccaaactcaaatgttctctgatgatgag- attctcaagaattgga cacaatgtaactttgctgatgagacaaatgtgtcc-SEQ ID NO:65). The primers were designed to allow the cloning of the fragments into the silencing vector pHANNIBAL (Table 5). The fragment was cloned into Xhol site in the sense direction in front of the pdk intron or the Xbal sites, after the pdk intron, in the antisense direction. Direction of the cloning was determined by PCR to ensure the fragment was in the correct orientation. The NotI fragment from MYB14pHANNIBAL containing the hpRNA cassette was subcloned into pHZBar (designated pHZBARSMYB (FIG. 13) and used in transformation experiments.
TABLE-US-00013 TABLE 8 Primers modified to include either an XbaI re- striction enzyme site (highlighted with italics) or a XhoI restriction enzyme site (highlighted with bold) at the 5'end of the primers to allow cloning. Primer Sequence MYB14F1 TCTAGACAATGCTGGTTGATGGTGTGGC (SEQ ID NO: 66) MYB14R TCTAGAGGACACATTTGTCTCATCAGC (SEQ ID NO: 67) MYB14F CTCGAGCAATGCTGGTTGATGGTGTGGC (SEQ ID NO: 68) MYB14R1 CTCGAGGGACACATTTGTCTCATCAGC (SEQ ID NO: 69)
T. arvense Transformation:
[0454] Cultivars of T. arvense were transformed with the pHZbarSMYB silencing binary vector, essentially as described for T. repens, with some minor modifications (Voisey et al., 1994). The ammonium glufosinate level was decreased to 1.25 mg/L; and plants were placed onto CR5 media for only a fortnight prior to placement onto CR0 medium for root regeneration.
Results
[0455] Molecular Analysis, DMACA Screen and Biochemistry of Transgenic Trifolium arvense. T. arvense Molecular Analysis
[0456] DNA extracted from transgenic T. arvense plants was tested for integration of the M14pHANNIBAL binary vector. PCR reactions were performed using primer sets designed to amplify a portion of the 35S promoter and the 3' end of the cDNA gene fragment. Results of this analysis indicated integration of the binary vector containing the hpRNA gene construct into the genome (FIG. 26).
T. arvense DMACA Analysis
[0457] Plant material from control T. arvense and some of the transformed plantlets have been stained using DMACA (FIG. 27) as described in Example 1. The transformed plants were compared to the wild type mature leaves also regenerated through tissue culture as tissue culture affects leaf regeneration and the onset of tannin production compared to naturally soil grown plants derived from seeds. Wild type T. arvense callus does not produce tannin (A), but cells start to accumulate tannin in tissue resembling leaves (B to D-purple colour). The transgenic plants also do not produce tannin in callus, but leaf tissue similarly stained with DMACA showed only a light blue stain (E-L), indicating the levels of CT were dramatically reduced in plants expressing the silencing construct.
T. arvense HPLC/LCMS Analysis
[0458] Flavonoids were extracted from transgenic and wild type control T. arvense plants, and processed via HPLC/LCMS, as described in Example 2. Wild type (non-transformed) T. arvense plantlets had high detectable levels of CT monomers. The majority of these monomers were catechin, with small amounts of gallocatechin monomers (FIG. 28). Dimers were also detected (FIG. 29). In contrast, only traces of these compounds were detected in the transformed plantlets, if at all. Therefore HPLC analysis of silenced T. arvense plantlets confirmed CT accumulation had been significantly reduced. These results confirm the absence of CT in leaf extracts from the transgenic T. arvense plants is associated with the presence of the vector designed to silence expression of TaMYB14.
Example 5
Use of the MYB14 Nucleic Acid Sequence of the Invention to Produce Condensed Tannins in Alfalfa (Medicago sativa)
Materials and Methods
Alfalfa Transformation by Microprojectile Bombardment
[0459] The cultivar Regen-SY was used for all transformation experiments (Bingham 1991). The transformation protocol was adapted from Samac et al (1995). Callus cultures were initiated from petiole explants and grown in the dark on Schenk and Hildebrandt media (Schenk and Hildebrandt, 1972) supplemented with 2, 4-Dichlorophenoxyacetic acid and Kinetin (SHDK). Developing cultures were passaged by regular subculture onto fresh media at four weekly intervals. Eight to twelve week old Regen Sy callus was transformed by microprojectile bombardment in a Bio-Rad PDS1000/He Biolisticยฎ Particle Delivery System apparatus. Callus cultures were incubated for a minimum of four hours on SHDK medium supplemented with a 0.7M concentration of sorbitol and mannitol to induce cell plasmolysis. Plasmid DNA (1 ฮผg/ฮผl) of p35STaMyb14A (containing the NotI fragment from M14ApHZBAR) and pCW122 (which contains an nptll gene for conferring resistance to the antibiotic kanamycin; Walter et al, 1998) were precipitated to tungsten particles (M17, Bio-Rad) as described by the manufacturer. Standard parameters (27''Hg vacuum, 1100 psi rupture, and 100 mm target distance) were used for transformation according to the instruction manual. Transformed tissues were rested overnight before transfer to SHDK medium. After two days, cultures were transferred to SHDK medium containing antibiotic selection (kanamycin 50 mg/L) for selection of transformed cells. This material was sub-cultured up to three times at three weekly intervals before transfer to hormone-free SH medium or Blaydes medium (Blaydes, 1966) and placed in the light for regeneration. Germinating somatic embryos were dissected from the callus mass and transferred to a half-strength Murashige and Skoog medium (Murashige and Skoog, 1962) for root and shoot development.
Aim
[0460] Transformation experiments were undertaken to introduce a plasmid containing the TaMyb14 gene under the control of the CaMV35S promoter into alfalfa. The objective was to generate plants expressing TaMyb14 and to screen for the accumulation of condensed tannins in foliar tissues.
Results
Molecular Analysis, DMACA Screen and Biochemistry of Transgenic Alfalfa.
Alfalfa Molecular Analysis
[0461] DNA extracted from transgenic alfalfa was tested for integration of the p35STaMyb14A vector. Primer sets designed to amplify product from either the nptII gene or TaMyb14A gene (SEQ ID NO:2) were used. Results of this analysis indicated integration of both plasmid constructs into the alfalfa genome (FIG. 30).
Alfalfa DMACA Analysis
[0462] To test for accumulation of condensed-tannins, DMACA analysis can be conducted for the Alfalfa plants as described for clover in Example 1.
Alfalfa HPLC/LCMS Analysis
[0463] HPLC/LCMS analysis as described for clover in Example 2 above can be used to accurately detect the presence of tannin monomers, dimers and trimers in transgenic alfalfa. To conduct the analysis, flavonoids are extracted from transgenic and wild type control alfalfa plants, as described for clover. Wild type alfalfa accumulates (in the seed coat) mainly cyanidin derived tannins and small amounts of delphinidin derived tannins (Pang et al., 2007). The leaves of transgenic medicago lines expressing TaMYB14 can be tested for production of epicatechin, catechin and epigallocatechin, and gallocatechin monomers as well as dimer and trimer combinations of these base units.
Example 6
Use of the MYB14 Nucleic Acid Sequence of the Invention to Produce Condensed Tannins in Brassica (Brassica oleracea)
Materials and Methods
Transformation of Brassica Lines
[0464] Seeds of Brassica oleracea var. acephala cv. Coleor (red forage kale) and Gruner (green forage kale) were germinated in vitro as described in Christey et al. (1997, 2006). Hypocotyl and cotyledonary petiole explants from 4-5 day old seedlings were co-cultivated briefly with a culture of Agrobacterium tumefaciens grown overnight in LB medium containing antibiotics prior to 1:10 dilution in antibiotic-free minimal medium (7.6 mM (NH4)2SO4, 1.7 mM sodium citrate, 78.7 mM K2HPO4, 0.33 M KH2PO4, 1 mM MgSO4, 0.2% sucrose) with growth for a further 4 hrs. Explants were cultured on Murashige-Skoog (MS, Murashige and Skoog, 1962) based medium with B5 vitamins and 2.5 mg/L BA and solidified with 10 gm/L Danisco standard agar. After 3 days co-cultivation, explants were transferred to the same medium with the addition of 300 mg/L Timentin (SmithKline Beecham) and 15/L kanamycin. Explants were transferred every 3-4 weeks to fresh selection medium. Green shoots were transferred as they appeared to hormone-free Linsmaier-Skoog based medium (LS, Linsmaier and Skoog, 1965) containing 50 mg/L kanamycin and solidified with 10 gm/L Danisco standard agar. Explants were cultured in tall Petri dishes (9 cm diameter, 2 cm tall) sealed with Micropore (3M) surgical tape. Shoots were cultured in clear plastic tubs (98 mm, 250 ml, Vertex). All plant culture manipulations were conducted at 25ยฐ C. with a 16 h/day photoperiod, provided by Cool White fluorescent lights, 20 uE/m2/s.
Results
Molecular Analysis, DMACA Screen and Biochemistry of Transgenic Brassica
Brassica Molecular Analysis
[0465] DNA extracted from transgenic brassica plants was tested for integration of the M14ApHZBAR binary vector. PCR reactions were performed using primer sets designed to amplify a portion of the 35S promoter and the majority of the gene. Results of this analysis indicated integration of the binary vector containing the TaMyb14A gene (SEQ ID NO:2) into the brassica genome (shown in FIG. 31).
Brassica DMACA Analysis
[0466] DMACA analysis was performed on the Brassica plants as described for clover in Example 1. Transgenic brassica plantlets expressing TaMYB14A (under the control of the cauliflower mosaic virus 35S promoter) were indistinguishable from the wild-type plants. Wild type untransformed cabbage of either cultivar that does not naturally accumulate CT in vegetative tissues, remained unstained. However, CT was detected in leaf tissue of transgenic brassica plantlets derived from the accumulating anthocyanin cultivars, as evidenced by the positive DMACA staining. The staining was not as intense as that noted for tobacco and clovers. In contrast transgenic plantlets derived from wild type green cultivar never stained with DMACA.
[0467] This indicates that the T. arvense MYB14 gene is able to activate a portion of the genes of the CT pathway in brassica, but may require an active anthocyanin pathway for CT production. Examples of the DMACA staining of transgenic brassica leaves are shown in the pictures below (FIG. 32). The CT specific stain, DMACA, stained the leaf blade of the transgenic brassica (B to D) compared to wild type leaves (A), which are always devoid of CT.
Brassica HPLC/LCMS Analysis
[0468] Flavonoids were extracted from transgenic and wild type control Brassica plants, and processed via HPLC as described for clover in Example 2. Results of these analyses confirmed the presence of CT in leaf extracts from one transgenic brassica sample. The brassica transformation was done with both normal green coloured brassica as well as with a brassica line accumulating anthocyanin. The HPLC analysis detected epicatechin in green coloured brassica but no tannin monomers in the anthocyanin accumulating lines. The transgenic brassica overexpressing TaMYB14 that accumulated CTs in the leaf was derived from an anthocyanin accumulating line. Only epicatechin monomers were detected in this transgenic line as shown in FIG. 33.
Example 6
To Demonstrate Modification of Condensed Tannin Production by MYB14 Variants
[0469] Any variant MYB sequences, which may be identified by methods described herein, can be tested for their ability to alter condensed tannins in plants using the methods described in Examples 2 to 5.
[0470] Briefly the coding sequences (such as but not limited to those of SEQ ID NO: 56-64) of the variant sequences can be cloned into a suitable expression construct (e.g. pHZBar, as described in Example 2) and transformed into a plant cell or plant. A particularly convenient and relatively simple approach is to use tobacco as a test plant as described in Example 3. DMACA analysis can be used as a quick and convenient test for alternations in condensed tannin production as described in Example 1.
[0471] In this way the function of MYB14 variants in regulating condensed tannin production can be quickly confirmed.
[0472] More detailed analysis of the condensed tannins can also be performed using HPLC/LCMS analysis as described in Example 2.
Example 7
Use of the MYB14 Nucleic Acid Sequence of the Invention to Produce Condensed Tannins in Medicago
Materials and Methods
Plant Materials and Histochemical Analysis
[0473] Seeds of M. Sativa (Alfalfa) were obtained from the Margot Forde Forage Germplasm Centre (Palmerston North, NZ). Seeds were germinated on seed trays and plants grown in a glass house. Plant tissues were harvested at various developmental stages and either immediately processed for histochemical staining or frozen in liquid nitrogen and stored at -80ยฐ C. for subsequent DNA, RNA, and PA isolation.
Genetic Constructs, Plant Transformation and Regeneration
[0474] For over-expression of TaMYB14 in Medicago, the same construct (M14ApHZBarP) and Agrobacterium strain used for clover in Example 2.
[0475] Leaf disks of M. sativa were transformed using Agrobacterium-mediated transformation and plant regeneration protocols as described (Blaydes, 1966; An, 1985; Bingham 1991; Shetty et al., 1993; Voisey et al., 1994; Austin et al., 1995).
[0476] A genotype of alfalfa (Medicago sativa L.) derived from Regen-SY (Bingham 1991) was used for Agrobacterium-mediated transformation. Vegetatively propagated plants, as a source of leaf explant material, were maintained under a standard greenhouse environment. Leaf disks were transformed with A. tumefaciens strain GV3101 containing the TaMyb14 over-expression construct using a protocol adapted from Austin et al. 1995. Briefly, young fully expanded trifoliate leaves were surfaced sterilised, cut into pieces and floated on SHO solution (Shenk and Hildebrenk basal medium, Duchefa) before inoculation in a suspension of Agrobacterium cells and co-cultivation for two days on SH4K medium (Shetty and McKersie 1993). Following co-cultivation leaf disks were cultured on SH4K supplemented with 25 mg/L Kanamycin and 300 mg/L Cefotaxime for four weeks, then transferred to Blaydes medium (Blaydes, 1966) with antibiotic selection for induction of somatic embryogenesis. Mature green embryos developing under selection were dissected from callus and placed upright in a half strength MS salts (Murashige and Skoog 1962) supplemented with Nitsch vitamins (Nitsch and Nitsch 1969) and 3% sucrose but without kanamycin for further development. Whole rooted plants were transferred to the greenhouse and potted into a peat-based growth medium for analysis.
Medicago DMACA Analysis
[0477] Fresh tissue samples (mature leaves, flowers, roots, immature/meristematic leaves, and trichomes) were collected from plants and PAs were histochemically analysed using the acidified DMACA (4-dimethylaminocinnamaldehyde; Sigma NZ Ltd., Auckland, NZ) method essentially as described in Example 1. Briefly, tissue samples were decolorised in ethanol: acetic acid (3:1) overnight, stained with DMACA (3 mg/ml, methanol: hydrochloric acid, 1: 1), and destained with several washes of 70% ethanol. Meristematic leaves and trichomes were dissected from end tips of stolons under a microscope.
Medicago LC-MS/MS Analysis and Quantitation of PAs in Plant Tissues
[0478] LC-MS/MS analysis and quantification of CTs was as described for white clover in Example 2.
Results
[0479] Functional Analysis of TaMYB14 in Transgenic M. sativa Plants
[0480] M. sativa plants were transformed with TaMYB14 under the control of the CaMV35S promoter to test the function of TaMYB14 in this legume; presence and expression of TaMYB14 was confirmed by (RT)-PCR (data not shown).
Medicago DMACA Analysis
[0481] Leaves from regenerated plantlets were screened for PA accumulation using DMACA staining and a number of plants transformed with TaMYB14 tested positive. Leaves from non-transformed wild type plants stained positive with DMACA in the trichomes on the abaxial leaf layers only, while plants transformed with TaMYB14 stained positive in epidermal leaf cells as well (FIG. 36).
Medicago LC-MS/MS Analysis
[0482] The presence of PA monomers (epicatechin and catechin, FIG. 37), PC:PC dimers (FIG. 38), PC:PC:PC and PC:PC:PD trimers (FIG. 39), and trace levels of tetramers in leaf extracts of M. sativa plants transformed with the TaMYB14 construct was confirmed by LC-MS/MS analysis, while PAs were undetectable in control plants. A glycosylated monomer, epicatechin-glycoside (Pang et al., 2008), was also detected by LC-MS/MS (MS1 m/z 453, MS2 m/z 291, MS3 m/z 123, 139, 151, 165) in TaMYB14 transformed plants only, with levels 10-fold lower relative to free epicatechin (data not shown).
[0483] Quantification of soluble PAs in leaves of CaMV35S::TaMYB14 transformed M. sativa plants using the butanol/HCl method (Terrill et al., 1992) showed accumulation of PAs up to 2.2% DW.
Summary of Examples
[0484] The examples clearly demonstrate that the MYB14 gene of the invention is useful for manipulating the production of flavonoids, specifically condensed tannins in a range of plant genera, including tobacco (Nicotiana tabacum; Solanaceae Family), and in the legumes white clover (Trifolium repens; Fabaceae Family) and Alfalfa (Medicago sativa) and brassica (Brassica oleracea, Brassicaceae Family).
[0485] The applicants have demonstrated both increase and decrease in the production of condensed tannins using the methods and polynucloetides of the invention.
[0486] It is not the intention to limit the scope of the invention to the above mentioned examples only. As would be appreciated by a skilled person in the art, many variations are possible without departing from the scope of the invention.
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Sequence CWU
1
1
10211243DNATrifolium arvense 1gaattcgccc ttaagcagtg gtatcaacgc agagtacgcg
ggggaagtta tttaatttta 60tctacatcaa acacttcaag aggttggaat acaagacaga
ctaattaaga ataacatcaa 120tggggagaag cccttgttgt gcaaaggaag gcttgaatag
aggtgcttgg acaactcaag 180aagacaaaat cctcactgaa tacattaagc tccatggtga
aggaaaatgg agaaaccttc 240caaaaagagc agatttaaaa agatgtggaa aaagttgtag
acttagatgg ttgaattatc 300taagaccaga tattaagcga ggtaatatat ccccggatga
agaagaactt attatccgac 360ttcacaaact actcggaaac agatggtctc taatagccgg
aagacttcca gggcgaacag 420acaatgaaat aaagaactac tggaacacaa atttaggaaa
aaaggttaag gatcttaatc 480aacaaaacac caacaattct tctcctacta aactttctgc
tcaaccaaaa aatgcaaaga 540tcaaacagaa acagatcaat cctaagccaa tgaagccaaa
ctcaaatgtt gtccgtacaa 600aagctaccaa gtgttctaag gtattgttca taaactcact
ccccaactca ccaatgcatg 660atttgcagaa caaagctgag gcagagacaa caacaaagcc
atcaatgctg gttgatggtg 720tggctagtga ttcaatgagt aacaacgaaa tggaacacgg
ttatggattt ttgtcatttt 780gcgatgaaga gaaagaacta tccgcagatt tgctagaaga
ttttaacatc gcggatgata 840tttgcttatc tgaacttttg aactctgatt tctcaaatgc
gtgcaatttc gattacaatg 900atctattgtc accttgttcg gaccaaactc aaatgttctc
tgatgatgag attctcaaga 960attggacaca atgtaacttt gctgatgaga caaatgtgtc
caacaacctt cattcttttg 1020cttcctttct tgaatccagt gaggaagtac taggagaatg
ataataaaaa ttcattttcc 1080aataaaatta actactctag gttttttttt ttttttttta
atttcaattt catgttaggg 1140tggtttaata aataaatata ttctatggtt taatattgca
aaaaaaaaaa aaaaaaaaaa 1200aaaaagtact ctgcgttgat accactgctt aagggcgaat
tcc 124321290DNATrifolium arvense 2gaattcgccc
ttaggttgga atacaagaca gactaattaa gaataacatc aatggggaga 60agcccttgtt
gtgcaaagga aggcttgaat agaggtgctt ggacaactca agaagacaaa 120atcctcactg
aatacattaa gctccatggt gaaggaaaat ggagaaacct tccaaaaaga 180gcaggttcat
tcattctagt atcttgcaat tatagatcaa tcactttcat acttttgttt 240gcttataaat
tttcttgcat tttttcttca attttccatg tgaaatgcaa attactagta 300cattattatg
gatatgtttt tgcaaatatg tgtatgccat gcaggtttaa aaagatgcgg 360aaaaagttgt
agacttagat ggttgaatta tctaagacca gatattaagc gaggtaatat 420atcctcggat
gaagaagaac ttatcatcag acttcacaaa ctactcggaa acaggtaaaa 480gtaccgacat
aatcactaac ttattaacat ttatctataa tttgtttttt ttgacaatta 540gtactactaa
tttaatttta taatgtgtgc taatttgctt tgcctttaat ttgtggtaga 600tggtctctaa
tagccggaag acttccagga cgaacagaca atgaaataaa gaactactgg 660aacacaaatt
taggaaaaaa ggttaaggat cttaatcaac aaaacaccaa caattcttct 720cctactaaac
tctctgctca accaaaaaat gcaaagatca aacagaaaca gatcaatcct 780aagccaatga
agccaaactc aaatgttgtc cgtacaaaag ctaccaagtg ttctaaggta 840ttgttcataa
actcactccc caactcacca atgcatgatt tgcagaacaa agctgaggca 900gagacaacaa
caaagccatc aatgctggtt gatggtgtgg ctagtgattc aatgagtaac 960aacgaaatgg
aacacggtta tggatttttg tcattttgcg atgaagagaa agaactatcc 1020gcagatttgc
tagaagattt taacatcgcg gatgatattt gcttatctga acttttgaac 1080tctgatttct
caaatgcgtg caatttcgat tacaatgatc tattgtcacc ttgttcggac 1140caaactcaaa
tgttctctga tgatgagatt ctcaagaatt ggacacaatg taactttgct 1200gatgagacaa
atgtgtccaa caaccttcat tcttttgctt cctttcttga atccagtgag 1260gaagtactag
gagaatgaaa gggcgaattc
129031296DNATrifolium arvense 3gaattcgccc ttaggttgga atacaagaca
gactaattaa gaataacatc aatggggaga 60agcccttgtt gtgcaaagga aggcttgaat
agaggtgctt ggacaactca agaagacaaa 120atcctcactg aatacattaa gctccatggt
gaaggaaaat ggagaaacct tccaaaaaga 180gcaggttcat tcattctgta tcttacaatt
atagattaac cactttcata cttttgtttg 240cttataaatt ttcttgtatt ttttcttcca
tttttcatga gaaatgcaaa ttactagtac 300attattatgg acatgttttg gcaaatatgt
ttatgccatg cagatttaaa aagatgtgga 360aaaagttgta gacttagatg gttgaattat
ctaagaccag atattaagcg aggtaatata 420tccccggatg aagaagaact tattatccga
cttcacaaac tactcggaaa caggtaaagt 480cctaacataa tcactaactt attaacgttt
gtctataatt tgtttttttt gaccattagt 540actactaatt taattttaca atgtgtgcta
atttgcttgt ctttaatttg tggtagatgg 600tctctaatag ccggaagact tccagggcga
acagacaatg aaataaagaa ctactggaac 660acaaatttag gaaaaaaggt taaggatctt
gatcaacaaa acaccaacaa ttcttctcct 720actaaactct ctgctcaacc aaaaaatgca
gagatcaaac agaaacagat caatcctaag 780ccaaactcat atgttgtccg tacaaaagct
accaagtgtt ctaaggtatt gttcataaac 840tcacccccca actcaccacc aatgcatgat
ttgcagagca aagctgaggc agagacaaca 900acaacaacaa agccatcaat gccatcaatg
ctggttgatg gtgtggctag tgattcaatg 960agtaacaacg aaatggaatg cggtaatgga
tttttgtcat tttgcgatga agagaaagaa 1020ctatccgcag atttgctaga agattttaac
atcgcggatg atatttgctt atctgaattt 1080ctaaacttcg atttctcaaa tgcgtgcgat
atcgattaca atgatctatt gtcgccttgt 1140tcggaccaaa ctcaaatgtt ccctgatgat
gagattctaa agaattggac acaatgtaac 1200tttgctgatg agacaaatgt gtccaacaac
cttcagtctt ctgcttcctt tcttgaatcc 1260agtgaggaag tactaggaga atgaaagggc
gaattc 129641239DNATrifolium affine
4gaattcgccc ttatggggag aagcccttgt tgtgcgaagg aaggcttgaa tagaggtgct
60tggacaactc aagaagacaa aatcctcact gaatacatta agctccatgg tgaaggaaaa
120tggagaaacc ttccaaaaag agcaggttca ttcattctgt atcttacaat tatagattaa
180ccactttcat acttttgttt gcttataaat tttcttgtat tttttcttcc atttttcatg
240agaaatgcaa attactagta cattattatg gacatgtttt tgcaaatatg tttatgccat
300gcaggtttaa aaagatgtgg aaaaagttgt agacttagat ggttgaatta tctaagacta
360gatattaagc gaggtaatat atcctcggat gaagaagaac ttatcatccg acttcacaaa
420ttactcggaa acaggtaaag tcctaacata atcactaact tattaacgtt tgtctataac
480ttgttttttt gacaattagt actactaatt taattttata atgtgtgcta atttgcttgt
540ctttaatttg tggtagatgg tctctaatag ccggaagact tccaggacga acagacaatg
600aaataaagaa ctactggaac acaaatttag gaaaaaaggt taaggatctt aatcaagaaa
660acaccaacaa ttcttctcct actaaacttt ctgctcaact aaaaaatgca aagatcaaac
720agaaacagat caatcctaag ccaatggagc caaactcaaa tgttgtccgt acaaaagcta
780ccaagtgttc taaggcattg ttcataaact caccccccaa ctcaccacca atgcatgatt
840tgcagaacaa agctgaggca gagacaacaa caaagtcatc aatgccatca atgctggttg
900atggcgtggc tagtgattca atgagtaaca acgaaatgga atacggtgat ggatttgttt
960cattttgcga tgacgataaa gaactatccg cagatttgct agaagatttt aacatctcgg
1020atgatatttg cttatccgaa tttctaaact tcgatttctc aaatgcgtgc aatttcgatt
1080acaacgatct attgtcgcct tgttcggacc aaacacaaat gttctctgat gatgagattc
1140tcaagaattc gacaccatgt aactttgctg ctgagacaaa ttatgtgtcc aacaaccaat
1200ccagtgagga agtactagga gaatgaaagg gcgaattct
12395933DNATrifolium affine 5atggggagaa gcccttgttg tgcgaaggaa ggcttgaata
gaggtgcttg gacaactcaa 60gaagacaaaa tcctcactga atacattaag ctccatggtg
aaggaaaatg gagaaacctt 120ccaaaaagag caggtttaaa aagatgtgga aaaagttgta
gacttagatg gttgaattat 180ctaagactag atattaagcg aggtaatata tcctcggatg
aagaagaact tatcatccga 240cttcacaaat tactcggaaa cagatggtct ctaatagccg
gaagacttcc aggacgaaca 300gacaatgaaa taaagaacta ctggaacaca aatttaggaa
aaaaggttaa ggatcttaat 360caagaaaaca ccaacaattc ttctcctact aaactttctg
ctcaactaaa aaatgcaaag 420atcaaacaga aacagatcaa tcctaagcca atggagccaa
actcaaatgt tgtccgtaca 480aaagctacca agtgttctaa ggcattgttc ataaactcac
cccccaactc accaccaatg 540catgatttgc agaacaaagc tgaggcagag acaacaacaa
agtcatcaat gccatcaatg 600ctggttgatg gcgtggctag tgattcaatg agtaacaacg
aaatggaata cggtgatgga 660tttgtttcat tttgcgatga cgataaagaa ctatccgcag
atttgctaga agattttaac 720atctcggatg atatttgctt atccgaattt ctaaacttcg
atttctcaaa tgcgtgcaat 780ttcgattaca acgatctatt gtcgccttgt tcggaccaaa
cacaaatgtt ctctgatgat 840gagattctca agaattcgac accatgtaac tttgctgctg
agacaaatta tgtgtccaac 900aaccaatcca gtgaggaagt actaggagaa tga
93361238DNATrifolium affine 6gaattcgccc ttatggggag
aagcccttgt tgtgcaaagg aaggcttgaa tagaggtgct 60tggacaactc aagaagacaa
aatcctcact gaatacatta agctccatgg tgaaggaaaa 120tggagaaacc ttccaaaaag
agcaggttca ttcattctgt atcttacaat tatagattaa 180ccactttcat acttttgttt
tcttataaat tttcttgtat tttttcttcc atttttcatg 240agaaatgcaa attactagta
cattattatg gacatgtttt tgcaaatatg tttatgccat 300gcaggtttaa aaagatgtgg
aaaaagttgt agacttagat ggttgaatta tctaagacca 360gatattaagc gaggtaatat
atcctcggat gaagaagaac ttatcatccg acttcacaaa 420ctactcggaa acaggtaaag
tcataacata atcattaatt tattaacggt tatctataat 480ttgttttttt gacaattatc
actacaaatt taattttata atgtgcgcta atttgcttgt 540ctttaatttg tggtagatgg
tctctaatag ccggaagact tccagggcga acagacaatg 600aaataaagaa ctactggaac
acaaatttag gaaaaaaggt taaggatctt aatcaagaaa 660acaccaacaa ttcttctcct
actaaacttt ctgctcaact aaaaaatgca aagatcaaac 720agaaacagat caatcctaag
ccaatggagc caaactcaaa tgttgtccgt acaaaagcta 780ccaagtgttc taaggcattg
ttcataaact caccccccaa ctcaccacca atgcatgatt 840tgcagaacaa agctgaggca
gagacaacaa caaagtcatc aatgccatca atgctggttg 900atggcgtggc tagtgattca
atgagtaaca acgaaatgga atacggtgat ggatttgttt 960cattttgcga tgacgataaa
gaactatccg cagatttgct agaagatttt aacatctcgg 1020atgatatttg cttatccgaa
tttctaaact tcgatttctc aaatgcgtgc aatttcgatt 1080acaacgatct attgtcgcct
tgttcggacc aaacacaaat gttctctggt gatgagattc 1140tcaagaattc gacacaatgt
aactttgctg ctgagacaaa ttatgtgtcc aacaaccaat 1200ccagtgagga agtactagga
gaatgaaagg gcgaattc 123871252DNATrifolium
occidentale 7gaattcgccc ttatggggag aagcccttgt tgtgcaaagg aaggcttgaa
tagaggtgct 60tggacaactc aagaagacaa aatcctcact gaatacatta agctccatgg
tgaaggaaaa 120tggagaaacc ttccaaaaag agcaggttca ttcattctag tatcttgcaa
ttatagatca 180atcactttca tacttttgtt tgcttataaa ttttcttgca ttttttcttc
aattttccat 240gtgaaatgca aattactagt acattattat ggatatgttt ttgcaaatat
gtgtatgcca 300tgcgaggttt aaaaagatgc ggaaaaagtt gtagacttag atggttgaat
tatctaagac 360cagatattaa gcgaggtaat atatcctcgg atgaagaaga acttatcatc
agacttcaca 420aactactcgg aaacaggtaa aagtaccgac ataatcacta acttattaac
atttatctat 480aatttgtttt ttttgacaat tagtactact aatttaattt tataatgtgt
gctaatttgc 540tttgccttta atttgtggta gatggtctct aatagccgga agacttccag
gacgaacaga 600caatgaaata aagaactact ggaacacaaa tttaggaaaa aaggttaagg
atcttaatca 660acaaaacacc aacaagtctt ctcctactaa actctctgct caaccaaaaa
atgcaaagat 720caaacagaaa cagatcaatc ctaagccaat gaagccaaac tcaaatgttg
tccgtacaag 780agctaccaag tgttctaagg tattgttcat aaactcactc cccaactcac
caatgcatga 840tttgcagaac aaagctgagg cagagacaac aacaaagcca tcaatgctgg
ttgatggtgt 900ggctagtgat tcaatgagta acaacgaaat ggaacacggt tatggatttt
tgtcattttg 960cgatgaagag aaagaactat ccgcagattt gctagaagat tttaacatcg
cggatgatat 1020ttgcttatct gaacttttga actctgattt ctcaaatgcg tgcaatttcg
attacaatga 1080tctattgtcm ccttgttcgg accaaactca aatgttctct gatgatgaga
ttctcaagaa 1140ttggacacaa tgtaactttg ctgatgagac aaatgtgtcc aacaaccttc
attcttttgc 1200ttcctttctt gaatccagtg aggaagtact aggagaatga aagggcgaat
tc 125281164DNATrifolium occidentale 8gaattcgccc ttatggggag
aagcccttgt tgtgcaaagg aaggtttgaa tagaggtgct 60tggacagctc atgaagacaa
aatcctcact gaatacatta agctccatgg tgaaggaaaa 120tggagaaacc ttccaaaaag
agcaggttca ttcattctgt atcttactat ttatagatca 180ataatcactt tcatgtattt
tttttccttc cattttccat tagaaatgca aattaatagt 240acattattat ggacatgttt
ttccaggttt aaaaagatgt ggaaaaagtt gtagacttag 300atggttgaat tatcttagac
cagatattaa gagaggtaat atatcgtccg atgaagaaga 360acttatcatt agacttcaca
aactacttgg aaaccggtaa agtatcgaca taatcactaa 420cttactaaca tttgtttata
atgtgtacta attgcgattc ctttgatttg tggtagatgg 480tctctaatag ccggaagact
tccagggcga acagacaatg aaataaaaaa ttactggaac 540acgaatttag gaaaaaaggt
taaggatctt aatcaacaaa acaccaacaa ttcttctcct 600actaaacctt ctgctcaacc
aaaaaatgca aagatcaaac agaaacaaca gatcaataat 660cctaagccaa tgaagccaaa
ctcgaatgtt gtccgtacaa aagctaccaa atgttctaag 720gtattgttca taaactcacc
accaatgcat aatttgcaga acaaagctga ggcagagaca 780aaaacaaaga catcaatgtt
ggttaatggt gtagctagtg attcaatgag taacaacgaa 840atggaacgag gtaatggatt
tttgtcattt cgcgatgaag agaaagaact atccgctgat 900ttgctagatg attttaacat
cgcggatgac atttgcttat ccgaatttct aaactccgat 960ttctcaaatg cgtgcaattt
cgattacaat gatctattgt caccttgttc ggatcaaact 1020caaatgttct ctgatgatga
gattctcaag aattggacac aatgtaactt tgctgatgag 1080acaaatgtgt ccaacaacct
tcattctttt gcttcctttc tcgaatccag tgaggaagta 1140ctaggagaat gaaagggcga
attc 116491205DNATrifolium
repens 9gaattcgccc ttatggggag aagcccttgt tgtgcaaaag aaggcttgaa tagaggtgct
60tggacagctc atgaagacaa aatcctcact gaatacatta agctccatgg tgaaggaaaa
120tggagaaacc ttccaaaaag agcaggttca ttcattctgt atcttactat tatagatcaa
180taatcacttt cacacttttt tttttactta taaattttca tgtatttttt cttccatttt
240ccattagaaa tgcaaattaa tagtacatta ttatggacat gttttttcaa aaatgtgtat
300tccatgcagg tttaaaaaga tgtggaaaaa gttgtagact aaggtggttg aattatctta
360gaccggatat taagagaggt aatatatcgt cggatgaaga agaacttatc attagacttc
420acaaactact cggaaaccgg taaagtatcg acataatcac tgacttacta acatttgttt
480ataatgtgtg ctaattgctc ttcctttgat ttgtggtaga tggtctctaa tagccggaag
540acttccaggg cgaacagaca atgaaataaa gaactactgg aacacaaatt taggaaaaaa
600agttaaggat cttaatcaac aaaacaccaa caattcttct cctactaaac cttctgctca
660accaaaaaat gcaaatatca aacagaaaca acagatcaat cctaagccaa tgaagccaaa
720ctcgaatgtt gtccgtacaa aagctaccaa atgttctaag gtattgttca taaactcacc
780accaatgcat aatttgcaga acaaagctga ggcagagaca aaaacaaagc cattaatgct
840ggttaatggt gtagctagtg attcaatgag taacaacgaa atggaacgcg gtaatggatt
900tttgtcattt tgcgacgaag agaaagaact atccgcagat ttgctagatg attttaacat
960cgcggatgat atttgcttat ctgaatttct aaactccgat ttctcaaatg cgtgcaattt
1020cgattacaat gatctattgt cgccttgttc ggatcaaact caaatgttct ctgatgatga
1080gattctcaag aattggacac aatgtaactt tgctgatgag acaaatgtgt ccaacaacct
1140taattctttt gcttcttttc tcgaatccag tgaggaagta ctaggagaat gaaagggcga
1200attct
1205101202DNATrifolium repens 10gaattcgccc ttatggggag aagcccttgt
tgtgcaaaag aaggcttgaa tagaggtgct 60tggacagctc atgaagacaa aatcctcact
gaatacatta agctccatgg tgaaggaaaa 120tggagaaacc ttccaaaaag agcaggttca
ttcattctgt atcttactat tatagatcaa 180taatcacttt cacacttttt tttacttata
aattttcatg tattttttct tccattttcc 240attagaaatg caaattaata gtacattatt
atggacatgt tttttcaaaa atgtgtattc 300catgcaggtt taaaaagatg tggaaaaagt
tgtagactaa ggtggttgaa ttatcttaga 360ccggatatta agagaggtaa tatatcgtcg
gatgaagaag aacttatcat tagacttcac 420aaactactcg gaaaccggta aagtatcgac
ataatcacta acttactaac atttgtttat 480aatgtgtgct aattgctctt cctttgattt
gtggtagatg gtctctaata gccggaagac 540ttccagggcg aacagacaat gaaataaaga
actactggaa cacaaattta ggaaaaaaag 600ttaaggatct taatcaacaa aacaccaaca
attcttctcc tactaaacct tctgctcaac 660caaaaaatgc aaatatcaaa cagaaacaac
agatcaatcc taagccaatg aagccaaact 720cgaatgttgt ccgtacaaaa gctaccaaat
gttctaaggt attgttcata aactcaccac 780caatgcataa tttgcagaac aaagctgagg
cagagacaaa aacaaagcca ttaatgctgg 840ttaatggtgt agctagtgat tcaatgagta
acaacgaaat ggaacgcggt aatggatttt 900tgtcattttg cgacgaagag aaagaactat
ccgcagattt gctagatgat tttaacatcg 960cggatgatat ttgcttacct gaatttctaa
actccgattt ctcaaatgcg tgcaatttcg 1020attacaatga tctattgtcg ccttgttcgg
atcaaactca aatgttctct gatgatgaga 1080ttctcaagaa ttggacacaa tgtaactttg
ctgatgagac aaatgtgtcc aacaacctta 1140attcttttgc ttcttttctc gaatccagtg
aggaagtact aggagaatga aagggcgaat 1200tc
1202111203DNATrifolium repens
11gaattcgccc ttatggggag aagcccttgt tgtgcaaaag aaggcttgaa tagaggtgct
60tggacagctc atgaagacaa aatcctcact gaatacatta agctccatgg tgaaggaaaa
120tggagaaacc ttccaaaaag agcaggttca ttcattctgt atcttactat tatagatcaa
180tagtcacttt cacacttttt ttttacttat aaattttcat gtattttttc ttccattttc
240cattagaaat gcaaattaat agtacattat tatggacatg ttttttcaaa aatgtgtatt
300ccatgcaggt ttaaaaagat gtggaaaaag ttgtagacta aggtggttga attatcttag
360accggatatt aagagaggta atatatcgtc ggatgaagaa gaacttatca ttagacttca
420caaactactc ggaaaccggt aaagtatcga cataatcact aacttactaa catttgttta
480taatgtgtgc taattgctct tcctttgatt tgtggtagat ggtctctaat agccggaaga
540cttccagggc gaacagacaa tgaaataaag aactactgga acacaaattt aggaaaaaaa
600gttaaggatc ttaatcaaca aaacaccaac aattcttctc ctactaaacc ttctgctcaa
660ccaaaaaatg caaatatcaa acagaaacaa cagatcaatc ctaagccaat gaagccaaac
720tcgaatgttg tccgtacaaa agctaccaaa tgttctaagg tattgttcat aaactcacca
780ccaatgcata atttgcagaa caaagctgag gcagagacaa agacaaagcc attaatgctg
840gttaatggtg tagctagtga ttcaatgagt aacaacgaaa tggaacgcgg taatggattt
900ttgtcatttt gcgacgaaga gaaagaacta tccgcagatt tgctagatga ttttaacatc
960gcggatgata tttgcttatc tgaatttcta aactccgatt tctcaaatgc gtgcaatttc
1020gattacaatg atctattgtc gccttgttcg gatcaaactc aaatgttctc tgatgatgag
1080attctcaaga attggacaca atgtaacttt gctgatgaga caaatgtgtc caacaacctt
1140cattcttttg cttcctttct cgaatccagt gaggaagtac taggagaatg aaagggcgaa
1200ttc
1203121206DNATrifolium repens 12gaattcgccc ttatggggag aagcccttgt
tgtgcaaaag aaggcttgaa tagaggtgct 60tggacagctc atgaggacaa aatcctcact
gaatacatta agctccatgg tgaaggaaaa 120tggagaaacc ttccaaaaag agcaggttca
ttcattctgt atcttactat tatagatcaa 180taatcacttt cacacttttt tttttttact
tataaatttt catgtatttt ttcttccatt 240ttccattaga aatgcaaatt aatagtacat
tattatggac atgttttttc aaaaatgtgt 300attccatgca ggtttaaaaa gatgtggaaa
aagttgtaga ctaaggtggt tgaattatct 360tagaccggat attaagagag gtaatatatc
gtcggatgaa gaagaactta tcattagact 420tcacaaacta ctcggaaacc ggtaaagtat
cgacataatc actaacttac taacatttgt 480ttataatgtg tgctaattgc tcttcctttg
atttgtggta gatggtctct aatagccgga 540agacttccag ggcgaacaga caatgaaata
aagaactact ggaacacaaa tttaggaaaa 600aaagttaagg atcttaatca acaaaacacc
aacaattctt ctcctactaa accttctgct 660caaccaaaaa atgcaaatat caaacagaaa
caacagatca atcctaagcc aatgaagcca 720aactcgaatg ttgtccgtac aaaagctacc
aaatgttcta aggtattgtt cataaactca 780ccaccaatgc ataatttgca gaacaaagct
gaggcagaga caaaaacaaa gccattaatg 840ctggttaatg gtgtagctag tgattcaatg
agtaacaacg aaatggaacg cggtaatgga 900tttttgtcat tttgcgacga agagaaagaa
ctatccgcag atttgctaga tgattttaac 960atcgcggatg atatttgctt atctgaattt
ctaaactccg atttctcaaa tgcgtgcaat 1020ttcgattaca atgatctatt gtcgccttgt
tcggatcaaa ctcaaatgtt ctctgatgat 1080gagattctca agaattggac acaatgtaac
tttgctgatg agacaaatgt gtccaacaac 1140cttaattctt ttgcttcttt tctcgaatcc
agtgaggaag tactaggaga atgaaagggc 1200gaattc
1206131243DNATrifolium arvense
13gaattcgccc ttaagcagtg gtatcaacgc agagtacgcg ggggaagtta tttaatttta
60tctacatcaa acacttcaag aggttggaat acaagacaga ctaattaaga ataacatcaa
120tggggagaag cccttgttgt gcaaaggaag gcttgaatag aggtgcttgg acaactcaag
180aagacaaaat cctcactgaa tacattaagc tccatggtga aggaaaatgg agaaaccttc
240caaaaagagc agatttaaaa agatgtggaa aaagttgtag acttagatgg ttgaattatc
300taagaccaga tattaagcga ggtaatatat ccccggatga agaagaactt attatccgac
360ttcacaaact actcggaaac agatggtctc taatagccgg aagacttcca gggcgaacag
420acaatgaaat aaagaactac tggaacacaa atttaggaaa aaaggttaag gatcttaatc
480aacaaaacac caacaattct tctcctacta aactttctgc tcaaccaaaa aatgcaaaga
540tcaaacagaa acagatcaat cctaagccaa tgaagccaaa ctcaaatgtt gtccgtacaa
600aagctaccaa gtgttctaag gtattgttca taaactcact ccccaactca ccaatgcatg
660atttgcagaa caaagctgag gcagagacaa caacaaagcc atcaatgctg gttgatggtg
720tggctagtga ttcaatgagt aacaacgaaa tggaacacgg ttatggattt ttgtcatttt
780gcgatgaaga gaaagaacta tccgcagatt tgctagaaga ttttaacatc gcggatgata
840tttgcttatc tgaacttttg aactctgatt tctcaaatgc gtgcaatttc gattacaatg
900atctattgtc accttgttcg gaccaaactc aaatgttctc tgatgatgag attctcaaga
960attggacaca atgtaacttt gctgatgaga caaatgtgtc caacaacctt cattcttttg
1020cttcctttct tgaatccagt gaggaagtac taggagaatg ataataaaaa ttcattttcc
1080aataaaatta actactctag gttttttttt ttttttttta atttcaattt catgttaggg
1140tggtttaata aataaatata ttctatggtt taatattgca aaaaaaaaaa aaaaaaaaaa
1200aaaaagtact ctgcgttgat accactgctt aagggcgaat tcc
124314313PRTTrifolium arvense 14Met Gly Arg Ser Pro Cys Cys Ala Lys Glu
Gly Leu Asn Arg Gly Ala 1 5 10
15 Trp Thr Thr Gln Glu Asp Lys Ile Leu Thr Glu Tyr Ile Lys Leu
His 20 25 30 Gly
Glu Gly Lys Trp Arg Asn Leu Pro Lys Arg Ala Gly Leu Lys Arg 35
40 45 Cys Gly Lys Ser Cys Arg
Leu Arg Trp Leu Asn Tyr Leu Arg Pro Asp 50 55
60 Ile Lys Arg Gly Asn Ile Ser Ser Asp Glu Glu
Glu Leu Ile Ile Arg 65 70 75
80 Leu His Lys Leu Leu Gly Asn Arg Trp Ser Leu Ile Ala Gly Arg Leu
85 90 95 Pro Gly
Arg Thr Asp Asn Glu Ile Lys Asn Tyr Trp Asn Thr Asn Leu 100
105 110 Gly Lys Lys Val Lys Asp Leu
Asn Gln Gln Asn Thr Asn Asn Ser Ser 115 120
125 Pro Thr Lys Leu Ser Ala Gln Pro Lys Asn Ala Lys
Ile Lys Gln Lys 130 135 140
Gln Ile Asn Pro Lys Pro Met Lys Pro Asn Ser Asn Val Val Arg Thr 145
150 155 160 Lys Ala Thr
Lys Cys Ser Lys Val Leu Phe Ile Asn Ser Leu Pro Asn 165
170 175 Ser Pro Met His Asp Leu Gln Asn
Lys Ala Glu Ala Glu Thr Thr Thr 180 185
190 Lys Pro Ser Met Leu Val Asp Gly Val Ala Ser Asp Ser
Met Ser Asn 195 200 205
Asn Glu Met Glu His Gly Tyr Gly Phe Leu Ser Phe Cys Asp Glu Glu 210
215 220 Lys Glu Leu Ser
Ala Asp Leu Leu Glu Asp Phe Asn Ile Ala Asp Asp 225 230
235 240 Ile Cys Leu Ser Glu Leu Leu Asn Ser
Asp Phe Ser Asn Ala Cys Asn 245 250
255 Phe Asp Tyr Asn Asp Leu Leu Ser Pro Cys Ser Asp Gln Thr
Gln Met 260 265 270
Phe Ser Asp Asp Glu Ile Leu Lys Asn Trp Thr Gln Cys Asn Phe Ala
275 280 285 Asp Glu Thr Asn
Val Ser Asn Asn Leu His Ser Phe Ala Ser Phe Leu 290
295 300 Glu Ser Ser Glu Glu Val Leu Gly
Glu 305 310 157PRTArtificialMotif 15Asp Asp
Glu Ile Leu Lys Asn 1 5 167PRTArtificialMotif
16Lys Pro Arg Pro Arg Ser Thr 1 5
1712PRTArtificialMotif 17Xaa Val Val Arg Thr Xaa Ala Xaa Lys Cys Ser Lys
1 5 10 1824DNAArtificialPrimer
sequence 18gacaatgaga taaagaatta cttg
241921DNAArtificialPrimer sequence 19aagagttgta gacttagmtg g
212017DNAArtificialPrimer
sequence 20ytkggsaaca ggttgtc
172123DNAArtificialPrimer sequence 21atggggagaa gcccttgttg tgc
232226DNAArtificialPrimer
sequence 22tcattctcct agtacttcct cactgg
262321DNAArtificialPrimer sequence 23ctctttttgg aaggtttctc c
212423DNAArtificialPrimer
sequence 24ttctccattt tccttcacca tgg
232522DNAArtificialPrimer sequence 25tccaagcacc tctattcaag cc
222631DNAArtificialPrimer
sequence 26ctcgagatgc aatgctggtt gatggtgtgg c
312723DNAArtificialPrimer sequence 27cattgcctgt agattctgta gcc
232822DNAArtificialPrimer
sequence 28tgaagattgt tggacacatt gg
222921DNAArtificialPrimer sequence 29aggttggaat acaagacaga c
213022DNAArtificialPrimer
sequence 30tctcctagta cttcctcact gg
223123DNAArtificialPrimer sequence 31ataatcatac taattaacat cac
233220DNAArtificialPrimer
sequence 32tgatagatca tgtcattgtg
203323DNAArtificialPrimer sequence 33gccttccttt gcacaacaag ggc
233421DNAArtificialPrimer
sequence 34gcacaacaag ggcttctccc c
213523DNAArtificialPrimer sequence 35atggggagaa gcccttgttg tgc
233622DNAArtificialPrimer
sequence 36tctcctagta cttcctcact gg
223728DNAArtificialPrimer sequence 37ctcgagcaat gctggttgat
ggtgtggc 283827DNAArtificialPrimer
sequence 38tctagaggac acatttgtct catcagc
273927DNAArtificialPrimer sequence 39tctagattga gtttggtccg aacaagg
274027DNAArtificialPrimer
sequence 40tctagaaatc ttctagcaaa tctgcgg
274116DNAArtificialPrimer sequence 41gtaaaacgac ggccag
164217DNAArtificialPrimer
sequence 42caggaaacag ctatgac
174330DNAArtificialPrimer sequence 43aagcagtggt atcaacgcag
agtacgcggg 304428DNAArtificialPrimer
sequence 44aagcagtggt atcaacgcag agtactvn
284523DNAArtificialPrimer sequence 45aagcagtggt atcaacgcag agt
2346316PRTTrifolium arvense 46Met
Gly Arg Ser Pro Cys Cys Ala Lys Glu Gly Leu Asn Arg Gly Ala 1
5 10 15 Trp Thr Thr Gln Glu Asp
Lys Ile Leu Thr Glu Tyr Ile Lys Leu His 20
25 30 Gly Glu Gly Lys Trp Arg Asn Leu Pro Lys
Arg Ala Gly Leu Lys Arg 35 40
45 Cys Gly Lys Ser Cys Arg Leu Arg Trp Leu Asn Tyr Leu Arg
Pro Asp 50 55 60
Ile Lys Arg Gly Asn Ile Ser Pro Asp Glu Glu Glu Leu Ile Ile Arg 65
70 75 80 Leu His Lys Leu Leu
Gly Asn Arg Trp Ser Leu Ile Ala Gly Arg Leu 85
90 95 Pro Gly Arg Thr Asp Asn Glu Ile Lys Asn
Tyr Trp Asn Thr Asn Leu 100 105
110 Gly Lys Lys Val Lys Asp Leu Asp Gln Gln Asn Thr Asn Asn Ser
Ser 115 120 125 Pro
Thr Lys Leu Ser Ala Gln Pro Lys Asn Ala Glu Ile Lys Gln Lys 130
135 140 Gln Ile Asn Pro Lys Pro
Asn Ser Tyr Val Val Arg Thr Lys Ala Thr 145 150
155 160 Lys Cys Ser Lys Val Leu Phe Ile Asn Ser Pro
Pro Asn Ser Pro Pro 165 170
175 Met His Asp Leu Gln Ser Lys Ala Glu Ala Glu Thr Thr Thr Thr Thr
180 185 190 Lys Pro
Ser Met Pro Ser Met Leu Val Asp Gly Val Ala Ser Asp Ser 195
200 205 Met Ser Asn Asn Glu Met Glu
Cys Gly Asn Gly Phe Leu Ser Phe Cys 210 215
220 Asp Glu Glu Lys Glu Leu Ser Ala Asp Leu Leu Glu
Asp Phe Asn Ile 225 230 235
240 Ala Asp Asp Ile Cys Leu Ser Glu Phe Leu Asn Phe Asp Phe Ser Asn
245 250 255 Ala Cys Asp
Ile Asp Tyr Asn Asp Leu Leu Ser Pro Cys Ser Asp Gln 260
265 270 Thr Gln Met Phe Pro Asp Asp Glu
Ile Leu Lys Asn Trp Thr Gln Cys 275 280
285 Asn Phe Ala Asp Glu Thr Asn Val Ser Asn Asn Leu Gln
Ser Ser Ala 290 295 300
Ser Phe Leu Glu Ser Ser Glu Glu Val Leu Gly Glu 305 310
315 47310PRTTrifolium affine 47Met Gly Arg Ser Pro
Cys Cys Ala Lys Glu Gly Leu Asn Arg Gly Ala 1 5
10 15 Trp Thr Thr Gln Glu Asp Lys Ile Leu Thr
Glu Tyr Ile Lys Leu His 20 25
30 Gly Glu Gly Lys Trp Arg Asn Leu Pro Lys Arg Ala Gly Leu Lys
Arg 35 40 45 Cys
Gly Lys Ser Cys Arg Leu Arg Trp Leu Asn Tyr Leu Arg Leu Asp 50
55 60 Ile Lys Arg Gly Asn Ile
Ser Ser Asp Glu Glu Glu Leu Ile Ile Arg 65 70
75 80 Leu His Lys Leu Leu Gly Asn Arg Trp Ser Leu
Ile Ala Gly Arg Leu 85 90
95 Pro Gly Arg Thr Asp Asn Glu Ile Lys Asn Tyr Trp Asn Thr Asn Leu
100 105 110 Gly Lys
Lys Val Lys Asp Leu Asn Gln Glu Asn Thr Asn Asn Ser Ser 115
120 125 Pro Thr Lys Leu Ser Ala Gln
Leu Lys Asn Ala Lys Ile Lys Gln Lys 130 135
140 Gln Ile Asn Pro Lys Pro Met Glu Pro Asn Ser Asn
Val Val Arg Thr 145 150 155
160 Lys Ala Thr Lys Cys Ser Lys Ala Leu Phe Ile Asn Ser Pro Pro Asn
165 170 175 Ser Pro Pro
Met His Asp Leu Gln Asn Lys Ala Glu Ala Glu Thr Thr 180
185 190 Thr Lys Ser Ser Met Pro Ser Met
Leu Val Asp Gly Val Ala Ser Asp 195 200
205 Ser Met Ser Asn Asn Glu Met Glu Tyr Gly Asp Gly Phe
Val Ser Phe 210 215 220
Cys Asp Asp Asp Lys Glu Leu Ser Ala Asp Leu Leu Glu Asp Phe Asn 225
230 235 240 Ile Ser Asp Asp
Ile Cys Leu Ser Glu Phe Leu Asn Phe Asp Phe Ser 245
250 255 Asn Ala Cys Asn Phe Asp Tyr Asn Asp
Leu Leu Ser Pro Cys Ser Asp 260 265
270 Gln Thr Gln Met Phe Ser Asp Asp Glu Ile Leu Lys Asn Ser
Thr Pro 275 280 285
Cys Asn Phe Ala Ala Glu Thr Asn Tyr Val Ser Asn Asn Gln Ser Ser 290
295 300 Glu Glu Val Leu Gly
Glu 305 310 48296PRTTrifolium affine 48Met Gly Arg Ser
Pro Cys Cys Ala Lys Glu Gly Leu Asn Arg Gly Ala 1 5
10 15 Trp Thr Thr Gln Glu Asp Lys Ile Leu
Thr Glu Tyr Ile Lys Leu His 20 25
30 Gly Glu Gly Lys Trp Arg Asn Leu Pro Lys Arg Ala Gly Leu
Lys Arg 35 40 45
Cys Gly Lys Ser Cys Arg Leu Arg Trp Leu Asn Tyr Leu Arg Pro Asp 50
55 60 Ile Lys Arg Gly Asn
Ile Ser Ser Asp Glu Glu Glu Leu Ile Ile Arg 65 70
75 80 Leu His Lys Leu Leu Gly Asn Arg Trp Ser
Leu Ile Ala Gly Arg Leu 85 90
95 Pro Gly Arg Thr Asp Asn Glu Ile Lys Asn Tyr Trp Asn Thr Asn
Leu 100 105 110 Gly
Lys Lys Val Lys Asp Leu Asn Gln Glu Asn Thr Asn Asn Ser Ser 115
120 125 Pro Thr Lys Leu Ser Ala
Gln Leu Lys Asn Ala Lys Ile Lys Gln Lys 130 135
140 Gln Ile Asn Pro Lys Pro Met Glu Pro Asn Ser
Asn Val Val Arg Thr 145 150 155
160 Lys Ala Thr Lys Cys Ser Lys Ala Leu Phe Ile Asn Ser Pro Pro Asn
165 170 175 Ser Pro
Pro Met His Asp Leu Gln Asn Lys Ala Glu Ala Glu Thr Thr 180
185 190 Thr Lys Ser Ser Met Pro Ser
Met Leu Val Asp Gly Val Ala Ser Asp 195 200
205 Ser Met Ser Asn Asn Glu Met Glu Tyr Gly Asp Gly
Phe Val Ser Phe 210 215 220
Cys Asp Asp Asp Lys Glu Leu Ser Ala Asp Leu Leu Glu Asp Phe Asn 225
230 235 240 Ile Ser Asp
Asp Ile Cys Leu Ser Glu Phe Leu Asn Phe Asp Phe Ser 245
250 255 Asn Ala Cys Asn Phe Asp Tyr Asn
Asp Leu Leu Ser Pro Cys Ser Asp 260 265
270 Gln Thr Gln Met Phe Ser Asp Asp Glu Ile Leu Lys Asn
Ser Thr Gln 275 280 285
Cys Asn Phe Ala Ala Glu Thr Asn 290 295
49313PRTTrifolium occidentale 49Met Gly Arg Ser Pro Cys Cys Ala Lys Glu
Gly Leu Asn Arg Gly Ala 1 5 10
15 Trp Thr Thr Gln Glu Asp Lys Ile Leu Thr Glu Tyr Ile Lys Leu
His 20 25 30 Gly
Glu Gly Lys Trp Arg Asn Leu Pro Lys Arg Ala Gly Leu Lys Arg 35
40 45 Cys Gly Lys Ser Cys Arg
Leu Arg Trp Leu Asn Tyr Leu Arg Pro Asp 50 55
60 Ile Lys Arg Gly Asn Ile Ser Ser Asp Glu Glu
Glu Leu Ile Ile Arg 65 70 75
80 Leu His Lys Leu Leu Gly Asn Arg Trp Ser Leu Ile Ala Gly Arg Leu
85 90 95 Pro Gly
Arg Thr Asp Asn Glu Ile Lys Asn Tyr Trp Asn Thr Asn Leu 100
105 110 Gly Lys Lys Val Lys Asp Leu
Asn Gln Gln Asn Thr Asn Lys Ser Ser 115 120
125 Pro Thr Lys Leu Ser Ala Gln Pro Lys Asn Ala Lys
Ile Lys Gln Lys 130 135 140
Gln Ile Asn Pro Lys Pro Met Lys Pro Asn Ser Asn Val Val Arg Thr 145
150 155 160 Arg Ala Thr
Lys Cys Ser Lys Val Leu Phe Ile Asn Ser Leu Pro Asn 165
170 175 Ser Pro Met His Asp Leu Gln Asn
Lys Ala Glu Ala Glu Thr Thr Thr 180 185
190 Lys Pro Ser Met Leu Val Asp Gly Val Ala Ser Asp Ser
Met Ser Asn 195 200 205
Asn Glu Met Glu His Gly Tyr Gly Phe Leu Ser Phe Cys Asp Glu Glu 210
215 220 Lys Glu Leu Ser
Ala Asp Leu Leu Glu Asp Phe Asn Ile Ala Asp Asp 225 230
235 240 Ile Cys Leu Ser Glu Leu Leu Asn Ser
Asp Phe Ser Asn Ala Cys Asn 245 250
255 Phe Asp Tyr Asn Asp Leu Leu Ser Pro Cys Ser Asp Gln Thr
Gln Met 260 265 270
Phe Ser Asp Asp Glu Ile Leu Lys Asn Trp Thr Gln Cys Asn Phe Ala
275 280 285 Asp Glu Thr Asn
Val Ser Asn Asn Leu His Ser Phe Ala Ser Phe Leu 290
295 300 Glu Ser Ser Glu Glu Val Leu Gly
Glu 305 310 50312PRTTrifolium occidentale
50Met Gly Arg Ser Pro Cys Cys Ala Lys Glu Gly Leu Asn Arg Gly Ala 1
5 10 15 Trp Thr Ala His
Glu Asp Lys Ile Leu Thr Glu Tyr Ile Lys Leu His 20
25 30 Gly Glu Gly Lys Trp Arg Asn Leu Pro
Lys Arg Ala Gly Leu Lys Arg 35 40
45 Cys Gly Lys Ser Cys Arg Leu Arg Trp Leu Asn Tyr Leu Arg
Pro Asp 50 55 60
Ile Lys Arg Gly Asn Ile Ser Ser Asp Glu Glu Glu Leu Ile Ile Arg 65
70 75 80 Leu His Lys Leu Leu
Gly Asn Arg Trp Ser Leu Ile Ala Gly Arg Leu 85
90 95 Pro Gly Arg Thr Asp Asn Glu Ile Lys Asn
Tyr Trp Asn Thr Asn Leu 100 105
110 Gly Lys Lys Val Lys Asp Leu Asn Gln Gln Asn Thr Asn Asn Ser
Ser 115 120 125 Pro
Thr Lys Pro Ser Ala Gln Pro Lys Asn Ala Lys Ile Lys Gln Lys 130
135 140 Gln Gln Ile Asn Asn Pro
Lys Pro Met Lys Pro Asn Ser Asn Val Val 145 150
155 160 Arg Thr Lys Ala Thr Lys Cys Ser Lys Val Leu
Phe Ile Asn Ser Pro 165 170
175 Pro Met His Asn Leu Gln Asn Lys Ala Glu Ala Glu Thr Lys Thr Lys
180 185 190 Thr Ser
Met Leu Val Asn Gly Val Ala Ser Asp Ser Met Ser Asn Asn 195
200 205 Glu Met Glu Arg Gly Asn Gly
Phe Leu Ser Phe Arg Asp Glu Glu Lys 210 215
220 Glu Leu Ser Ala Asp Leu Leu Asp Asp Phe Asn Ile
Ala Asp Asp Ile 225 230 235
240 Cys Leu Ser Glu Phe Leu Asn Ser Asp Phe Ser Asn Ala Cys Asn Phe
245 250 255 Asp Tyr Asn
Asp Leu Leu Ser Pro Cys Ser Asp Gln Thr Gln Met Phe 260
265 270 Ser Asp Asp Glu Ile Leu Lys Asn
Trp Thr Gln Cys Asn Phe Ala Asp 275 280
285 Glu Thr Asn Val Ser Asn Asn Leu His Ser Phe Ala Ser
Phe Leu Glu 290 295 300
Ser Ser Glu Glu Val Leu Gly Glu 305 310
51311PRTTrifolium repens 51Met Gly Arg Ser Pro Cys Cys Ala Lys Glu Gly
Leu Asn Arg Gly Ala 1 5 10
15 Trp Thr Ala His Glu Asp Lys Ile Leu Thr Glu Tyr Ile Lys Leu His
20 25 30 Gly Glu
Gly Lys Trp Arg Asn Leu Pro Lys Arg Ala Gly Leu Lys Arg 35
40 45 Cys Gly Lys Ser Cys Arg Leu
Arg Trp Leu Asn Tyr Leu Arg Pro Asp 50 55
60 Ile Lys Arg Gly Asn Ile Ser Ser Asp Glu Glu Glu
Leu Ile Ile Arg 65 70 75
80 Leu His Lys Leu Leu Gly Asn Arg Trp Ser Leu Ile Ala Gly Arg Leu
85 90 95 Pro Gly Arg
Thr Asp Asn Glu Ile Lys Asn Tyr Trp Asn Thr Asn Leu 100
105 110 Gly Lys Lys Val Lys Asp Leu Asn
Gln Gln Asn Thr Asn Asn Ser Ser 115 120
125 Pro Thr Lys Pro Ser Ala Gln Pro Lys Asn Ala Asn Ile
Lys Gln Lys 130 135 140
Gln Gln Ile Asn Pro Lys Pro Met Lys Pro Asn Ser Asn Val Val Arg 145
150 155 160 Thr Lys Ala Thr
Lys Cys Ser Lys Val Leu Phe Ile Asn Ser Pro Pro 165
170 175 Met His Asn Leu Gln Asn Lys Ala Glu
Ala Glu Thr Lys Thr Lys Pro 180 185
190 Leu Met Leu Val Asn Gly Val Ala Ser Asp Ser Met Ser Asn
Asn Glu 195 200 205
Met Glu Arg Gly Asn Gly Phe Leu Ser Phe Cys Asp Glu Glu Lys Glu 210
215 220 Leu Ser Ala Asp Leu
Leu Asp Asp Phe Asn Ile Ala Asp Asp Ile Cys 225 230
235 240 Leu Ser Glu Phe Leu Asn Ser Asp Phe Ser
Asn Ala Cys Asn Phe Asp 245 250
255 Tyr Asn Asp Leu Leu Ser Pro Cys Ser Asp Gln Thr Gln Met Phe
Ser 260 265 270 Asp
Asp Glu Ile Leu Lys Asn Trp Thr Gln Cys Asn Phe Ala Asp Glu 275
280 285 Thr Asn Val Ser Asn Asn
Leu Asn Ser Phe Ala Ser Phe Leu Glu Ser 290 295
300 Ser Glu Glu Val Leu Gly Glu 305
310 52311PRTTrifolium repens 52Met Gly Arg Ser Pro Cys Cys Ala
Lys Glu Gly Leu Asn Arg Gly Ala 1 5 10
15 Trp Thr Ala His Glu Asp Lys Ile Leu Thr Glu Tyr Ile
Lys Leu His 20 25 30
Gly Glu Gly Lys Trp Arg Asn Leu Pro Lys Arg Ala Gly Leu Lys Arg
35 40 45 Cys Gly Lys Ser
Cys Arg Leu Arg Trp Leu Asn Tyr Leu Arg Pro Asp 50
55 60 Ile Lys Arg Gly Asn Ile Ser Ser
Asp Glu Glu Glu Leu Ile Ile Arg 65 70
75 80 Leu His Lys Leu Leu Gly Asn Arg Trp Ser Leu Ile
Ala Gly Arg Leu 85 90
95 Pro Gly Arg Thr Asp Asn Glu Ile Lys Asn Tyr Trp Asn Thr Asn Leu
100 105 110 Gly Lys Lys
Val Lys Asp Leu Asn Gln Gln Asn Thr Asn Asn Ser Ser 115
120 125 Pro Thr Lys Pro Ser Ala Gln Pro
Lys Asn Ala Asn Ile Lys Gln Lys 130 135
140 Gln Gln Ile Asn Pro Lys Pro Met Lys Pro Asn Ser Asn
Val Val Arg 145 150 155
160 Thr Lys Ala Thr Lys Cys Ser Lys Val Leu Phe Ile Asn Ser Pro Pro
165 170 175 Met His Asn Leu
Gln Asn Lys Ala Glu Ala Glu Thr Lys Thr Lys Pro 180
185 190 Leu Met Leu Val Asn Gly Val Ala Ser
Asp Ser Met Ser Asn Asn Glu 195 200
205 Met Glu Arg Gly Asn Gly Phe Leu Ser Phe Cys Asp Glu Glu
Lys Glu 210 215 220
Leu Ser Ala Asp Leu Leu Asp Asp Phe Asn Ile Ala Asp Asp Ile Cys 225
230 235 240 Leu Pro Glu Phe Leu
Asn Ser Asp Phe Ser Asn Ala Cys Asn Phe Asp 245
250 255 Tyr Asn Asp Leu Leu Ser Pro Cys Ser Asp
Gln Thr Gln Met Phe Ser 260 265
270 Asp Asp Glu Ile Leu Lys Asn Trp Thr Gln Cys Asn Phe Ala Asp
Glu 275 280 285 Thr
Asn Val Ser Asn Asn Leu Asn Ser Phe Ala Ser Phe Leu Glu Ser 290
295 300 Ser Glu Glu Val Leu Gly
Glu 305 310 53311PRTTrifolium repens 53Met Gly Arg
Ser Pro Cys Cys Ala Lys Glu Gly Leu Asn Arg Gly Ala 1 5
10 15 Trp Thr Ala His Glu Asp Lys Ile
Leu Thr Glu Tyr Ile Lys Leu His 20 25
30 Gly Glu Gly Lys Trp Arg Asn Leu Pro Lys Arg Ala Gly
Leu Lys Arg 35 40 45
Cys Gly Lys Ser Cys Arg Leu Arg Trp Leu Asn Tyr Leu Arg Pro Asp 50
55 60 Ile Lys Arg Gly
Asn Ile Ser Ser Asp Glu Glu Glu Leu Ile Ile Arg 65 70
75 80 Leu His Lys Leu Leu Gly Asn Arg Trp
Ser Leu Ile Ala Gly Arg Leu 85 90
95 Pro Gly Arg Thr Asp Asn Glu Ile Lys Asn Tyr Trp Asn Thr
Asn Leu 100 105 110
Gly Lys Lys Val Lys Asp Leu Asn Gln Gln Asn Thr Asn Asn Ser Ser
115 120 125 Pro Thr Lys Pro
Ser Ala Gln Pro Lys Asn Ala Asn Ile Lys Gln Lys 130
135 140 Gln Gln Ile Asn Pro Lys Pro Met
Lys Pro Asn Ser Asn Val Val Arg 145 150
155 160 Thr Lys Ala Thr Lys Cys Ser Lys Val Leu Phe Ile
Asn Ser Pro Pro 165 170
175 Met His Asn Leu Gln Asn Lys Ala Glu Ala Glu Thr Lys Thr Lys Pro
180 185 190 Leu Met Leu
Val Asn Gly Val Ala Ser Asp Ser Met Ser Asn Asn Glu 195
200 205 Met Glu Arg Gly Asn Gly Phe Leu
Ser Phe Cys Asp Glu Glu Lys Glu 210 215
220 Leu Ser Ala Asp Leu Leu Asp Asp Phe Asn Ile Ala Asp
Asp Ile Cys 225 230 235
240 Leu Ser Glu Phe Leu Asn Ser Asp Phe Ser Asn Ala Cys Asn Phe Asp
245 250 255 Tyr Asn Asp Leu
Leu Ser Pro Cys Ser Asp Gln Thr Gln Met Phe Ser 260
265 270 Asp Asp Glu Ile Leu Lys Asn Trp Thr
Gln Cys Asn Phe Ala Asp Glu 275 280
285 Thr Asn Val Ser Asn Asn Leu His Ser Phe Ala Ser Phe Leu
Glu Ser 290 295 300
Ser Glu Glu Val Leu Gly Glu 305 310 54311PRTTrifolium
repens 54Met Gly Arg Ser Pro Cys Cys Ala Lys Glu Gly Leu Asn Arg Gly Ala
1 5 10 15 Trp Thr
Ala His Glu Asp Lys Ile Leu Thr Glu Tyr Ile Lys Leu His 20
25 30 Gly Glu Gly Lys Trp Arg Asn
Leu Pro Lys Arg Ala Gly Leu Lys Arg 35 40
45 Cys Gly Lys Ser Cys Arg Leu Arg Trp Leu Asn Tyr
Leu Arg Pro Asp 50 55 60
Ile Lys Arg Gly Asn Ile Ser Ser Asp Glu Glu Glu Leu Ile Ile Arg 65
70 75 80 Leu His Lys
Leu Leu Gly Asn Arg Trp Ser Leu Ile Ala Gly Arg Leu 85
90 95 Pro Gly Arg Thr Asp Asn Glu Ile
Lys Asn Tyr Trp Asn Thr Asn Leu 100 105
110 Gly Lys Lys Val Lys Asp Leu Asn Gln Gln Asn Thr Asn
Asn Ser Ser 115 120 125
Pro Thr Lys Pro Ser Ala Gln Pro Lys Asn Ala Asn Ile Lys Gln Lys 130
135 140 Gln Gln Ile Asn
Pro Lys Pro Met Lys Pro Asn Ser Asn Val Val Arg 145 150
155 160 Thr Lys Ala Thr Lys Cys Ser Lys Val
Leu Phe Ile Asn Ser Pro Pro 165 170
175 Met His Asn Leu Gln Asn Lys Ala Glu Ala Glu Thr Lys Thr
Lys Pro 180 185 190
Leu Met Leu Val Asn Gly Val Ala Ser Asp Ser Met Ser Asn Asn Glu
195 200 205 Met Glu Arg Gly
Asn Gly Phe Leu Ser Phe Cys Asp Glu Glu Lys Glu 210
215 220 Leu Ser Ala Asp Leu Leu Asp Asp
Phe Asn Ile Ala Asp Asp Ile Cys 225 230
235 240 Leu Ser Glu Phe Leu Asn Ser Asp Phe Ser Asn Ala
Cys Asn Phe Asp 245 250
255 Tyr Asn Asp Leu Leu Ser Pro Cys Ser Asp Gln Thr Gln Met Phe Ser
260 265 270 Asp Asp Glu
Ile Leu Lys Asn Trp Thr Gln Cys Asn Phe Ala Asp Glu 275
280 285 Thr Asn Val Ser Asn Asn Leu His
Ser Phe Ala Ser Phe Leu Glu Ser 290 295
300 Ser Glu Glu Val Leu Gly Glu 305 310
55942DNATrifolium arvense 55atggggagaa gcccttgttg tgcaaaggaa
ggcttgaata gaggtgcttg gacaactcaa 60gaagacaaaa tcctcactga atacattaag
ctccatggtg aaggaaaatg gagaaacctt 120ccaaaaagag caggtttaaa aagatgcgga
aaaagttgta gacttagatg gttgaattat 180ctaagaccag atattaagcg aggtaatata
tcctcggatg aagaagaact tatcatcaga 240cttcacaaac tactcggaaa cagatggtct
ctaatagccg gaagacttcc aggacgaaca 300gacaatgaaa taaagaacta ctggaacaca
aatttaggaa aaaaggttaa ggatcttaat 360caacaaaaca ccaacaattc ttctcctact
aaactctctg ctcaaccaaa aaatgcaaag 420atcaaacaga aacagatcaa tcctaagcca
atgaagccaa actcaaatgt tgtccgtaca 480aaagctacca agtgttctaa ggtattgttc
ataaactcac tccccaactc accaatgcat 540gatttgcaga acaaagctga ggcagagaca
acaacaaagc catcaatgct ggttgatggt 600gtggctagtg attcaatgag taacaacgaa
atggaacacg gttatggatt tttgtcattt 660tgcgatgaag agaaagaact atccgcagat
ttgctagaag attttaacat cgcggatgat 720atttgcttat ctgaactttt gaactctgat
ttctcaaatg cgtgcaattt cgattacaat 780gatctattgt caccttgttc ggaccaaact
caaatgttct ctgatgatga gattctcaag 840aattggacac aatgtaactt tgctgatgag
acaaatgtgt ccaacaacct tcattctttt 900gcttcctttc ttgaatccag tgaggaagta
ctaggagaat ga 94256933DNATrifolium arvense
56atggggagaa gcccttgttg tgcaaaggaa ggcttgaata gaggtgcttg gacaactcaa
60gaagacaaaa tcctcactga atacattaag ctccatggtg aaggaaaatg gagaaacctt
120ccaaaaagag caggtttaaa aagatgtgga aaaagttgta gacttagatg gttgaattat
180ctaagaccag atattaagcg aggtaatata tcctcggatg aagaagaact tatcatccga
240cttcacaaac tactcggaaa cagatggtct ctaatagccg gaagacttcc agggcgaaca
300gacaatgaaa taaagaacta ctggaacaca aatttaggaa aaaaggttaa ggatcttaat
360caagaaaaca ccaacaattc ttctcctact aaactttctg ctcaactaaa aaatgcaaag
420atcaaacaga aacagatcaa tcctaagcca atggagccaa actcaaatgt tgtccgtaca
480aaagctacca agtgttctaa ggcattgttc ataaactcac cccccaactc accaccaatg
540catgatttgc agaacaaagc tgaggcagag acaacaacaa agtcatcaat gccatcaatg
600ctggttgatg gcgtggctag tgattcaatg agtaacaacg aaatggaata cggtgatgga
660tttgtttcat tttgcgatga cgataaagaa ctatccgcag atttgctaga agattttaac
720atctcggatg atatttgctt atccgaattt ctaaacttcg atttctcaaa tgcgtgcaat
780ttcgattaca acgatctatt gtcgccttgt tcggaccaaa cacaaatgtt ctctggtgat
840gagattctca agaattcgac acaatgtaac tttgctgctg agacaaatta tgtgtccaac
900aaccaatcca gtgaggaagt actaggagaa tga
93357933DNATrifolium affine 57atggggagaa gcccttgttg tgcgaaggaa ggcttgaata
gaggtgcttg gacaactcaa 60gaagacaaaa tcctcactga atacattaag ctccatggtg
aaggaaaatg gagaaacctt 120ccaaaaagag caggtttaaa aagatgtgga aaaagttgta
gacttagatg gttgaattat 180ctaagactag atattaagcg aggtaatata tcctcggatg
aagaagaact tatcatccga 240cttcacaaat tactcggaaa cagatggtct ctaatagccg
gaagacttcc aggacgaaca 300gacaatgaaa taaagaacta ctggaacaca aatttaggaa
aaaaggttaa ggatcttaat 360caagaaaaca ccaacaattc ttctcctact aaactttctg
ctcaactaaa aaatgcaaag 420atcaaacaga aacagatcaa tcctaagcca atggagccaa
actcaaatgt tgtccgtaca 480aaagctacca agtgttctaa ggcattgttc ataaactcac
cccccaactc accaccaatg 540catgatttgc agaacaaagc tgaggcagag acaacaacaa
agtcatcaat gccatcaatg 600ctggttgatg gcgtggctag tgattcaatg agtaacaacg
aaatggaata cggtgatgga 660tttgtttcat tttgcgatga cgataaagaa ctatccgcag
atttgctaga agattttaac 720atctcggatg atatttgctt atccgaattt ctaaacttcg
atttctcaaa tgcgtgcaat 780ttcgattaca acgatctatt gtcgccttgt tcggaccaaa
cacaaatgtt ctctgatgat 840gagattctca agaattcgac accatgtaac tttgctgctg
agacaaatta tgtgtccaac 900aaccaatcca gtgaggaagt actaggagaa tga
93358891DNATrifolium affine 58atggggagaa
gcccttgttg tgcaaaggaa ggcttgaata gaggtgcttg gacaactcaa 60gaagacaaaa
tcctcactga atacattaag ctccatggtg aaggaaaatg gagaaacctt 120ccaaaaagag
caggtttaaa aagatgtgga aaaagttgta gacttagatg gttgaattat 180ctaagaccag
atattaagcg aggtaatata tcctcggatg aagaagaact tatcatccga 240cttcacaaac
tactcggaaa cagatggtct ctaatagccg gaagacttcc agggcgaaca 300gacaatgaaa
taaagaacta ctggaacaca aatttaggaa aaaaggttaa ggatcttaat 360caagaaaaca
ccaacaattc ttctcctact aaactttctg ctcaactaaa aaatgcaaag 420atcaaacaga
aacagatcaa tcctaagcca atggagccaa actcaaatgt tgtccgtaca 480aaagctacca
agtgttctaa ggcattgttc ataaactcac cccccaactc accaccaatg 540catgatttgc
agaacaaagc tgaggcagag acaacaacaa agtcatcaat gccatcaatg 600ctggttgatg
gcgtggctag tgattcaatg agtaacaacg aaatggaata cggtgatgga 660tttgtttcat
tttgcgatga cgataaagaa ctatccgcag atttgctaga agattttaac 720atctcggatg
atatttgctt atccgaattt ctaaacttcg atttctcaaa tgcgtgcaat 780ttcgattaca
acgatctatt gtcgccttgt tcggaccaaa cacaaatgtt ctctgatgat 840gagattctca
agaattcgac acaatgtaac tttgctgctg agacaaatta a
89159942DNATrifolium occidentale 59atggggagaa gcccttgttg tgcaaaggaa
ggcttgaata gaggtgcttg gacaactcaa 60gaagacaaaa tcctcactga atacattaag
ctccatggtg aaggaaaatg gagaaacctt 120ccaaaaagag caggtttaaa aagatgcgga
aaaagttgta gacttagatg gttgaattat 180ctaagaccag atattaagcg aggtaatata
tcctcggatg aagaagaact tatcatcaga 240cttcacaaac tactcggaaa cagatggtct
ctaatagccg gaagacttcc aggacgaaca 300gacaatgaaa taaagaacta ctggaacaca
aatttaggaa aaaaggttaa ggatcttaat 360caacaaaaca ccaacaagtc ttctcctact
aaactctctg ctcaaccaaa aaatgcaaag 420atcaaacaga aacagatcaa tcctaagcca
atgaagccaa actcaaatgt tgtccgtaca 480agagctacca agtgttctaa ggtattgttc
ataaactcac tccccaactc accaatgcat 540gatttgcaga acaaagctga ggcagagaca
acaacaaagc catcaatgct ggttgatggt 600gtggctagtg attcaatgag taacaacgaa
atggaacacg gttatggatt tttgtcattt 660tgcgatgaag agaaagaact atccgcagat
ttgctagaag attttaacat cgcggatgat 720atttgcttat ctgaactttt gaactctgat
ttctcaaatg cgtgcaattt cgattacaat 780gatctattgt cmccttgttc ggaccaaact
caaatgttct ctgatgatga gattctcaag 840aattggacac aatgtaactt tgctgatgag
acaaatgtgt ccaacaacct tcattctttt 900gcttcctttc ttgaatccag tgaggaagta
ctaggagaat ga 94260939DNATrifolium occidentale
60atggggagaa gcccttgttg tgcaaaggaa ggtttgaata gaggtgcttg gacagctcat
60gaagacaaaa tcctcactga atacattaag ctccatggtg aaggaaaatg gagaaacctt
120ccaaaaagag caggtttaaa aagatgtgga aaaagttgta gacttagatg gttgaattat
180cttagaccag atattaagag aggtaatata tcgtccgatg aagaagaact tatcattaga
240cttcacaaac tacttggaaa ccgatggtct ctaatagccg gaagacttcc agggcgaaca
300gacaatgaaa taaaaaatta ctggaacacg aatttaggaa aaaaggttaa ggatcttaat
360caacaaaaca ccaacaattc ttctcctact aaaccttctg ctcaaccaaa aaatgcaaag
420atcaaacaga aacaacagat caataatcct aagccaatga agccaaactc gaatgttgtc
480cgtacaaaag ctaccaaatg ttctaaggta ttgttcataa actcaccacc aatgcataat
540ttgcagaaca aagctgaggc agagacaaaa acaaagacat caatgttggt taatggtgta
600gctagtgatt caatgagtaa caacgaaatg gaacgaggta atggattttt gtcatttcgc
660gatgaagaga aagaactatc cgctgatttg ctagatgatt ttaacatcgc ggatgacatt
720tgcttatccg aatttctaaa ctccgatttc tcaaatgcgt gcaatttcga ttacaatgat
780ctattgtcac cttgttcgga tcaaactcaa atgttctctg atgatgagat tctcaagaat
840tggacacaat gtaactttgc tgatgagaca aatgtgtcca acaaccttca ttcttttgct
900tcctttctcg aatccagtga ggaagtacta ggagaatga
93961936DNATrifolium repens 61atggggagaa gcccttgttg tgcaaaagaa ggcttgaata
gaggtgcttg gacagctcat 60gaagacaaaa tcctcactga atacattaag ctccatggtg
aaggaaaatg gagaaacctt 120ccaaaaagag caggtttaaa aagatgtgga aaaagttgta
gactaaggtg gttgaattat 180cttagaccgg atattaagag aggtaatata tcgtcggatg
aagaagaact tatcattaga 240cttcacaaac tactcggaaa ccgatggtct ctaatagccg
gaagacttcc agggcgaaca 300gacaatgaaa taaagaacta ctggaacaca aatttaggaa
aaaaagttaa ggatcttaat 360caacaaaaca ccaacaattc ttctcctact aaaccttctg
ctcaaccaaa aaatgcaaat 420atcaaacaga aacaacagat caatcctaag ccaatgaagc
caaactcgaa tgttgtccgt 480acaaaagcta ccaaatgttc taaggtattg ttcataaact
caccaccaat gcataatttg 540cagaacaaag ctgaggcaga gacaaaaaca aagccattaa
tgctggttaa tggtgtagct 600agtgattcaa tgagtaacaa cgaaatggaa cgcggtaatg
gatttttgtc attttgcgac 660gaagagaaag aactatccgc agatttgcta gatgatttta
acatcgcgga tgatatttgc 720ttatctgaat ttctaaactc cgatttctca aatgcgtgca
atttcgatta caatgatcta 780ttgtcgcctt gttcggatca aactcaaatg ttctctgatg
atgagattct caagaattgg 840acacaatgta actttgctga tgagacaaat gtgtccaaca
accttaattc ttttgcttct 900tttctcgaat ccagtgagga agtactagga gaatga
93662936DNATrifolium repens 62atggggagaa
gcccttgttg tgcaaaagaa ggcttgaata gaggtgcttg gacagctcat 60gaagacaaaa
tcctcactga atacattaag ctccatggtg aaggaaaatg gagaaacctt 120ccaaaaagag
caggtttaaa aagatgtgga aaaagttgta gactaaggtg gttgaattat 180cttagaccgg
atattaagag aggtaatata tcgtcggatg aagaagaact tatcattaga 240cttcacaaac
tactcggaaa ccgatggtct ctaatagccg gaagacttcc agggcgaaca 300gacaatgaaa
taaagaacta ctggaacaca aatttaggaa aaaaagttaa ggatcttaat 360caacaaaaca
ccaacaattc ttctcctact aaaccttctg ctcaaccaaa aaatgcaaat 420atcaaacaga
aacaacagat caatcctaag ccaatgaagc caaactcgaa tgttgtccgt 480acaaaagcta
ccaaatgttc taaggtattg ttcataaact caccaccaat gcataatttg 540cagaacaaag
ctgaggcaga gacaaaaaca aagccattaa tgctggttaa tggtgtagct 600agtgattcaa
tgagtaacaa cgaaatggaa cgcggtaatg gatttttgtc attttgcgac 660gaagagaaag
aactatccgc agatttgcta gatgatttta acatcgcgga tgatatttgc 720ttacctgaat
ttctaaactc cgatttctca aatgcgtgca atttcgatta caatgatcta 780ttgtcgcctt
gttcggatca aactcaaatg ttctctgatg atgagattct caagaattgg 840acacaatgta
actttgctga tgagacaaat gtgtccaaca accttaattc ttttgcttct 900tttctcgaat
ccagtgagga agtactagga gaatga
93663936DNATrifolium repens 63atggggagaa gcccttgttg tgcaaaagaa ggcttgaata
gaggtgcttg gacagctcat 60gaagacaaaa tcctcactga atacattaag ctccatggtg
aaggaaaatg gagaaacctt 120ccaaaaagag caggtttaaa aagatgtgga aaaagttgta
gactaaggtg gttgaattat 180cttagaccgg atattaagag aggtaatata tcgtcggatg
aagaagaact tatcattaga 240cttcacaaac tactcggaaa ccgatggtct ctaatagccg
gaagacttcc agggcgaaca 300gacaatgaaa taaagaacta ctggaacaca aatttaggaa
aaaaagttaa ggatcttaat 360caacaaaaca ccaacaattc ttctcctact aaaccttctg
ctcaaccaaa aaatgcaaat 420atcaaacaga aacaacagat caatcctaag ccaatgaagc
caaactcgaa tgttgtccgt 480acaaaagcta ccaaatgttc taaggtattg ttcataaact
caccaccaat gcataatttg 540cagaacaaag ctgaggcaga gacaaagaca aagccattaa
tgctggttaa tggtgtagct 600agtgattcaa tgagtaacaa cgaaatggaa cgcggtaatg
gatttttgtc attttgcgac 660gaagagaaag aactatccgc agatttgcta gatgatttta
acatcgcgga tgatatttgc 720ttatctgaat ttctaaactc cgatttctca aatgcgtgca
atttcgatta caatgatcta 780ttgtcgcctt gttcggatca aactcaaatg ttctctgatg
atgagattct caagaattgg 840acacaatgta actttgctga tgagacaaat gtgtccaaca
accttcattc ttttgcttcc 900tttctcgaat ccagtgagga agtactagga gaatga
93664936DNATrifolium repens 64atggggagaa
gcccttgttg tgcaaaagaa ggcttgaata gaggtgcttg gacagctcat 60gaagacaaaa
tcctcactga atacattaag ctccatggtg aaggaaaatg gagaaacctt 120ccaaaaagag
caggtttaaa aagatgtgga aaaagttgta gactaaggtg gttgaattat 180cttagaccgg
atattaagag aggtaatata tcgtcggatg aagaagaact tatcattaga 240cttcacaaac
tactcggaaa ccgatggtct ctaatagccg gaagacttcc agggcgaaca 300gacaatgaaa
taaagaacta ctggaacaca aatttaggaa aaaaagttaa ggatcttaat 360caacaaaaca
ccaacaattc ttctcctact aaaccttctg ctcaaccaaa aaatgcaaat 420atcaaacaga
aacaacagat caatcctaag ccaatgaagc caaactcgaa tgttgtccgt 480acaaaagcta
ccaaatgttc taaggtattg ttcataaact caccaccaat gcataatttg 540cagaacaaag
ctgaggcaga gacaaagaca aagccattaa tgctggttaa tggtgtagct 600agtgattcaa
tgagtaacaa cgaaatggaa cgcggtaatg gatttttgtc attttgcgac 660gaagagaaag
aactatccgc agatttgcta gatgatttta acatcgcgga tgatatttgc 720ttatctgaat
ttctaaactc cgatttctca aatgcgtgca atttcgatta caatgatcta 780ttgtcgcctt
gttcggatca aactcaaatg ttctctgatg atgagattct caagaattgg 840acacaatgta
actttgctga tgagacaaat gtgtccaaca accttcattc ttttgcttcc 900tttctcgaat
ccagtgagga agtactagga gaatga
93665299DNATrifolium arvense 65caatgctggt tgatggtgtg gctagtgatt
caatgagtaa caacgaaatg gaacacggtt 60atggattttt gtcattttgc gatgaagaga
aagaactatc cgcagatttg ctagaagatt 120ttaacatcgc ggatgatatt tgcttatctg
aacttttgaa ctctgatttc tcaaatgcgt 180gcaatttcga ttacaatgat ctattgtcac
cttgttcgga ccaaactcaa atgttctctg 240atgatgagat tctcaagaat tggacacaat
gtaactttgc tgatgagaca aatgtgtcc 2996628DNAArtificialPrimer sequence
66tctagacaat gctggttgat ggtgtggc
286727DNAArtificialPrimer sequence 67tctagaggac acatttgtct catcagc
276828DNAArtificialPrimer sequence
68ctcgagcaat gctggttgat ggtgtggc
286927DNAArtificialPrimer sequence 69ctcgagggac acatttgtct catcagc
2770912DNALotus japonicus 70atgggaagaa
gcccttgttg ttcaaagcag ggtttgaacc gaggtgcctg gacagcacag 60gaagaccaaa
tcctccgaga ctatgttcat ctccatggcc aaggaaaatg gaggaacctt 120cctcaaagtg
caggtttgaa acgttgtggc aaaagctgta gacttagatg gttgaattat 180ctaagaccag
atatcaaaag aggcaatata tccagagatg aagaagagct tatcatccga 240cttcacaagc
tcctaggaaa cagatggtct ctaatagctg gaaggcttcc aggaagaaca 300gacaatgaga
taaagaacta ctggaacacc aatctatgta aaagagttca agatggtgtt 360gatgttggtg
actccaaaac cccatcttca caagaaaaga acaatcacca tgatcagaaa 420gcaaagcctc
aatctgttac tccctcagta ttctcctcat cacagcctaa aaacaataat 480gtgattcgta
caaaggcatc gaagtgctcc aaggtgctgc tccgggatcc tcttctccct 540tgcccgccaa
tgcaaacgca gagcgacgat ttcatcgcaa aattattaga agaagcagaa 600ggagagccat
tgctttctgc tgtggccaat gattttacta gtggcgacga agacggggtt 660ctttcatttg
atccttgtgg aaatgagaag gaactctcca cggatttgct cttggatttg 720gacattggtg
aaatttgctt gcctgaattt atcaactcag atttttcata tgtgtgtgac 780ttcagctaca
acactcatga ggatctaatg cttttttccg agaacacact tgtccaggca 840cagaagtacc
tcggtgatga aacaaatttg gtaaataatt gttttaatga ggagaaggat 900aatggttgct
aa
912711143DNATrifolium affine 71ctaattaaga ataacatcaa tggggagaag
cccttgttgt gcaaaggaag gcttgaatag 60aggtgcttgg acaactcaag aagacaaaat
cctcactgaa tacattaagc tccatggtga 120aggaaaatgg agaaaccttc caaaaagagc
agatttaaaa agatgtggaa aaagttgtag 180acttagatgg ttgaattatc taagaccaga
tattaagcga ggtaatatat ccccggatga 240agaagaactt attatccgac ttcacaaact
actcggaaac agatggtctc taatagccgg 300aagacttcca gggcgaacag acaatgaaat
aaagaactac tggaacacaa atttaggaaa 360aaaggttaag gatcttaatc aacaaaacac
caacaattct tctcctacta aactttctgc 420tcaaccaaaa aatgcaaaga tcaaacagaa
acagatcaat cctaagccaa tgaagccaaa 480ctcaaatgtt gtccgtacaa aagctaccaa
gtgttctaag gtattgttca taaactcact 540ccccaactca ccaatgcatg atttgcagaa
caaagctgag gcagagacaa caacaaagcc 600atcaatgctg gttgatggtg tggctagtga
ttcaatgagt aacaacgaaa tggaacacgg 660ttatggattt ttgtcatttt gcgatgaaga
gaaagaacta tccgcagatt tgctagaaga 720ttttaacatc gcggatgata tttgcttatc
tgaacttttg aactctgatt tctcaaatgc 780gtgcaatttc gattacaatg atctattgtc
accttgttcg gaccaaactc aaatgttctc 840tgatgatgag attctcaaga attggacaca
atgtaacttt gctgatgaga caaatgtgtc 900caacaacctt cattcttttg cttcctttct
tgaatccagt gaggaagtac taggagaatg 960ataataaaaa ttcattttcc aataaaatta
actactctag gttttttttt ttttttttta 1020atttcaattt catgttaggg tggtttaata
aataaatata ttctatggtt taatattgca 1080aaaaaaaaaa aaaaaaaaaa aaaaagtact
ctgcgttgat accactgctt aagggcgaat 1140tcc
1143721049DNAGlycine max 72gcaaaaaatg
ggaagggctc cttgttgttc caaagtgggg ttgcacaaag gtccatggac 60tcctaaagaa
gatgcattgc ttaccaagta tatccaagct catggagaag gccaatggaa 120atcactaccc
aaaaaagcag ggcttcttag atgtggaaaa agttgtagat tgagatggat 180gaactatctg
agaccagaca taaagagagg gaacatagca ccagaagaag atgatcttat 240aatcagaatg
cattcacttt tgggaaacag atggtccctc atagcaggaa ggttaccagg 300gagaacagac
aatgaaataa agaactactg gaacacccat ctaagcaaaa agctgaaaat 360tcaaggaaca
gaagacacag acacacacaa aatgttagag aatcctcaag aagaggctgc 420aagtgatggt
ggcaacaaca acaaaaagaa gaagaagaag aagaacggtg gcaaaaagaa 480caagcagaag
aacaaaggca aagaaaatga tgagccgcca aagacacaag tttacctacc 540aaaaccaatt
agagtgaagg caatgtattt acaaagaacg gatagtaaca ccttcacctt 600tgattccaat
tcagctagtg gatcaacaag ccaagagaag gaggaaagcc ccgtgacaaa 660agaatcaaac
gtggttagtg aagttggtaa tgtgggagaa gaaagtgatg gttttggctt 720cttcagtgag
gaccatgact tagtcaacgt ctcagatatt gaatgccact cttattttcc 780cacagatcat
ggcaacctac agcaattgta tgaagaatat ttccagctct tgaacatgga 840ccatggccaa
ttcgaactga attcatttgc agaatcttta ttagattaaa agaatatcaa 900caaagatttg
ttcagttcat gaagatcaca ttgcttacat ataaactttg ttgatagatc 960atatgtaaat
atatctgtaa atgatctctg agttatgaga tcttttttgt ctttaataaa 1020tatcgccatc
taactcaaaa aaaaaaaaa
1049731000DNADaucus carota 73gaagaatggg aaggagccct tgttgctcaa aagttgggct
gaacaaagga gcctggacca 60ctgctgagga caaaattctc actgatttca ttcatcttca
tggtgaaggt ggatggagaa 120accttcccaa aagagcaggt ttgaagagat gcggaaagag
ttgcaggctg agatggttga 180attatttgag accggatatc aagagaggca acatttctga
tgatgaagaa gacctcatca 240ttcgtcttca caagcttctc ggtaataggt ggtctttaat
agctggaagg ctccctggcc 300gaacagacaa tgaaatcaag aactactgga acacgacatt
gaggaaaaag gctcatgata 360atcacacttc atctgcagct gctccaaaga ccccgactaa
acaatgcaac aacaagaaga 420cgaagaaaca caagaagaag cgcgagaaat ctgagccaat
taaaccggaa atcaaggcca 480atgcatccga tgttagggcc aaggccgctc tggacgaggc
tgatcatcaa ctcataacta 540gtactagtac catggagcca ttggttcaac aagcattaca
aaataagact actgatcaat 600cttcggatct ggtccctggc gttgactcca gcgacatgtg
cttaacggat tttcttaatt 660atgatttctc aggtttgtta aacactgata ttaatcacca
ggattacgac atggagagcg 720cgtcgccttg ttcgtcgtcg gagaagccta taatgcagat
actggaggag ttctggaatg 780cagaggaacc atgtctggtt tctaactcta atctttattt
tacctcatta tcagagtgtt 840tagtgggtga ttggttggcc taatatgtga aaactgggaa
gtgtacattt tactgttgtt 900cattttactt aacttcccgg aaataaagat gcatgtatca
tagttcaaat aatgactact 960tctgatgtgt tgaattgttg taaaaaaaaa aaaaaaaaaa
100074909DNAGossypium hirsutum 74atgggaagga
gtccttgttg ttctaaggaa ggccttaaca gaggagcttg gactgctctt 60gaagacaaaa
ttcttaaaga ttatatcaaa gtacacggtg aaggtcgttg gagaaatctc 120cccaaaagag
ctggtcttaa gagatgtggg aaaagttgta ggcttcggtg gttgaattat 180ttgagacctg
atattaaaag aggtaacata tcacctgacg aggaagagct tatcatcaaa 240ctccacaaac
tcttgggaaa cagatggtct ttgatagctg ggaggcttcc aggacgaaca 300gacaatgaaa
taaagaatta ctggaacacc aacttaagta aaagagtttc cgatcgtcaa 360aagtcacccg
ccgctccttc gaaaaaaccc gaggcggctc gacgaggaac tgctggtaat 420ggcaatacca
atggtaatgg tagtggtagt tcctcgacac acgtggtgcg gacaagggcg 480acaaggtgct
ccaaggtttt cataaaccct catcaccaca cacaaaacag acacccaaag 540ccttcctcaa
cttgttcaaa tcatggggat caccgggaac ctaaaacaat gaatgagttg 600ttattaccga
taatgtcaga atccgagaat gaagggacga ccgatcatat atcatcggat 660tttacatttg
acttcaacat gggagagttt tgtttatcgg atcttttgaa ttccgatttc 720tgcgatgtaa
acgagcttaa ttacagcaat ggttttgatt cgtcaccctc accggatcag 780cctcctatgg
atttctccga cgaaatgcta aaagagtgga cggccgccgc ctccactcac 840tgctgtcacc
aaagtgcggc ttccaatctc cagtccttgc ctccatttat tgaaaatgga 900attgaatga
90975938DNABrassica napus 75aatctattct caacacaacg ctaaagacaa gtctaccaac
cacacaacaa caagagagat 60gatgagaaag agagaaagta gtaaggtgaa gaaagaggag
ttaaacagag gggcttggac 120cgatcaagaa gacaagatcc ttaaagacta tatcatgttc
cacggcgaag gaaaatggag 180cacactccca aaccaagctg gtctcaagag gtgtggcaaa
agctgcagac ttcggtggaa 240gaactacttg agaccaggca taaagcgcgg aaacatctca
tctgatgaag aagaacttat 300aatccgcctc cataatctcc ttggaaacag atggtcgttg
atagctggga ggcttccagg 360gcgaacagac aatgaaataa agaaccactg gaactcaaac
ctccgcaaaa gacttccaaa 420atctcaaacc aaccaacaga aaagtcgaaa acattccaac
aacaacaaca tgaataaagt 480atgtgttata cgtccaaagg cgattaggat cccaaaggct
ctgacatttc agaatcagag 540tagtattggt agtaccagtc ttcttactgt gaaggaaaac
gtgattgatc atcaagctgg 600ttctccttcg ttgttgggag atcttaaaat cgattttgat
aaaattcagt ctgagtatct 660cttctctgat ttaatgggct ttgatggttt gggttgtgga
aacgtaatgt ctcttgtttc 720atctgacgag gtgctaggag attatgtttc ggctgatgct
tcttgtctgg gtaatcttga 780tcttaataga cctttcactt cttgtcttca agaagattgt
ctctgggact ttaattgtta 840gaccctatcg taaatcttca tatattacgt ctacctctgt
acgaacaaaa gtatatattt 900atattctgtt tgaacgcttc taattacaag taatatct
93876816DNAGossypium hirsutum 76atgggaagaa
gtccatgttg ctccaaggaa ggactcaaca aaggagcttg gactgcttta 60gaagataaaa
tacttgcatc atatattcat gttcatggtg aaggcaaatg gagaaacctc 120cccaagagag
ctggtttgaa gagatgtggc aaaagttgca gacttagatg gctgaattat 180cttagaccag
atattaaaag aggcaacatc tctcatgatg aagaagaact cattataaga 240ctccataatc
ttcttggcaa cagatggtct ttaatagctg gaaggctacc cgggcgaaca 300gacaatgaaa
tcaagaacta ctggaacact actttaggta agagagctaa agctcaagca 360tccattgatg
ctaaaacgat accaaccgag tctaggctca atgaaccctc gaaaagttca 420actaaaatcg
aagtgattcg aactaaagct attaggtgta gcagcaaggt gatggtccca 480ttacaaccac
ctgcaactca tcaacatggt caacatcact gtacaaataa taatgaagaa 540atgggtggtg
gtattgcaac aattgaagct cacaatggaa ttcaaatgct cgagtcattg 600tacagtgatg
gcggctcaaa tttgttgagc ttcgagatca atgaactgtt gaaatcacac 660gatggtggag
aatttgagga gaatcctatg cagcagcact ttccgttggg tgaggcaatg 720cttaaggatt
ggtctacatg tcattgtctt gatgacaatg gtgccactga tttggaatca 780ttggcctttt
tgcttgacac tgatgaatgg ccatga
81677258PRTArabidopsis thaliana 77Met Gly Lys Arg Ala Thr Thr Ser Val Arg
Arg Glu Glu Leu Asn Arg 1 5 10
15 Gly Ala Trp Thr Asp His Glu Asp Lys Ile Leu Arg Asp Tyr Ile
Thr 20 25 30 Thr
His Gly Glu Gly Lys Trp Ser Thr Leu Pro Asn Gln Ala Gly Leu 35
40 45 Lys Arg Cys Gly Lys Ser
Cys Arg Leu Arg Trp Lys Asn Tyr Leu Arg 50 55
60 Pro Gly Ile Lys Arg Gly Asn Ile Ser Ser Asp
Glu Glu Glu Leu Ile 65 70 75
80 Ile Arg Leu His Asn Leu Leu Gly Asn Arg Trp Ser Leu Ile Ala Gly
85 90 95 Arg Leu
Pro Gly Arg Thr Asp Asn Glu Ile Lys Asn His Trp Asn Ser 100
105 110 Asn Leu Arg Lys Arg Leu Pro
Lys Thr Gln Thr Lys Gln Pro Lys Arg 115 120
125 Ile Lys His Ser Thr Asn Asn Glu Asn Asn Val Cys
Val Ile Arg Thr 130 135 140
Lys Ala Ile Arg Cys Ser Lys Thr Leu Leu Phe Ser Asp Leu Ser Leu 145
150 155 160 Gln Lys Lys
Ser Ser Thr Ser Pro Leu Pro Leu Lys Glu Gln Glu Met 165
170 175 Asp Gln Gly Gly Ser Ser Leu Met
Gly Asp Leu Glu Phe Asp Phe Asp 180 185
190 Arg Ile His Ser Glu Phe His Phe Pro Asp Leu Met Asp
Phe Asp Gly 195 200 205
Leu Asp Cys Gly Asn Val Thr Ser Leu Val Ser Ser Asn Glu Ile Leu 210
215 220 Gly Glu Leu Val
Pro Ala Gln Gly Asn Leu Asp Leu Asn Arg Pro Phe 225 230
235 240 Thr Ser Cys His His Arg Gly Asp Asp
Glu Asp Trp Leu Arg Asp Phe 245 250
255 Thr Cys 78260PRTBrassica napus 78Met Met Arg Lys Arg
Glu Ser Ser Lys Val Lys Lys Glu Glu Leu Asn 1 5
10 15 Arg Gly Ala Trp Thr Asp Gln Glu Asp Lys
Ile Leu Lys Asp Tyr Ile 20 25
30 Met Phe His Gly Glu Gly Lys Trp Ser Thr Leu Pro Asn Gln Ala
Gly 35 40 45 Leu
Lys Arg Cys Gly Lys Ser Cys Arg Leu Arg Trp Lys Asn Tyr Leu 50
55 60 Arg Pro Gly Ile Lys Arg
Gly Asn Ile Ser Ser Asp Glu Glu Glu Leu 65 70
75 80 Ile Ile Arg Leu His Asn Leu Leu Gly Asn Arg
Trp Ser Leu Ile Ala 85 90
95 Gly Arg Leu Pro Gly Arg Thr Asp Asn Glu Ile Lys Asn His Trp Asn
100 105 110 Ser Asn
Leu Arg Lys Arg Leu Pro Lys Ser Gln Thr Asn Gln Gln Lys 115
120 125 Ser Arg Lys His Ser Asn Asn
Asn Asn Met Asn Lys Val Cys Val Ile 130 135
140 Arg Pro Lys Ala Ile Arg Phe Pro Lys Ala Leu Thr
Phe Gln Asn Gln 145 150 155
160 Ser Ser Ile Gly Ser Thr Ser Leu Leu Thr Val Lys Glu Asn Val Ile
165 170 175 Asp His Gln
Ala Gly Ser Pro Ser Leu Leu Gly Asp Leu Lys Ile Asp 180
185 190 Phe Asp Lys Ile Gln Ser Glu Tyr
Leu Phe Ser Asp Leu Met Asp Phe 195 200
205 Asp Gly Leu Gly Cys Gly Asn Val Met Ser Leu Val Ser
Ser Asp Glu 210 215 220
Val Leu Gly Asp Tyr Val Ser Thr Asp Thr Ser Cys Leu Gly Asn Leu 225
230 235 240 Asp Leu Asn Arg
Pro Phe Thr Ser Cys Leu Gln Glu Asp Cys Leu Trp 245
250 255 Asp Phe Asn Cys 260
79266PRTZea mays 79Met Gly Arg Arg Ala Cys Cys Ala Lys Glu Gly Val Lys
Arg Gly Ala 1 5 10 15
Trp Thr Ala Lys Glu Asp Asp Thr Leu Ala Ala Tyr Val Lys Ala His
20 25 30 Gly Glu Gly Lys
Trp Arg Glu Val Pro Gln Lys Ala Gly Leu Arg Arg 35
40 45 Cys Gly Lys Ser Cys Arg Leu Arg Trp
Leu Asn Tyr Leu Arg Pro Asn 50 55
60 Ile Lys Arg Gly Asn Ile Ser Tyr Asp Glu Glu Asp Leu
Ile Val Arg 65 70 75
80 Leu His Lys Leu Leu Gly Asn Arg Trp Ser Leu Ile Ala Gly Arg Leu
85 90 95 Pro Gly Arg Thr
Asp Asn Glu Ile Lys Asn Tyr Trp Asn Ser Thr Leu 100
105 110 Gly Arg Arg Ala Gly Ala Ala Gly Ala
Ser Arg Val Val Phe Ala Pro 115 120
125 Asp Thr Gly Ser His Ala Thr Pro Ala Ala Ser Gly Ser Arg
Glu Met 130 135 140
Thr Gly Gly Gln Lys Gly Ala Ala Pro Arg Ala Asp Leu Gly Ser Pro 145
150 155 160 Gly Ser Ala Ala Val
Val Trp Ala Pro Lys Ala Ala Arg Cys Thr Gly 165
170 175 Gly Leu Phe Phe His Arg Asp Thr Pro His
Ala Gly Glu Thr Glu Thr 180 185
190 Pro Thr Pro Met Met Met Ala Gly Gly Gly Gly Gly Glu Ala Arg
Ser 195 200 205 Ser
Asp Asp Cys Ser Ser Ala Ala Ser Val Ser Pro Leu Val Gly Ser 210
215 220 Ser Gln His Asp Pro Cys
Phe Ser Gly Asp Gly Asp Gly Asp Trp Met 225 230
235 240 Asp Asp Val Arg Ala Leu Ala Ser Phe Leu Glu
Ser Asp Glu Glu Trp 245 250
255 Leu Arg Cys His Thr Ala Glu Gln Leu Val 260
265 80302PRTGossypium hirsutum 80Met Gly Arg Ser Pro Cys Cys
Ser Lys Glu Gly Leu Asn Arg Gly Ala 1 5
10 15 Trp Thr Ala Leu Glu Asp Lys Ile Leu Lys Asp
Tyr Ile Lys Val His 20 25
30 Gly Glu Gly Arg Trp Arg Asn Leu Pro Lys Arg Ala Gly Leu Lys
Arg 35 40 45 Cys
Gly Lys Ser Cys Arg Leu Arg Trp Leu Asn Tyr Leu Arg Pro Asp 50
55 60 Ile Lys Arg Gly Asn Ile
Ser Pro Asp Glu Glu Glu Leu Ile Ile Lys 65 70
75 80 Leu His Lys Leu Leu Gly Asn Arg Trp Ser Leu
Ile Ala Gly Arg Leu 85 90
95 Pro Gly Arg Thr Asp Asn Glu Ile Lys Asn Tyr Trp Asn Thr Asn Leu
100 105 110 Ser Lys
Arg Val Ser Asp Arg Gln Lys Ser Pro Ala Ala Pro Ser Lys 115
120 125 Lys Pro Glu Ala Ala Arg Arg
Gly Thr Ala Gly Asn Gly Asn Thr Asn 130 135
140 Gly Asn Gly Ser Gly Ser Ser Ser Thr His Val Val
Arg Thr Arg Ala 145 150 155
160 Thr Arg Cys Ser Lys Val Phe Ile Asn Pro His His His Thr Gln Asn
165 170 175 Arg His Pro
Lys Pro Ser Ser Thr Cys Ser Asn His Gly Asp His Arg 180
185 190 Glu Pro Lys Thr Met Asn Glu Leu
Leu Leu Pro Ile Met Ser Glu Ser 195 200
205 Glu Asn Glu Gly Thr Thr Asp His Ile Ser Ser Asp Phe
Thr Phe Asp 210 215 220
Phe Asn Met Gly Glu Phe Cys Leu Ser Asp Leu Leu Asn Ser Asp Phe 225
230 235 240 Cys Asp Val Asn
Glu Leu Asn Tyr Ser Asn Gly Phe Asp Ser Ser Pro 245
250 255 Ser Pro Asp Gln Pro Pro Met Asp Phe
Ser Asp Glu Met Leu Lys Glu 260 265
270 Trp Thr Ala Ala Ala Ser Thr His Cys Cys His Gln Ser Ala
Ala Ser 275 280 285
Asn Leu Gln Ser Leu Pro Pro Phe Ile Glu Asn Gly Ile Glu 290
295 300 81286PRTVitis vinifera 81Met Gly
Arg Ala Pro Cys Cys Ser Lys Val Gly Leu His Arg Gly Ser 1 5
10 15 Trp Thr Ala Arg Glu Asp Thr
Leu Leu Thr Lys Tyr Ile Gln Ala Lys 20 25
30 Gly Glu Gly His Trp Arg Ser Leu Pro Lys Lys Ala
Gly Leu Leu His 35 40 45
Cys Gly Lys Ser Cys Arg Leu Arg Trp Met Asn Tyr Leu Arg Pro Asp
50 55 60 Ile Lys Arg
Gly Asn Ile Thr Pro Asp Lys Asp Asp Leu Ile Ile Arg 65
70 75 80 Leu Lys Ser Leu Leu Gly Asn
Arg Trp Ser Leu Ile Ala Gly Arg Leu 85
90 95 Pro Gly Arg Thr Asp Asn Ser Ile Lys Asn Tyr
Trp Asn Thr His Leu 100 105
110 Ser Lys Lys Leu Arg Ser Gln Gly Thr Asp Pro Asn Thr His Lys
Lys 115 120 125 Met
Thr Glu Pro Pro Glu Pro Lys Arg Arg Lys Asn Thr Arg Thr Arg 130
135 140 Thr Asn Asn Gly Gly Gly
Ser Lys Arg Val Lys Ile Ser Lys Asp Glu 145 150
155 160 Glu Asn Ser Asn His Lys Val His Leu Pro Lys
Pro Val Arg Val Thr 165 170
175 Ser Leu Ile Ser Met Ser Arg Asn Asn Ser Phe Glu Ser Asn Thr Val
180 185 190 Ser Gly
Gly Ser Gly Ser Ser Ser Gly Gly Asn Gly Glu Ser Leu Pro 195
200 205 Trp Pro Ser Phe Arg Asp Ile
Arg Asp Asp Lys Val Ile Gly Val Asp 210 215
220 Gly Val Asp Phe Phe Ile Gly Asp Asp Gln Gly Gln
Asp Leu Val Ala 225 230 235
240 Ser Ser Asp Pro Glu Ser Gln Ser Lys Met Pro Pro Thr Asp Asn Ser
245 250 255 Leu Asp Lys
Leu Tyr Glu Glu Tyr Leu Gln Leu Leu Glu Arg Glu Asp 260
265 270 Thr Gln Val Gln Leu Asp Ser Phe
Ala Glu Ser Leu Leu Ile 275 280
285 82303PRTLotus japonicus 82Met Gly Arg Ser Pro Cys Cys Ser Lys Gln
Gly Leu Asn Arg Gly Ala 1 5 10
15 Trp Thr Ala Gln Glu Asp Gln Ile Leu Arg Asp Tyr Val His Leu
His 20 25 30 Gly
Gln Gly Lys Trp Arg Asn Leu Pro Gln Ser Ala Gly Leu Lys Arg 35
40 45 Cys Gly Lys Ser Cys Arg
Leu Arg Trp Leu Asn Tyr Leu Arg Pro Asp 50 55
60 Ile Lys Arg Gly Asn Ile Ser Arg Asp Glu Glu
Glu Leu Ile Ile Arg 65 70 75
80 Leu His Lys Leu Leu Gly Asn Arg Trp Ser Leu Ile Ala Gly Arg Leu
85 90 95 Pro Gly
Arg Thr Asp Asn Glu Ile Lys Asn Tyr Trp Asn Thr Asn Leu 100
105 110 Cys Lys Arg Val Gln Asp Gly
Val Asp Val Gly Asp Ser Lys Thr Pro 115 120
125 Ser Ser Gln Glu Lys Asn Asn His His Asp Gln Lys
Ala Lys Pro Gln 130 135 140
Ser Val Thr Pro Ser Val Phe Ser Ser Ser Gln Pro Lys Asn Asn Asn 145
150 155 160 Val Ile Arg
Thr Lys Ala Ser Lys Cys Ser Lys Val Leu Leu Arg Asp 165
170 175 Pro Leu Leu Pro Cys Pro Pro Met
Gln Thr Gln Ser Asp Asp Phe Ile 180 185
190 Ala Lys Leu Leu Glu Glu Ala Glu Gly Glu Pro Leu Leu
Ser Ala Val 195 200 205
Ala Asn Asp Phe Thr Ser Gly Asp Glu Asp Gly Val Leu Ser Phe Asp 210
215 220 Pro Cys Gly Asn
Glu Lys Glu Leu Ser Thr Asp Leu Leu Leu Asp Leu 225 230
235 240 Asp Ile Gly Glu Ile Cys Leu Pro Glu
Phe Ile Asn Ser Asp Phe Ser 245 250
255 Tyr Val Cys Asp Phe Ser Tyr Asn Thr His Glu Asp Leu Met
Leu Phe 260 265 270
Ser Glu Asn Thr Leu Val Gln Ala Gln Lys Tyr Leu Gly Asp Glu Thr
275 280 285 Asn Leu Val Asn
Asn Cys Phe Asn Glu Glu Lys Asp Asn Gly Cys 290 295
300 83281PRTGlycine max 83Met Gly Arg Ala Pro
Cys Cys Ser Lys Val Gly Leu His Arg Gly Pro 1 5
10 15 Trp Thr Pro Arg Glu Asp Ala Leu Leu Thr
Lys Tyr Ile Gln Thr His 20 25
30 Gly Glu Gly Gln Trp Arg Ser Leu Pro Lys Arg Ala Gly Leu Leu
Arg 35 40 45 Cys
Gly Lys Ser Cys Arg Leu Arg Trp Met Asn Tyr Leu Arg Pro Asp 50
55 60 Ile Lys Arg Gly Asn Ile
Thr Pro Glu Glu Asp Asp Leu Ile Val Arg 65 70
75 80 Met His Ser Leu Leu Gly Asn Arg Trp Ser Leu
Ile Ala Gly Arg Leu 85 90
95 Pro Gly Arg Thr Asp Asn Glu Ile Lys Asn Tyr Trp Asn Thr His Leu
100 105 110 Ser Lys
Lys Leu Arg Asn Gln Gly Thr Asp Pro Lys Thr His Asp Lys 115
120 125 Leu Thr Glu Ala Pro Glu Lys
Lys Lys Gly Lys Lys Lys Asn Lys Gln 130 135
140 Lys Asn Glu Asn Asn Lys Gly Ser Glu Lys Thr Leu
Val Tyr Leu Pro 145 150 155
160 Lys Pro Ile Arg Val Lys Ala Leu Ser Ser Cys Ile Pro Arg Thr Asp
165 170 175 Ser Thr Leu
Thr Leu Asn Ser Asn Ser Ala Thr Ala Ser Thr Ser Glu 180
185 190 Glu Lys Val Gln Ser Pro Glu Ala
Glu Val Lys Glu Val Asn Met Val 195 200
205 Trp Gly Val Gly Asp Asp Ala Asp Asn Gly Gly Ile Glu
Ile Phe Phe 210 215 220
Gly Glu Asp His Asp Leu Val Asn Asn Thr Ala Ser Tyr Glu Glu Cys 225
230 235 240 Tyr Ser Asp Val
His Thr Asp Asp His Gly Thr Leu Glu Lys Leu Tyr 245
250 255 Glu Glu Tyr Leu Gln Leu Leu Asn Val
Glu Glu Lys Pro Asp Glu Leu 260 265
270 Asp Ser Phe Ala Gln Ser Leu Leu Val 275
280 84286PRTMalus domestica 84Met Gly Arg Ser Pro Cys Cys Ser
Lys Asp Glu Gly Leu Asn Arg Gly 1 5 10
15 Ala Trp Thr Ala Met Glu Asp Lys Val Leu Thr Glu Tyr
Ile Gly Asn 20 25 30
His Gly Glu Gly Lys Trp Arg Asn Leu Pro Lys Arg Ala Gly Leu Lys
35 40 45 Arg Cys Gly Lys
Ser Cys Arg Leu Arg Trp Leu Asn Tyr Leu Arg Pro 50
55 60 Asp Ile Lys Arg Gly Asn Ile Thr
Arg Asp Glu Glu Glu Leu Ile Ile 65 70
75 80 Arg Leu His Lys Leu Leu Gly Asn Arg Trp Ser Leu
Ile Ala Gly Arg 85 90
95 Leu Pro Gly Arg Thr Asp Asn Glu Ile Lys Asn Tyr Trp Asn Thr Thr
100 105 110 Ile Gly Lys
Arg Ile Gln Val Glu Gly Arg Ser Cys Ser Asp Gly Asn 115
120 125 Arg Arg Pro Thr Gln Glu Lys Pro
Lys Pro Thr Leu Ser Pro Lys Pro 130 135
140 Ser Thr Asn Ile Ser Cys Thr Lys Val Val Arg Thr Lys
Ala Ser Arg 145 150 155
160 Cys Thr Lys Val Val Leu Pro His Glu Ser Gln Lys Phe Gly Tyr Ser
165 170 175 Thr Glu Gln Val
Val Asn Ala Ala Pro Thr Leu Asp Gln Ala Val Asn 180
185 190 Asn Pro Met Val Gly Ile Asp Asp Pro
Leu Leu Pro Met Ser Phe Leu 195 200
205 Asp Asp Glu Asn Asn Asn Ser Cys Glu Phe Leu Val Asp Phe
Lys Met 210 215 220
Asp Glu Asn Phe Leu Ser Asp Phe Leu Asn Val Asp Phe Ser Val Leu 225
230 235 240 Tyr Asn Asn Glu Gly
Ala Gly Lys Ala Ala Ala Ala Ala Thr Thr Glu 245
250 255 Asp Thr Ser Asn Lys Leu His Gly Pro Asp
Leu Arg Ser Ser Lys Ala 260 265
270 Pro Ile Ile Glu Ser Glu Leu Asp Cys Trp Leu Val Asp Asn
275 280 285 85316PRTTrifolium
arvense 85Met Gly Arg Ser Pro Cys Cys Ala Lys Glu Gly Leu Asn Arg Gly Ala
1 5 10 15 Trp Thr
Thr Gln Glu Asp Lys Ile Leu Thr Glu Tyr Ile Lys Leu His 20
25 30 Gly Glu Gly Lys Trp Arg Asn
Leu Pro Lys Arg Ala Gly Leu Lys Arg 35 40
45 Cys Gly Lys Ser Cys Arg Leu Arg Trp Leu Asn Tyr
Leu Arg Pro Asp 50 55 60
Ile Lys Arg Gly Asn Ile Ser Pro Asp Glu Glu Glu Leu Ile Ile Arg 65
70 75 80 Leu His Lys
Leu Leu Gly Asn Arg Trp Ser Leu Ile Ala Gly Arg Leu 85
90 95 Pro Gly Arg Thr Asp Asn Glu Ile
Lys Asn Tyr Trp Asn Thr Asn Leu 100 105
110 Gly Lys Lys Val Lys Asp Leu Asp Gln Gln Asn Thr Asn
Asn Ser Ser 115 120 125
Pro Thr Lys Leu Ser Ala Gln Pro Lys Asn Ala Glu Ile Lys Gln Lys 130
135 140 Gln Ile Asn Pro
Lys Pro Asn Ser Tyr Val Val Arg Thr Lys Ala Thr 145 150
155 160 Lys Cys Ser Lys Val Leu Phe Ile Asn
Ser Pro Pro Asn Ser Pro Pro 165 170
175 Met His Asp Leu Gln Ser Lys Ala Glu Ala Glu Thr Thr Thr
Thr Thr 180 185 190
Lys Pro Ser Met Pro Ser Met Leu Val Asp Gly Val Ala Ser Asp Ser
195 200 205 Met Ser Asn Asn
Glu Met Glu Cys Gly Asn Gly Phe Leu Ser Phe Cys 210
215 220 Asp Glu Glu Lys Glu Leu Ser Ala
Asp Leu Leu Glu Asp Phe Asn Ile 225 230
235 240 Ala Asp Asp Ile Cys Leu Ser Glu Phe Leu Asn Phe
Asp Phe Ser Asn 245 250
255 Ala Cys Asp Ile Asp Tyr Asn Asp Leu Leu Ser Pro Cys Ser Asp Gln
260 265 270 Thr Gln Met
Phe Pro Asp Asp Glu Ile Leu Lys Asn Trp Thr Gln Cys 275
280 285 Asn Phe Ala Asp Glu Thr Asn Val
Ser Asn Asn Leu Gln Ser Ser Ala 290 295
300 Ser Phe Leu Glu Ser Ser Glu Glu Val Leu Gly Glu 305
310 315 86311PRTTrifolium repens
86Met Gly Arg Ser Pro Cys Cys Ala Lys Glu Gly Leu Asn Arg Gly Ala 1
5 10 15 Trp Thr Ala His
Glu Asp Lys Ile Leu Thr Glu Tyr Ile Lys Leu His 20
25 30 Gly Glu Gly Lys Trp Arg Asn Leu Pro
Lys Arg Ala Gly Leu Lys Arg 35 40
45 Cys Gly Lys Ser Cys Arg Leu Arg Trp Leu Asn Tyr Leu Arg
Pro Asp 50 55 60
Ile Lys Arg Gly Asn Ile Ser Ser Asp Glu Glu Glu Leu Ile Ile Arg 65
70 75 80 Leu His Lys Leu Leu
Gly Asn Arg Trp Ser Leu Ile Ala Gly Arg Leu 85
90 95 Pro Gly Arg Thr Asp Asn Glu Ile Lys Asn
Tyr Trp Asn Thr Asn Leu 100 105
110 Gly Lys Lys Val Lys Asp Leu Asn Gln Gln Asn Thr Asn Asn Ser
Ser 115 120 125 Pro
Thr Lys Pro Ser Ala Gln Pro Lys Asn Ala Asn Ile Lys Gln Lys 130
135 140 Gln Gln Ile Asn Pro Lys
Pro Met Lys Pro Asn Ser Asn Val Val Arg 145 150
155 160 Thr Lys Ala Thr Lys Cys Ser Lys Val Leu Phe
Ile Asn Ser Pro Pro 165 170
175 Met His Asn Leu Gln Asn Lys Ala Glu Ala Glu Thr Lys Thr Lys Pro
180 185 190 Leu Met
Leu Val Asn Gly Val Ala Ser Asp Ser Met Ser Asn Asn Glu 195
200 205 Met Glu Arg Gly Asn Gly Phe
Leu Ser Phe Cys Asp Glu Glu Lys Glu 210 215
220 Leu Ser Ala Asp Leu Leu Asp Asp Phe Asn Ile Ala
Asp Asp Ile Cys 225 230 235
240 Leu Ser Glu Phe Leu Asn Ser Asp Phe Ser Asn Ala Cys Asn Phe Asp
245 250 255 Cys Asn Asp
Leu Leu Ser Pro Cys Ser Asp Gln Thr Gln Met Phe Ser 260
265 270 Asp Asp Glu Ile Leu Lys Asn Trp
Thr Gln Cys Asn Phe Ala Asp Glu 275 280
285 Thr Asn Val Ser Asn Asn Leu Asn Ser Phe Ala Ser Phe
Leu Glu Ser 290 295 300
Ser Glu Glu Val Leu Gly Glu 305 310
87313PRTTrifolium occidentale 87Met Gly Arg Ser Pro Cys Cys Ala Lys Glu
Gly Leu Asn Arg Gly Ala 1 5 10
15 Trp Thr Thr Gln Glu Asp Lys Ile Leu Thr Glu Tyr Ile Lys Leu
His 20 25 30 Gly
Glu Gly Lys Trp Arg Asn Leu Pro Lys Arg Ala Gly Leu Lys Arg 35
40 45 Cys Gly Lys Ser Cys Arg
Leu Arg Trp Leu Asn Tyr Leu Arg Pro Asp 50 55
60 Ile Lys Arg Gly Asn Ile Ser Ser Asp Glu Glu
Glu Leu Ile Ile Arg 65 70 75
80 Leu His Lys Leu Leu Gly Asn Arg Trp Ser Leu Ile Ala Gly Arg Leu
85 90 95 Pro Gly
Arg Thr Asp Asn Glu Ile Lys Asn Tyr Trp Asn Thr Asn Leu 100
105 110 Gly Lys Lys Val Lys Asp Leu
Asn Gln Gln Asn Thr Asn Lys Ser Ser 115 120
125 Pro Thr Lys Leu Ser Ala Gln Pro Lys Asn Ala Lys
Ile Lys Gln Lys 130 135 140
Gln Ile Asn Pro Lys Pro Met Lys Pro Asn Ser Asn Val Val Arg Thr 145
150 155 160 Arg Ala Thr
Lys Cys Ser Lys Val Leu Phe Ile Asn Ser Leu Pro Asn 165
170 175 Ser Pro Met His Asp Leu Gln Asn
Lys Ala Glu Ala Glu Thr Thr Thr 180 185
190 Lys Pro Ser Met Leu Val Asp Gly Val Ala Ser Asp Ser
Met Ser Asn 195 200 205
Asn Glu Met Glu His Gly Tyr Gly Phe Leu Ser Phe Cys Asp Glu Glu 210
215 220 Lys Glu Leu Ser
Ala Asp Leu Leu Glu Asp Phe Asn Ile Ala Asp Asp 225 230
235 240 Ile Cys Leu Ser Glu Leu Leu Asn Ser
Asp Phe Ser Asn Ala Cys Asn 245 250
255 Phe Asp Tyr Asn Asp Leu Leu Ser Pro Cys Ser Asp Gln Thr
Gln Met 260 265 270
Phe Ser Asp Asp Glu Ile Leu Lys Asn Trp Thr Gln Cys Asn Phe Ala
275 280 285 Asp Glu Thr Asn
Val Ser Asn Asn Leu His Ser Phe Ala Ser Phe Leu 290
295 300 Glu Ser Ser Glu Glu Val Leu Gly
Glu 305 310 88314PRTArtificial
sequenceConsensus sequence of MYB14 protein sequences 88Met Gly Arg Ser
Pro Cys Cys Ala Lys Glu Gly Leu Asn Arg Gly Ala 1 5
10 15 Trp Thr Thr Gln Glu Asp Lys Ile Leu
Thr Glu Tyr Ile Lys Leu His 20 25
30 Gly Glu Gly Lys Trp Arg Asn Leu Pro Lys Arg Ala Gly Leu
Lys Arg 35 40 45
Cys Gly Lys Ser Cys Arg Leu Arg Trp Leu Asn Tyr Leu Arg Pro Asp 50
55 60 Ile Lys Arg Gly Asn
Ile Ser Ser Asp Glu Glu Glu Leu Ile Ile Arg 65 70
75 80 Leu His Lys Leu Leu Gly Asn Arg Trp Ser
Leu Ile Ala Gly Arg Leu 85 90
95 Pro Gly Arg Thr Asp Asn Glu Ile Lys Asn Tyr Trp Asn Thr Asn
Leu 100 105 110 Gly
Lys Lys Val Lys Asp Leu Asn Gln Gln Asn Thr Asn Asn Ser Ser 115
120 125 Pro Thr Lys Leu Ser Ala
Gln Pro Lys Asn Ala Lys Ile Lys Gln Lys 130 135
140 Gln Ile Asn Pro Lys Pro Met Lys Pro Asn Ser
Asn Val Val Arg Thr 145 150 155
160 Lys Ala Thr Lys Cys Ser Lys Val Leu Phe Ile Asn Ser Pro Pro Asn
165 170 175 Ser Pro
Met His Asp Leu Gln Asn Lys Ala Glu Ala Glu Thr Thr Thr 180
185 190 Lys Pro Ser Met Leu Val Asp
Gly Val Ala Ser Asp Ser Met Ser Asn 195 200
205 Asn Glu Met Glu His Gly Asn Gly Phe Leu Ser Phe
Cys Asp Glu Glu 210 215 220
Lys Glu Leu Ser Ala Asp Leu Leu Glu Asp Phe Asn Ile Ala Asp Asp 225
230 235 240 Ile Cys Leu
Ser Glu Phe Leu Asn Ser Asp Phe Ser Asn Ala Cys Asn 245
250 255 Phe Asp Tyr Asn Asp Leu Leu Ser
Pro Cys Ser Asp Gln Thr Gln Met 260 265
270 Phe Ser Asp Asp Glu Ile Leu Lys Asn Trp Thr Gln Cys
Asn Phe Ala 275 280 285
Asp Glu Thr Asn Val Val Ser Asn Asn Leu His Ser Phe Ala Ser Phe 290
295 300 Leu Glu Ser Ser
Glu Glu Val Leu Gly Glu 305 310
891203DNATrifolium repens 89gaattcgccc ttatggggag aagcccttgt tgtgcaaaag
aaggcttgaa tagaggtgct 60tggacagctc atgaagacaa aatcctcact gaatacatta
agctccatgg tgaaggaaaa 120tggagaaacc ttccaaaaag agcaggttca ttcattctgt
atcttactat tatagatcaa 180taatcacttt cacacttttt ttttacttat aaattttcat
gtattttttc ttccattttc 240cattagaaat gcaaattaat agtacattat tatggacatg
ttttttcaaa aatgtgtatt 300ccatgcaggt ttaaaaagat gtggaaaaag ttgtagacta
aggtggttga attatcttag 360accggatatt aagagaggta atatatcgtc ggatgaagaa
gaacttatca ttagacttca 420caaactactc ggaaaccggt aaagtatcga cataatcact
aacttactaa catttgttta 480taatgtgtgc taattgctct tcctttgatt tgtggtagat
ggtctctaat agccggaaga 540cttccagggc gaacagacaa tgaaataaag aactactgga
acacaaattt aggaaaaaaa 600gttaaggatc ttaatcaaca aaacaccaac aattcttctc
ctactaaacc ttctgctcaa 660ccaaaaaatg caaatatcaa acagaaacaa cagatcaatc
ctaagccaat gaagccaaac 720tcgaatgttg tccgtacaaa agctaccaaa tgttctaagg
tattgttcat aaactcacca 780ccaatgcata atttgcagaa caaagctgag gcagagacaa
aaacaaagcc attaatgctg 840gttaatggtg tagctagtga ttcaatgagt aacaacgaaa
tggaacgcgg taatggattt 900ttgtcatttt gcgacgaaga gaaagaacta tccgcagatt
tgctagatga ttttaacatc 960gcggatgata tttgcttatc tgaatttcta aactccgatt
tctcaaatgc gtgcaatttc 1020gattgcaatg atctattgtc gccttgttcg gatcaaactc
aaatgttctc tgatgatgag 1080attctcaaga attggacaca atgtaacttt gctgatgaga
caaatgtgtc caacaacctt 1140aattcttttg cttcttttct cgaatccagt gaggaagtac
taggagaatg aaagggcgaa 1200ttc
1203901205DNATrifolium repens 90gaattcgccc
ttatggggag aagcccttgt tgtgcaaaag aaggcttgaa tagaggtgct 60tggacagctc
atgaagacaa aatcctcact gaatacatta agctccatgg tgaaggaaaa 120tggagaaacc
ttccaaaaag agcaggttca ttcattctgt atcttactat tatagatcaa 180taatcacttt
cacacttttt ttttttactt ataaattttc atgtattttt tcttccattt 240tccattagaa
atgcaaatta atagtacatt attatggaca tgttttttca aaaatgtgta 300ttccatgcag
gtttaaaaag atgtggaaaa agttgtagac taaggtggtt gaattatctt 360agaccggata
ttaagagagg taatatatcg tcggatgaag aagaacttat cattagactt 420cacaaactac
tcggaaaccg gtaaagtatc gacataatca ctaacttact aacatttgtt 480tataatgtgt
gctaattgct cttcctttga tttgtggtag atggtctcta atagccggaa 540gacttccagg
gcgaacagac aatgaaataa agaactactg gaacacaaat ttaggaaaaa 600aagttaagga
tcttaatcaa caaaacacca acaattcttc tcctactaaa ccttctgctc 660aaccaaaaaa
tgcaaatatc aaacagaaac aacagatcaa tcctaagcca atgaagccaa 720actcgaatgt
tgtccgtaca aaagctacca attgttctaa ggtattgttc ataaactcac 780caccaatgca
taatttgcag aacaaagctg aggcagagac aaaaacaaag ccattaatgc 840tggttaatgg
tgtagctagt gattcaatga gtaacaacga aatggaacgc ggtaatggat 900ttttgtcatt
ttgcgacgaa gagaaagaac tatccgcaga tttgctagat gattttaaca 960tcgcggatga
tatttgctta tctgaatttc taaactccga tttctcaaat gcgtgcaatt 1020tcgattacaa
tgatctattg tcgccttgtt cggatcaaac tcaaatgttc tctgatgatg 1080agattctcaa
gaattggaca caatgtaact ttgctgatga gacaaatgtg tccaacaacc 1140ttaattcttt
tgcttctttt ctcgaatcca gtgaggaagt actaggagaa tgaaagggcg 1200aattc
1205911164DNATrifolium occidentale 91gaattcgccc ttatggggag aagcccttgt
tgtgcaaagg aaggtttgaa tagaggtgct 60tggacagctc atgaagacaa aatcctcact
gaatacatta agctccatgg tgaaggaaaa 120tggagaaacc ttccaaaaag agcaggttca
ttcattctgt atcttactat ttatagatca 180ataatcactt tcatgtattt tttttccttc
cattttccat tagaaatgca aattaatagt 240acattattat ggacatgttt ttccaggttt
aaaaagatgt ggaaaaagtt gtagacttag 300atggttgaat tatcttagac cagatattaa
gagaggtaat atatcgtccg atgaagaaga 360acttatcatt agacttcaca aactacttgg
aaaccggtaa agtatcgaca taatcactaa 420cttactaaca tttgtttata atgtgtacta
attgcgattc ctttgatttg tggtagatgg 480tctctaatag ccggaagact tccagggcga
acagacaatg aaataaaaaa ttactggaac 540acgaatttag gaaaaaaggt taaggatctt
tatcaacaaa acaccaacaa ttcttctcct 600actaaacctt ctgctcaacc aaaaaatgca
aagatcaaac agaaacaaca gatcaataat 660cctaagccaa tgaagccaaa ctcgaatgtt
gtccgtacaa aagctaccaa atgttctaag 720gtattgttca taaactcacc accaatgcat
aatttgcaga acaaagctga ggcagagaca 780aaaacaaaga catcaatgtt ggttaatggt
gtagctagtg attcaatgag taacaacgaa 840atggaacgag gtaatggatt tttgtcattt
cgcgatgaag agaaagaact atccgctgat 900ttgctagatg attttaacat cgcggatgac
atttgcttat ccgaatttct aaactccgat 960ttctcaaatg cgtgcaattt cgattacaat
gatctattgt caccttgttc ggatcaaact 1020caaatgttct ctgatgatga gattctcaag
aattggacac aatgtaactt tgctgatgag 1080acaaatgtgt ccaacaacct tcattctttt
gcttcctttc tcgaatccag tgaggaagta 1140ctaggagaat gaaagggcga attc
1164921164DNATrifolium occidentale
92gaattcgccc ttatggggag aagcccttgt tgtgcaaagg aaggtttgaa tagaggtgct
60tggacagctc atgaagacaa aatcctcact gaatacatta agctccatgg tgaaggaaaa
120tggagaaacc ttccaaaaag agcaggttca ttcattctgt atcttactat ttatagatca
180ataatcactt tcatgtattt tttttccttc cattttccat tagaaatgca aattaatagt
240acattattat ggacatgttt ttccaggttt aaaaagatgt ggaaaaagtt gtagacttag
300atggttgaat tatcttagac cagatattaa gagaggtaat atatcgtccg atgaagaaga
360acttatcatt agacttcaca aactacttgg aaaccggtaa agtatcgaca taatcactaa
420cttactaaca tttgtttata atgtgtacta attgcgattc ctttgatttg tggtagatgg
480tctctaatag ccggaagact tccagggcga acagacaatg aaataaaaaa ttactggaac
540acgaatttag gaaaaaaggt taaggatctt aatcaacaaa acaccaacaa ttcttctcct
600actaaacctt ctgctcaacc aaaaaatgca aagatcaaac agaaacaaca gatcaataat
660cctaagccaa tgaagccaaa ctcgaatgtt gtccgtacaa aagctaccaa atgttctaag
720gtattgttca taaactcacc accaatgcat aatttgcaga acaaagctga ggcagagaca
780aaaacaaaga catcaatgtt ggttaatggt gtagctagtg attcaatgag taacaacgaa
840atggaacggg gtaatggatt tttgtcattt cgcgatgaag agaaagaact atccgctgat
900ttgctagatg attttaacat cgcggatgac atttgcttat ccgaatttct aaactccgat
960ttctcaaatg cgtgcaattt cgattacaat gatctattgt caccttgttc ggatcaaact
1020caaatgttct ctgatgatga gattctcaag aattggacac aatgtaactt tgctgatgag
1080acaaatgtgt ccaacaacct tcattctttt gcttcctttc tcgaatccag tgaggaagta
1140ctaggagaat gaaagggcga attc
1164931240DNATrifolium affine 93gaattcgccc ttatggggag aagcccttgt
tgtgcgaagg aaggcttgaa tagaggtgct 60tggacaactc aagaagacaa aatcctcact
gaatacatta agctccatgg tgaaggaaaa 120tggagaaacc ttccaaaaag agcaggttca
ttcattctgt atcttacaat tatagattaa 180ccactttcat acttttgttt gcttataaat
tttcttgtat tttttcttcc atttttcatg 240agaaatgcaa attactagta cattattatg
gacatgtttt tgcaaatatg tttatgccat 300gcaggtttaa aaagatgtgg aaaaagttgt
agacttagat ggttgaatta tctaagacta 360gatattaagc gaggtaatat atcctcggat
gaagaagaac ttatcatccg acttcacaaa 420ttactcggaa acaggtaaag tcctaacata
atcactaact tattaacgtt tgtctataac 480ttgttttttt gacaattagt actactaatt
taattttata atgtgtgcta atttgcttgt 540ctttaatttg tggtagatgg tctctaatag
ccggaagact tccaggacga acagacaatg 600aaataaagaa ctactggaac acaaatttag
gaaaaaaggt taaggatctt aatcaagaaa 660acaccaacaa ttcttctcct actaaacttt
ctgctcaact aaaaaatgca aagatcaaac 720agaaacagat caatcctaag ccaatggagc
caaactcaaa tgttgtccgt acaaaagcta 780ccaagtgttc taaggcattg ttcataaact
caccccccaa ctcaccacca atgcatgatt 840tgcagaacaa agctgaggca gagacaacaa
caaagtcatc aatgccatca atgctggttg 900atggcgtggc tagtgattca atgagtaaca
acgaaatgga atacggtgat ggatttgttt 960cattttgcga tgacgataaa gaactatccg
cagatttgct agaagatttt aacatctcgg 1020atgatatttg cttatccgaa tttctaaact
tcgatttctc aaatgcgtgc aatttcgatt 1080acaacgatct attgtcgcct tgttcggacc
aaacacaaat gttctctgat gatgagattc 1140tcaagaattc gacaccatgt aactttgctg
ctgagacaaa ttaatgtgtc caacaaccaa 1200tccagtgagg aagtactagg agaatgaaag
ggcgaattct 1240941240DNATrifolium affine
94ggaattcgcc cttatgggga gaagcccttg ttgtgcaaag gaaggcttga atagaggtgc
60ttggacaact caagaagaca aaatcctcac tgaatacatt aagctccatg gtgaaggaaa
120atggagaaac cttccaaaaa gagcaggttc attcattctg tatcttacaa ttatagatta
180accactttca tacttttgtt ttcttataaa ttttcttgta ttttttcttc catttttcat
240gagaaatgca aattactagt acattattat ggacatgttt ttgcaaatat gtttatgcca
300tgcaggttta aaaagatgtg gaaaaagttg tagacttaga tggttgaatt atctaagacc
360agatattaag cgaggtaata tatcctcgga tgaagaagaa cttatcatcc gacttcacaa
420actactcgga aacaggtaaa gtcataacat aatcattaat ttattaacgg ttatctataa
480tttgtttttt tgacaattat tactacaaat ttaattttat aatgtgtgct aatttgcttg
540tctttaattt gtggtagatg gtctctaata gccggaagac ttccagggcg aacagacaat
600gaaataaaga actactggaa cacaaattta ggaaaaaagg ttaaggatct taatcaagaa
660aacaccaaca attcttctcc tactaaactt tctgctcaac taaaaaatgc aaagatcaaa
720caaaaacaga tcaatcctaa gccaatgaag ccaaactcaa atgttgtccg tacaaaagct
780accaagtgtt ctaaggtatt gttcataaac tcacccccca actcaccacc aatgcatgat
840ttgcagaaca aagctgaggc agagacaaca acaaagccat caatgccatc aatgctggtt
900gatggcgtgg ctagtgattc aatgagtaac aacgaaatgg gatacggtga tggatttgtt
960tcattttgcg atgacgataa agaactatcc gcagatttgc tagaagattt taacatctcg
1020gatgatattt gcttatccga atttctaaac ttcgatttct caaatgcgtg caatttcgat
1080tacaacgatc tattgtcgcc ttgttcggac caaacacaaa tgttctctga tgatgagatt
1140ctcaagaatt cgacacaatg taactttgct gctgagacaa attaatgtgt ccaacaacca
1200atccagtgag gaagtactag gagaatgaaa gggcgaattc
1240951239DNATrifolium affine 95gaattcgccc ttatggggag aagcccttgt
tgtgcaaagg aaggcttgaa tagaggtgct 60tggacaactc aagaagacaa aatcctcact
gaatacatta agctccatgg tgaaggaaaa 120tggagaaacc ttccaaaaag agcaggttca
ttcattctgt atcttacaat tatagattaa 180ccactttcat acttttgttt tcttataaat
tttcttgtat tttttcttcc atttttcatg 240agaaatgcaa attactagta cattattatg
gacatgtttt tgcaaatatg tttatgccat 300gcaggtttaa aaagatgtgg aaaaagttgt
agacttagat ggttgaatta tctaagacca 360gatattaagc gaggtaatat atcctcggat
gaagaagaac ttatcatccg acttcacaaa 420ctactcggaa acaggtaaag tcataacatg
atcattaatt tattaacggt tatctataat 480ttgttttttt gacaattatc actacaaatt
taattttata atgtgcgcta atttgcttgt 540ctttaatttg tggtagatgg tctctaatag
ccggaagact tccagggcga acaaacaatg 600aaataaagaa ctactggaac acaaatttag
gaaaaaaggt taaggatctt aatcaagaaa 660acaccaacaa ttcttctcct actaaacttt
ctgctcaact aaaaaatgca aagatcaaac 720agaaacagat caatcctaag ccaatggagc
caaactcaaa tgttgtccgt acaaaagcta 780ccaagtgttc taaggcattg ttcataaact
caccccccaa ctcaccacca atgcatgatt 840tgcagaacaa agctgaggca gagacaacaa
caaagtcatc aatgccatca atgctggttg 900atggcgtggc tagtgattca gtgagtaaca
acgaaatgga atacggtgat ggatttgttt 960cattttgcga tgacgataaa gaactatccg
cagatttgct agaagatttt aacatctcgg 1020atgatatttg cttatccgaa tttctaaact
tcgatttctc aaatgcgtgc aatttcgatt 1080acaacgatct attgtcgcct tgttcggacc
aaacacaaat gttctctgat gatgagattc 1140tcaagaattc gacacaatgt aactttgctg
ctgagacaaa ttaatgtgtc caacaaccaa 1200tccagtgagg aagtactagg agaatgaaag
ggcgaattc 1239961239DNATrifolium affine
96gaattcgccc ttatggggag aagcccttgt tgtgcaaagg aaggcttgaa tagaggtgct
60tggacaactc aagaagacaa aatcctcact gaatacatta agctccatgg tgaaggaaaa
120tggagaaacc ttccaaaaag agcaggttca ttcattctgt atcttacaat tatagattaa
180ccactttcat acttttgttt tcttataaat tttcttgtat tttttcttcc atttttcatg
240agaaatgcaa attactagta cattattatg gacatgtttt tgcaaatatg tttatgccat
300gcaggtttaa aaagatgtgg aaaaagttgt agacttagat ggttgaatta tctaagacca
360gatattaagc gaggtaatat atcctcggat gaagaagaac ttatcatccg acttcacaaa
420ctactcggaa acaggtaaag tcataacata atcattaatt tattaacggt tatctataat
480ttgttttttt gacaattatc actacaaatt taattttata atgtgcgcta atttgcttgt
540ctttaatttg tggtagatgg tctctaatag ccggaagact tccagggcga acagacaatg
600aaataaagaa ctactggaac acaaatttag gaaaaaaggt taaggatctt aatcaagaaa
660acaccaacaa ttcttctcct actaaacttt ctgctcaact aaaaaatgca aagatcaaac
720agaaacagat caatcctaag ccaatggagc caaactcaaa tgttgtccgt acaaaagcta
780ccaagtgttc taaggcattg ttcataaact caccccccaa ctcaccacca atgcatgatt
840tgcagaacaa agctgaggca gagacaacaa caaagtcatc aatgccatca atgctggttg
900atggcgtggc tagtgattca atgagtaaca acgaaatgga atacggtgat ggatttgttt
960cattttgcga tgacgataaa gaactatccg cagatttgct agaagatttt aacatctcgg
1020atgatatttg cttatccgaa tttctaaact tcgatttctc aaatgcgtgc aatttcgatt
1080acaacgatct attgtcgcct tgttcggacc aaacacaaat gttctctgat gatgagattc
1140tcaagaattc gacacaatgt aactttgctg ctgagacaaa ttaatgtgtc caacaaccaa
1200tccagtgagg aagtactagg agaatgaaag ggcgaattc
1239971239DNATrifolium affine 97gaattcgccc ttatggggag aagcccttgt
tgtgcaaagg aaggcttgaa tagaggtgct 60tggacaactc aagaagacaa aatcctcact
gaatacatta agctccatgg tgaaggaaaa 120tggagaaacc ttccaaaaag agcaggttca
ttcattctgt atcttacaat tatagattaa 180ccactttcat acttttgttt tcttataaat
tttcttgtat tttttcttcc atttttcatg 240agaaatgcaa attactagta cattattatg
gacatgtttt tgcaaatatg tttatgccat 300gcaggtttaa aaagatgtgg aaaaagttgt
agacttagat ggttgaatta tctaagacca 360gatattaagc gaggtaatat atcctcggat
gaagaagaac ttatcatccg acttcacaaa 420ctactcggaa acaggtaaag tcataacata
atcattaatt tattaacggt tatctataat 480ttgttttttt gacaattatc actacaaatt
taattttata atgtgcgcta atttgcttgt 540ctttaatttg tggtagatgg tctctaatag
ccggaagact tccagggcga acagacaatg 600aaataaagaa ctactggaac acaaatttag
gaaaaaaggt taaggatctt aatcaagaaa 660acaccaacaa ttcttctcct actaaacttt
ctgctcaact aaaaaatgca aagatcaaac 720agaaacagat caatcctaag ccaatggagc
caaactcaaa tgttgtccgt acaaaagcta 780ccaagtgttc taaggcattg ttcataaact
caccccccaa ctcaccacca atgcatgatt 840tgcagaacaa agctgaggca gagacaacaa
caaagtcatc aatgccatca atgctggttg 900atggcgtggc tagtgattca atgagtaaca
acgaaatgga atacggtgat ggatttgttt 960cattttgcga tgacgataaa gaactatccg
cagatttgct agaagatttt aacatctcgg 1020atgatatttg cttatccgaa tttctaaact
tcgatttctc aaatgcgtgc aatttcgatt 1080acaacgatct attgtcgcct tgttcggacc
aaacacaaat gttctctgat gatgagattc 1140tcaagaattc gacacaatgt aactttgctg
ctgagacaaa ttaatgtgtc caacaaccaa 1200tccagtgagg aagtactagg agaatgaaag
ggcgaattc 1239981239DNATrifolium affine
98gaattcgccc ttatggggag aagcccttgt tgtgcaaagg aaggcttgaa tagaggtgct
60tggacaactc aagaagacaa aatcctcact gaatacatta agctccatgg tgaaggaaaa
120tggagaaacc ttccaaaaag agcaggttca ttcattctgt atcttacaat tatagattaa
180ccactttcat acttttgttt tcttataaat tttcttgtat tttttcttcc atttttcatg
240agaaatgcaa attactagta cattattatg gacatgtttt tgcaaatatg tttatgccat
300gcaggtttaa aaagatgtgg aaaaagttgt agacttagat ggttgaatta tctaagacca
360gatattaagc gaggtaatat atcctcggat gaagaagaac ttatcatccg acttcacaaa
420ctactcggaa acaggtaaag tcataacata atcattaatt tattaacggt tatctataat
480ttgttttttt gacaattatc actacaaatt taattttata atgtgcgcta atttgcttgt
540ctttaatttg tggtagatgg tctctaatag ccggaagact tccagggcga acagacaatg
600aaataaagaa ctactggaac acaaatttag gaaaaaaggt taaggatctt aatcaagaaa
660acaccaacaa ttcttctcct actaaacttt ctgctcaact aaaaaatgca aagatcaaac
720agaaacagat caatcctaag ccaatggagc caaactcaaa tgttgtccgt acaaaagcta
780ccaagtgttc taaggcattg ttcataaact caccccccaa ctcaccacca atgcatgatt
840tgcagaacaa agctgaggca gagacaacaa caaagtcatc aatgccatca atgctggttg
900atggcgtggc tagtgattca atgagtaaca acgaaatgga atacggtgat ggatttgttt
960cattttgcga tgacgataaa gaactatccg cagatttgct agaagatttt aacatctcgg
1020atgatatttg cttatccgaa tttctaaact tcgatttctc aaatgcgtgc aatttcgatt
1080acaacgatct attgtcgcct tgttcggacc aaacacaaat gttctctggt gatgagattc
1140tcaagaattc gacacaatgt aactttgctg ctgagacaaa ttaatgtgtc caacaaccaa
1200tccagtgagg aagtactagg agaatgaaag ggcgaattc
123999300PRTTrifolium occidentale 99Met Gly Arg Ser Pro Cys Cys Ala Lys
Glu Gly Leu Asn Arg Gly Ala 1 5 10
15 Trp Thr Ala His Glu Asp Lys Ile Leu Thr Glu Tyr Ile Lys
Leu His 20 25 30
Gly Glu Gly Lys Trp Arg Asn Leu Pro Lys Arg Ala Gly Leu Lys Arg
35 40 45 Cys Gly Lys Ser
Cys Arg Leu Arg Trp Leu Asn Tyr Leu Arg Pro Asp 50
55 60 Ile Lys Arg Gly Asn Ile Ser Ser
Asp Glu Glu Glu Leu Ile Ile Arg 65 70
75 80 Leu His Lys Leu Leu Gly Asn Arg Trp Ser Leu Ile
Ala Gly Arg Leu 85 90
95 Pro Gly Arg Thr Asp Asn Glu Ile Lys Asn Tyr Trp Asn Thr Asn Leu
100 105 110 Gly Lys Lys
Val Lys Asp Leu Asn Gln Gln Asn Thr Asn Asn Ser Ser 115
120 125 Pro Thr Lys Pro Ser Ala Gln Pro
Lys Asn Ala Lys Ile Lys Gln Lys 130 135
140 Gln Gln Ile Asn Asn Pro Lys Pro Met Lys Pro Asn Ser
Asn Val Val 145 150 155
160 Arg Thr Lys Ala Thr Lys Cys Ser Lys Val Leu Phe Ile Asn Ser Pro
165 170 175 Pro Met His Asn
Leu Gln Asn Lys Ala Glu Ala Glu Thr Lys Thr Lys 180
185 190 Thr Ser Met Leu Val Asn Gly Val Ala
Ser Asp Ser Met Ser Asn Asn 195 200
205 Glu Met Glu Arg Gly Asn Gly Phe Leu Ser Phe Arg Asp Glu
Glu Lys 210 215 220
Glu Leu Ser Ala Asp Leu Leu Asp Asp Phe Asn Ile Ala Asp Asp Ile 225
230 235 240 Cys Leu Ser Glu Phe
Leu Asn Ser Asp Phe Ser Asn Ala Cys Asn Phe 245
250 255 Asp Tyr Asn Asp Leu Leu Ser Pro Cys Ser
Asp Gln Thr Gln Met Phe 260 265
270 Ser Asp Asp Glu Ile Leu Lys Asn Trp Thr Gln Cys Asn Phe Ala
Asp 275 280 285 Glu
Thr Asn Val Ser Asn Asn Leu His Ser Phe Ala 290 295
300 100314PRTArtificial sequenceConsensus sequence of MYB14
protein sequences 100Met Gly Arg Ser Pro Cys Cys Ala Lys Glu Gly Leu Asn
Arg Gly Ala 1 5 10 15
Trp Thr Thr Gln Glu Asp Lys Ile Leu Thr Glu Tyr Ile Lys Leu His
20 25 30 Gly Glu Gly Lys
Trp Arg Asn Leu Pro Lys Arg Ala Gly Leu Lys Arg 35
40 45 Cys Gly Lys Ser Cys Arg Leu Arg Trp
Leu Asn Tyr Leu Arg Pro Asp 50 55
60 Ile Lys Arg Gly Asn Ile Ser Ser Asp Glu Glu Glu Leu
Ile Ile Arg 65 70 75
80 Leu His Lys Leu Leu Gly Asn Arg Trp Ser Leu Ile Ala Gly Arg Leu
85 90 95 Pro Gly Arg Thr
Asp Asn Glu Ile Lys Asn Tyr Trp Asn Thr Asn Leu 100
105 110 Gly Lys Lys Val Lys Asp Leu Asn Gln
Gln Asn Thr Asn Asn Ser Ser 115 120
125 Pro Thr Lys Pro Ser Ala Gln Pro Lys Asn Ala Lys Ile Lys
Gln Lys 130 135 140
Gln Gln Ile Asn Pro Lys Pro Met Lys Pro Asn Ser Asn Val Val Arg 145
150 155 160 Thr Lys Ala Thr Lys
Cys Ser Lys Val Leu Phe Ile Asn Ser Pro Pro 165
170 175 Asn Ser Pro Met His Asn Leu Gln Asn Lys
Ala Glu Ala Glu Thr Thr 180 185
190 Thr Lys Pro Ser Met Leu Val Asn Gly Val Ala Ser Asp Ser Met
Ser 195 200 205 Asn
Asn Glu Met Glu Arg Gly Asn Gly Phe Leu Ser Phe Cys Asp Glu 210
215 220 Glu Lys Glu Leu Ser Ala
Asp Leu Leu Asp Asp Phe Asn Ile Ala Asp 225 230
235 240 Asp Ile Cys Leu Ser Glu Phe Leu Asn Ser Asp
Phe Ser Asn Ala Cys 245 250
255 Asn Phe Asp Tyr Asn Asp Leu Leu Ser Pro Cys Ser Asp Gln Thr Gln
260 265 270 Met Phe
Ser Asp Asp Glu Ile Leu Lys Asn Trp Thr Gln Cys Asn Phe 275
280 285 Ala Asp Glu Thr Asn Val Ser
Asn Asn Leu His Ser Phe Ala Ser Phe 290 295
300 Leu Glu Ser Ser Glu Glu Val Leu Gly Glu 305
310 10113PRTArtificial sequenceMotif
associated with MYB TFs that regulate CT pathways 101Val Ile Val Arg
Thr Lys Ala Xaa Arg Lys Xaa Ser Lys 1 5
10 1029PRTArtificial sequenceMotif of subgroup 5 common to
previously known CT MYB activators 102Asp Glu Xaa Trp Arg Leu Xaa
Xaa Thr 1 5
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