CN114805510B - Gene for regulating and controlling aluminum-toxicity-resistant transcription factor STOP-1 protein and application thereof - Google Patents

Gene for regulating and controlling aluminum-toxicity-resistant transcription factor STOP-1 protein and application thereof Download PDF

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CN114805510B
CN114805510B CN202210220377.2A CN202210220377A CN114805510B CN 114805510 B CN114805510 B CN 114805510B CN 202210220377 A CN202210220377 A CN 202210220377A CN 114805510 B CN114805510 B CN 114805510B
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CN114805510A (en
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黄朝锋
张阳
张�杰
郭金两
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Center for Excellence in Molecular Plant Sciences of CAS
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Abstract

The invention relates to a gene for regulating and controlling an aluminum toxicity resisting transcription factor STOP-1 protein and application thereof. Specifically, the RAE1 gene or its encoded protein, or its mutein, or its promoter or inhibitor can be used to (1) regulate STOP-1 protein expression or degradation; (2) regulating and controlling the aluminum toxicity resistance or aluminum resistance of the plants; and/or (3) regulating expression of a gene selected from the group consisting of: atALMT1, atm mate, ALS3, or a combination thereof. The expression level and/or activity of RAE1 protein in plants are down regulated, so that the degradation of STOP-1 can be obviously inhibited, the expression level of AtALMT is up regulated, the secretion of organic acid (such as malic acid) is increased, and the aluminum toxicity resistance (or tolerance to aluminum) of the plants is enhanced. In contrast, the expression level and/or activity of RAE1 protein in the over-expressed plants can significantly promote STOP-1 degradation, down-regulate AtALMT expression level, reduce secretion of organic acids (e.g., malic acid) and enhance plant sensitivity to aluminum toxicity.

Description

Gene for regulating and controlling aluminum-toxicity-resistant transcription factor STOP-1 protein and application thereof
The application relates to a divisional application of an application patent application of which the application date is 2019, 3 and 25, the application number is 201910228864.1 and the application name is 'a gene for regulating and controlling an anti-aluminum toxin transcription factor STOP1 protein' and application thereof.
Technical Field
The invention relates to the field of agriculture, in particular to a gene for regulating and controlling the stability of an aluminum toxicity resisting transcription factor STOP-1 protein and application thereof.
Background
30% Of cultivated land worldwide is acid soil and with excessive cultivation and excessive use of nitrogen fertilizer, the soil is being further acidified. Aluminum is a major factor of limiting plant growth and crop yield in acid soil, and aluminum toxicity mainly acts on root tip transition areas and is combined with cell walls, cell membranes and intracellular targets to generate toxicity. Since plants cannot move like animals, many plants evolved a variety of detoxification mechanisms to cope with aluminum stress in order to survive, fully explore the potential of excavating plants, and make use of and improve the way and strategy to solve the aluminum poisoning of acid soil.
The secretion of organic acid is a key anti-aluminum toxicity mechanism, and the toxicity of aluminum to plants can be obviously reduced by the secretion of a small amount of organic acid by plants. Arabidopsis thaliana malate secretion protein AtALMT plays an important role in the anti-aluminum toxicity process, atALMT1 expression is induced by Al and is also influenced by a plurality of factors such as low pH, ABA, H 2O2 and IAA, the transcription factor STOP1 directly regulates AtALMT1 expression, but STOP1 mRNA expression is constitutive and cannot explain AtALMT1 expression regulation, and the study of STOP1 protein level regulation mechanism is particularly important.
Therefore, there is an urgent need in the art to develop functional studies for regulating genes related to aluminum toxicity resistance so as to solve the aluminum toxicity of acidic soil.
Disclosure of Invention
The invention aims to provide a gene for regulating and controlling the stability of an aluminum toxicity resisting transcription factor STOP-1 protein and application thereof.
In a first aspect of the invention there is provided the use of a RAE1 gene or a RAL1 gene, or a protein encoded thereby, a mutein, an enhancer or an inhibitor thereof, for one or more uses selected from the group consisting of:
(1) Regulating and controlling the expression quantity of STOP-1 protein;
(2) Regulating expression of a gene selected from the group consisting of: atALMT1, atm mate, ALS3, or a combination thereof;
(3) Regulating and controlling the aluminum toxicity resistance or aluminum resistance of the plants; and/or
(4) Preparing a preparation or an agricultural composition, wherein the preparation or the agricultural composition is used for regulating and controlling the expression quantity of STOP-1 protein, regulating and controlling the aluminum toxicity resistance or aluminum resistance of plants, or regulating and controlling the expression of genes selected from the following groups: atALMT1, atm mate, ALS3, or a combination thereof.
In another preferred embodiment, the modulating the aluminum toxicity resistance of the plant comprises: regulating and controlling the aluminium toxicity resistance of the plant in the growing period and/or the reproductive period.
In another preferred embodiment, the modulating the aluminum toxicity resistance of the plant comprises: regulate the secretion level of organic acid in plants (such as roots).
In another preferred embodiment, said modulating the amount of expression of the STOP-1 protein comprises mediating ubiquitination of the STOP-1 protein, thereby modulating the amount of expression of the STOP-1 protein.
In another preferred embodiment, the RAE1 gene or RAL1 gene, or a protein encoded thereby, a mutein, or an enhancer or inhibitor thereof, is also used to regulate the secretion level of organic acids in plants.
In another preferred example, the promoter or inhibitor is a promoter or inhibitor of RAE1 or RAL1, and RAE1 comprises the RAE1 gene, or a protein encoded by the same, a mutein; RAL1 includes the RAL1 gene, or a protein encoded by the RAL1 gene, and a mutein.
In another preferred embodiment, the promoter or inhibitor comprises a RAE1 gene or a RAL1 gene, or a promoter or inhibitor of a protein encoded thereby.
In another preferred example, the plant comprises a crop, a forestry plant, a vegetable, a melon, a flower, a pasture (including turf grass).
In another preferred embodiment, the plant is selected from the group consisting of: gramineae, crucifers, or a combination thereof.
In another preferred embodiment, the plant is selected from the group consisting of: arabidopsis, tobacco, rice, wheat, corn, sorghum, barley, brassica, soybean, or a combination thereof.
In another preferred embodiment, the plant is Arabidopsis thaliana.
In another preferred embodiment, the RAE1 gene or RAL1 gene is selected from the group consisting of: a cDNA sequence, a genomic sequence, or a combination thereof.
In another preferred embodiment, the RAE1 gene or RAL1 gene, or a protein encoded thereby, is derived from a crucifer.
In another preferred embodiment, the RAE1 gene or RAL1 gene, or a protein encoded thereby, is derived from Arabidopsis thaliana (e.g., col-0 ecological Arabidopsis thaliana) or a variant thereof.
In another preferred embodiment, the RAE1 gene, or a protein encoded thereby, is derived from monocots (e.g., rice, maize, barley, etc.) and dicots (e.g., brassica cabbage, soybean, etc.).
In another preferred embodiment, the RAE1 gene, or a protein encoded thereby, is derived from Arabidopsis thaliana, rice (OsRAE1.1, osRAE1.2), maize (XM_ 008677340.2), barley (AK 372025.1), brassica (XM_ 009132381.2), soybean (XM_ 003521974.4), or a combination thereof.
In another preferred embodiment, the amino acid sequence of the RAE1 protein is selected from the group consisting of:
(i) A polypeptide having the amino acid sequence set forth in any one of SEQ ID nos. 35, 37-42;
(ii) A polypeptide derived from (i) having the same function as the polypeptide shown in SEQ ID NO. 35, wherein the polypeptide is formed by substitution, deletion or addition of one or more (e.g., 1 to 10) amino acid residues to the amino acid sequence shown in any one of SEQ ID NO. 35, 37 to 42; or (b)
(Iii) A polypeptide derived from (i) having the same function as the polypeptide shown in any one of SEQ ID No. 35, 37-42, and having an amino acid sequence homology of 80% or more (preferably 90% or more, more preferably 95% or 98% or more) to the amino acid sequence shown in any one of SEQ ID No. 35, 37-42.
In another preferred embodiment, the nucleotide sequence of the RAE1 gene is selected from the group consisting of:
(a) A polynucleotide encoding a polypeptide as set forth in any one of SEQ ID nos. 35, 37-42;
(b) A polynucleotide having a sequence as set forth in any one of SEQ ID NOs 36, 43-49;
(c) A polynucleotide having a nucleotide sequence which has a homology of greater than or equal to 75% (preferably greater than or equal to 85%, more preferably greater than or equal to 90% or greater than or equal to 95%) to any of the sequences set forth in SEQ ID No. 36, 43-49;
(d) A polynucleotide truncated or added at the 5 'and/or 3' end of the polynucleotide shown in any one of SEQ ID nos. 36, 43-49, with 1-60 (preferably 1-30, more preferably 1-10) nucleotides; or (b)
(E) A polynucleotide complementary to the polynucleotide of any one of (a) - (d).
In another preferred embodiment, the coding gene encodes a RAE1 protein selected from (i), (ii) or (iii) above.
In another preferred embodiment, the RAL1 gene, or a protein encoded thereby, is derived from monocotyledonous (e.g., rice, maize, barley, etc.) and dicotyledonous plants (e.g., brassica cabbage, soybean, etc.).
In another preferred embodiment, the RAL1 gene, or a protein encoded thereby, is derived from Arabidopsis thaliana (AT 5G 27920), rice (LOC_Os 12G 36670), maize (XM_ 008664387.2), barley (AK 358574.1), brassica (XM_ 009113489.2), soybean (XM_ 003521631.4), or a combination thereof.
In another preferred embodiment, the amino acid sequence of the RAL1 protein is selected from the group consisting of:
(i) A polypeptide having the amino acid sequence set forth in any one of SEQ ID nos. 50 to 55;
(ii) A polypeptide derived from (i) having the same function as the polypeptide shown in any one of SEQ ID No. 50-55, which is formed by substitution, deletion or addition of one or more (e.g., 1-10) amino acid residues to the amino acid sequence shown in any one of SEQ ID No. 50-55; or (b)
(Iii) A polypeptide derived from (i) having the same function as the polypeptide shown in any one of SEQ ID No. 50-55, wherein the homology of the amino acid sequence to the amino acid sequence shown in any one of SEQ ID No. 50-55 is 80% or more, preferably 90% or more, 95% or 98% or more.
In another preferred embodiment, the nucleotide sequence of the RAL1 gene is selected from the group consisting of:
(a) A polynucleotide encoding a polypeptide as set forth in any one of SEQ ID nos. 50-55;
(b) A polynucleotide having a sequence as set forth in any one of SEQ ID NOs 56-61;
(c) A polynucleotide having a nucleotide sequence which has a homology of greater than or equal to 75% (preferably greater than or equal to 85%, more preferably greater than or equal to 90% or greater than or equal to 95%) to any of the sequences set forth in SEQ ID No.;
(d) A polynucleotide truncated or added at the 5 'and/or 3' end of the polynucleotide shown in any one of SEQ ID nos. 56 to 61 with 1 to 60 (preferably 1 to 30, more preferably 1 to 10) nucleotides; or (b)
(E) A polynucleotide complementary to the polynucleotide of any one of (a) - (d).
In another preferred embodiment, the coding gene encodes a RAL1 protein selected from (i), (ii) or (iii) above.
In a second aspect of the invention there is provided the use of a RAE1 gene or RAL1 gene, or a protein encoded thereby, a mutein of enhanced activity, or an enhancer thereof, for one or more uses selected from the group consisting of:
(1) Reducing the expression level of STOP-1 protein;
(2) Reducing or down-regulating expression of a gene selected from the group consisting of: atALMT1, atm mate, ALS3, or a combination thereof;
(3) Reducing the aluminum toxicity resistance of plants; and/or
(4) A formulation or agricultural composition is prepared and used to reduce the expression level of a STOP1 protein, reduce the aluminum toxicity resistance of a plant, or down-regulate the expression of a gene selected from the group consisting of: atALMT1, atm mate, ALS3, or a combination thereof.
In another preferred embodiment, the RAE1 gene or RAL1 gene, or encoded protein thereof, mutant protein with enhanced activity, or promoter thereof is degraded by mediating ubiquitination modification of STOP-1 protein, thereby reducing the expression level of STOP-1 protein.
In another preferred embodiment, the activity is one that mediates STOP-1 protein degradation, preferably by ubiquitination.
In another preferred embodiment, the activity enhancement means that the ratio of STOP-1 protein degradation-mediating activity A1 of the mutant protein to STOP-1 protein degradation-mediating activity A0 of the wild-type RAE1 protein or the wild-type RAL1 protein is not less than 1.2, preferably not less than 1.5.
In another preferred embodiment, said reducing the aluminum toxicity resistance of a plant comprises down-regulating gene expression selected from the group consisting of: atALMT1, atm mate, ALS3, or a combination thereof.
In another preferred embodiment, the promoter is a promoter of RAE1 or RAL1, wherein RAE1 comprises a RAE1 gene, or a protein encoded thereby, or a mutant protein with enhanced activity; RAL1 includes the RAL1 gene, or a protein encoded thereby, or a mutant protein having enhanced activity.
In another preferred embodiment, the promoter comprises a promoter of the RAE1 gene or the RAL1 gene, or a protein encoded thereby, or a mutant protein having enhanced activity.
In another preferred embodiment, the promoter comprises a compound or formulation that promotes expression of the RAE1 gene or its encoded protein, or increases the amount of RAE1 protein expressed, or enhances the activity of RAE1 protein; and/or
The promoter includes a compound or preparation that promotes expression of the RAL1 gene or a protein encoded by the RAL1 gene, or increases expression level of the RAL1 protein, or enhances activity of the RAL1 protein.
In another preferred embodiment, the accelerator is selected from the group consisting of: a small molecule compound, a nucleic acid molecule, a polypeptide, a small molecule ligand, or a combination thereof.
In another preferred embodiment, the nucleic acid molecule is selected from the group consisting of: miRNA, shRNA, siRNA, or a combination thereof.
In a third aspect of the invention there is provided a RAE1 mutein or a RAL1 mutein, or a RAE1 gene or an inhibitor of a RAL1 gene or a protein encoding the same, said RAE1 mutein being a RAE1 mutein having reduced activity compared to a wild type RAE1 protein, said RAL1 mutein being a RAL1 mutein having reduced activity compared to a wild type RAL1 protein, for one or more uses selected from the group consisting of:
(1) Increasing the expression level of STOP-1 protein;
(2) Increasing or up-regulating expression of a gene selected from the group consisting of: atALMT1, atm mate, ALS3, or a combination thereof;
(3) Enhancing the aluminum toxicity resistance of plants; and/or
(4) A formulation or agricultural composition is prepared and used to increase the expression level of a STOP1 protein, enhance the aluminum toxicity resistance of a plant, or up-regulate the expression of a gene selected from the group consisting of: atALMT1, atm mate, ALS3, or a combination thereof.
In another preferred embodiment, the enhancing the aluminum toxicity resistance (or tolerance to aluminum) of the plant comprises: improving the survival rate of plants under the condition of aluminum or reducing the toxicity of aluminum toxicity to plants (such as roots).
In another preferred embodiment, said enhancing the aluminum toxicity resistance of a plant comprises up-regulating gene expression selected from the group consisting of: atALMT1, atm mate, ALS3, or a combination thereof.
In another preferred embodiment, the RAE1 mutein, or an inhibitor of the RAE1 gene or its encoded protein, is also used to increase the secretion level of a plant organic acid, such as malic acid.
In another preferred embodiment, the RAL1 mutein, or an inhibitor of the RAL1 gene or its encoded protein, is also used to increase the secretion level of a plant organic acid (e.g., malic acid).
In another preferred embodiment, the inhibitor comprises a compound or formulation that inhibits the expression of the RAE1 gene or its encoded protein, or reduces the amount of RAE1 protein expressed, or reduces the activity of RAE1 protein; and/or
The inhibitor includes a compound or preparation that inhibits the expression of the RAL1 gene or a protein encoded thereby, or reduces the amount of expression of the RAL1 protein, or reduces the activity of the RAL1 protein.
In another preferred embodiment, the inhibitor is selected from the group consisting of: a small molecule compound, a nucleic acid molecule, a polypeptide, a small molecule ligand, or a combination thereof.
In another preferred embodiment, the nucleic acid molecule is selected from the group consisting of: miRNA, shRNA, siRNA, or a combination thereof.
In another preferred embodiment, the inhibitor is selected from the group consisting of: a small molecule compound, an antisense nucleic acid, microRNA, siRNA, RNAi, crispr reagents, or a combination thereof.
In another preferred embodiment, the RAE1 mutein is a mutein with reduced or lost RAE1 activity.
In another preferred embodiment, the RAL1 mutein is a mutein with reduced or lost RAL1 activity.
In another preferred embodiment, the activity is one that mediates STOP-1 protein degradation.
In another preferred embodiment, the decrease in activity refers to a ratio of STOP-1 protein degradation-mediating activity A1 of the mutant protein to STOP-1 protein degradation-mediating activity A0 of the wild-type RAE1 protein or the wild-type RAL1 protein of 0.8 or less, preferably 0.6 or less.
In another preferred embodiment, the RAE1 mutein is a reduced or lost activity mutein.
In another preferred embodiment, the RAE1 mutein is a RAE1 protein lacking the F-box domain.
In another preferred embodiment, the RAL1 mutein is a reduced or lost activity mutein.
In another preferred embodiment, the RAL1 mutein is a RAL1 protein lacking the F-box domain.
In another preferred embodiment, the RAE1 mutein comprises a mutation in an amino acid corresponding to the wild-type RAE1 protein selected from the group consisting of: 167 th bit (G), 439 th bit (G), 466 th bit (R), 524 th bit (Q), 568 th bit (S), 116 th bit (G), 193 th bit (G), 400 th bit (W), or a combination thereof.
In another preferred embodiment, the RAE1 mutein has a mutation in the RAE1 protein corresponding to the wild type selected from the group consisting of: G167R, G439R, R466K, Q524STOP, S568L, G116R, G193R, W400STOP, or combinations thereof.
In another preferred embodiment, the amino acid sequence of the wild-type RAE1 protein is as set forth in any one of SEQ ID NOs.35, 37-42.
In another preferred embodiment, the amino acid sequence of the wild-type RAL1 protein is as set forth in any one of SEQ ID NOs.50-55.
In a fourth aspect of the present invention, there is provided a method of modulating the aluminium toxicity resistance of a plant, the method comprising the steps of: regulating the expression quantity and/or activity of RAE1 protein or RAL1 protein in the plant, thereby regulating the aluminum toxicity resistance of the plant.
In another preferred example, the plants suitable for use in the method include crops, forestry plants, vegetables, fruits, flowers, pastures (including turf grass).
In another preferred embodiment, the modulation of the anti-aluminum toxicity capability of a plant is the down-regulation of the expression and/or activity of a RAE1 protein or a RAL1 protein in a plant or the inactivation of a RAE1 protein or a RAL1 protein when the anti-aluminum toxicity capability of a plant is enhanced.
In another preferred embodiment, the modulation of the plant's ability to resist aluminum toxicity is an up-regulation of the expression and/or activity of RAE1 protein or RAL1 protein in the plant when the plant's ability to resist aluminum toxicity is reduced.
In another preferred embodiment, the method is also used to regulate the level of organic acids secreted by plants (e.g., roots).
In another preferred embodiment, said modulating the ability of a plant to resist aluminum toxicity comprises modulating the level of organic acids secreted by the plant (e.g., root).
In a fifth aspect of the present invention, there is provided a method of regulating expression of a gene, the method comprising the steps of:
(i) Regulating the expression level and/or activity of RAE1 protein or RAL1 protein in the plant, thereby regulating the expression of the gene;
wherein the gene is selected from the group consisting of: atALMT1, atm mate, ALS3, or a combination thereof.
In another preferred embodiment, step (i) comprises: up-regulating the expression level and/or activity of a RAE1 protein or a RAL1 protein in a plant, thereby down-regulating the expression of a gene selected from the group consisting of: atALMT1, atm mate, ALS3, or a combination thereof.
In another preferred embodiment, step (i) comprises: down-regulating the expression level and/or activity of a RAE1 protein or a RAL1 protein in a plant, thereby up-regulating the expression of a gene selected from the group consisting of: atALMT1, atm mate, ALS3, or a combination thereof.
In another preferred embodiment, the method further comprises the steps of:
(ii) Regulating the expression level and/or activity of STOP-1 protein in said plant, thereby regulating the expression of RAE1 gene.
In a sixth aspect of the present invention, there is provided a method of modulating STOP-1 protein levels, comprising the steps of:
(a) Regulating the expression level and/or activity of RAE1 protein or RAL1 protein in said plant, thereby regulating STOP-1 protein level.
In another preferred embodiment, step (a) comprises: up-regulating the expression level and/or activity of RAE1 protein or RAL1 protein in plants, thereby down-regulating STOP-1 protein levels.
In another preferred embodiment, step (a) comprises: down-regulating the expression level and/or activity of RAE1 protein or RAL1 protein in plants, thereby up-regulating STOP-1 protein levels.
In another preferred embodiment, said down-regulating STOP-1 protein level comprises promoting degradation of the STOP-1 protein.
In another preferred embodiment, said up-regulating STOP-1 protein level comprises reducing or inhibiting degradation of STOP-1 protein.
In a seventh aspect of the invention, there is provided an isolated mutant RAE1 protein or mutant RAL1 protein, said mutant RAE1 protein or mutant RAL1 protein being a non-native protein, and said mutant RAE1 protein or mutant RAL1 protein for use in one or more applications selected from the group consisting of:
(1) Regulating the expression or degradation of STOP-1 protein;
(2) Regulating and controlling the aluminum toxicity resistance or aluminum resistance of the plants; and/or
(3) Regulating expression of a gene selected from the group consisting of: atALMT1, atm mate, ALS3, or a combination thereof.
In another preferred embodiment, the mutant RAE1 protein or mutant RAL1 protein comprises a mutant protein with increased activity or a mutant protein with decreased activity.
In another preferred embodiment, the mutant RAE1 protein or mutant RAL1 protein is a mutant protein with reduced or lost activity.
In another preferred embodiment, the RAE1 mutein comprises a mutation in an amino acid corresponding to the wild-type RAE1 protein selected from the group consisting of: 167 th bit (G), 439 th bit (G), 466 th bit (R), 524 th bit (Q), 568 th bit (S), 116 th bit (G), 193 th bit (G), 400 th bit (W), or a combination thereof.
In another preferred embodiment, the RAE1 mutein has a mutation in the RAE1 protein corresponding to the wild type selected from the group consisting of: G167R, G439R, R466K, Q524STOP, S568L, G116R, G193R, W400STOP, or combinations thereof.
In another preferred embodiment, the amino acid sequence of the wild-type RAE1 protein is as set forth in any one of SEQ ID NOs.35, 37-42.
In another preferred embodiment, the amino acid sequence of the wild-type RAL1 protein is as set forth in any one of SEQ ID NOs.50-55.
In another preferred embodiment, the mutant RAE1 protein has the same or substantially the same amino acid sequence as any one of SEQ ID NOS.35, 37-42, except for the mutation.
In another preferred embodiment, said substantial identity is a variation of up to 50 (preferably 1-20, more preferably 1-10, more preferably 1-5) amino acids, wherein said variation comprises amino acid substitutions, deletions or additions and said mutant RAE1 protein does not have activity in mediating STOP-1 protein degradation.
In another preferred embodiment, the mutant RAE1 protein or mutant RAL1 protein has one or more uses selected from the group consisting of:
(1) Increasing the expression level of STOP-1 protein;
(2) Enhancing the aluminum toxicity resistance of plants; and/or
(3) Increasing or up-regulating expression of a gene selected from the group consisting of: atALMT1, atm mate, ALS3, or a combination thereof.
In an eighth aspect of the invention, there is provided a polynucleotide encoding a mutant RAE1 protein or a mutant RAL1 protein according to the seventh aspect of the invention.
In a ninth aspect, the present invention provides an use of RAE1 protein or RAL1 protein, or a coding gene thereof, for screening a drug capable of controlling aluminum toxicity resistance of plants.
In another preferred embodiment, the drug is selected from the group consisting of: a small molecule compound, polypeptide, nucleic acid, small molecule ligand, or a combination thereof.
In a tenth aspect of the present invention, there is provided a method of identifying a modulator of the anti-aluminium toxicity ability of a plant, the method comprising the steps of:
(a) Providing a modulator to be identified, and a RAE1 gene or a RAL1 gene or a protein encoded thereby;
(b) Contacting the compound to be identified with the RAE1 gene or RAL1 gene or a protein encoded thereby;
(c) Determining the activity or expression level of said RAE1 protein or RAL1 protein;
(d) Identifying the compound as a plant aluminium toxicity resistance enhancer or inhibitor based on the results of step (c).
In another preferred embodiment, the compound to be identified is a plant aluminium toxicity resistance enhancer if the activity and/or expression level of the RAE1 protein is reduced or down-regulated as a result of step (c).
In another preferred embodiment, the compound to be identified is a plant aluminium toxicity inhibitor, if the result of step (c) is an increase or up-regulation of the activity and/or expression of the RAE1 protein.
In another preferred embodiment, the compound to be identified is a plant aluminium toxicity resistance enhancer if the activity and/or expression level of the RAL1 protein is reduced or down-regulated as a result of step (c).
In another preferred embodiment, the compound to be identified is a plant aluminium toxicity inhibitor, if the result of step (c) is an increase or up-regulation of the activity and/or expression of the RAL1 protein.
In an eleventh aspect of the invention, there is provided a method of modulating expression of RAE1, the method comprising the steps of:
(a) Regulating the expression level and/or activity of STOP-1 protein in said plant, thereby regulating the expression of RAE1 gene.
In another preferred embodiment, the method is a method of up-regulating expression of RAE1, the method comprising the steps of:
(a) Down-regulating the expression level and/or activity of STOP-1 protein in said plant.
In another preferred embodiment, the method is for non-therapeutic and non-diagnostic purposes.
The invention also provides an inhibitor of RAE1 gene or RAE1 protein, which contains STOP-1 protein or encoding gene thereof as an active ingredient.
It is understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.
Drawings
FIG. 1 shows enhanced expression of pAtALMT1:LUC in mutant rae 1. Wherein (A) 8 pAtALMT LUC expression enhancement mutants are obtained by screening; (B) mutation site of mutant rae1 on protein sequence; (C) pRAE 1A RAE1 transgenic line complemented mutant RAE-1 in pAtALMT A LUC expression.
FIG. 2 shows the expression levels of the genes in qPCR analysis WT, rae1-1, rae1-2, stop 1-3. Wherein, (a) reporter gene LUC; (B) AtALMT a 1; (C) AtMATE; (D) ALS3; (E) AtSTAR a 1; (F) ALS1.
FIG. 3 shows qPCR analysis of expression levels of each gene in WT, over-expressed RAE1 lines RAE1-OX1 and RAE1-OX 2. Wherein, (a) RAE1; (B) AtALMT a 1; (C) AtMATE; (D) ALS3; (E) AtSTAR a 1; (F) ALS1.
FIG. 4 shows the expression control mechanism of RAE 1. (A) qPCR analysis of RAE1 expression in tissues (B) RAE1 expression was induced by Al treatment and regulated by STOP-1, expression level was up-regulated in RAE A (C) GUS Activity analysis pRAE1: expression of GUS (D, E, F, G, H, I) GUS staining analysis pRAE1: expression of GUS (J) protoplast expression system showed STOP-1 to regulate pRAE expression of LUC, and mutation of the binding region would regulate attenuation (K) EMSA to verify that STOP-1 binds directly to the binding region on the RAE1 promoter.
FIG. 5 shows RAE1 and STOP-1 interaction both in vivo and in vitro. (A) In vitro verification of RAE1 interaction with STOP-1 by Pull-down test, the combination of GST-STOP-1 and RAE1-His can Pull down RAE1 protein, the combination of GST-RAE1 and His-Trx-STOP-1 can Pull down STOP-1 protein, and other combinations are negative controls; (B) In vivo verification of RAE1 interaction with STOP-1 by Split-LUC assays, STOP-1-nLUC and cLUC-RAE1-1/cLUC-RAE1 DeltaF produced LUC fluorescence signals, no LUC fluorescence signals were produced by the combination of STOP-1-nLUC and cLUC-RAE1, other combinations were negative controls; (C) Co-IP experiments in vivo demonstrated that RAE1 interacted with STOP-1, FLAG-RAE1/FLAG-RAE1-1/FLAG-RAE1 DeltaF and STOP-1-HA were Co-expressed in protoplasts and crude protein extracts were immunoprecipitated with FLAG beads and the STOP-1-HA protein was detected with HA antibody.
FIG. 6 shows that STOP-1 protein accumulates in mutant RAE, while overexpression of RAE1 promotes STOP-1 protein degradation. Western blot analysis pSTOP STOP-1 protein levels in mutant RAE and over-expressed RAE1 strains of the STOP-1-HA transgenic lines.
FIG. 7 shows that the proteasome inhibitor MG132 stabilizes the STOP-1 protein. (A) the STOP-1 protein is stabilized by MG132 treatment for 1 and 3 hours; (B) STOP-1 protein is stabilized by MG132 under both aluminum-free and aluminum-bearing conditions; (C) GUS staining analysis pSTOP1 STOP-1 protein levels in STOP-1-GUS transgenic lines; (D) pSTOP 1A STOP-1-GUS transgenic line was analyzed for GUS activity.
FIG. 8 shows that RAE1 degrades by mediating ubiquitination modification of STOP-1 proteins. (A) STOP-1 protein in the protoplasm expression system is degraded along with the increase of RAE1 expression, and mutant proteins RAE-1 and RAE1 delta F can not degrade STOP-1; (B) Verifying that the STOP-1 protein is ubiquitinated modified in the protoplast expression system; (C) Mutant proteins RAE-1 and RAE 1. DELTA.F in the protoplast expression system were not able to mediate ubiquitination modification of STOP-1.
FIG. 9 shows that mutant RAE1 has increased aluminum tolerance, while the over-expressed RAE1 strain has decreased aluminum tolerance. Wherein, (a) malic acid secretion of rae under aluminum toxin treatment conditions is increased compared with wild type; (B) aluminum accumulation in rae roots was reduced compared to wild type; (C) Root length photographs of rae and wild type under aluminum toxin treatment conditions; (D) Root relative growth of rae1 under aluminum toxin treatment conditions was longer than that of wild type; (E) Root length photograph of over-expressed RAE1 strain under aluminum toxicity treatment conditions and wild type (F) root relative growth of over-expressed RAE1 strain under aluminum toxicity treatment conditions is shorter than that of wild type.
FIG. 10 shows AtALMT in RAL1 expression is up-regulated and that RAL1 interacts with STOP-1. (A) AtALMT in ral1 expression is up-regulated; (B) Up-regulation of LUC expression in pAtALMT 1:3835 in ral1, up-regulation of LUC expression in pAtALMT 1:5248 in rae1 and dual mutants of ral 1; (C) RAL1 expression is induced by Al treatment and regulated by STOP1, and the expression level is up-regulated in rae; (D) GUS staining analysis pRAL1: GUS expression (E) Split-LUC assay in vivo demonstrated that RAL1 interacted with STOP-1, STOP-1-nLUC and cLUC-RAL 1. DELTA.F produced LUC fluorescent signals, and the combination of STOP-1-nLUC and cLUC-RAL1 did not produce LUC fluorescent signals, as RAL1 mediated degradation of STOP-1 protein.
FIG. 11 shows the expression pattern of OsRAE1 and OsRAE1 interacts with ART 1. (A) OsRAE1.1 and OsRAE1.2 are expressed in root tips and root base parts, and the expression is induced by Al; (B) decreased expression of OsRAE1.1 in art 1; (C) In vivo experiments on Split-LUC demonstrated that OsRAE1.1 interacted with ARTT1, ART1-nLUC and cLUC-OsRAE1.1. DELTA.F produced LUC fluorescent signals.
Detailed Description
The inventors have conducted extensive and intensive studies to find, for the first time, unexpectedly a gene RAE1 and RAL1 gene regulating the stability of the anti-aluminum transcription factor STOP-1 protein by a large number of mutagenesis, screening and analysis. The RAE1 gene or RAL1 gene or encoded protein thereof, or mutein thereof, or promoter or inhibitor thereof can be used to (1) regulate expression or degradation of STOP-1 protein; (2) regulating and controlling the aluminum toxicity resistance or aluminum resistance of the plants; and/or (3) regulating expression of a gene selected from the group consisting of: atALMT1, atm mate, ALS3, or a combination thereof. Experiments have shown that RAE1 or RAL1 mediates the degradation of STOP-1 proteins. Furthermore, rae-1 mutant proteins and the removal of F-box domain proteins lost the function of mediating STOP-1 degradation. Furthermore, the inventors have unexpectedly found for the first time that down-regulating the expression level and/or activity of RAE1 protein in plants can significantly inhibit STOP-1 degradation, up-regulating AtALMT's 1 expression level, increasing the secretion of organic acids (e.g., malic acid) and enhancing the plant's resistance to aluminum toxicity (or tolerance to aluminum). In contrast, the expression level and/or activity of RAE1 protein in the over-expressed plants can significantly promote STOP-1 degradation, down-regulate AtALMT expression level, reduce secretion of organic acids (e.g., malic acid) and enhance sensitivity of the plants to aluminum toxin. On this basis, the present inventors have completed the present invention.
A LUC reporter gene system (pAtALMT 1:LUC) fused with AtALMT promoter and luciferase gene is constructed, and EMS mutagenesis is performed by using the material to screen mutants affecting the expression of the LUC reporter gene. A novel gene for negative control AtALMT1 Expression was cloned using one of the LUC Expression-enhancing mutants and designated RAE1 (Regulation of AtALMT1 Expression 1), and mutants with different site mutations on 8 RAE1 were obtained by a total selection (RAE 1-1 to RAE 1-8). Construction of vector pRAE1 RAE 1A complementation transgenic line was obtained by infection of RAE1-1, 2 lines each complemented RAE1-1 fluorescent phenotype.
In addition to the LUC reporter gene and AtALMT1, expression of other STOP1 downstream regulatory genes AtMATE and ALS3 was increased in mutant rae compared to Wild Type (WT) both in the absence of aluminum and in the presence of aluminum. Conversely, overexpression of RAE1 reduces the expression of AtALTM, atMATE, and ALS 3. RAE1 expression was induced by aluminum treatment and regulated by STOP-1, and RAE1 expression levels were also up-regulated in RAE. Protoplast expression systems showed STOP-1 to regulate pRAE expression of LUC, and mutation of the binding region reduced regulation, EMSA verified that STOP-1 directly bound to the binding region on the RAE1 promoter. RAE1 expression is expressed in various tissues of plants and is mainly expressed in vascular tissues.
RAE1 was shown to be capable of interacting with STOP-1 protein by in vitro Pull-Down test, tobacco Split-LUC test, and in vivo Co-IP test. RAE1-1 mutant proteins and F-box domain-deleted proteins did not lose the ability to bind STOP-1, whereas RAE1 interaction with STOP-1 in vivo was probably due to RAE1 mediated degradation of STOP-1 protein.
Construction pSTOP1 STOP-1-HA transgenic lines STOP-1 protein was detected and aluminum treatment allowed accumulation of STOP-1 protein, and in the rae-1 mutant STOP-1 protein was increased over wild-type, both in the absence of aluminum and in the presence of aluminum. The proteasome inhibitor MG132 treatment can inhibit the degradation of STOP-1 in the absence of aluminum or in the presence of aluminum, resulting in increased protein. Further, RAE1 mediates ubiquitination modification and degradation of STOP-1 proteins as demonstrated by the protoplast expression system. Furthermore, rae-1 mutant proteins and F-box domain-deleted proteins lose the function of allowing STOP-1 to be modified and degraded by ubiquitination. In contrast, overexpression of RAE1 promotes degradation of STOP-1 protein.
The mutant rae1 was analyzed for an aluminum resistant phenotype, with increased malate secretion of rae1 compared to the wild type, reduced accumulation of aluminum on the root, and longer root length of rae1 than the wild type under aluminum toxin treatment conditions. In contrast, overexpressing RAE1 has shorter root length than wild-type under aluminum toxin treatment conditions.
RAE1 has a homologous gene RAL1 (RAE 1 Like 1) in Arabidopsis thaliana, and expression of AtALMT1, pAtALMT1: LUC is up-regulated in a T-DNA knockout mutant RAL1 of RAL1, but up-regulated to a lower extent than in RAE 1-1. pAtALMT.LUC expression was higher in RAE-1 RAL1 double mutants than in each single mutant, indicating that RAE1 is functionally redundant with RAL 1. Expression of RAL1 is also induced by aluminum toxicity, but the tissue site of RAL1 expression in roots is different from that of RAE 1. The tobacco Split-LUC test demonstrates that RAL1 is capable of interacting with STOP-1 protein. As shown above, RAL1 has the same function as RAE1 for degrading STOP-1 protein, but RAE1 plays an important role in plant resistance to aluminum toxicity because it is not expressed in root tip meristems and elongation regions.
RAE1 and RAL1 have corresponding homologous genes in most monocots (rice, maize, barley, etc.) and dicots (Brassica cabbage, soybean, etc.). The homologous genes of RAE1 in rice are OsRAE1.1 and OsRAE1.2, the amino acid sequences of the two homologous genes are 97.5% similar, the OsRAE1.1 and the OsRAE1.2 are expressed in root tips and root base parts, and the expression is induced by Al and regulated by ART1 (genes homologous to STOP1 in rice). Tobacco Split-LUC experiments demonstrated that osrae1.1 is capable of interacting with ART1 protein. Therefore, we consider that RAE1 is conserved in the mechanism for regulating the stability of plant anti-aluminum toxicity transcription factor STOP1, and it is possible to improve the anti-aluminum toxicity capability of crops by performing function deletion mutation on RAE1 homologous genes in crops.
Terminology
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
As used herein, when used in reference to a specifically recited value, the term "about" means that the value can vary no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes 99 and 101 and all values therebetween (e.g., 99.1, 99.2, 99.3, 99.4, etc.).
As used herein, the term "comprising" or "including" can be open, semi-closed, and closed. In other words, the term also includes "consisting essentially of …", or "consisting of …".
As used herein, the term "AxxB" means that amino acid a at position xx is changed to amino acid B, e.g. "G167R" means that amino acid G at position 167 is mutated to R, and so on.
As used herein, the term "AxxSTOP" means that amino acid a at position xx is changed to a STOP codon, e.g. "Q524STOP" means that amino acid Q at position 524 is mutated to a STOP codon, and so on.
As used herein, the terms "resistance to aluminum toxicity," "aluminum resistance," "aluminum phenotype," "resistance to aluminum," and "resistance to aluminum" are used interchangeably and refer to the resistance to aluminum.
As used herein, the term "plant" is not particularly limited, including (but not limited to): flower plants, fruit plants, forestry plants, vegetables, crops, and the like, such as rice, wheat, corn, soybean, sorghum, brassica, barley, and the like.
Fruit plants include (but are not limited to): plants of Citrus, rosaceae, cucurbitaceae, musaceae, etc.
Vegetable plants include (but are not limited to): plants of Compositae, solanaceae, labiatae, umbelliferae, and Brassicaceae.
Crops such as but not limited to: plants of Gramineae and Amaryllidaceae, etc.
In a preferred embodiment of the invention, the plant is selected from the crucifers family, more preferably from the genus arabidopsis.
As used herein, the present RAE1 proteins include wild-type RAE1 proteins and mutant RAE1 proteins.
As used herein, the RAL1 proteins of the invention include wild-type RAL1 proteins and mutant RAL1 proteins.
Muteins of the invention and nucleic acids encoding same
As used herein, the term "mutein", "mutein of the present invention" includes mutant RAE1 proteins and mutant RAL1 proteins.
As used herein, the terms "mutant RAE1 proteins", "mutant RAE1 proteins of the invention" are used interchangeably, all refer to non-naturally occurring RAE1 mutant proteins, and the mutant proteins are proteins engineered based on any of the proteins shown in SEQ ID No.:35, 37-42. The muteins contain core amino acids associated with activity in mediating STOP-1 degradation.
In another preferred embodiment, the mutant RAE1 protein comprises a mutant protein with increased activity or a mutant protein with decreased activity.
As used herein, the terms "mutant RAL1 proteins", "mutant RAL1 proteins of the invention" are used interchangeably, all refer to non-naturally occurring RAL1 muteins, and are proteins that have been engineered based on any of the proteins set forth in SEQ ID No.: 50-55. The muteins contain core amino acids associated with activity in mediating STOP-1 degradation.
In another preferred embodiment, the mutant RAL1 protein comprises a mutant protein with increased activity or a mutant protein with decreased activity.
In another preferred embodiment, the activity is one that mediates STOP-1 protein degradation.
In another preferred embodiment, the mutant RAE1 protein or mutant RAL1 protein is a mutant protein with reduced or lost activity.
In another preferred embodiment, at least one of said core amino acids of said mutein is engineered and has reduced or lost activity in mediating STOP-1 degradation.
Preferably, in the present invention, the RAE1 mutein is mutated at an amino acid corresponding to the wild-type RAE1 protein selected from the group consisting of: 167 th bit (G), 439 th bit (G), 466 th bit (R), 524 th bit (Q), 568 th bit (S), 116 th bit (G), 193 th bit (G), 400 th bit (W), or a combination thereof. Such mutations may result in a substantial decrease or even loss of activity of the RAE1 protein.
It will be appreciated that where the amino acid numbering in a mutein of the invention is based on SEQ ID NO. 35, when a particular mutein has 80% or more homology to the sequence shown in SEQ ID NO. 35, the amino acid numbering of the mutein may be shifted, e.g., 1-5 to the N-or C-terminus of the amino acid, relative to the amino acid numbering of SEQ ID NO. 35, and such shifting is generally understood by those skilled in the art to be within reasonable limits using conventional sequence alignment techniques and muteins having the same or similar activity that do not have 80% (e.g., 90%, 95%, 98%) homology due to amino acid numbering are not within the scope of the muteins of the invention.
The muteins of the present invention are synthetic or recombinant proteins, i.e., can be the product of chemical synthesis, or can be produced from a prokaryotic or eukaryotic host (e.g., bacteria, yeast, plants) using recombinant techniques. Depending on the host used in the recombinant production protocol, the muteins of the present invention may be glycosylated or may be non-glycosylated. The muteins of the present invention may or may not also include an initial methionine residue.
The invention also includes fragments, derivatives and analogues of the muteins. As used herein, the terms "fragment," "derivative," and "analog" refer to a protein that retains substantially the same biological function or activity of the mutein.
The mutein fragment, derivative or analogue of the present invention may be (i) a mutein having one or more conserved or non-conserved amino acid residues, preferably conserved amino acid residues, substituted, which may or may not be encoded by the genetic code, or (ii) a mutein having a substituent in one or more amino acid residues, or (iii) a mutein formed by fusion of a mature mutein with another compound, such as a compound that extends the half-life of the mutein, e.g. polyethylene glycol, or (iv) a mutein formed by fusion of an additional amino acid sequence to the mutein sequence, such as a leader or secretory sequence or a sequence used to purify the mutein or a pro-protein sequence, or a fusion protein formed with an antigen IgG fragment. Such fragments, derivatives and analogs are within the purview of one skilled in the art and would be well known in light of the teachings herein. In the present invention, conservatively substituted amino acids are preferably generated by amino acid substitution according to Table I.
TABLE I
Initial residues Representative substitution Preferred substitution
Ala(A) Val；Leu；Ile Val
Arg(R) Lys；Gln；Asn Lys
Asn(N) Gln；His；Lys；Arg Gln
Asp(D) Glu Glu
Cys(C) Ser Ser
Gln(Q) Asn Asn
Glu(E) Asp Asp
Gly(G) Pro；Ala Ala
His(H) Asn；Gln；Lys；Arg Arg
Ile(I) Leu；Val；Met；Ala；Phe Leu
Leu(L) Ile；Val；Met；Ala；Phe Ile
Lys(K) Arg；Gln；Asn Arg
Met(M) Leu；Phe；Ile Leu
Phe(F) Leu；Val；Ile；Ala；Tyr Leu
Pro(P) Ala Ala
Ser(S) Thr Thr
Thr(T) Ser Ser
Trp(W) Tyr；Phe Tyr
Tyr(Y) Trp；Phe；Thr；Ser Phe
Val(V) Ile；Leu；Met；Phe；Ala Leu
The mutant proteins of the present invention having reduced activity do not have the activity of mediating STOP-1 protein degradation.
In addition, the mutant proteins of the present invention may be modified. Modified (typically without altering the primary structure) forms include: chemically derivatized forms of muteins such as acetylated or carboxylated in vivo or in vitro. Modifications also include glycosylation, such as those resulting from glycosylation modifications during synthesis and processing of the mutein or during further processing steps. Such modification may be accomplished by exposing the mutein to an enzyme that performs glycosylation (e.g., mammalian glycosylase or deglycosylase). Modified forms also include sequences having phosphorylated amino acid residues (e.g., phosphotyrosine, phosphoserine, phosphothreonine). Also included are muteins modified to enhance their proteolytic resistance or to optimize their solubility properties.
The invention also provides polynucleotide sequences encoding RAE1 polypeptides, proteins or variants thereof. The polynucleotides of the invention may be in the form of DNA or RNA. The DNA forms include: DNA, genomic DNA or synthetic DNA, which may be single-stranded or double-stranded. The DNA may be a coding strand or a non-coding strand.
The term "polynucleotide encoding a mutein" may include polynucleotides encoding the muteins of the present invention, as well as polynucleotides further comprising additional coding and/or non-coding sequences.
The invention also relates to variants of the above polynucleotides which encode fragments, analogs and derivatives of the polypeptides or muteins having the same amino acid sequence as the invention. Such nucleotide variants include substitution variants, deletion variants and insertion variants. As known in the art, an allelic variant is a substitution of a polynucleotide, which may be a substitution, deletion, or insertion of one or more nucleotides, without substantially altering the function of the mutein encoded thereby.
The invention also relates to polynucleotides which hybridize to the sequences described above and which have at least 50%, preferably at least 70%, more preferably at least 80% identity between the two sequences. The invention relates in particular to polynucleotides which hybridize under stringent conditions (or stringent conditions) to the polynucleotides of the invention. In the present invention, "stringent conditions" means: (1) Hybridization and elution at lower ionic strength and higher temperature, e.g., 0.2 XSSC, 0.1% SDS,60 ℃; or (2) adding denaturing agents such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll,42℃and the like during hybridization; or (3) hybridization only occurs when the identity between the two sequences is at least 90% or more, more preferably 95% or more.
The muteins and polynucleotides of the invention are preferably provided in isolated form, and more preferably purified to homogeneity.
It should be understood that while the RAE1 gene of the present invention is preferably derived from Arabidopsis thaliana, other genes from other plants that are highly homologous (e.g., have more than 80%, such as 85%,90%,95%, or even 98% sequence identity) to the RAE1 gene are also within the contemplation of the present invention. Methods and tools for aligning sequence identity are also well known in the art, such as BLAST.
The full-length polynucleotide sequence of the present invention can be obtained by PCR amplification, recombinant methods or artificial synthesis. For the PCR amplification method, primers can be designed according to the nucleotide sequences disclosed in the present invention, particularly the open reading frame sequences, and amplified to obtain the relevant sequences using a commercially available cDNA library or a cDNA library prepared according to a conventional method known to those skilled in the art as a template. When the sequence is longer, it is often necessary to perform two or more PCR amplifications, and then splice the amplified fragments together in the correct order.
Once the relevant sequences are obtained, recombinant methods can be used to obtain the relevant sequences in large quantities. This is usually done by cloning it into a vector, transferring it into a cell, and isolating the relevant sequence from the propagated host cell by conventional methods.
Furthermore, the sequences concerned, in particular fragments of short length, can also be synthesized by artificial synthesis. In general, fragments of very long sequences are obtained by first synthesizing a plurality of small fragments and then ligating them.
At present, it is already possible to obtain the DNA sequences encoding the proteins of the invention (or fragments or derivatives thereof) entirely by chemical synthesis. The DNA sequence can then be introduced into a variety of existing DNA molecules (or vectors, for example) and cells known in the art. In addition, mutations can be introduced into the protein sequences of the invention by chemical synthesis.
Methods of amplifying DNA/RNA using PCR techniques are preferred for obtaining polynucleotides of the invention. In particular, when it is difficult to obtain full-length cDNA from a library, it is preferable to use RACE method (RACE-cDNA end rapid amplification method), and primers for PCR can be appropriately selected according to the sequence information of the present invention disclosed herein and synthesized by a conventional method. The amplified DNA/RNA fragments can be isolated and purified by conventional methods, such as by gel electrophoresis.
Wild-type RAE1 protein and RAE1 gene
As used herein, wild-type RAE1 protein refers to a naturally occurring, plant-derived (preferably from arabidopsis, rice, maize, barley, brassica, soybean, or the like), non-engineered RAE1 protein that negatively regulates AtALMT1 expression. The nucleotide can be obtained by genetic engineering techniques such as genome sequencing, polymerase Chain Reaction (PCR), etc., and the amino acid sequence can be deduced from the nucleotide sequence.
In another preferred embodiment, the RAE1 protein is derived from Arabidopsis thaliana, and the amino acid sequence is shown in SEQ ID NO. 35.
MKKVKQIRVLKPFDLLSEELVFIILDLISPNPSDLKSFSLTCKSFYQLESKHRGSLKPLRSDYLPRILTRYRNTTDLDLTFCPRVTDYALSVVGCLSGPTLRSLDLSRSGSFSAAGLLRLALKCVNLVEIDLSNATEMRDADAAVVAEARSLERLKLGRCKMLTDMGIGCIAVGCKKLNTVSLKWCVGVGDLGVGLLAVKCKDIRTLDLSYLPITGKCLHDILKLQHLEELLLEGCFGVDDDSLKSLRHDCKSLKKLDASSCQNLTHRGLTSLLSGAGYLQRLDLSHCSSVISLDFASSLKKVSALQSIRLDGCSVTPDGLKAIGTLCNSLKEVSLSKCVSVTDEGLSSLVMKLKDLRKLDITCCRKLSRVSITQIANSCPLLVSLKMESCSLVSREAFWLIGQKCRLLEELDLTDNEIDDEGLKSISSCLSLSSLKLGICLNITDKGLSYIGMGCSNLRELDLYRSVGITDVGISTIAQGCIHLETINISYCQDITDKSLVSLSKCSLLQTFESRGCPNITSQGLAAIAVRCKRLAKVDLKKCPSINDAGLLALAHFSQNLKQINVSDTAVTEVGLLSLANIGCLQNIAVVNSSGLRPSGVAAALLGCGGLRKAKLHASLRSLLPLSLIHHLEARGCAFLWKDNTLQAELDPKYWKQQLEEMAP(SEQ ID NO.:35)
In another preferred embodiment, the RAE1 protein is derived from rice (OsRAE 1.1) and has an amino acid sequence shown in SEQ ID NO. 37.
MASPMPTPTHRVKRRRLDLSPPPHLNDLADELLFLILDRAAAHDPRALKSFSLVSRACHAAESRHRRVLRPFRPDLLPAALARYPALSRLDLSLCPRLPDAALAALPAAPSVSAVDLSRSRGFGAAGLAALVAACPNLTDLDLSNGLDLGDAAAAEVAKARRLQRLSLSRCKRITDMGLGCIAVGCPDLRELSLKWCIGVTHLGLDLLALKCNKLNILDLSYTMIVKKCFPAIMKLQSLQVLLLVGCNGIDDDALTSLDQECSKSLQVLDMSNYYNVTHVGVLSIVKAMPNLLELNLSYCSPVTPSMSSSFEMIHKLQTLKLDGCQFMDDGLKSIGKSCVSLRELSLSKCSGVTDTDLSFVVPRLKNLLKLDVTCCRKITDVSLAAITTSCPSLISLRMESCSLVSSKGLQLIGRRCTHLEELDLTDTDLDDEGLKALSGCSKLSSLKIGICLRITDEGLRHVSKSCPDLRDIDLYRSGAISDEGVTHIAQGCPMLESINLSYCTKLTDCSLRSLSKCIKLNTLEIRGCPMVSSAGLSEIATGCRLLSKLDIKKCFEINDMGMIFLSQFSHNLRQINLSYCSVTDIGLISLSSICGLQNMTIVHLAGVTPNGLIAALMVCGLRKVKLHEAFKSMVPSHMLKVVEARGCLFQWINKPYQVAVEPCDVWKQQSQDLLVQ(SEQ ID NO.:37)
In another preferred embodiment, the RAE1 protein is derived from rice (OsRAE 1.2) and has the amino acid sequence shown in SEQ ID NO. 38.
MASPAPTHHAKRRRLALPPPPPPHLNDLADELLFLILDRAAAHDPRALKSFSLVSRACHAAESRHRRVLRPFRPDLLPAALARYPAISHLDLSLCPRLPDAALAALPAAPFVSAVDLSRSRGFGAAGLAALVAAFPNLTDLDLSNGLDLGDAAAAEVAKARRLQRLSLSRCKRITDMGLGCIAVGCPDLRELSLKWCIGVTHLGLDLLALKCNKLNILDLSYTMIVKKCFPAIMKLQNLQVLLLVGCNGIDDDALTSLDQECSKSLQVLDMSNSYNVTHVGVLSIVKAMPNLLELNLSYCSPVTPSMSSSFEMIHKLQKLKLDGCQFMDDGLKSIGKSCVSLRELSLSKCSGVTDTDLSFVVPRLKNLLKLDVTCCRKITDVSLAAITTSCPSLISLRMESCSLVSSKGLQLIGRRCTHLEELDLTDTDLDDEGLKALSGCSKLSSLKIGICLRITDEGLRHVSKSCPDLRDIDLYRSGAISDEGVTHIAQGCPMLESINMSYCTKLTDCSLRSLSKCIKLNTLEIRGCPMVSSAGLSEIATGCRLLSKLDIKKCFEINDMGMIFLSQFSHNLRQINLSYCSVTDIGLISLSSICGLQNMTIVHLAGVTPNGLIAALMVCGLRKVKLHEAFKSMVPSHMLKVVEARGCLFQWINKPYQVAVEPCDVWKQQSQDLLVQ(SEQ ID NO.:38)
In another preferred embodiment, the RAE1 protein is derived from maize and has the amino acid sequence shown in SEQ ID NO. 39.
MAMAAQQHRHHKRRRIALSPSPSPSLAPIPGAPTPPLDSLADELLFLVLDRVAQADPRALKSFALASRACHAAESRHRRTLRPLRADLLPAALARYPSATRLDLTLCARVPDAALASAAVSGSSALRAVDLSRSRGFGAAGVAALAAACPDLADLDLSNGVHLGDAAAAEVARARALRRLSLVRWKPLTDMGLGCVAVGCTELKDLSLKWCLGLTDLGIQLLALKCRKLTSLDLSYTMITKDSLPSIMKLPNLQELTLVGCIGIDDGALVSLERECSKSLQVLDMSQCQNITDVGVSSILKSVPNLLELDLSYCCPVTPSMVRNFQKLPKLQALKLEGCKFMANGLKAIGTSCVSLRELSLSKSSGVTDTELSFVVSRLKNLLKLDITCCRSITDVSLAAITSSCTSLISLRMESCSHVSSGALQLIGKHCSHLEELDLTDSDLDDEGLKALARCSELSSLKIGICLKISDEGLSHIGRSCPKLREIDLYRCGVISDDGIIQIAQGCPMLESINLSYCTEITDRSLISLSKCAKLNTLEIRGCPSVSSIGLSEIAMGCRLLSKLDIKKCFGINDVGMLYLSQFAHSLRQINLSYCSVTDVGLLSLSSISGLQNMTIVHLAGITPNGLTATLMVCGGLTKVKLHEAFRSMMPPHTIKNVEARGCVFQWIDKPFKVEVEPCDVWKQQSQDVLVR(SEQ ID NO.:39)
In another preferred embodiment, the RAE1 protein is derived from barley and has the amino acid sequence shown in SEQ ID NO. 40.
ISKDCLPAIMELPNLEVLALVGCVGIDDDALSGLENESSKSLRVLDMSTCRNVTHTGVSSVVKALPNLLELNLSYCCNVTASMGKCFQMLPKLQTLKLEGCKFMADGLKHIGISCVSLRELSLSKCSGVTDTDLSFVVSRLKNLLKLDITCNRNITDVSLAAITSSCHSLISLRIESCSHFSSEGLRLIGKRCCHLEELDITDSDLDDEGLKALSGCSKLSSLKIGICMRISDQGLIHIGKSCPELRDIDLYRSGGISDEGVTQIAQGCPMLESINLSYCTEITDVSLMSLSKCAKLNTLEIRGCPSISSAGLSEIAIGCRLLAKLDVKKCFAINDVGMFFLSQFSHSLRQINLSYCSVTDIGLLSLSSICGLQNMTIVHLAGITPNGLLAALMVSGGLTRVKLHAAFRSMMPPHMLKVVEARGCAFQWIDKPFKVEQERCDIWQQQSRDVLVR(SEQ ID NO.:40)
In another preferred embodiment, the RAE1 protein is derived from Brassica and has the amino acid sequence set forth in SEQ ID NO. 41.
MKKAKQIQHMISKPFDLLSEELVFIILDLVAQNPSDLKSFSLTCKWFYQVEARHRRSLKPLRAEYLPRILTRYRNTADLDLSFCPRVTDYALSVVGCLSGPTLRSVDLSRSFSFSAAGLLRLAVKCVSLVEIDLSNATEMRDAAAAVVAEAKSLERLKLGRCKKLTDMGIGCIAVGCRKLKRVSLKWCVGVGDLGVGLLAVKCKDIRSLDLSYLPITGKCLHDVLKLQHLEELLLQGCFGVDDDSLKSLTHHCNSLKNLDASSCQNLTQRGLTSLLSGAGCLERLDLAHSSSVISLDFASSLNKVSSALQSIRLDGCAVTCDGLKAIGTLCISLREVSLSKCVTVTDEGLSCLVMKLKDLRKLDITCCRKLTGVSITQVASSCPLLVSLKMESCSLVSRDAFWLIGHKCRLLEELDFTDNEIDDEGLKSISSCRSLSSLKLGICLNITDRGLSYIGMGCSNLRELDLYRSVGITDVGISSIAQGCCHLETINISYCKDITDKSLVSLSKCSMLQTFESRGCPHITCQGLAAIAVRCKRLSKLDLKKCPFINDSGLLTLAHFSQGLKQISVSETGVTDVGLVSLANIGCLQNIAAVNTRGLSPSGVAAALVGCGGLRKVKLHASLRSLLPSSLINHMEARGCSFLWKDYNNHNNSNTLQAELDPKYWKV(SEQ ID NO.:41)
In another preferred embodiment, the RAE1 protein is derived from soybean and has the amino acid sequence shown in SEQ ID NO. 42.
MSRIYSSNIETKQTNPSLLSPQLFIINLQNDQPNAKRRTMKKQKLSEPQNDTTNPFEVLSEELMFVILDFLQTTSLDKKSFSLTCKLFYSVEAKHRRLLRPLRAEHLPALAARYPNVTELDLSLCPRVGDGALGLVAGAYAATLRRMDLSRSRRFTATGLLSLGARCEHLVELDLSNATELRDAGVAAVARARNLRKLWLARCKMVTDMGIGCIAVGCRKLRLLCLKWCVGIGDLGVDLVAIKCKELTTLDLSYLPITEKCLPSIFKLQHLEDLVLEGCFGIDDDSLDVDLLKQGCKTLKRLDISGCQNISHVGLSKLTSISGGLEKLILADGSPVTLSLADGLNKLSMLQSIVLDGCPVTSEGLRAIGNLCISLRELSLSKCLGVTDEALSFLVSKHKDLRKLDITCCRKITDVSIASIANSCTGLTSLKMESCTLVPSEAFVLIGQKCHYLEELDLTDNEIDDEGLMSISSCSWLTSLKIGICLNITDRGLAYVGMRCSKLKELDLYRSTGVDDLGISAIAGGCPGLEMINTSYCTSITDRALIALSKCSNLETLEIRGCLLVTSIGLAAIAMNCRQLSRLDIKKCYNIDDSGMIALAHFSQNLRQINLSYSSVTDVGLLSLANISCLQSFTLLHLQGLVPGGLAAALLACGGLTKVKLHLSLRSLLPELLIRHVEARGCVFEWRDKEFQAELDPKCWKLQLEDVI(SEQ ID NO.:42)
As used herein, the terms "the present RAE1 gene", "RAE1 gene" are used interchangeably.
In the present invention, the gene of RAE1 includes genomic gene, cDNA sequence, mRNA sequence.
In the present invention, the RAE1 gene is derived from Arabidopsis thaliana, and the nucleic acid sequence is shown as SEQ ID NO. 36 (DNA sequence) or SEQ ID NO. 43 (mRNA sequence).
GGGTTTTTTCCAAAATCCTCAAAGAGAGACTATACTTCTCTCGGATCCTTCCTGATTTCAAAATTTCTTCAACCCACCAGCAGCAGCAGCAGATGAAGAAGGTTAAACAGATTCGTGTCTTAAAGCCTTTCGATCTTCTCTCGGAAGAGCTCGTCTTTATCATCCTTGACCTCATCTCTCCCAACCCTTCCGATCTCAAATCCTTCTCTCTTACTTGCAAATCTTTCTACCAGCTCGAGTCCAAGCACCGCGGATCCTTAAAACCTCTCCGCTCCGACTATCTCCCTCGCATCTTGACTCGTTACCGGAACACCACCGATCTCGATCTTACCTTCTGCCCGCGTGTCACTGACTACGCGCTTAGCGTCGTTGGCTGTCTCTCCGGACCTACGCTTCGCTCCCTCGACCTCTCGCGCTCTGGCTCCTTCTCCGCCGCGGGACTACTGCGATTGGCCCTCAAATGTGTCAATTTAGTCGAGATTGACCTGTCCAATGCGACGGAGATGAGAGACGCCGATGCTGCGGTGGTGGCGGAGGCGAGGAGTCTGGAGAGGCTGAAGCTGGGCAGATGCAAGATGCTGACGGACATGGGAATCGGATGTATAGCAGTTGGATGTAAGAAGCTCAATACGGTTAGCTTGAAATGGTGTGTTGGCGTCGGAGATTTAGGGGTTGGCTTGCTTGCCGTCAAATGCAAGGACATTCGCACCTTAGACCTCTCCTACTTGCCGGTAATTAACTTCTCTCTTCACATTTTCTTAATCCGCAGAGCCAAATTTCGATTAATATGGTAAGTTTGTCGAATTGGAATTATTTCTAAATTTGTTCTTCCTGAGACCTGTTGGTTGATTTTTCAAGTTAACAACAAGTTTTGGTTGAACTTTTGGTCTTTCAACATCTGTGGATCATTATCTATCTGAATTCGACACTTTAAGTGCCATATGTTTGGATTATATCTTCAGAGGAGCTGGAAATTCAAACCTATTCTTTTGTTCCGTATTCTACAGATCACAGGAAAGTGTTTACATGACATTCTGAAACTTCAACACCTTGAAGAACTTCTTCTAGAAGGGTGCTTTGGAGTAGATGATGACAGTCTTAAATCACTCAGACATGATTGCAAGTCATTGAAGGTAACCTCTATTCCAAGAAATTTGTTTGATTCTTATACATGAGATCACATAATAGTTTTGGAATCGACTTGACAGACAAACTTTTTAGTTGATATTTTAGAACAGAAAACTTGGTATTCTCAGAATATGGCTTGGAGAAGATGTTACGCTTCTTCTTGCTCGCTGCATTTATTCTGGAAACATTTAACTTTTATGAGAGAAGATGTATAAACAGGTTTCATGTTTTCTTAGACTCAGAAAGCTATTTATCATTGAAAGTCTAATGCTGGAAGACTATATAGTTGATTGTTTTCATCATCATCTTATCCATAGACATTTGAGATTTTACTTACCAAAACCTGTGTTCTGCAGAAGCTTGATGCATCCAGCTGCCAGAATTTAACTCATAGAGGTTTAACCTCACTTTTAAGCGGGGCAGGATATCTTCAGCGACTTGATCTATCACACTGTTCTTCTGTAAGTTCTCCGTTTTCTCATTATCATCAGAACACATGCTTTCCTTGCTTTGTTCTCTGGTTATGGCCTGTATGTAAGTATTTGCTAAATGGCTGAGTCAACTTAGTTTGGCACTAAAACTCTTTAGTAGTAGCAAAACCAGTAATTGCACATACCACCGAATAGACCATTTGTGTCATGGATCGATAGCAGTACACAGACCCACCAAGTTCTATGATGCCTTCTTATATGAGGTTTATTTACTGATTTAGGTGATATCATTGGATTTTGCGAGTAGCTTAAAGAAGGTTTCAGCATTACAGTCGATCAGGTTGGATGGTTGTTCTGTTACGCCTGATGGTTTGAAGGCGATAGGCACATTGTGCAATTCCCTGAAAGAGGTTAGCCTAAGCAAATGCGTGAGCGTAACTGATGAAGGTCTTTCTTCTCTAGTAATGAAACTGAAAGACCTCAGAAAACTTGACATCACATGTTGCCGGAAACTAAGTAGAGTTTCAATCACCCAAATCGCCAATTCGTGTCCTTTACTAGTCTCTTTGAAGATGGAGTCTTGTTCTCTTGTTTCCAGAGAAGCCTTTTGGTTGATCGGACAAAAGTGTCGGCTACTTGAAGAGCTTGACTTAACTGACAACGAGATTGATGATGAAGGTTCTTTATTTTCTTCAGATCAATTCTTAGAACCGTGGTTTTTGGATTATACCTTATCTTGTTAGTCTTTCCTCGTATGATGCAGGACTGAAATCCATATCTAGTTGTCTGAGTCTTTCCTCGTTAAAGCTGGGAATTTGTCTTAACATAACAGACAAAGGGCTCTCGTACATCGGGATGGGCTGTTCAAATCTCCGTGAACTTGATCTCTATAGGTATCTCGCGTAGAAATTTATTTTTCAGTCGGGTCAATAATAAAATTCTCCGTTCTCAATCTAGTCTTTATCTACTCCAGGTCAGTGGGAATAACAGACGTAGGCATCTCCACAATTGCCCAAGGCTGCATTCATCTGGAAACAATAAACATTTCATACTGCCAAGACATAACAGACAAGTCCCTGGTTTCATTGTCCAAATGCTCGTTGTTACAAACATTCGAGAGCAGGGGATGTCCTAACATCACGTCCCAAGGACTTGCAGCCATTGCTGTTCGGTGCAAGCGACTCGCCAAGGTTGACTTGAAGAAGTGCCCATCCATCAATGATGCGGGGTTGCTCGCTCTGGCTCACTTCTCTCAGAATCTCAAACAGGTAATAAACCGTTGAATCCTTCACTGACAGCTGAAAAAACTACAATATACTGGTTGAATCTGTTATCTGATTGCGATCCCCATGTTGCAGATAAACGTGTCAGACACAGCTGTGACTGAAGTGGGACTTCTCTCCCTAGCCAACATAGGGTGTTTACAGAACATAGCGGTTGTGAACTCGAGTGGTTTAAGACCGAGCGGAGTAGCAGCAGCATTGCTGGGGTGTGGAGGATTAAGGAAAGCGAAACTACATGCGTCCCTAAGATCACTGCTTCCTCTTTCTCTAATCCACCACTTGGAAGCTCGTGGTTGTGCGTTCCTCTGGAAAGACAATACCCTTCAGGTGAATATATATACAAGTATATGTAGATTACTACTTACTACACTAAATAGCCGCAAAAGGTTGGTGTAGCTTTTTATGATTACAAATGAAAATCTTAGAGCGAAGGAGTCTGACATATGGAAATGTAAATGATGTGGGAACAGGCGGAGTTAGATCCCAAGTACTGGAAGCAACAGCTGGAAGAGATGGCGCCTTAAATTAAAAGTGAGAAGAAACATTCTGAAATGGGAAAGAGGAGTTCCTATGGGAGCCAGCAGACAGGCCCTGTTTTGGGCCCAGTGTGGATTAGCTGGAACAATTTTGGTCTCTTTTGTGTTGTTGTTGACGGCGCGTGTTCTAACAACCCCACACAACCTTACATTATAAGTCTAGTCACATGGTGGGTGACATGGTACCGTTGTATATGTAGTTTTTGTTTCTTTTTTTTTTTTTTTTGGTTGGGGAGCCGAAATTAACGGACGTAACAGAGTCACAAGGGGATGCCTTCTCTGTGCCTTTTTGACTTTTTCCTCCTTTCTTTTTTTTCTGTAATCATATGAGTTTTATGTAATTTAATGCCTCATCAACTTGCCTGGATAGGGCCGGCCTTTCC(SEQ ID NO.:36)
GGGTTTTTTCCAAAATCCTCAAAGAGAGACTATACTTCTCTCGGATCCTTCCTGATTTCAAAATTTCTTCAACCCACCAGCAGCAGCAGCAGATGAAGAAGGTTAAACAGATTCGTGTCTTAAAGCCTTTCGATCTTCTCTCGGAAGAGCTCGTCTTTATCATCCTTGACCTCATCTCTCCCAACCCTTCCGATCTCAAATCCTTCTCTCTTACTTGCAAATCTTTCTACCAGCTCGAGTCCAAGCACCGCGGATCCTTAAAACCTCTCCGCTCCGACTATCTCCCTCGCATCTTGACTCGTTACCGGAACACCACCGATCTCGATCTTACCTTCTGCCCGCGTGTCACTGACTACGCGCTTAGCGTCGTTGGCTGTCTCTCCGGACCTACGCTTCGCTCCCTCGACCTCTCGCGCTCTGGCTCCTTCTCCGCCGCGGGACTACTGCGATTGGCCCTCAAATGTGTCAATTTAGTCGAGATTGACCTGTCCAATGCGACGGAGATGAGAGACGCCGATGCTGCGGTGGTGGCGGAGGCGAGGAGTCTGGAGAGGCTGAAGCTGGGCAGATGCAAGATGCTGACGGACATGGGAATCGGATGTATAGCAGTTGGATGTAAGAAGCTCAATACGGTTAGCTTGAAATGGTGTGTTGGCGTCGGAGATTTAGGGGTTGGCTTGCTTGCCGTCAAATGCAAGGACATTCGCACCTTAGACCTCTCCTACTTGCCGATCACAGGAAAGTGTTTACATGACATTCTGAAACTTCAACACCTTGAAGAACTTCTTCTAGAAGGGTGCTTTGGAGTAGATGATGACAGTCTTAAATCACTCAGACATGATTGCAAGTCATTGAAGAAGCTTGATGCATCCAGCTGCCAGAATTTAACTCATAGAGGTTTAACCTCACTTTTAAGCGGGGCAGGATATCTTCAGCGACTTGATCTATCACACTGTTCTTCTGTGATATCATTGGATTTTGCGAGTAGCTTAAAGAAGGTTTCAGCATTACAGTCGATCAGGTTGGATGGTTGTTCTGTTACGCCTGATGGTTTGAAGGCGATAGGCACATTGTGCAATTCCCTGAAAGAGGTTAGCCTAAGCAAATGCGTGAGCGTAACTGATGAAGGTCTTTCTTCTCTAGTAATGAAACTGAAAGACCTCAGAAAACTTGACATCACATGTTGCCGGAAACTAAGTAGAGTTTCAATCACCCAAATCGCCAATTCGTGTCCTTTACTAGTCTCTTTGAAGATGGAGTCTTGTTCTCTTGTTTCCAGAGAAGCCTTTTGGTTGATCGGACAAAAGTGTCGGCTACTTGAAGAGCTTGACTTAACTGACAACGAGATTGATGATGAAGGACTGAAATCCATATCTAGTTGTCTGAGTCTTTCCTCGTTAAAGCTGGGAATTTGTCTTAACATAACAGACAAAGGGCTCTCGTACATCGGGATGGGCTGTTCAAATCTCCGTGAACTTGATCTCTATAGGTCAGTGGGAATAACAGACGTAGGCATCTCCACAATTGCCCAAGGCTGCATTCATCTGGAAACAATAAACATTTCATACTGCCAAGACATAACAGACAAGTCCCTGGTTTCATTGTCCAAATGCTCGTTGTTACAAACATTCGAGAGCAGGGGATGTCCTAACATCACGTCCCAAGGACTTGCAGCCATTGCTGTTCGGTGCAAGCGACTCGCCAAGGTTGACTTGAAGAAGTGCCCATCCATCAATGATGCGGGGTTGCTCGCTCTGGCTCACTTCTCTCAGAATCTCAAACAGATAAACGTGTCAGACACAGCTGTGACTGAAGTGGGACTTCTCTCCCTAGCCAACATAGGGTGTTTACAGAACATAGCGGTTGTGAACTCGAGTGGTTTAAGACCGAGCGGAGTAGCAGCAGCATTGCTGGGGTGTGGAGGATTAAGGAAAGCGAAACTACATGCGTCCCTAAGATCACTGCTTCCTCTTTCTCTAATCCACCACTTGGAAGCTCGTGGTTGTGCGTTCCTCTGGAAAGACAATACCCTTCAGGCGGAGTTAGATCCCAAGTACTGGAAGCAACAGCTGGAAGAGATGGCGCCTTAAATTAAAAGTGAGAAGAAACATTCTGAAATGGGAAAGAGGAGTTCCTATGGGAGCCAGCAGACAGGCCCTGTTTTGGGCCCAGTGTGGATTAGCTGGAACAATTTTGGTCTCTTTTGTGTTGTTGTTGACGGCGCGTGTTCTAACAACCCCACACAACCTTACATTATAAGTCTAGTCACATGGTGGGTGACATGGTACCGTTGTATATGTAGTTTTTGTTTCTTTTTTTTTTTTTTTTGGTTGGGGAGCCGAAATTAACGGACGTAACAGAGTCACAAGGGGATGCCTTCTCTGTGCCTTTTTGACTTTTTCCTCCTTTCTTTTTTTTCTGTAATCATATGAGTTTTATGTAATTTAATGCCTCATCAACTTGCCTGGATAGGGCCGGCCTTTCC(SEQ ID NO.:43)
In the invention, the RAE1 gene is derived from rice (OsRAE1.1, LOC_Os11g 01780), and the nucleic acid sequence is shown as SEQ ID NO. 44.
ATTATTACTACCTTCCCTTCCCTTCTTCTTGTTGCAGTCAACCTCGCCATGGCCTCGCCTATGCCAACGCCTACCCACCGCGTCAAGCGCCGCCGCCTCGACCTCTCCCCGCCCCCGCACCTCAACGACCTCGCCGACGAGCTCCTCTTCCTCATCCTCGACCGTGCCGCCGCCCATGACCCCCGCGCCCTCAAGTCCTTCTCCCTCGTCTCCCGCGCCTGCCACGCCGCCGAGTCGCGCCACCGCCGCGTACTCCGCCCCTTCCGCCCCGACCTCCTCCCCGCCGCGCTCGCCCGCTACCCCGCCCTCTCCCGCCTCGATCTCTCCCTCTGCCCGCGCCTCCCCGACGCCGCCCTCGCCGCGCTCCCCGCCGCGCCGTCCGTCTCCGCCGTCGACCTCTCCCGCTCCCGGGGGTTCGGCGCCGCCGGCCTCGCCGCGCTCGTCGCCGCGTGCCCCAATCTCACGGACCTCGACCTCTCCAATGGCCTCGACCTCGGGGATGCCGCGGCGGCGGAGGTGGCCAAGGCGCGCCGCCTGCAGAGGCTCTCGCTGTCGCGATGCAAGCGCATCACTGACATGGGGCTCGGATGCATCGCCGTCGGATGCCCCGACCTGCGCGAGCTCTCGCTCAAGTGGTGCATCGGGGTCACTCATCTCGGACTAGACCTCCTCGCCCTCAAGTGCAACAAGCTCAACATCCTGGATCTCTCCTACACCATGATTGTAAAAAAATGCTTTCCAGCCATCATGAAGCTACAAAGTCTACAAGTGTTACTACTGGTGGGATGTAATGGAATTGATGATGATGCCCTTACTAGTCTTGATCAAGAATGCAGCAAATCACTACAGGTTCTTGATATGTCAAATTATTACAATGTCACTCATGTCGGTGTTCTGTCCATTGTGAAGGCAATGCCGAATCTGTTGGAACTCAATCTATCATACTGCTCTCCTGTTACTCCTTCTATGTCAAGCAGCTTCGAAATGATTCATAAATTGCAGACACTGAAGCTGGATGGTTGCCAATTCATGGATGATGGATTAAAATCCATTGGGAAATCCTGTGTTTCTTTGAGGGAGTTAAGTCTGAGCAAATGTTCTGGAGTGACAGACACAGATCTTTCTTTTGTCGTGCCAAGACTGAAAAATTTGCTGAAGCTGGATGTTACTTGTTGTCGCAAAATCACTGATGTTTCATTAGCTGCCATCACAACCTCATGCCCCTCCCTCATCTCTCTGAGAATGGAGTCCTGTAGCCTTGTTTCCAGCAAAGGACTACAGCTGATTGGAAGGCGCTGCACTCACTTGGAGGAATTGGATCTTACTGACACTGATTTGGATGATGAAGGTTTGAAAGCTCTCTCTGGATGCAGCAAACTTTCAAGCCTAAAAATTGGCATATGCTTGAGGATAACTGATGAGGGCCTTAGACACGTTAGCAAGTCCTGTCCAGATCTCCGAGATATCGATTTGTACAGGTCTGGGGCGATCAGTGATGAAGGGGTTACTCATATAGCTCAAGGATGCCCAATGTTAGAGTCTATCAATTTGTCCTACTGCACAAAATTAACAGACTGTTCACTGAGATCACTTTCAAAATGCATAAAGCTGAACACATTGGAGATTCGTGGCTGCCCCATGGTTTCATCTGCTGGTCTCTCGGAAATTGCCACAGGATGCAGGCTACTTTCTAAGCTTGATATCAAGAAATGCTTTGAGATCAATGACATGGGAATGATTTTCCTTTCCCAATTCTCTCACAACCTCCGGCAGATAAACTTGTCATATTGTTCGGTCACCGACATTGGGCTTATATCCCTTTCAAGCATATGTGGCTTGCAGAACATGACCATTGTGCATTTAGCGGGTGTTACGCCTAATGGACTGATAGCTGCTCTTATGGTCTGTGGTTTGAGAAAAGTGAAGCTTCATGAAGCATTCAAATCCATGGTGCCATCACATATGCTCAAAGTTGTTGAAGCCCGTGGTTGTCTTTTCCAGTGGATTAATAAACCCTACCAGGTTGCGGTAGAACCGTGTGATGTATGGAAGCAGCAGTCGCAGGATTTGCTTGTACAGTGAAATGTTTCAAAGATAAACGTTGTGGAAACTGGGGCGTGTTTTGTGGTGTTGAATTTATCTAGAGCAATATCTCCAGTCCTAGAGAATGAGCTCCAAAAGTTTTGTGCCATAACTGGCTGAATAGTGTATTGAATTGCTGACGGTAGTATTGTCAGAACAACATACTAGTACTGGTATTTTGCTTGTATGCCAGTGGAGGCGAGGGAGGTTATATTCTTGCTGTGTTGTATATAGCGCAGGTAGGAATCAACAATCAAAGAGAGATCATTGGGGTAAAGCATGTTGTAAGTAGTGCGGAGTATGTGATATGCCTTGCTGTGCCTTTTTGATGCACAATTTGATTAATGGAATGGAACATTGCATTCTCACT(SEQ ID NO.:44)
In the invention, the RAE1 gene is derived from rice (OsRAE1.2, LOC_Os12g 01760), and the nucleic acid sequence is shown as SEQ ID NO. 45.
GCCAAACGCCCACCATTATTACTACCTTCCCTTCCCTTCTCTCGTTTCAGTCAACTTCGCCATGGCCTCGCCTGCGCCCACCCACCACGCCAAGCGCCGCCGCCTCGCCCTGCCCCCGCCCCCGCCCCCGCACCTCAACGACCTCGCCGACGAGCTCCTCTTCCTCATCCTCGACCGTGCCGCCGCCCATGACCCACGCGCCCTCAAGTCCTTCTCCCTCGTCTCCCGCGCCTGCCACGCCGCCGAGTCGCGCCACCGCCGCGTCCTCCGCCCCTTCCGCCCCGACCTCCTCCCCGCCGCGCTCGCCCGCTACCCCGCCATCTCCCACCTCGATCTCTCCCTCTGCCCCCGCCTCCCCGACGCCGCCCTCGCCGCGCTCCCCGCCGCGCCGTTCGTCTCCGCCGTCGACCTCTCCCGCTCCCGCGGGTTCGGCGCCGCCGGCCTCGCCGCGCTCGTCGCCGCGTTCCCCAATCTCACGGACCTCGACCTCTCCAATGGCCTCGACCTCGGGGATGCCGCGGCGGCGGAGGTGGCCAAGGCGCGCCGCCTCCAGAGGCTCTCGCTGTCGCGATGCAAGCGCATCACTGACATGGGGCTCGGATGCATCGCCGTCGGATGCCCCGACCTGCGCGAGCTCTCGCTCAAGTGGTGCATCGGGGTCACTCATCTGGGACTAGACCTCCTTGCCCTCAAGTGCAACAAGCTCAACATCCTGGATCTCTCCTACACCATGATAGTAAAAAAATGCTTTCCAGCCATCATGAAGCTACAAAATCTACAAGTGTTACTACTGGTGGGATGTAATGGAATTGATGATGATGCCCTTACTAGTCTTGATCAAGAATGCAGCAAATCACTACAGGTTCTTGATATGTCAAACTCTTACAATGTCACTCATGTCGGTGTTCTGTCCATTGTGAAGGCAATGCCGAATCTGTTGGAACTCAATCTATCATACTGCTCTCCTGTTACTCCTTCTATGTCAAGCAGCTTCGAAATGATTCATAAATTGCAGAAACTGAAGCTGGATGGTTGCCAATTCATGGATGATGGATTAAAATCCATTGGGAAATCCTGTGTTTCTTTGAGGGAGTTAAGTCTGAGCAAATGTTCTGGAGTGACAGACACAGACCTTTCTTTTGTCGTGCCAAGACTGAAAAATTTGCTGAAGCTGGATGTTACTTGTTGTCGCAAAATCACTGATGTTTCATTAGCTGCCATCACAACCTCATGCCCATCCCTCATCTCTCTGAGAATGGAGTCCTGTAGCCTTGTTTCCAGCAAAGGACTACAGCTGATTGGAAGGCGCTGCACTCACTTGGAGGAATTGGATCTTACTGACACTGATTTGGATGATGAAGGTTTGAAAGCTCTCTCTGGATGCAGCAAACTTTCAAGCCTAAAAATTGGCATATGCTTGAGGATAACTGATGAGGGCCTTAGACACGTTAGCAAGTCCTGTCCAGATCTCCGAGATATCGATTTGTACAGGTCTGGGGCGATCAGTGATGAAGGGGTTACTCATATAGCTCAAGGATGCCCAATGTTAGAGTCTATCAATATGTCCTACTGCACAAAATTAACAGACTGTTCACTGAGATCACTTTCAAAATGCATAAAGCTGAACACATTGGAGATTCGTGGCTGCCCCATGGTTTCATCTGCTGGTCTCTCGGAAATTGCAACAGGATGCAGGCTACTTTCTAAGCTTGATATCAAGAAATGCTTTGAGATCAATGACATGGGAATGATTTTCCTTTCCCAATTCTCTCACAACCTCCGGCAGATAAACTTGTCATATTGTTCGGTCACCGACATTGGGCTTATATCCCTTTCAAGCATATGTGGCTTGCAGAACATGACCATTGTGCATTTAGCGGGTGTTACGCCTAATGGACTGATAGCTGCTCTTATGGTCTGTGGTTTGAGAAAAGTGAAGCTTCATGAAGCATTCAAATCCATGGTGCCATCACATATGCTCAAAGTTGTTGAAGCCCGTGGTTGTCTTTTCCAGTGGATTAATAAACCCTACCAGGTTGCGGTAGAACCGTGTGACGTATGGAAGCAGCAGTCGCAGGATTTGCTTGTACAGTGAAATGTTTCAAAGATAAACGTTGTGGAAACTGGGGCGTGTTTTGTGGTGTTGAATTTATCTTAGAGCAATATCTCCAGTCCTAGAGAATGAGCTCCAAAAGTTTTGTGCCATAACTGGCTGAATAGTGTATTGAATTACTGACGGTAGTATTGTCAGAACAACATACTAGTACTGGTATTTTGCTTGTATGCCAGTGGAGGCGAGGGAGGTTATATTCTTGATGTGTTGTATATAGCGCAGGTAGGAATCAACAATCAAAGAGAGATTATTGGGGTAAAGCATGTAGTAAGTGTGGAGTATGTGATATGCCTTGTTGTGCCTTTTTGATGCACAATTTGATTAATGGAATGGAACATTGCATTCGCACT(SEQ ID NO.:45)
In the invention, the RAE1 gene is derived from corn (XM_ 008677340.2 predicts: zea mays F-box/LRR-repeat protein 3 (LOC 103651656)), and the nucleic acid sequence is shown as SEQ ID NO. 46.
CTCCATCTCCATCGCCGGCCTCCATTTCTTGCCTCCCGTGCCAGCCAGTCACTCTCGTCCGCCGCAGATCGAGAAGCCACCACCACCACCACCACCACCGCATGGCCATGGCAGCCCAGCAGCACCGGCACCACAAGCGCCGCCGCATCGCCCTCTCCCCCTCCCCGTCCCCGTCCCTCGCGCCCATCCCCGGCGCCCCCACGCCGCCGCTCGACTCGCTGGCCGACGAGCTCCTCTTCCTGGTCCTGGACCGCGTGGCCCAGGCCGACCCGCGGGCGCTCAAGTCCTTCGCGCTGGCCTCCCGCGCCTGCCACGCCGCGGAGTCACGGCACCGCCGGACGCTCCGCCCGCTCCGCGCGGACCTCCTGCCCGCCGCGCTGGCGCGGTACCCGTCCGCGACCCGCCTCGACCTCACCCTCTGCGCGCGCGTCCCCGACGCCGCCCTCGCCTCCGCCGCCGTCTCCGGCTCCTCCGCCCTCCGCGCCGTCGACCTCTCCCGCTCCCGCGGGTTCGGCGCCGCGGGCGTCGCCGCGCTCGCCGCCGCGTGCCCGGACCTCGCCGACCTCGACCTCTCCAATGGGGTCCACCTCGGGGACGCCGCGGCGGCCGAGGTAGCGCGGGCCAGGGCGCTGCGGAGGCTCTCGCTGGTCCGCTGGAAGCCGCTCACCGACATGGGCCTCGGATGCGTCGCCGTCGGGTGCACGGAGCTGAAGGACCTCTCGCTCAAGTGGTGCCTTGGACTCACGGATCTGGGGATCCAGCTCCTCGCCCTCAAGTGCAGGAAGCTCACCAGCCTGGATCTCTCCTACACCATGATCACAAAGGATAGCTTGCCTTCTATCATGAAGCTACCCAATCTTCAAGAGCTGACACTGGTGGGGTGTATTGGAATCGATGATGGTGCTCTTGTTAGTCTTGAGAGAGAATGCAGTAAATCACTACAGGTGCTTGATATGTCTCAGTGTCAGAATATCACCGATGTAGGAGTTTCATCCATCCTGAAGTCGGTACCCAATCTATTGGAACTGGATCTTTCATACTGCTGTCCTGTTACTCCTTCTATGGTGAGAAACTTCCAGAAGCTTCCTAAACTGCAGGCCCTGAAGCTGGAAGGCTGCAAATTCATGGCCAATGGACTAAAAGCCATTGGGACCTCTTGTGTTTCTTTAAGGGAGCTAAGTCTTAGCAAGTCATCTGGAGTGACAGATACAGAACTCTCTTTTGTTGTGTCAAGGCTAAAGAACCTGCTGAAGCTGGACATTACCTGTTGTCGCAGTATTACTGATGTTTCACTAGCGGCCATAACTAGTTCGTGCACTTCCCTCATCTCTCTGAGGATGGAGTCTTGTAGCCATGTTTCCAGTGGAGCACTCCAACTGATTGGGAAGCACTGTTCTCACTTGGAAGAGTTGGACCTTACTGACAGTGATTTGGATGATGAAGGATTGAAAGCTCTTGCCAGATGTAGCGAACTTTCGAGCCTAAAAATTGGCATTTGCTTGAAGATAAGTGATGAAGGTCTTAGCCACATTGGAAGGTCTTGCCCAAAACTCCGCGAGATTGATTTGTACAGGTGTGGAGTTATTAGCGATGATGGAATTATTCAAATTGCGCAGGGTTGTCCGATGCTAGAGTCTATCAACCTATCATACTGCACAGAAATAACAGACCGTTCACTGATTTCACTCTCAAAATGCGCAAAGCTGAATACTTTGGAGATCCGTGGCTGCCCCAGTGTTTCATCGATTGGGCTCTCAGAAATAGCGATGGGGTGCAGGCTGCTTTCCAAGCTTGATATTAAGAAATGCTTCGGGATTAATGATGTTGGAATGCTTTACCTTTCCCAGTTCGCTCATAGCCTCCGTCAGATAAACTTGTCATACTGTTCAGTCACCGATGTTGGGCTCCTTTCCCTTTCTAGCATATCCGGCCTGCAGAACATGACCATCGTCCATTTGGCGGGTATAACACCCAATGGCTTGACAGCAACTCTTATGGTTTGCGGTGGGTTGACGAAAGTGAAGCTTCATGAAGCATTCAGATCCATGATGCCTCCTCATACGATAAAAAATGTTGAGGCACGTGGCTGTGTTTTCCAGTGGATCGATAAACCGTTCAAGGTTGAGGTGGAGCCTTGTGATGTATGGAAGCAACAGTCACAAGATGTGCTTGTGCGATGAGAATACAGGAGACCTCGAGCGTAGCCGCTATCGTGGAAATCGTGGCATGCATCGTATGGATGGATGAGTTGTTGTTGGCTGGCAATGGCATCCAGAAGTGAAGTCTGATGGGGGGAGCTCCAAACTCAGCGTTGTAATCAGTGCGATTCGGCACAAAGATACCAGCATTTGAGCAGAGGTGTATGTATGTCTTGATGTTGTTTTATACTGCTATAGATGTGTAGATCCCATTGTTTGGTGAGGTGATCATTTGCGAGGAAGTTGACTATTAGCATGTATTAAGATAAAAAGGAAAGAGATGAGAAAATGTTTTTGAAATTTAGTACGATATGCCTTGCAGTGCCTGTATCTGTTGCATTCATATTTGTGATCAGCTGGAATGAAGTGGCTTGCATTCATGTTTTA(SEQ ID NO.:46)
In the present invention, the RAE1 gene is derived from barley (AK372025.1 Hordeum vulgare subsp. Vulgare mRNA, part of CDS, clone: NIASHv2145B 10) of the predicted protein, and the nucleic acid sequence is shown in SEQ ID NO. 47.
GAGGTTGGCCGTGGCCAGCCATTGCTACTCCTCCCTCCCTTCTTGGTGTCGCCGCAGCGGACAACCAACTGCCAACTTGCCTTGCCCGTCATGGCCATGGCGACCCACAGCCACCTCCCCAAGCGCCGACGCGTATGCCCTGCCGCGGGCGCGCCGATCGACGAGCTGCCCGACGAGCTCCTCTTCTTGGTCCTGGACCGGGTGGCGGCCGCCGATCCGCGCGCGCTCAAGTCCTTCGCGCTGGCCTCCCGCGCCTGCCACGCCGCCGAGTCGCGCCACCGCCGCGTGCTCCGCCCGTACCGCGCCGACCTACTCCGCGCCGCGCTTGCCCGCTACCCCACCGCCGCCCGCCTCGACCTCACCCTCTGCGCGCGCGTGCCCGGCGCGGCCCTCTCCTCCGCGCCCGTGCCTTCCCTCCGCGCCGTCGACCTCTCCCGCTCCCGCGGCTTCGGGGCGCCCGGCCTCGCCGCGCTCGTCGCCGCCTGCCCCGCCCTGGCCGACCTCGACCTCTCCAATGGGGTCGACCTCGGGGACGCGGCGGCCGCGGAGCTGGCGCGGGCGCGGGGCCTGCAGAGGCTCTGCCTCTCGCGCTGCAAGCCCATCACGGACATGGGCCTCGGCTGCATCGCCGTCGGCTGCCCAGACCTGCGGGACCTCACGCTCAACTGGTGCCTCGGGATCACGGATTTGGGGATCCAGCTCCTCGCCCTCAAGTGCAACAAACTCAGGAACCTGCATCTTTCCTACACCATGGTTAGATCTCCAAAGACTGCCTTCCAGCCATCATGGAGCTACCCAATCTTGAGGTGTTGGCACTGGTGGGATGTGTTGGAATAGATGATGATGCCCTTAGTGGTCTTGAGAATGAAAGCAGCAAATCACTACGGGTTCTCGATATGTCTACCTGTCGAAATGTCACTCATACGGGAGTTTCATCAGTTGTGAAGGCACTGCCAAATCTCTTGGAGTTGAATCTGTCCTACTGCTGTAATGTTACTGCATCTATGGGAAAATGCTTCCAAATGCTTCCTAAATTGCAGACCTTGAAATTGGAAGGCTGCAAGTTCATGGCTGATGGACTAAAACACATTGGAATTTCTTGTGTCTCTTTAAGAGAGTTGAGCCTGAGCAAGTGCTCAGGAGTGACAGATACTGATCTGTCTTTCGTTGTGTCAAGACTAAAGAATTTGCTGAAGCTGGACATTACTTGCAATCGCAATATCACTGATGTTTCGTTAGCTGCCATCACTAGCTCATGCCATTCCCTCATCTCTCTAAGAATAGAGTCCTGTAGCCATTTTTCTAGTGAAGGGCTCCGACTTATTGGGAAGCGATGTTGCCATTTGGAAGAGTTGGATATCACCGACAGTGATTTGGACGATGAAGGTTTGAAAGCTTTGTCTGGATGCAGCAAACTGTCAAGCTTAAAAATTGGAATATGCATGAGGATAAGTGACCAAGGCCTTATCCACATTGGGAAGTCTTGTCCAGAACTCCGAGATATTGATTTGTATAGGTCTGGGGGTATTAGTGATGAGGGGGTTACTCAAATTGCCCAAGGTTGTCCAATGCTAGAGTCTATCAACCTGTCGTACTGTACAGAAATAACAGATGTCTCGTTGATGTCGCTCTCAAAATGTGCAAAGCTAAACACACTGGAGATCCGTGGTTGCCCCAGTATTTCATCTGCTGGGCTCTCAGAAATAGCAATCGGATGCAGGCTACTTGCCAAGCTTGATGTCAAGAAGTGCTTTGCGATCAATGATGTGGGGATGTTTTTTCTTTCCCAGTTCTCTCATAGCCTCCGTCAGATAAACTTGTCATACTGTTCGGTCACCGATATTGGGCTTCTGTCCCTCTCTAGCATATGCGGGCTTCAGAACATGACGATTGTACACTTGGCGGGTATTACGCCTAATGGCTTGCTGGCTGCTCTGATGGTCTCTGGTGGTTTGACAAGGGTGAAGCTTCATGCAGCGTTCAGATCTATGATGCCCCCGCATATGCTCAAAGTCGTTGAGGCTCGCGGCTGTGCTTTCCAGTGGATTGATAAACCATTCAAGGTCGAGCAAGAACGATGCGACATATGGCAACAACAGTCTCGAGACGTGCTTGTACGATGAGAAATGTTTGACGTCGTCGTCTTGGCTGTCAGCCTACAGGATATGGTGTAGAACGAGGCTTTGCACCAAGGTGGACTTGTGTACAGAACAGACATTATCGGAACAATGTTGTATACTCATGTTTCCTTCAGTGCACTAGGAGGTTCTTTTGTTGTGCCGTATCCTGTATATAGAGCAGCAGGAGATGGATGAAATCATAGAGGAATTGCTGGAGGTCGACGTGTACTTAATAGAAACTCACCGATCAATGTGAGAGTGTGCACAGTTCTATTACCTGTCCAATGCGCTCTTGTTTCGAATGAGTAGTAGTTTAGCCTGTGTTTCTCCTGGTGTCC(SEQ ID NO.:47)
In the present invention, the RAE1 gene is derived from Brassica (XM_ 009132381.2. Sup. Predicts. Brassica rapa F-box/LRR-repeat protein 3 (LOC 103855400)), and the nucleic acid sequence is shown in SEQ ID NO. 48.
CTGTCCTTTTTTATTCTCTGTTTCCCATAGAAACTGTGAATCTGTGGGTTTTTAGCAAATCCTCATCGAAACTGTTTCTCCATTTGATAAAAAAAAAAATCAGTAAGTAGTTGGGTTCGAATGAAGAAGGCGAAGCAGATTCAACACATGATCTCAAAGCCTTTCGATCTTCTGTCGGAGGAGCTGGTCTTCATAATCCTAGACCTCGTCGCTCAGAACCCTTCCGATCTCAAATCCTTCTCTCTTACCTGCAAATGGTTCTACCAAGTGGAGGCCAGGCACCGGAGATCCCTGAAACCTCTCAGAGCGGAGTATCTTCCTCGAATCCTGACAAGGTACCGGAATACCGCCGATCTCGATCTTAGCTTCTGCCCGCGCGTCACGGACTACGCGCTTAGCGTGGTCGGGTGCCTCTCCGGACCGACGCTGAGGTCAGTAGACTTATCGAGGTCATTCTCCTTCTCGGCGGCGGGGCTGCTGAGACTGGCCGTGAAATGTGTGAGTCTGGTGGAGATAGACCTGTCGAACGCGACGGAGATGAGGGACGCGGCAGCGGCGGTGGTGGCGGAGGCGAAGAGCCTGGAGAGGCTGAAGCTGGGGAGATGCAAGAAGCTGACGGACATGGGAATAGGATGCATAGCGGTGGGATGTAGGAAGCTGAAGAGGGTGAGCTTGAAGTGGTGTGTAGGCGTCGGAGATTTAGGAGTCGGTCTGCTTGCCGTCAAATGCAAGGACATTCGCTCCTTAGACCTCTCCTACTTGCCGATCACAGGCAAGTGTTTGCATGATGTTCTCAAACTTCAACACCTTGAAGAACTTCTTCTACAAGGTTGCTTCGGAGTCGATGATGACTCTCTTAAATCACTCACTCATCATTGCAACTCCTTAAAGAACCTGGATGCATCGAGCTGCCAGAATTTAACTCAGAGAGGTTTAACCTCACTTTTAAGCGGGGCCGGATGTCTTGAGCGCCTTGATCTAGCACACTCTTCTTCTGTGATCTCATTGGATTTTGCAAGTAGCCTGAACAAGGTTTCTTCAGCATTACAGTCGATCAGGTTGGATGGTTGTGCGGTAACTTGTGATGGGTTGAAGGCGATAGGGACACTGTGCATTTCCCTCAGAGAGGTTAGCCTAAGCAAATGTGTCACCGTAACTGATGAAGGTCTCTCTTGTCTTGTTATGAAACTCAAAGACCTTAGAAAACTTGACATCACCTGTTGCCGGAAACTAACTGGAGTTTCAATCACCCAAGTCGCCAGTTCTTGTCCCTTACTAGTCTCCTTGAAGATGGAGTCTTGTTCTCTTGTTTCCAGAGATGCCTTTTGGTTGATCGGACACAAGTGTCGCCTACTTGAAGAGCTTGACTTTACTGACAATGAGATTGATGATGAAGGACTAAAATCCATATCCAGTTGTCGTAGTCTTTCCTCGTTGAAGTTGGGAATATGTCTGAATATAACAGACAGAGGACTCTCGTACATTGGTATGGGCTGCTCAAATCTCCGTGAACTTGATCTCTACAGGTCGGTGGGAATAACAGACGTAGGAATCTCATCGATCGCTCAAGGCTGCTGTCATCTCGAAACAATAAACATATCATACTGCAAAGACATCACAGACAAGTCGTTGGTGTCATTGTCAAAATGCTCAATGTTACAAACATTCGAGAGCCGGGGATGTCCACACATTACTTGCCAAGGACTTGCCGCCATTGCTGTTCGATGTAAGCGGCTCAGCAAGCTCGACTTGAAAAAGTGTCCTTTCATCAATGACTCCGGTTTGCTCACTCTAGCTCACTTTTCACAGGGCCTCAAACAGATAAGCGTGTCCGAAACGGGGGTGACAGACGTGGGACTTGTGTCGCTAGCAAACATAGGGTGTTTGCAGAACATAGCGGCAGTGAACACAAGGGGTTTAAGCCCGAGTGGAGTAGCAGCAGCGTTGGTTGGGTGTGGAGGTTTAAGGAAAGTGAAACTCCACGCTTCCCTCAGATCACTACTTCCTTCGTCTCTTATAAACCACATGGAAGCTCGTGGCTGCTCCTTCCTGTGGAAAGACTATAACAACCATAATAATAGTAATACACTTCAGGCGGAGTTAGATCCCAAGTACTGGAAGGTATAGCCGGAAGAAGATATTGAGCTTTAGAAGAAAGAAACATCCACAAGGGGAAAGAGACGGAGCCACCATACAGGCCAAGGCCCCATCCATTGTTTGGATTTAGAAAGAGGAACTAAAGCTTGGTATCTATCTATGTCGATGGCGCGTGTTGCCTCACACACATACACGTATATCCATCCTTACGTATGTAGTATATGGTTGTGACATGGATTGGTGGTTAGTTTGTATGTACCCTTTTGTAATTTCATTTCCTTTTTTTGGATGCCAAAGTTAACGGACGTCACAAGAGATTGCATTTGTGTCCTTTTTTTGAGTTTTTCTTTTCTGGATGGTTCTATCAA(SEQ ID NO.:48)
In the invention, the RAE1 gene is derived from soybean (XM_ 003521974.4 predicts that Glycine max F-box/LRR-repeat protein 3 (LOC 100803617)), and the nucleic acid sequence is shown as SEQ ID NO. 49.
ACTCATTCAATATTAATAAATGTCTCGCATTTATTCCTCAAACATAGAAACCAAACAAACAAACCCTTCCCTTCTCTCCCCACAGTTATTCATCATTAACCTCCAAAACGACCAACCAAACGCAAAACGCAGAACCATGAAGAAGCAGAAGCTCTCCGAGCCTCAAAACGACACCACCAACCCCTTCGAGGTTCTCTCCGAGGAGCTAATGTTCGTCATCCTCGACTTCCTCCAAACGACGTCGTTGGACAAAAAATCTTTCTCGCTCACGTGTAAGTTGTTTTACTCCGTCGAGGCCAAGCACCGTCGTTTGCTTCGCCCGCTACGTGCGGAACACCTGCCCGCGCTCGCTGCCCGCTACCCGAACGTAACGGAATTGGATCTTTCCTTGTGTCCGCGCGTGGGCGACGGCGCGCTGGGGCTCGTCGCTGGCGCGTACGCGGCGACGCTGCGGCGAATGGACCTGTCGCGGTCGCGGCGGTTCACGGCGACCGGGCTGCTAAGCCTCGGCGCACGGTGCGAGCACCTGGTGGAGCTGGACTTGTCGAACGCGACGGAGCTGAGGGACGCCGGCGTCGCCGCGGTGGCGCGTGCGCGGAACTTGCGGAAGCTGTGGCTGGCGAGGTGCAAGATGGTGACGGATATGGGGATTGGGTGTATTGCGGTGGGGTGCAGGAAGCTGAGGCTGCTTTGCTTGAAGTGGTGTGTGGGAATTGGGGATTTGGGTGTGGATTTGGTTGCGATTAAGTGTAAGGAGCTCACAACATTGGATCTCTCTTATTTGCCTATCACGGAGAAATGTCTACCGTCAATCTTCAAATTGCAACATCTTGAAGATTTGGTCCTTGAAGGATGCTTTGGCATTGATGACGACAGCCTTGATGTTGATCTCTTAAAACAAGGGTGCAAGACATTGAAGAGACTTGATATCTCAGGTTGTCAAAACATAAGTCATGTTGGGTTATCAAAGCTTACAAGCATTTCTGGAGGTTTAGAGAAACTCATTTTAGCAGATGGCTCTCCTGTCACCCTTTCTCTTGCTGATGGTTTGAATAAACTTTCCATGTTGCAATCAATTGTATTAGATGGCTGCCCTGTTACATCTGAAGGATTACGGGCCATTGGAAATTTGTGCATTTCACTTAGGGAGCTTAGTCTAAGCAAATGTTTGGGAGTGACAGATGAGGCACTCTCATTTCTTGTGTCAAAACACAAAGATTTAAGGAAACTTGACATCACATGCTGTCGCAAGATAACTGATGTTTCCATTGCCAGCATTGCAAATTCATGCACAGGTCTAACTTCTCTCAAAATGGAGTCATGTACACTAGTTCCAAGTGAAGCATTTGTCTTGATTGGACAGAAATGCCATTATCTTGAGGAGCTTGACCTAACAGATAATGAAATTGATGATGAAGGTCTTATGTCCATTTCTTCTTGTTCTTGGCTTACCAGCTTGAAAATAGGAATATGCCTGAACATAACTGACAGAGGACTTGCCTATGTTGGCATGCGTTGCTCAAAATTAAAGGAGCTGGATCTATACAGGTCTACTGGAGTAGATGATTTGGGCATTTCAGCAATTGCTGGTGGTTGCCCTGGCCTTGAGATGATAAACACATCCTATTGTACTAGCATTACTGACAGGGCACTAATTGCCTTGTCAAAATGTTCAAATTTGGAGACACTTGAAATTCGAGGATGTCTTCTCGTTACATCCATAGGTCTGGCAGCTATTGCAATGAATTGCAGACAACTAAGTCGTCTAGACATAAAAAAGTGTTACAACATTGATGACAGTGGGATGATTGCTCTGGCTCATTTCTCCCAAAATCTAAGACAGATAAATTTGTCATATAGCTCAGTTACAGATGTGGGGCTTCTGTCACTTGCTAATATCAGTTGCCTTCAAAGCTTTACCTTGCTTCACCTGCAAGGCTTGGTTCCAGGAGGACTGGCGGCAGCCTTATTAGCTTGTGGAGGGCTAACAAAAGTGAAGCTCCATCTTTCACTAAGATCTCTGTTACCTGAGCTACTTATCAGACATGTGGAAGCACGTGGCTGTGTATTTGAATGGAGAGATAAAGAGTTTCAGGCTGAATTGGACCCCAAGTGTTGGAAATTACAGTTGGAAGATGTGATATAATAGGATTTTTCTCAGGTTCTTTGAAGTTTTGATAAAGACAACTCTGCTGCATGGCTCATGAAATTCAATTCGGAAGTCATCATCTTCTCTCTCTTCTCTGTTCACCTAACCTACACTACAAGAAATGAAGAAAGCAGCATGAAAAATGCCAGAAGTATTCTGTTTAGTCCATTGGAGGCTACACAGGCATTTGGAATGGAGATTGTTGATCATCCTTCCTTAAGTCTCGCTCGCCTAACTAGGATGAGTTTTGTTTATTCTTTTTTCTTTTTTTTGCTGCAAATAGCAGGATTAGTGAAGTACTTAAAGTGTTACAAACCCCATCTAACGTCTTTCATTTATTTATTTTGGGAGTACAATTGGAAATGTTCAGAGAATGGAGAAGCAGTGGCTATCGAATGTATAAAAACACTAACTTCTGTTCAATCTTTTCTTTTGACAGTGAAAAGCAAGATTAAGTGTGTAAAACTTAGAAACCTGTTTTGTTTATATATGATTAGAGCTGTTCATGGACATCCAGAATAGAAGTTTCAAAA(SEQ ID NO.:49)
Wild-type RAL1 protein and RAL1 gene
As used herein, wild-type RAL1 protein refers to a naturally occurring, plant-derived (preferably from arabidopsis, rice, maize, barley, brassica, soybean, or the like), non-engineered RAL1 protein that negatively regulates AtALMT expression. The nucleotide can be obtained by genetic engineering techniques such as genome sequencing, polymerase Chain Reaction (PCR), etc., and the amino acid sequence can be deduced from the nucleotide sequence.
In another preferred embodiment, the RAL1 protein is derived from Arabidopsis thaliana, and the amino acid sequence is set forth in SEQ ID NO. 50.
MSTSPSILSVLSEDLLVRVYECLDPPCRKTWRLISKDFLRVDSLTRTTIRILRVEFLPTLLFKYPNLSSLDLSVCPKLDDDVVLRLALDGAISTLGIKSLNLSRSTAVRARGLETLARMCHALERVDVSHCWGFGDREAAALSSATGLRELKMDKCLSLSDVGLARIVVGCSNLNKISLKWCMEISDLGIDLLCKICKGLKSLDVSYLKITNDSIRSIALLVKLEVLDMVSCPLIDDGGLQFLENGSPSLQEVDVTRCDRVSLSGLISIVRGHPDIQLLKASHCVSEVSGSFLKYIKGLKHLKTIWIDGAHVSDSSLVSLSSSCRSLMEIGLSRCVDVTDIGMISLARNCLNLKTLNLACCGFVTDVAISAVAQSCRNLGTLKLESCHLITEKGLQSLGCYSMLVQELDLTDCYGVNDRGLEYISKCSNLQRLKLGLCTNISDKGIFHIGSKCSKLLELDLYRCAGFGDDGLAALSRGCKSLNRLILSYCCELTDTGVEQIRQLELLSHLELRGLKNITGVGLAAIASGCKKLGYLDVKLCENIDDSGFWALAYFSKNLRQINLCNCSVSDTALCMLMSNLSRVQDVDLVHLSRVTVEGFEFALRACCNRLKKLKLLAPLRFLLSSELLETLHARGCRIRWD(SEQ ID NO.:50)
In another preferred embodiment, the RAL1 protein is derived from rice, and the amino acid sequence is shown in SEQ ID NO. 51.
MSEEVQRYGGGGGGGGGGVAALSLDLLGQVLDRVREPRDRKACRLVSRAFARAEAAHRRALRVLRREPLARLLRAFRALERLDLSACASLDDASLAAALSGADLAGVRRVCLARASGVGWRGLDALVAACPRLEAVDLSHCVGAGDREAAALAAATGLRELSLEKCLGVTDMGLAKVVVGCPRLEKLSLKWCREISDIGIDLLSKKCHELRSLDISYLKVGNESLRSISSLEKLEELAMVCCSCIDDDGLELLGKGSNSLQSVDVSRCDHVTSQGLASLIDGHNFLQKLNAADSLHEMRQSFLSNLAKLKDTLTVLRLDGLEVSSSVLLAIGGCNNLVEIGLSKCNGVTDEGISSLVTQCSHLRVIDLTCCNLLTNNALDSIAENCKMVEHLRLESCSSISEKGLEQIATSCPNLKEIDLTDCGVNDAALQHLAKCSELLVLKLGLCSSISDKGLAFISSSCGKLIELDLYRCNSITDDGLAALANGCKKIKMLNLCYCNKITDSGLGHLGSLEELTNLELRCLVRITGIGISSVAIGCKNLIEIDLKRCYSVDDAGLWALARYALNLRQLTISYCQVTGLGLCHLLSSLRCLQDVKMVHLSWVSIEGFEMALRAACGRLKKLKMLSGLKSVLSPELLQMLQACGCRIRWVNKPLVYKD(SEQ ID NO.:51)
In another preferred embodiment, the RAL1 protein is derived from maize and has the amino acid sequence shown in SEQ ID NO. 52.
MSREAQKLDCAAGAGGIGVLSLDLLGQVLEHLREPRDRKTCRLVSRAFERAEAAHRRALRVLRREPLPRLLRAFPALERLDLSACASLDDASLAAAVADAGGGLAGLRSVCLARANGVGWRGLEALVAACPKLAAVDLSHCVTAGDREAAALAAASELRDLRLDKCLAVTDMGLAKVAVGCPKLEKLSLKWCREISDIGIDLLAKKCPELRSLNISYLKVGNGSLGSISSLERLEELAMVCCSGIDDEGLELLSKGSDSLQSVDVSRCDHVTSEGLASLIDGRNFLQKLYAADCLHEIGQRFLSKLARLKETLTLLKLDGLEVSDSLLQAIGESCNKLVEIGLSKCSGVTDGGISSLVARCSDLRTIDLTCCNLITNNALDSIADNCKMLECLRLESCSLINEKGLERITTCCPNLKEIDLTDCGVDDAALQHLAKCSELRILKLGLCSSISDRGIAFISSNCGKLVELDLYRCNSITDDGLAALANGCKRIKLLNLCYCNKITDTGLGHLGSLEELTNLELRCLVRVTGIGISSVAIGCKNLIELDLKRCYSVDDAGLWALARYALNLRQLTISYCQVTGLGLCHLLSSLRCLQDIKMVHLSWVSIEGFEMALRAACGRLKKLKMLCGLKTVLSPELLQMLQACGCRIRWVNKPLVYKD(SEQ ID NO.:52)
In another preferred embodiment, the RAL1 protein is derived from barley and has the amino acid sequence shown in SEQ ID NO. 53.
MSEEEAQRYGGGTGGGGVGALSVDLLGQVLDRVLERRDRKACRLVSRAFARAEAAHRRALRVLRREPLPRLLRAFPALERLDLSACASLDDASLAAALAGADLGTVRQVCLARASGVGWRGLEALVAACPRLEAVDLSHCVGAGDREAAALAAASGLRELNLEKCLGVTDMGLAKVAVGCPRLETLSFKWCREISDIGVDLLVKKCRDLRSLDISYLKVSNESLRSISTLEKLEELAMVACSCIDDEGLELLSRGSNSLQSVDVSRCNHVTSQGLASLIDGHSFLQKLNAADSLHEIGQNFLSKLVTLKATLTVLRLDGFEVSSSLLSAIGEGCTNLVEIGLSKCNGVTDEGISSLVARCSYLRKIDLTCCNLVTNDSLDSIADNCKMLECLRLESCSSINEKGLERIASCCPNLKEIDLTDCGVNDEALHHLAKCSELLILKLGLSSSISDKGLGFISSKCGKLIELDLYRCSSITDDGLAALANGCKKIKLLNLCYCNKITDSGLSHLGALEELTNLELRCLVRITGIGISSVVIGCKSLVELDLKRCYSVNDSGLWALARYALNLRQLTISYCQVTGLGLCHLLSSLRCLQDVKMVHLSWVSIEGFEMALRAACGRLKKLKILGGLKSVLSPDLLQLLQACGCRIRWVNKPLVYKDAI(SEQ ID NO.:53)
In another preferred embodiment, the RAL1 protein is derived from Brassica and has the amino acid sequence set forth in SEQ ID NO. 54.
MPLSPSILSVLSEDLLVRVYGFLDPPCRKKWRLVSKEFHRVDSLSRTSIRILRVEFLPALLSNYPHLSSLDLSVCPKLDDDVVLRLASYGAVSIKSLNLSRATALRARGLETLARLCRGLERVDVSHCWGFGDREAAALSVAAGLREVRLDKCLSLSDVGLARIVLGCSNLSKISLKWCMEISDLGIDLLCKKCKDLKSLDVSYLKITNDSIRSIALLPKLEVLEMVNCPLVDDDGLQYLENGCPSLQEIDVTRCERVSLSGVVSIVRGHPDLQHLKASHCVSEVSLSFLHNIKALKHLKTLWIDGARVSDSSLLTLSSSCRPLTDIGVSKCVGVTDIGITGLARNCINLKTLNLACCGFVTDAAISAVAQSCRNLETLKLESCHMITEKGLQSLGCYSKHLQELDLTDCYGVNDRGLEYISKCSNLLRLKLGLCTNISDKGMFHIGSKCSKLLELDLYRCGGFGDDGLAAISRGCKSLNRLIISYCGELTDTGVEQIRQLEHLSHLELRGLKNITGAGLAAVACGCKKLDYLDLKKCENIDDSGFWALAYFARNLRQINLCYCSVSDTALCMLMSNLSRVQDVDLVNLNRVTVEGSEFALRACCNRLKKLKLFAPLRFLLSSELLEMLHARGCRIRWD(SEQ ID NO.:54)
In another preferred embodiment, the RAL1 protein is derived from soybean and has the amino acid sequence shown in SEQ ID NO. 55.
MLSESVFCLLTEDLLIRVLEKLGPDRKPWRLVCKEFLRVESSTRKKIRILRIEFLLGLLEKFCNIETLDLSMCPRIEDGAVSVVLSQGSASWTRGLRRLVLSRATGLGHVGLEMLIRACPMLEAVDVSHCWGYGDREAAALSCAARLRELNMDKCLGVTDIGLAKIAVGCGKLERLSLKWCLEISDLGIDLLCKKCLDLKFLDVSYLKVTSESLRSIASLLKLEVFVMVGCSLVDDVGLRFLEKGCPLLKAIDVSRCDCVSSSGLISVISGHGGLEQLDAGYCLSELSAPLVKCLENLKQLRIIRIDGVRVSDFILQTIGTNCKSLVELGLSKCVGVTNKGIVQLVSGCGYLKILDLTCCRFISDAAISTIADSCPDLVCLKLESCDMVTENCLYQLGLNCSLLKELDLTDCSGVDDIALRYLSRCSELVRLKLGLCTNISDIGLAHIACNCPKMTELDLYRCVRIGDDGLAALTSGCKGLTNLNLSYCNRITDRGLEYISHLGELSDLELRGLSNITSIGIKAVAISCKRLADLDLKHCEKIDDSGFWALAFYSQNLRQINMSYCIVSDMVLCMLMGNLKRLQDAKLVCLSKVSVKGLEVALRACCGRIKKVKLQRSLRFSLSSEMLETMHARGCKIRWD(SEQ ID NO.:55)
As used herein, the terms "RAL1 gene of the invention", "RAL1 gene" are used interchangeably.
In the present invention, the genes of RAL1 include genomic genes, cDNA sequences, mRNA sequences.
In the invention, the RAL1 gene is derived from Arabidopsis thaliana (AT 5G 27920), and the nucleic acid sequence is shown as SEQ ID NO. 56.
CTTTCTTTATTTTATTTACCTTTTACCAAAGTTACACTTCTACCCCTATTTTTCTTCAATAAGAGATGGAAGAGCAAGGGCAATTAAGTAATTTGACAATATCCTTCTCCTTCAAGACTCTCTCTCTCCCACAGAAAACAAACTGTTTGTTGTTAATAACTGTGGGTTTTTTCCAAAATCCTCAAAGAGAGACTATACTTCTCTCGGATCCTTCCTGATTTCAAAATTTCTTCAACCCACCAGCAGCAGCAGCAGATGAAGAAGGTTAAACAGATTCGTGTCTTAAAGCCTTTCGATCTTCTCTCGGAAGAGCTCGTCTTTATCATCCTTGACCTCATCTCTCCCAACCCTTCCGATCTCAAATCCTTCTCTCTTACTTGCAAATCTTTCTACCAGCTCGAGTCCAAGCACCGCGGATCCTTAAAACCTCTCCGCTCCGACTATCTCCCTCGCATCTTGACTCGTTACCGGAACACCACCGATCTCGATCTTACCTTCTGCCCGCGTGTCACTGACTACGCGCTTAGCGTCGTTGGCTGTCTCTCCGGACCTACGCTTCGCTCCCTCGACCTCTCGCGCTCTGGCTCCTTCTCCGCCGCGGGACTACTGCGATTGGCCCTCAAATGTGTCAATTTAGTCGAGATTGACCTGTCCAATGCGACGGAGATGAGAGACGCCGATGCTGCGGTGGTGGCGGAGGCGAGGAGTCTGGAGAGGCTGAAGCTGGGCAGATGCAAGATGCTGACGGACATGGGAATCGGATGTATAGCAGTTGGATGTAAGAAGCTCAATACGGTTAGCTTGAAATGGTGTGTTGGCGTCGGAGATTTAGGGGTTGGCTTGCTTGCCGTCAAATGCAAGGACATTCGCACCTTAGACCTCTCCTACTTGCCGATCACAGGAAAGTGTTTACATGACATTCTGAAACTTCAACACCTTGAAGAACTTCTTCTAGAAGGGTGCTTTGGAGTAGATGATGACAGTCTTAAATCACTCAGACATGATTGCAAGTCATTGAAGAAGCTTGATGCATCCAGCTGCCAGAATTTAACTCATAGAGGTTTAACCTCACTTTTAAGCGGGGCAGGATATCTTCAGCGACTTGATCTATCACACTGTTCTTCTGTGATATCATTGGATTTTGCGAGTAGCTTAAAGAAGGTTTCAGCATTACAGTCGATCAGGTTGGATGGTTGTTCTGTTACGCCTGATGGTTTGAAGGCGATAGGCACATTGTGCAATTCCCTGAAAGAGGTTAGCCTAAGCAAATGCGTGAGCGTAACTGATGAAGGTCTTTCTTCTCTAGTAATGAAACTGAAAGACCTCAGAAAACTTGACATCACATGTTGCCGGAAACTAAGTAGAGTTTCAATCACCCAAATCGCCAATTCGTGTCCTTTACTAGTCTCTTTGAAGATGGAGTCTTGTTCTCTTGTTTCCAGAGAAGCCTTTTGGTTGATCGGACAAAAGTGTCGGCTACTTGAAGAGCTTGACTTAACTGACAACGAGATTGATGATGAAGGACTGAAATCCATATCTAGTTGTCTGAGTCTTTCCTCGTTAAAGCTGGGAATTTGTCTTAACATAACAGACAAAGGGCTCTCGTACATCGGGATGGGCTGTTCAAATCTCCGTGAACTTGATCTCTATAGGTCAGTGGGAATAACAGACGTAGGCATCTCCACAATTGCCCAAGGCTGCATTCATCTGGAAACAATAAACATTTCATACTGCCAAGACATAACAGACAAGTCCCTGGTTTCATTGTCCAAATGCTCGTTGTTACAAACATTCGAGAGCAGGGGATGTCCTAACATCACGTCCCAAGGACTTGCAGCCATTGCTGTTCGGTGCAAGCGACTCGCCAAGGTTGACTTGAAGAAGTGCCCATCCATCAATGATGCGGGGTTGCTCGCTCTGGCTCACTTCTCTCAGAATCTCAAACAGATAAACGTGTCAGACACAGCTGTGACTGAAGTGGGACTTCTCTCCCTAGCCAACATAGGGTGTTTACAGAACATAGCGGTTGTGAACTCGAGTGGTTTAAGACCGAGCGGAGTAGCAGCAGCATTGCTGGGGTGTGGAGGATTAAGGAAAGCGAAACTACATGCGTCCCTAAGATCACTGCTTCCTCTTTCTCTAATCCACCACTTGGAAGCTCGTGGTTGTGCGTTCCTCTGGAAAGACAATACCCTTCAGGCGGAGTTAGATCCCAAGTACTGGAAGCAACAGCTGGAAGAGATGGCGCCTTAAATTAAAAGTGAGAAGAAACATTCTGAAATGGGAAAGAGGAGTTCCTATGGGAGCCAGCAGACAGGCCCTGTTTTGGGCCCAGTGTGGATTAGCTGGAACAATTTTGGTCTCTTTTGTGTTGTTGTTGACGGCGCGTGTTCTAACAACCCCACACAACCTTACATTATAAGTCTAGTCACATGGTGGGTGACATGGTACCGTTGTATATGTAGTTTTTGTTTCTTTTTTTTTTTTTTTTGGTTGGGGAGCCGAAATTAACGGACGTAACAGAGTCACAAGGGGATGCCTTCTCTGTGCCTTTTTGACTTTTTCCTCCTTTCTTTTTTTTCTGTAATCATATGAGTTTTATGTAATTTAATGCCTCATCAACTTGCCTGGATAGGGCCGGCCTTTCCACGTCACCTTAGTCGCGTTTTGCGGTAAAAAAAGTAAAAAGAAAGGCCGTGACTCAGTAGGGCCCATTACTGGTCCAGTCCAGGCTCTCACCGTCTGTACAAAAATAGCGAGTCAGCGACTCGGTGTGACAGCAACAACCCTCTCTCGTTGAAAAACGGCTAATGCGCCCGACACTTCCATCCTCATATTCCATTACAAAAGAAATCCAGCTATGTAGTATCAAACTACCGAAAACATACACATGTCAATCAAGTCCAAAAAGTATCACCGTCAAATGAGTGATTCAGATTTTGTTTGCTCATGAAAGATAAACCAAATGATTACTTCCTTAATCACCTGCCCCTTGTTTTCCATTTTTTAATTACATAATGGAACCCCCCATTATTAAAACATGATCATTTTCTTTCGTAATAATTAAACTTCCCAACTAAATGCCTCTTAATA(SEQ ID NO.:56)
In the invention, the RAL1 gene is derived from rice (LOC_Os12g 36670), and the nucleic acid sequence is shown as SEQ ID NO. 57.
ATGAGCGAGGAGGTGCAGAGGTATGGAGGTGGTGGGGGTGGGGGAGGAGGTGGGGTGGCGGCGCTGTCGCTGGATTTGCTCGGCCAGGTGCTCGACCGGGTGCGGGAGCCGCGGGACCGCAAGGCGTGCAGGCTCGTGAGCCGCGCCTTCGCCCGCGCCGAGGCCGCGCACCGCCGCGCGCTGCGGGTGCTCCGCCGCGAGCCCCTGGCGCGCCTCCTCCGCGCGTTCCGGGCGCTGGAGCGGCTCGACCTCTCCGCCTGCGCCTCCCTCGACGACGCCTCCCTCGCCGCCGCGCTCTCCGGCGCGGATCTCGCCGGGGTGCGCCGGGTCTGCCTCGCCCGCGCCAGCGGCGTCGGGTGGCGCGGCCTCGACGCGCTCGTGGCGGCGTGCCCGAGGCTGGAGGCCGTCGACCTGTCGCACTGCGTCGGCGCCGGCGACCGCGAGGCCGCCGCGCTGGCCGCGGCGACGGGGCTGAGGGAATTGAGCCTCGAGAAGTGCCTCGGCGTCACGGACATGGGGCTCGCCAAGGTGGTGGTCGGGTGCCCGAGGCTGGAGAAGCTGAGCCTCAAGTGGTGCCGCGAGATCTCCGACATCGGCATCGATTTGCTCTCCAAGAAGTGCCACGAGCTCCGGAGCCTCGACATCTCCTACCTCAAGGTTGGAAATGAATCCCTTAGATCAATATCCTCACTTGAGAAACTTGAGGAGTTGGCAATGGTTTGTTGCTCATGCATAGATGATGATGGCCTGGAATTACTAGGCAAGGGGAGCAACTCACTGCAGAGCGTTGATGTTTCAAGATGTGATCATGTAACCTCCCAGGGATTAGCTTCACTCATAGATGGTCACAATTTTCTCCAGAAGTTAAATGCTGCTGATAGTTTGCATGAGATGAGACAGTCGTTTCTGTCCAACTTGGCAAAACTGAAGGATACCTTGACAGTGCTTAGACTTGATGGTCTTGAAGTCTCATCCTCTGTTCTTCTGGCCATTGGTGGTTGTAATAACTTGGTCGAGATTGGCCTTAGCAAATGCAATGGCGTTACAGATGAAGGAATCTCTTCACTTGTAACTCAATGCAGCCACTTAAGGGTTATTGATCTCACATGCTGTAACCTCCTTACCAACAATGCCCTTGATTCAATAGCTGAGAACTGTAAGATGGTTGAACATCTCCGTTTGGAATCCTGTTCTTCCATAAGCGAAAAGGGACTGGAGCAGATTGCAACCTCCTGCCCCAATCTAAAGGAGATAGACCTCACTGACTGTGGAGTGAATGATGCAGCATTGCAGCACTTGGCCAAGTGCTCTGAACTGCTTGTACTGAAATTAGGCCTGTGCTCAAGTATTTCTGACAAAGGTCTTGCTTTTATCAGTTCAAGCTGTGGAAAGCTGATTGAGCTTGATCTCTATCGCTGCAATTCTATTACCGATGATGGGCTGGCAGCTTTAGCTAATGGCTGCAAGAAGATTAAGATGCTAAACCTATGCTACTGCAACAAGATTACCGACAGTGGTTTGGGCCACCTAGGCTCTCTAGAGGAGCTCACAAACCTTGAACTGAGGTGCTTGGTCCGTATAACAGGTATTGGAATCTCATCAGTTGCCATCGGCTGCAAGAACCTGATAGAGATAGACTTGAAGCGTTGCTATTCTGTCGATGATGCTGGCTTGTGGGCTCTTGCCCGATATGCACTAAACCTTAGACAGCTTACTATATCATACTGCCAAGTCACTGGCCTGGGCCTGTGTCACCTGTTAAGCTCCCTGAGGTGCCTCCAGGATGTTAAGATGGTACACCTCTCATGGGTCTCTATAGAAGGGTTCGAGATGGCACTGAGAGCGGCTTGCGGGAGGCTGAAGAAACTGAAGATGCTGAGTGGGCTGAAGTCTGTGCTGTCTCCTGAGCTGCTCCAGATGCTGCAGGCCTGCGGCTGCCGCATCCGTTGGGTCAACAAGCCTCTTGTCTACAAGGACTAAACCTGCTCTCATATCATCATCTGTGCAGTGTTAATATCCTCGGATCATACACCATGGAGCCCTTACCAAGTCTAGCATGCTTACAAGCCATGTTAGATACCCGCAATAATCGTCAATTTTCATACCCAAGATGTACTGACGATGGTGTCTCTGCATTGCTGCAGTGCCATCCTTATGTTTGGCAACTCCTAGACCAATGTTGTCATCTTCAGATTAAAATCCAACATTTGCAAACATGAGGCATCGAAACAGTTATGTTCTGAGCAAGTGATCCTTGTACCAGACTGCAATGAAATGCTCTACACTTTCAATTGGTATATGCAAAGAACAATGGCATGTTTGCCTTTGTCATTGCTCCAACTCCAGGCTTGCTGACCGATGCATCTTTTAGAGTTTAAGAACAGCTTTAGATCGCAGAAAAACTCTTCCTTGATGCTCCAGGATCTGGGCTGGGACACTGGTTTCATGGTTGTCTGTATTGTATGACTGGTGGACCCGTCCTTTTTTTTTTACCTTGATTACAATAATCTCGATGTACTCATTCAGGCAGAGATTTGAAGTAATGTATTGAATCAGTTTTTAT(SEQ ID NO.:57)
In the invention, the RAL1 gene is derived from corn (XM_ 008664387.2 predicts: zea mays F-box/LRR-repeat protein 3 (LOC 103640946)), and the nucleic acid sequence is shown as SEQ ID NO. 58.
CGGCATCACCTATCCACATCTTTCTCCCTCTCTCTCTCTCTCCTCTTCCGTCCCTCCTCTCTCCCTCTCTACGCCACGCCTCCACCTCCACACCTGCACGCCTCTCGCTCTCTCTCCTCACCTGTCGAGCTGCCCACAGCCCGTACGTACCTGCCCCCAGTCCCAGTCCCCGCCGCCCCGCCTGCCCGACCCGCGGCTTATCCACCACGTCCGCCGCTGGGCTGGATCGATCGGGACCGGTATCGCGCCGCCGCCGCTGCCCTCGGCACTCGCGGGATCCTGTTCCTACCATGCAGCAGCGCTGCGCCCTTGCCCGACCGCGCCGGCCCGCCGAACCGCGCGTTCCGCTCCCCGACGATCACTCCCTCGTCGCCGTCTTCTCCTTCCTCTTCATCCCCACCTAAATCAACCTCCTCGCCGCGAAATTCTGCTACAATTTCTTCATCTTCCCTTCCCGCCGGGAGCGGACGGATCGCCGCTTCCTCGGCCGGCGGGATGAGCCGGGAGGCGCAGAAACTCGACTGCGCCGCCGGCGCCGGGGGGATCGGGGTCCTGTCGCTGGACCTACTGGGCCAGGTGCTGGAACACCTGCGGGAGCCCCGGGACCGCAAGACGTGCCGCCTCGTCAGCCGCGCCTTCGAGCGCGCCGAGGCCGCGCACCGCCGCGCGCTTCGGGTGCTCCGACGCGAGCCGCTCCCGCGCCTGCTCCGCGCGTTCCCGGCGCTCGAGCGGCTCGACCTCTCCGCCTGCGCCTCGCTCGACGACGCCTCCCTCGCCGCGGCCGTCGCCGACGCCGGCGGAGGGCTCGCCGGCCTCCGCAGCGTGTGCCTCGCGCGGGCCAATGGTGTCGGCTGGCGCGGCCTCGAGGCGCTCGTCGCGGCCTGCCCCAAGCTCGCGGCCGTCGACCTGTCGCACTGCGTCACCGCCGGGGACCGCGAGGCCGCCGCGTTGGCGGCGGCGTCCGAGCTCAGGGACCTGAGGCTGGACAAATGCCTTGCCGTCACCGACATGGGGCTCGCCAAGGTGGCTGTTGGGTGCCCCAAGCTGGAGAAGCTCAGCCTCAAGTGGTGCCGTGAGATCTCTGACATTGGAATTGATCTGCTGGCCAAGAAGTGCCCTGAGCTCCGCAGCCTCAACATATCCTACCTCAAGGTGGGCAATGGATCCCTTGGATCAATTTCGTCACTTGAGAGGCTTGAGGAATTGGCAATGGTTTGTTGTTCAGGTATAGATGACGAAGGCTTGGAATTGCTGAGCAAGGGGAGCGATTCGCTGCAGAGTGTTGATGTGTCAAGATGTGATCATGTGACTTCCGAGGGGTTAGCTTCACTCATAGATGGTCGCAATTTTCTTCAGAAGTTATATGCTGCAGATTGTTTGCATGAGATAGGACAGCGTTTTCTATCCAAGTTGGCAAGACTGAAGGAAACCTTGACATTGCTGAAACTCGACGGTCTTGAGGTCTCGGACTCTCTTCTTCAAGCCATTGGTGAAAGCTGTAACAAATTGGTTGAGATTGGGCTTAGCAAATGCAGTGGTGTTACTGATGGAGGAATCTCATCTCTTGTAGCTCGGTGTAGTGACCTAAGAACAATTGATCTCACATGCTGCAATCTCATTACCAACAATGCGCTTGATTCAATAGCTGACAACTGTAAGATGCTTGAATGTTTGCGGTTGGAATCATGCTCTTTGATAAATGAGAAGGGACTAGAGCGAATTACAACTTGTTGCCCCAACCTTAAGGAGATAGACCTCACTGACTGCGGAGTAGATGATGCAGCATTGCAGCACTTGGCTAAATGCTCTGAATTGCGAATATTGAAATTAGGCCTATGCTCAAGTATTTCTGACAGAGGCATTGCATTTATCAGTTCGAATTGTGGAAAGCTCGTGGAGCTTGATCTCTACCGGTGCAACTCTATTACTGATGATGGGTTGGCAGCTTTAGCAAATGGGTGCAAGAGGATTAAGTTACTGAACTTGTGTTACTGCAACAAGATCACTGATACTGGTTTGGGTCACCTAGGCTCCCTGGAGGAGCTCACAAACCTTGAACTGAGGTGCTTGGTCCGCGTAACAGGCATTGGGATCTCCTCAGTTGCAATCGGATGCAAGAACCTGATAGAGCTGGACTTGAAACGATGCTATTCGGTTGATGACGCTGGCCTGTGGGCCCTTGCTCGTTATGCTTTGAACCTTAGACAGCTTACGATATCGTACTGCCAAGTCACGGGGTTGGGCCTGTGCCACCTGCTGAGCTCCCTGCGATGCCTCCAGGACATTAAGATGGTGCACCTCTCATGGGTCTCAATAGAAGGGTTTGAGATGGCTCTGCGAGCAGCCTGCGGGAGGCTGAAGAAGCTGAAGATGCTCTGCGGGCTGAAGACCGTGCTCTCCCCTGAGCTCCTCCAGATGCTGCAGGCTTGTGGCTGCCGAATAAGATGGGTCAACAAGCCTCTTGTCTACAAGGACTAGACAGACTCGTGTTCCTCATTGTGTGTGCAGTGTCAGCATCAGCATGTATTGTCAAGATCTTGCAGGTTTGCCCTGGCTCACGTTGTGAGTCCGCAATAATCGTTGGGGGCGTAAGCTCCCGTCCCCCAGATGTGGTTATGGTGCCTTTGGGGCACTTCTTGCGCCATTTGTCACTCTCTGCTCCGGTGTATCTGTGTATGCAAACGGAGGCACCGGTAATGTTCATGTGTATATAGCGTGCAGTAGAATGATCCCATTTTATGGGATTAGTTAGTGTGTAATTGTTACCTCATTTCGCATGATTCGAATCTTGAGATGAAAGAATCATAAGGGGTATGATCGATGCATTGCATTCACCGGTTCTTA(SEQ ID NO.:58)
In the present invention, the RAL1 gene is derived from barley (AK358574.1 Hordeum vulgare subsp. Vulgare mRNA of predicted protein, all CDS, clone: NIASHv1079C 11), and the nucleic acid sequence is shown as SEQ ID NO. 59.
GGGGTGCTACCTATCCACATCTTTCTCCTCCCATCCTCCCTCCCTCTCCCCACTCCCCACCCATCGACCCACCTTCCTCCCTCCGTACCTTCGCCACCACCGGCCGTCGCCTTGCCGCTCCTCCGCCGCCCGGATCAGGAACGATCGCACCGTGCGGCCGCCTCCCGCCCTGCGCGCGGATCCTACCATGCTGAGGCGCTGCGCCCCATACCGACCCCGCAGGGCCGCCGCCGTACCGCGCCTCCCGATCCCCGGCGGCCACCCCTACCTCGTCGCCCTCTCCTTCCTCTTCATCCCGACCTAAGCTCCACGCCTCGCCGACAGGCCGGCCCTCGCCGCCGGAGCTGCCCGGTGGAATCGTCAGCCGCGATGAGCGAGGAGGAGGCGCAGAGGTACGGCGGCGGGACCGGCGGCGGCGGCGTCGGTGCGCTGTCCGTGGATCTGCTCGGCCAGGTGCTCGATCGCGTGCTGGAGCGGCGGGACCGCAAGGCCTGCCGCCTCGTCAGCCGCGCCTTCGCGCGCGCTGAGGCCGCGCACCGCCGGGCCCTCCGGGTGCTCCGCCGGGAGCCGCTCCCTCGCCTGCTCCGCGCCTTCCCGGCGCTCGAGCGCCTCGATCTCTCAGCCTGTGCCTCGCTCGACGACGCGTCTCTTGCAGCCGCTTTAGCCGGCGCGGACCTCGGCACCGTCCGACAGGTCTGCCTGGCGCGGGCCAGCGGAGTGGGCTGGCGCGGGCTGGAAGCCCTCGTGGCCGCCTGCCCCAGGCTCGAGGCTGTCGACCTGTCGCACTGCGTCGGTGCTGGGGATAGGGAGGCTGCCGCCCTGGCCGCCGCCTCTGGGCTGAGGGAGCTAAATCTGGAAAAGTGCCTTGGGGTCACTGATATGGGGCTCGCCAAGGTAGCCGTGGGCTGCCCCAGACTGGAGACTCTGAGCTTCAAGTGGTGCCGTGAAATCTCTGACATCGGCGTCGATCTGCTTGTCAAAAAGTGCCGCGACCTCCGCAGCCTTGACATCTCCTACCTAAAGGTGAGCAATGAGTCCCTTAGATCAATATCGACTCTTGAGAAGCTAGAGGAGTTGGCCATGGTTGCTTGCTCATGTATAGATGATGAAGGCCTGGAATTGCTTAGCAGAGGAAGCAATTCATTGCAGAGTGTTGATGTCTCAAGATGCAATCACGTGACTTCCCAGGGGTTAGCTTCACTGATAGATGGTCACAGTTTTCTCCAGAAGTTAAATGCCGCAGATAGTTTGCATGAGATTGGACAGAATTTTCTATCCAAGTTGGTAACACTGAAGGCAACCTTGACCGTGTTGAGACTTGACGGCTTTGAAGTGTCATCCTCTCTTCTTTCAGCGATTGGTGAAGGTTGTACCAACTTGGTTGAGATTGGACTAAGCAAATGCAACGGTGTTACAGATGAAGGCATCTCTTCGCTTGTAGCTCGCTGTAGCTACCTAAGGAAAATTGATCTCACATGCTGCAATCTAGTCACAAACGATTCCCTTGATTCAATAGCTGACAACTGTAAGATGCTTGAATGCCTCCGGTTGGAGTCCTGCTCTTCTATAAACGAGAAAGGACTAGAGAGAATTGCAAGCTGTTGCCCCAATCTAAAGGAGATAGATCTCACTGATTGTGGAGTGAACGACGAAGCGTTGCATCATTTGGCGAAGTGCTCTGAACTGCTGATATTGAAATTAGGCCTGAGCTCAAGTATTTCGGACAAAGGCCTTGGTTTTATTAGTTCAAAGTGTGGGAAGCTCATTGAACTTGACCTCTATCGCTGCAGTTCTATCACTGATGATGGGCTGGCAGCCTTAGCCAACGGCTGCAAGAAAATTAAGCTGCTGAACCTTTGTTACTGCAACAAGATAACTGATAGTGGTTTGAGCCACCTGGGCGCTCTTGAGGAGCTCACAAACCTTGAGCTGAGGTGCCTCGTTCGCATTACAGGCATCGGAATTTCTTCCGTTGTCATTGGCTGTAAGAGCCTGGTAGAACTTGACTTGAAGCGCTGCTATTCTGTCAATGATTCTGGGCTATGGGCTCTTGCCCGATATGCTCTAAACCTTAGACAGCTCACCATATCATACTGCCAAGTTACTGGCCTAGGCTTGTGCCACCTGCTTAGCTCCTTGAGGTGCCTCCAGGACGTGAAGATGGTGCACCTGTCATGGGTTTCCATAGAAGGGTTCGAGATGGCTCTGCGAGCCGCTTGCGGGAGGCTGAAGAAGCTGAAGATACTCGGCGGTTTGAAGTCCGTGCTATCCCCTGACCTGCTCCAGCTTCTGCAAGCCTGCGGCTGCCGCATCAGATGGGTCAACAAGCCTCTTGTCTACAAGGATGCCATCTGATCTGAGAAGTACCATATTGTTCATCCTTTCGGCAAGTTTGTCATCGCTGCGTAGCCCTGTTAGAAAATTCACAATAACTACACGCCTCGTCAATGCCATGCCTAAGGTGTGGTCAAGATCATGTCTGTTTGAGATGTCGAGTAAACATGGCAGCTGCTGTCGAATATATGTCATGTACAGTGAAGCCTTTTTTTTTGGACGGGATGTACAGTGAAGCCCTTTTGCATAGCAGAGTTGTGCAACTGGAGTTCTCTACTTGATGTGAAGGAACCTGAAAAATATGCTGT(SEQ ID NO.:59)
In the present invention, the RAL1 gene is derived from Brassica (XM_ 009113489.2. Sup. Predicts. Brassica rapa F-box/LRR-repeat protein 3-l ike (LOC 103837159)), and the nucleic acid sequence is shown in SEQ ID NO. 60.
GGGAAACAATTGAAGTAAAGAGAGAGAGGTTCGGTTTTGTGTTTCCAACCGGAAAAAAAACCCCGAAGGTTTGACGATGAACCCGAATCTAATAACCCGTAACCCAATTCGGATCCTGGATCAACCGGTTCACCACTCTCTCCTCTATCTCCGAGCTGTCTTTCTCTTCATCCCGACTTAAAAGATCCGTACTTTTTTTTTTTTGAAACTTTCCGACCAAACTAAAAGTAAACACATACTTGTCTTTTTTTTTCTTGTTCTGTCGCGAAATGCCGCTGTCTCCATCCATTCTATCCGTTCTGTCGGAAGATCTTCTAGTTCGTGTCTACGGGTTCCTAGACCCGCCCTGTCGGAAAAAATGGCGACTCGTCAGCAAAGAGTTTCACCGAGTCGACTCACTGAGCCGCACATCGATCCGAATCCTCCGAGTCGAGTTCCTCCCCGCGCTTCTCTCCAACTACCCTCACCTCTCCTCCCTCGACCTCTCCGTCTGCCCCAAACTCGACGACGACGTCGTTCTGCGCCTAGCGTCCTACGGCGCCGTTTCGATAAAGTCGCTGAACCTGAGCCGCGCCACCGCGCTCCGAGCGAGGGGACTGGAGACGCTGGCTCGCTTGTGCCGAGGCCTCGAGCGAGTCGACGTGTCTCACTGCTGGGGGTTCGGAGACAGGGAAGCGGCGGCGCTCTCCGTCGCGGCGGGGCTGAGGGAGGTGAGGTTGGACAAGTGCTTGAGCCTAAGCGACGTCGGATTGGCGAGGATCGTCCTCGGGTGTAGTAATCTGAGCAAGATTAGTTTGAAGTGGTGTATGGAGATCTCTGATCTAGGGATCGATCTTCTCTGTAAGAAATGCAAAGACTTGAAGTCTCTCGACGTCTCTTATCTTAAGATCACGAATGATTCGATCCGGTCCATAGCTTTGTTGCCAAAGCTCGAGGTTTTAGAGATGGTGAACTGTCCGTTGGTAGATGATGATGGGTTACAGTATCTTGAGAATGGTTGTCCTTCGTTACAGGAGATTGATGTCACAAGGTGTGAGCGTGTGAGTTTGTCTGGCGTTGTCTCCATTGTCAGAGGCCATCCTGATCTTCAACACCTGAAAGCCAGTCACTGTGTATCAGAAGTATCTCTGAGCTTCTTACATAACATCAAAGCTTTGAAGCATCTCAAGACTCTATGGATCGATGGAGCTCGTGTCTCTGACTCCTCTCTCTTAACCCTAAGCTCCAGCTGTAGACCCTTAACAGATATCGGAGTGAGCAAATGTGTGGGTGTGACGGATATTGGCATCACAGGACTAGCACGCAACTGCATAAACCTGAAAACCCTAAACCTAGCGTGCTGCGGGTTTGTGACTGATGCAGCCATCTCTGCGGTAGCTCAGTCTTGCCGCAATCTGGAGACTCTTAAGTTAGAGTCTTGTCATATGATAACCGAGAAAGGTCTTCAATCACTCGGATGTTACTCCAAGCATCTTCAAGAACTCGATCTTACCGACTGTTATGGCGTCAATGACAGAGGGCTAGAATATATCTCAAAGTGTTCGAATCTTCTAAGGTTGAAACTTGGCCTCTGCACAAATATCTCAGACAAAGGGATGTTTCATATCGGTTCCAAATGTTCCAAGCTTCTAGAACTTGATCTATACCGCTGTGGTGGTTTTGGAGATGACGGTTTAGCAGCTATATCCCGAGGTTGCAAGAGCTTGAACCGGCTCATTATATCGTACTGTGGTGAGCTAACAGACACAGGGGTTGAACAAATCCGCCAGCTTGAACATCTAAGCCATCTTGAACTTCGAGGGCTAAAGAATATAACCGGTGCTGGTCTAGCTGCAGTTGCGTGCGGCTGCAAGAAATTGGATTACTTGGACCTCAAGAAGTGCGAGAATATAGATGACTCAGGCTTCTGGGCGCTTGCTTACTTTGCAAGAAACCTAAGACAGATAAACTTGTGCTATTGCTCGGTTTCTGATACGGCTCTATGCATGTTAATGAGCAATCTAAGTCGGGTTCAAGACGTTGACTTAGTCAACCTGAACCGTGTGACAGTGGAAGGGTCTGAGTTTGCTCTAAGAGCCTGTTGCAATAGGCTCAAGAAGCTTAAACTTTTCGCTCCTCTCAGATTCTTGCTCTCATCTGAATTGCTTGAGATGCTTCATGCTCGTGGTTGCCGCATTAGATGGGACTGAAAGACGAAACTTTCTTCAATGGAACTTTACTAGTAACACTTTAACGTGTTCATATGTCTACTTGTTGTATCTATAAAAAATTCGACTATGTTTTTAAATTCGGTTTACCAATTATATGTCTACCGCTTTGGTATTATCTTCCAACATAAACAATAGTAGTTTTAGAAAACAAAAATATTATTTTGGTATTCTATGCAAAA(SEQ ID NO.:60)
In the invention, the RAL1 gene is derived from soybean (XM_ 003521631.4 predicts that Glycine max F-box/LRR-repeat protein 3 (LOC 100802904), TRANSCRIPT VARIANT X1), and the nucleic acid sequence is shown as SEQ ID NO. 61.
TTTCAACAATTTTTCTCTCATGTCCAAGTTTTGTCCTGAGCAGGACAACTTAATTGGCGGTTCCCTTCTCGCCCTTGGATCTTTAATTGGTTGTTCCCTCCGCTCCAAGAAAAAAAAGGGCATTTATGGCAAAAAAGAGCAGCAGCATTCAACAAGTTGCGAGAGGAACACAGGACTCAATTCACATGGCCGTACCCGAAGAAAGCGAAAAGTGTGAAACACGCACCCTCTGAAATCTAAAATTCAATTTTCCCTTCCCTGACCCTGTTTCTTCTATTCACATTGTATTCTCCTTGTTTTTTCGCTTTTCAATTTTCCATTGAACTCAGACAAAAACAGAAAGAAAGGGGAAAAAAATGAACCCAGAGAAATCTCGTTGCAGGTTGCCACTTCGCACGCTCTCTAAACACACGCACCACCCTCTTCTCCACCTCCGCCTCCTTTTCCTCTTCGTTCCCACCTAAACTGTTTCCCTTCAGTTCCACGTCACATTAACAAACCTTTTTTTTTCTTAAAAAAAAAACTTTAGTAATTACGAAATTTCTGATACCTTAATGTTGTCTGAATCCGTTTTCTGCCTCTTGACCGAGGACCTGCTCATCCGGGTCCTCGAAAAGCTCGGGCCGGATCGGAAACCGTGGCGGCTGGTGTGCAAGGAGTTTCTCCGGGTCGAATCGTCGACCCGGAAGAAGATTCGGATCCTCCGAATCGAGTTTCTGCTTGGGTTGTTGGAGAAGTTCTGCAACATTGAGACGCTGGACCTGTCGATGTGTCCGCGGATCGAGGACGGAGCTGTGTCGGTTGTGCTGAGTCAGGGATCGGCGAGTTGGACTCGGGGACTGAGGAGACTCGTGCTGAGTCGCGCCACCGGGTTGGGGCATGTGGGCTTGGAGATGCTGATTCGGGCGTGTCCCATGTTGGAGGCCGTGGATGTGTCCCATTGTTGGGGGTATGGCGACAGAGAGGCTGCGGCGCTATCGTGCGCCGCGAGGTTGAGGGAACTCAACATGGATAAGTGTTTGGGAGTTACTGATATTGGGTTGGCCAAGATTGCTGTCGGGTGTGGGAAATTGGAGAGGCTGAGTTTGAAGTGGTGCTTGGAGATTTCTGATCTGGGGATTGATCTTCTTTGCAAAAAGTGCTTGGATTTGAAATTTCTCGACGTGTCATATCTCAAGGTAACAAGTGAATCTTTGAGATCAATAGCTTCTCTGTTAAAGCTTGAGGTTTTTGTTATGGTTGGCTGCTCTTTAGTGGATGACGTTGGATTGCGGTTTCTTGAAAAAGGGTGTCCACTGCTTAAGGCAATTGATGTATCAAGGTGTGATTGTGTTAGCTCTTCCGGTTTAATATCTGTAATTAGTGGACATGGAGGTCTTGAGCAGTTGGATGCAGGATATTGCCTCTCTGAGCTTTCAGCACCTCTTGTTAAATGCTTGGAGAATTTAAAGCAGCTGAGAATAATTAGAATTGATGGTGTTCGAGTTTCTGACTTTATCCTCCAGACAATTGGCACCAATTGCAAGTCTTTAGTGGAACTTGGTTTAAGCAAATGCGTTGGAGTGACCAACAAGGGAATTGTGCAGCTAGTATCTGGCTGTGGCTATTTGAAGATACTTGATTTGACTTGTTGTCGGTTCATATCTGATGCAGCAATCTCTACTATAGCAGACTCTTGTCCAGACCTTGTCTGTCTGAAGCTAGAATCTTGTGATATGGTGACTGAGAATTGTCTTTATCAACTTGGATTAAATTGCTCGCTTCTCAAAGAGCTTGATCTTACTGATTGCTCTGGTGTTGATGACATAGCTCTAAGATATCTATCAAGATGTTCAGAACTTGTAAGATTGAAATTAGGATTATGCACAAATATATCAGACATAGGATTGGCACACATTGCTTGTAACTGCCCAAAAATGACTGAACTTGATCTCTATCGATGTGTACGTATTGGAGATGATGGGCTAGCGGCACTAACGAGTGGATGCAAGGGGTTGACAAACCTCAACTTGTCATATTGCAATAGAATTACAGACAGAGGGTTGGAGTATATCAGCCATCTTGGTGAACTATCTGATCTGGAGTTGCGTGGGCTTTCTAATATCACAAGCATTGGTATAAAAGCAGTTGCAATAAGTTGCAAGAGATTGGCAGATTTAGATTTGAAACATTGCGAAAAAATTGATGATTCAGGTTTCTGGGCCCTTGCTTTTTATTCGCAAAACCTGCGGCAGATAAATATGAGCTACTGTATCGTGTCAGATATGGTGTTGTGCATGCTTATGGGTAACCTGAAACGCCTGCAAGATGCCAAACTGGTTTGTCTTTCTAAAGTGAGTGTAAAAGGATTGGAAGTTGCCCTTAGAGCTTGCTGTGGTCGGATTAAAAAGGTTAAACTGCAGAGGTCCCTCAGGTTCTCGCTTTCCTCTGAAATGCTCGAGACAATGCATGCACGAGGGTGCAAGATCAGATGGGATTAGCCAATTGTCACGCTATTAATCTGTTATACTTTCTTTTCTTGGAAGGGTGCTGCTTCATTAAGATCTTTTTTATTCAAAATTTTATATTTCTCTAGCATTTTCTTTTCTTTAAACTCTGTTACCAGATTTCTACGGAAGGTATTTTTTTCACCTTCTTAAGTTACATCATCTCCAATAAAGCATGAGGTTACTTATCTTCGAATTAAA(SEQ ID NO.:61)
The invention also provides a recombinant vector comprising the gene of the invention. As a preferred mode, the promoter downstream of the recombinant vector comprises a multiple cloning site or at least one cleavage site. When it is desired to express the gene of interest of the present invention, the gene of interest is ligated into a suitable multiple cloning site or cleavage site, thereby operably linking the gene of interest to a promoter. As another preferred mode, the recombinant vector comprises (from 5 'to 3') the following: promoters, genes of interest, and terminators. The recombinant vector may further comprise, if desired, an element selected from the group consisting of: a 3' polynucleotide acidification signal; an untranslated nucleic acid sequence; transport and targeting nucleic acid sequences; resistance selection markers (dihydrofolate reductase, neomycin resistance, hygromycin resistance, green fluorescent protein, etc.); an enhancer; or an operator.
Methods for preparing recombinant vectors are well known to those of ordinary skill in the art. The expression vector may be a bacterial plasmid, phage, yeast plasmid, plant cell virus, mammalian cell virus, or other vector. In general, any plasmid or vector may be used as long as it is capable of replication and stability in a host.
One of ordinary skill in the art can construct expression vectors containing the genes of the present invention using well known methods. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like. When the gene of the present invention is used to construct a recombinant expression vector, any one of enhanced, constitutive, tissue-specific or inducible promoters such as cauliflower mosaic virus (CAMV) 35S promoter, ubiquitin (Ubiquitin) gene promoter (pUbi) and the like may be added before the transcription initiation nucleotide thereof, and they may be used alone or in combination with other promoters.
Vectors comprising the genes, expression cassettes or the invention may be used to transform an appropriate host cell to allow the host to express the protein. The host cell may be a prokaryotic cell, such as E.coli, streptomyces, agrobacterium: or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as plant cells. It will be clear to one of ordinary skill in the art how to select appropriate vectors and host cells. Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryote (e.g., E.coli), the treatment may be performed by CaCl 2 or electroporation. When the host is eukaryotic, the following DNA transfection methods may be used: calcium phosphate co-precipitation, conventional mechanical methods (e.g., microinjection, electroporation, liposome encapsulation, etc.). The transformed plant may also be transformed by Agrobacterium or gene gun, such as leaf disc method, embryo transformation method, flower bud soaking method, etc. Plants can be regenerated from the transformed plant cells, tissues or organs by conventional methods to obtain transgenic plants.
As a preferred mode of the present invention, the method for preparing a transgenic plant is: the vector carrying the promoter and the gene of interest (both operably linked) is transferred into agrobacterium, which in turn integrates the vector fragment containing the promoter and the gene of interest into the chromosome of the plant. Transgenic recipient plants are involved, for example, arabidopsis thaliana, nicotiana tabacum, fruit trees, etc.
In order to facilitate the identification and selection of transgenic plant cells or plants, the plant expression vectors used may be processed, for example, by adding genes (GUS gene, GFP gene, luciferase gene, etc.) which express enzymes or luminescent compounds capable of producing color changes in plants, antibiotic markers (gentamicin markers, kanamycin markers, etc.) which are resistant, or marker genes which are resistant to chemical agents (e.g., herbicide genes), etc. From the safety of transgenic plants, transformed plants can be screened directly in stress without adding any selectable marker gene.
RAE1 protein or RAL1 protein and its coding gene have various uses. For example, for screening compounds, polypeptides or other ligands having the ability to modulate anti-aluminum toxicity. Screening polypeptide libraries using expressed recombinant RAE1 proteins or RAL1 proteins can be used to find valuable polypeptide molecules that inhibit, or promote, the ability of plants to resist aluminum toxicity.
Method for improving aluminium toxicity resistance of plant
The invention also provides a method for regulating and controlling the aluminum toxicity resistance of plants, which comprises the following steps:
(i) Regulating the expression quantity and/or activity of RAE1 protein or RAL1 protein in the plant, thereby regulating the aluminum toxicity resistance of the plant.
In another preferred example, the plants suitable for use in the method include crops, forestry plants, vegetables, fruits, flowers, pastures (including turf grass).
In another preferred embodiment, the modulation of the anti-aluminum toxicity capability of a plant is the down-regulation of the expression and/or activity of a RAE1 protein or a RAL1 protein in a plant or the inactivation of a RAE1 protein or a RAL1 protein when the anti-aluminum toxicity capability of a plant is enhanced.
In another preferred embodiment, the modulation of the plant's ability to resist aluminum toxicity is an up-regulation of the expression and/or activity of RAE1 protein in the plant when the plant's ability to resist aluminum toxicity is reduced.
In another preferred embodiment, the method is also used to regulate the level of organic acids secreted by plants (e.g., roots).
In another preferred embodiment, the method of down-regulating the expression level and/or activity of RAE1 protein or RAL1 protein in a plant includes (but is not limited to): down-regulating RAE1 or RAL1 expression by RNAi technology or modifying RAE1 or RAL1 by CRISPR technology.
The main advantages of the invention include:
(1) The RAE1 gene or RAL1 gene or the encoding protein thereof, or the mutein thereof, or the promoter thereof, or the inhibitor thereof can regulate the expression or degradation of STOP-1 protein, the aluminum toxicity resistance or the aluminum resistance of plants, and can also regulate the expression of genes selected from the following groups: atALMT1, atm mate, ALS3, or a combination thereof.
(2) The invention discovers for the first time that RAE1 mediates STOP-1 protein degradation through ubiquitination modification, RAE mutant protein and F-box domain protein are removed to lose the function of mediating STOP-1 degradation, and conversely, overexpression of RAE1 can promote STOP-1 protein degradation.
(3) The method can effectively and remarkably improve the aluminum toxicity resistance of plants, thereby solving the problem of aluminum toxicity of acidic soil.
(4) The RAE1 gene or the RAL1 gene or the encoded protein thereof can be used for screening compounds, polypeptides or other ligands with the ability of regulating and controlling the aluminum toxicity resistance, and finding valuable polypeptide molecules capable of inhibiting or promoting the aluminum toxicity resistance of plants.
The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental procedure, which does not address the specific conditions in the examples below, is generally followed by routine conditions such as Sambrook et al, molecular cloning: conditions described in the laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989) or as recommended by the manufacturer. Unless otherwise specified, the materials and reagents used in the examples were all commercially available products.
General method
1. Experimental materials
Plant material: arabidopsis wild type Col-0; RAE1 mutant materials RAE1-1 to RAE1-8 and STOP1 mutant materials STOP1-3 (mutation site is H352Y, STOP1 protein is inactivated) obtained from EMS mutagenesis library screening; ordering a T-DNA knockout mutant SALK_114341C of RAL1 from ARBC; transgenic material pAtALMT1:LUC, pSTOP1:STOP1-HA, pSTOP1:STOP1-GUS, pRAE1:GUS, pUBQ10:RAE1 and pRAL1:GUS.
And (3) strain: coli DH 5. Alpha. BL21 (DE 3); agrobacterium GV3101.
2. Experimental method
1) EMS mutagenesis pAtALMT LUC transgenic seed Bank and mutant screening
PAtALMT1 construction of the LUC vector the 1.76kb AtALMT1 promoter was ligated into vector pGWB by gateway recombination kit (Thermofisher). Agrobacterium transformation and arabidopsis infection: agrobacterium GV3101 was transformed by heat shock, positive clones were picked up and cultured with LB liquid, then the cells were resuspended with 5% sucrose solution, and Silwet-L77 was added to a final concentration of 0.05% to complete the preparation of the infection suspension of agrobacterium. The method comprises the steps of infecting arabidopsis thaliana by a flower dipping method, cutting off pods of the arabidopsis thaliana, soaking flowers in an infection suspension for 1min, moisturizing with a preservative film, removing the preservative film from the sun for 24h, re-receiving light, and harvesting the seeds of the T0 generation after the pods are ripe. T0 generation seeds are planted on a hygromycin-containing 1/2MS flat-plate culture medium to screen T1 generation transgenic positive lines, single copy inserted transgenic lines are selected according to whether T2 generation shows a 3:1 separation ratio, and planting is continued until homozygous T3 generation is obtained for subsequent experiments.
Carrying out EMS mutagenesis on pAtALMT 1:1 LUC transgenic seed library, soaking seeds in 100mM phosphate buffer solution at 4 ℃ for overnight, sterilizing the seeds with 8% NaClO for 10min, washing the seeds with sterilized water for 4 times, putting the seeds into 40mL of newly sterilized 100mM phosphate buffer solution, turning the seeds up and down for 8h at room temperature by using an turning instrument, thoroughly rinsing M1 seeds with sterilized water for at least 20 times, then growing the seeds on a 1/2MS flat plate culture medium, and transplanting the seeds into soil for planting.
Screening M2 generation seeds, dibbling in a 1/2MS vertical plate culture medium, culturing for 7-8d in a 16h illumination/8 h darkness and 22 ℃ illumination incubator, uniformly spraying 1mM fluorescein working solution, placing in the darkness to react for 10min in a dark place, placing in the middle of the darkness of a LUC fluorescence imaging system, photographing by a CDD camera precooled to minus 110 ℃, exposing for 3min, and screening seedlings which are brighter than wild type contrast fluorescence.
Construction pAtALMT1 LUC primer:
F:5’-GGGGACAAGTTTGTACAAAAAAGCAGGCTCCTGGCTCCTTTTGGTTGTCTA-3’(SEQ ID NO.:1)
R:5’-GGGGACCACTTTGTACAAGAAAGCTGGGTAACACCTTTTGATGGTCACTCAGCT-3’(SEQ ID NO.:2)
2) mRNA expression analysis of anti-aluminum toxin genes
Changes in gene expression in wild type and mutant were studied by aluminum treatment, arabidopsis thaliana cultivated in water for 4 weeks, pretreated with 0.5mM CaCl 2 solution (pH 4.8) for 6h, followed by treatment with 0 or 30 μm CaCl 3 CaCl 2 solution (pH 4.8) for 12h. The whole root system was cut, RNA extracted and DNA digested with RNA extraction kit (9769; taKaRa, dalian, china), reverse transcribed with 1. Mu.g RNA, and then qRT-PCR (5. Mu.l system) with 5. Mu.l 1/25cDNA (Bio-Rad CFX ConnectTM Real-TIME SYSTEM; bio-Rad, singapore). AtUBQ10 as reference gene, the result is an average.+ -. SD,3 biological replicates.
3) Construction of transgenic Material for anaplerotic RAE1, detection of STOP-1 protein and detection of RAE1/RAL1 tissue expression Pattern
To make up for mutant rae a vector containing the 2.43kb RAE1 promoter, genomic fragment and 1.44kb gene downstream was ligated into vector pCAMBIA3301. For detection of STOP-1 protein in plants, a vector comprising a 2.79kb STOP-1 promoter and genomic fragment fused with 3 XHA or GUS was ligated into vector pCAMBIA1305 or pORE-R2 to give vector pSTOP 1:STOP-1-HA and pSTOP 1:STOP-1-GUS, respectively. The above vectors were constructed by recombinant kit (C112; vazyme Biotech co., ltd, south tokyo, china). To detect the tissue expression pattern of RAE1, a vector containing the 2.43kb RAE1 promoter linked to GUS was ligated into the pORE-R2 vector. For the overexpression of RAE1, the 2.55kb AtUBQ10 promoter was ligated into the pCAMBIA3301 vector in conjunction with the RAE1 CDS.
Construction pRAE1 RAE1 primer:
F:5’-GACCTGCAGGCATGCAAGCTTTGTTTCAGCATATTTGCATGTTTGAT-3’(SEQ ID NO.:3)
R:5’-CACCTGTAATTCACACGTGGTGTGCTACAGAGGAAAGAAGGTTTG-3’(SEQ ID NO.:4)
Construction pSTOP1 STOP-1-3 XHA primer:
F:5’-GGTACCCGGGGATCCTCTAGATTTAGGGCTTCAAACTCTTTACCC-3’(SEQ ID NO.:5)
R:5’-GGAACATCGTATGGGTAAAGCTTGAGACTAGTATCTGAAACAGACTCAC-3’(SEQ ID NO.:6)
Construction pSTOP1 STOP1-GUS primer:
F:5’-TTGGGGCCCAACGTTCTCGAGTTTAGGGCTTCAAACTCTTTACCC-3’(SEQ ID NO.:7)
R:5’-CGGCCGCAAAGTCGACGAATTCGAGACTAGTATCTGAAACAGACTCAC-3’(SEQ ID NO.:8)
Construction pRAE1 GUS primer:
F:5’-TTGGGGCCCAACGTTCTCGAGTGTTTCAGCATATTTGCATGTTTGA-3’(SEQ ID NO.:62)
R:5’-CGGCCGCAAAGTCGACGAATTCTGCTGCTGCTGCTGGTGGGT-3’(SEQ ID NO.:63)
construction pAtUBQ10 RAE1 primer:
F:5’-CGACCTGCAGGCATGCAAGCTTAAAGTCTGTATATATGACACAGAAGAAACC-3’(SEQ ID NO.:64)
F:5’-CACCTGTAATTCACACGTGGTGTTAAGGCGCCATCTCTTCCAG(SEQ ID NO.:65)
Construction pRAL A GUS primer
F 5’-TTGGGGCCCAACGTTCTCGAGTGACCTGAGGCTGAAAATCG-3’(SEQ ID NO.:66)
R 5’-CGGCCGCAAAGTCGACGAATTCTTTGCAACAGAACCAGAAATAAACA-3’(SEQ ID NO.:67)
4) GUS dyeing method
The pSTOP 1:1 STOP-GUS transgenic lines were incubated in water for 1 week, pretreated with 0.5mM CaCl 2 solution (pH 4.8) for 12h, followed by treatment with 0 or 15. Mu.M AlCl 3 in combination with 50. Mu.M MG132 in CaCl 2 solution (pH 4.8) for 12h. The cells were stained with GUS stain (161031;o'Biolab Co, ltd, beijing, china) for 2h at 37℃and then photographed with a stereoscope (SZX 7; olympus, japan).
5) LUC Activity assay for RAE1 promoter Regulation and EMSA assay for STOP-1 binding to RAE1 promoter
LUC activity assay: pRAE1 LUC or mpRAE.LUC, 35S: STOP-1-2 xFLAG or 35S:2 xFLAG, pZmUBQ: GUS (internal reference) were co-transformed into Arabidopsis protoplast and expressed for 20h at room temperature, followed by detection of LUC activity (RG 006-2;Beyot ime Biotechnology, china) and GUS activity (A602251; sangon Biotech Co., ltd., china), respectively.
EMSA test:
The CDS sequence of STOP-1 was constructed by ligating into pET29a (+) vector, and transferred into E.coli BL21 (DE 3) for expression and purification of the protein. The biotin-labeled DNA probe was synthesized and chemiluminescent detection was performed using the EMSA kit (GS 009; beyotime Biotechnology, china).
Construction pRAE1 LUC primer:
F:5’-GCCTGCAGGCTCTAGAGGATCCTGTTTCAGCATATTTGCATGTTTGA-3’(SEQ ID NO.:68)
R:5’-ATGTTTTTGGCGTCTTCCATGGTGCTGCTGCTGCTGGTGGGT-3’(SEQ ID NO.:69)
construction mpRAE1 LUC primer:
1F:5’-GCCTGCAGGCTCTAGAGGATCCTGTTTCAGCATATTTGCATGTTTGA-3’(SEQ ID NO.:70)
1R:5’-AGACTATCTCGGTTAAAGACTGCCTCGATTAACAGATTCGAGTGCGGACAGTACGAT-3’(SEQ ID NO.:71)
2F:5’-TAATCGAGGCAGTCTTTAACCGAGATAGTCTTCTACTACCAATGGCCTAAC(SEQ ID NO.:72)
TTGTT-3’
2R:5’-ATGTTTTTGGCGTCTTCCATGGTGCTGCTGCTGCTGGTGGGT-3’(SEQ ID NO.:73)
pRAE1-EMSA sequence was synthesized:
F:5'-TCAATCTATCGTACTGTCCGCACTCGAATCTGTCCTTCCTCGCATGCTTACTCCACCATATA CTTCTACTACCAATGGCCTAACTTGTTGATTGCG-3'(SEQ ID NO.:74)
R:5'-CGCAATCAACAAGTTAGGCCATTGGTAGTAGAAGTATATGGTGGAGTAAGCATGCGAGGAAG GACAGATTCGAGTGCGGACAGTACGATAGATTGA-3'(SEQ ID NO.:75)
mpRAE1-EMSA sequence was synthesized:
F:5'-TCAATCTATCGTACTGTCCGCACTCGAATCTGTTAATCGAGGCAGTCTTTAACCGAGATAGT CTTCTACTACCAATGGCCTAACTTGTTGATTGCG-3'(SEQ ID NO.:76)
R:5'-CGCAATCAACAAGTTAGGCCATTGGTAGTAGAAGACTATCTCGGTTAAAGACTGCCTCGATT AACAGATTCGAGTGCGGACAGTACGATAGATTGA-3'(SEQ ID NO.:77)
6) Pull Down test
The CDS sequences of RAE1 and STOP-1 are linked to pET29a (+), pGEX4T-1 or pET-H6Trx vector to construct RAE1-His, GST-RAE1, GST-STOP-1 or His-Trx-STOP-1, and the vector is transferred to E.coli BL21 (DE 3) to express the protein. BL21 (DE 3) was grown to OD 600 at 37℃to 0.6, and protein expression was induced at 25℃for 6h with the addition of 0.1mM IPTG. Bacterial lysates containing GST-STOP-1 or GST-RAE1 and control GST proteins were incubated with GST agarose beads (C60031; sangon Biotech Co., ltd, shanghai, china) for 1h on a shaker at 4 ℃. Then washed 4 times with buffer and incubated with lysates containing RAE1-His or His-Trx-STOP-1 protein for 2h on a shaker at 4 ℃. Immunoblotting was performed with 10% SDS-PAGE after 5 washes with buffer.
Construction of RAE1-His
F:5’-TCCATGGCTGATATCGGATCCATGAAGAAGGTTAAACAGATTCGT-3’(SEQ ID NO.:9)
R:5’-GTGGTGGTGGTGGTGCTCGAGAGGCGCCATCTCTTCCAG-3’(SEQ ID NO.:10)
Construction of GST-RAE1
F:5’-TCCGCGTGGATCCCCGGAATTCATGAAGAAGGTTAAACAGATTCGT-3’(SEQ ID NO.:11)
R:5’-GTCACGATGCGGCCGCTCGAGTTAAGGCGCCATCTCTTCCAG-3’(SEQ ID NO.:12)
Construction of GST-STOP-1
F:5’-TCCGCGTGGATCCCCGGAATTCATGGAAACTGAAGACGATTTGTG-3’(SEQ ID NO.:13)
R:5’-GTCACGATGCGGCCGCTCGAGTTAGAGACTAGTATCTGAAACAGAC-3’(SEQ ID NO.:14)
Construction of His-Trx-STOP-1
F:5’-GGTCTGGTGCCACGCGGATCCATGGAAACTGAAGACGATTTGTG-3’(SEQ ID NO.:15)
R:5’-ACTTAAGCATTATGCGGCCGCTTAGAGACTAGTATCTGAAACAGAC-3’(SEQ ID NO.:16)
7) Split-LUC test
CDS sequences of RAE1, RAE1-1, RAE1ΔF and STOP1 were ligated into pCAMBIA1-cLUC or pCAMBIA1-nLUC vectors to construct cLUC-RAE1, cLUC-RAE1-1, cLUC-RAE1ΔF and STOP1-nLUC.
The construct was transferred to agrobacterium GV3101, and injected into tobacco n.benthamiana leaves according to different combinations, cultured in the dark for 24h and then placed in a light incubator for 48d, after which the leaves were photographed with LUC imaging system.
Construction cLUC-RAE1/cLUC-RAE1-1
F:5’-GTACGCGTCCCGGGGCGGTACCATGAAGAAGGTTAAACAGATTCG-3’(SEQ ID NO.:17)
R:5’-GAACGAAAGCTCTGCAGGTCGACTTAAGGCGCCATCTCTTCCAG-3’(SEQ ID NO.:18)
Construction cLUC-RAE1ΔF
F:5’-GTACGCGTCCCGGGGCGGTACCCTCGATCTTACCTTCTGCCC-3’(SEQ ID NO.:19)
R:5’-GAACGAAAGCTCTGCAGGTCGACTTAAGGCGCCATCTCTTCCAG-3’(SEQ ID NO.:20)
Construction of STOP-1-nLUC
F:5’-ACGAGCTCGGTACCCGGGATCCATGGAAACTGAAGACGATTTGTG-3’(SEQ ID NO.:21)
R:5’-GACGCGTACGAGATCTGGTCGACGAGACTAGTATCTGAAACAGACT-3’(SEQ ID NO.:22)
8) Protein extraction, coIP assay and protein degradation assay and ubiquitination modification detection assay
Arabidopsis thaliana, which had been hydroponic for 4 weeks, was pretreated with 0.5mM CaCl 2 solution (pH 4.8) for 6h, followed by treatment with 0 or 30. Mu.M AlCl 3 in combination with 50. Mu.M MG132 in CaCl 2 solution (pH 4.8) for 12h. The whole root system was excised and the protein extracted with protein extract (20 mM Tris-HCl pH 7.5,300mM NaCl,5mM MgCl 2, 5mM DTT, 50. Mu.M MG132,0.5% NP-40, and 1X complete protease inhibitor mixture).
CoIP test: as controls, 2mL Arabidopsis protoplasts were co-transformed with 100. Mu.g 35S: STOP1-3 XHA and 100. Mu.g 35S: RAE1-2 XFLAG or 50. Mu.g 35S: RAE1-1-2 XFLAG or 35S: RAE1ΔF-2 XFLAG, 35S: STOP1-3 XHA and 35S:2 XFLAG. The protein was then extracted with 100. Mu.L of protein extract (20 mM Tris-HCl pH 7.4,150mM NaCl,1mM MgCl 2, 1mM DTT, 50. Mu.M MG132,0.25% NP-40, and 1 Xcomplete protease inhibitor cocktail (complete protease inhibitor mixture)), and 20. Mu.L of protein extract was used as input. The total protein extract was diluted to 1ml and incubated with 20. Mu.L of anti-FLAG M2 magnetic beads on a shaker at 4℃for 3h, washed 3 times with buffer, eluted with 100mM glycine (pH 2.5) and neutralized with 1M Tris-HCl (pH 9.0), followed by immunoblotting.
Protein degradation assay: the protein was extracted with 50. Mu.g 35S: STOP-1-2 xFLAG and 25. Mu.g 35S: RAE1-3 xHA or 50. Mu.g 35S: RAE1-3 xHA (35S: RAE1-1-3 xHA or 35S: RAE1ΔF-3 xHA kept the same) and then with 50. Mu.L of protein extract and 20. Mu.L of protein extract as input.
Ubiquitination modification detection assay:
The protein was extracted with 35S: myc-UBQ10, 35S: STOP1-2×FLAG,35S: RAE1-3×HA (35S: RAE1-1-3×HA or 35S: RAE1ΔF-3×HA) into Arabidopsis protoplasts, incubated at room temperature for 16h, then 50 μM MG132 was treated for 8h, and protein extract (20 mM Tris-HCl pH 7.4,150mM NaCl,1mM MgCl 2, 1mM DTT,50 μM MG132,0.25% NP-40, and 1× complete protease inhibitor mixture) and 20 μL protein extract was used as input. The total protein extract was diluted to 1ml and incubated with 20. Mu.L of anti-FLAG M2 beads on a shaker at 4℃for 2h, washed 5 times with buffer and then ubiquitinated modification was detected with anti-Myc antibody.
Construction 35S STOP-1-3 XHA
F:5’-CTTGCTCCGTGGATCCTCTAGAATGGAAACTGAAGACGATTTGTG-3’(SEQ ID NO.:23)
R:5’-GAACATCGTATGGGTATCTAGAGAGACTAGTATCTGAAACAGACT-3’(SEQ ID NO.:24)
Construction 35S RAE1/rae1-1-3 XHA
F:5’-CTTGCTCCGTGGATCCTCTAGAATGAAGAAGGTTAAACAGAT-3’(SEQ ID NO.:25)
R:5’-GAACATCGTATGGGTATCTAGAAGGCGCCATCTCTTCC-3’(SEQ ID NO.:26)
Construction 35S RAE1ΔF-3 XHA
F:5’-CTTGCTCCGTGGATCCTCTAGAATGCTCGATCTTACCTTCTGCCC-3’(SEQ ID NO.:27)
R:5’-GAACATCGTATGGGTATCTAGAAGGCGCCATCTCTTCC-3’(SEQ ID NO.:28)
Construction 35S STOP1-2 xFLAG
F:5’-CTTGCTCCGTGGATCCTCTAGAATGGAAACTGAAGACGATTTGTG-3’(SEQ ID NO.:29)
R:5’-TGTAGTCAGAAGGCCTGGTACCGAGACTAGTATCTGAAACAGACT-3’(SEQ ID NO.:30)
Construction 35S RAE1/rae1-1-2 xFLAG
F:5’-CTTGCTCCGTGGATCCTCTAGAATGAAGAAGGTTAAACAGAT-3’(SEQ ID NO.:31)
R:5’-TGTAGTCAGAAGGCCTGGTACCAGGCGCCATCTCTTCC-3’(SEQ ID NO.:32)
Construction 35S RAE1ΔF-2×FLAG
F:5’-CTTGCTCCGTGGATCCTCTAGAATGCTCGATCTTACCTTCTGCCC-3’(SEQ ID NO.:33)
R:5’-TGTAGTCAGAAGGCCTGGTACCAGGCGCCATCTCTTCC-3’(SEQ ID NO.:34)
9) Evaluation of aluminum toxicity resistance ability
To measure malic acid secreted from the rhizosphere, arabidopsis thaliana was hydroponic cultured for 4 weeks, pretreated with 0.5mM CaCl 2 solution (pH 4.8) for 6h, followed by treatment with 0 or 30 μm AlCl 3 CaCl 2 solution (pH 4.8) for 12h, and the rhizosphere secretions were collected and malic acid was determined by NAD/NADH enzymatic cycling (Hampp et al, 1984).
The aluminum content in Arabidopsis roots was determined according to the method of Ligaba-Osena et al (2017). The root surface was treated with 0.5mM citric acid solution (pH 4.2) at 4℃for 30min to remove aluminum, then washed three times with 18. OMEGA. Ultra-pure water and blotted dry, and the sample was then dried in an oven at 60 ℃. Digestion was performed with 1ml of a mixed acid of HNO 3 and HClO 4, diluted with 2% HNO 3, and then the aluminum content was determined by ICP-MS.
Arabidopsis aluminum tolerance was determined according to the aluminum soak medium method of Larsen et al (2005). The medium matrix formulation was ：1mM KNO3,0.2mM KH2PO4,2mM MgSO4,0.25mM(NH4)2SO4,1mM Ca(NO3)2,1mM CaSO4,1mM K2SO4,1μM MnSO4,5μM H3BO3,0.05μM CuSO4,0.2μM ZnSO4,0.02μM NaMoO4,0.1μM CaCl2,0.001μM CoCl2,1％sucrose and 0.3% Gellan gum (G1910; sigma-Aldrich) as follows. Then 50ml of the substrate was soaked for one day with 40ml of a culture broth containing 0, 0.75, 1, 1.25mM AlCl 3 of the same formulation (without Gellan gum), the broth was removed and the seeds were spotted and cultured vertically in a light incubator for seven days. Plants were photographed and root length was measured with Image J.
EXAMPLE 1 mutant mutagenesis screening and Gene complementation verification
In order to study the expression regulation of AtALMT1 and the STOP-1 protein level regulation mechanism, a 1.76kb AtALMT1 promoter is fused with a luciferase gene, a pAtALMT1 LUC transgenic reporter gene system is constructed by infecting Arabidopsis wild Col-0 with agrobacterium, EMS mutagenesis is carried out by using the material, and mutants affecting LUC reporter gene expression are screened by LUC fluorescence detection.
By screening about 10000M 2 strains, 17 mutants affecting LUC reporter gene Expression were obtained in total, one of the LUC Expression enhancement mutants was used to clone the target gene, designated RAE1 (Regulation of AtALMT.sup.1 Expression 1), and sequencing the rest of the mutants revealed that mutants with different site mutations on 8 RAE1 were screened in total (RAE-1 to RAE.sup.1-8), and that the LUC fluorescence of both root and aerial parts in mutants RAE-1 to RAE-8 was increased compared to the wild type (FIGS. 1A, 1B).
To understand the function of RAE1, first the secondary structure of RAE1 was analyzed to find that RAE1 encodes a protein containing an F-box domain and 18 LRR repeats (FIG. 1B).
Construction of vector pRAE1 RAE 1A complementation transgenic line was obtained by Agrobacterium infection of mutant RAE-1, and 2 independent lines were selected for LUC fluorescence detection, which both complemented the RAE1-1 fluorescent phenotype (FIG. 1C). A novel gene for negative regulation AtALMT <1 > expression is cloned by the method, and the gene is subjected to compensation verification.
EXAMPLE 2 expression of Albumt 1 et al anti-Albumin Gene in mutant RAE1 and over-expressed RAE1
Further analysis of LUC reporter and AtALMT1 expression in mutant rae1, selection of WT, rae1-1, rae1-2, and stop1-3 for testing, and qPCR analysis of expression by cutting root extracted RNA after 12h treatment without or with Al. The expression of the LUC reporter gene and AtALMT1 in rae-fold higher than that of the wild type in rae-fold without Al, and the expression of the LUC reporter gene and AtALMT1 in the Al treatment was 3-5-fold higher than that of the wild type (fig. 2A, 2B). LUC reporter and AtALMT were not expressed at all in STOP1-3, indicating that STOP1 is a transcription factor necessary for AtALMT1 expression (FIGS. 2A, 2B).
On the other hand, analysis of the expression of other anti-aluminum genes in mutant rae1 revealed that the expression of the other 2 STOP-1 downstream regulatory genes AtMATE and ALS3 was increased in mutant rae1 over wild-type (WT) both in the absence of aluminum and in the presence of aluminum (FIGS. 2C, 2D), whereas the expression of STOP-1-unregulated genes AtSTAR and ALS1 was unaffected in mutant rae1 (FIGS. 2E, 2F).
In contrast, overexpression of RAE1 reduced the expression of AtALTM, atMATE, and ALS3 (FIG. 3). RAE1 expression was induced by aluminum treatment and regulated by STOP-1, and RAE1 expression levels were also up-regulated in RAE.
The above results indicate that RAE1 affects STOP-1 regulated gene expression, further suggesting that RAE1 may affect STOP-1 protein stability.
Example 3 expression of RAE1 was induced by aluminum treatment and directly regulated by STOP-1
RAE1 expression was induced by aluminum treatment and regulated by STOP-1, and RAE1 expression levels were also up-regulated in RAE. Protoplast expression systems showed STOP-1 to regulate pRAE expression of LUC, and mutation of the binding region reduced regulation, EMSA verified that STOP-1 directly bound to the binding region on the RAE1 promoter. RAE1 expression was expressed in various tissues of the plant and was mainly expressed in vascular tissues (FIG. 4). STOP-1 regulates RAE1 expression by direct binding to the RAE1 promoter, thereby forming a circular negative feedback regulation mechanism.
EXAMPLE 4 binding relationship of RAE1 to STOP1
To analyze the binding relationship of RAE1 to STOP-1, an in vitro Pull-Down assay was first performed. GST-STOP-1 can specifically pull down RAE1-His, which in turn GST-RAE1 can specifically pull down His-Trx-STOP-1, indicating that RAE1 and STOP-1 can bind directly in vitro (FIG. 5A).
To analyze the interaction of RAE1 with STOP-1 in plants, a tobacco Split-LUC assay was performed, where the N-terminus of RAE1/RAE1-1 (mutein)/RAE 1 ΔF (F-box domain protein removed) was fused to cLUC, the C-terminus of STOP-1 was fused to nLUC, fluorescence was seen when cLUC-RAE-1 and STOP-1-nLUC were co-expressed in tobacco leaves, and cLUC-RAE1 was co-expressed with STOP-1-nLUC without fluorescence (FIG. 3B). cLUC-RAE 1. DELTA.F was also co-expressed with STOP-1-nLUC and found to interact to produce fluorescence (FIG. 5B). The Split-LUC test results show that RAE1 can interact with STOP-1 in vivo, and that binding of functional RAE1 to STOP-1 promotes STOP-1 degradation, and that RAE1-1 muteins do not affect interaction with STOP-1, but rather affect the function of promoting STOP-1 degradation.
Further in vivo Co-IP experiments were performed, FLAG-tagged RAE1/RAE1-1/RAE1 DeltaF and HA-tagged STOP-1 were Co-expressed in Arabidopsis protoplasts and immunoprecipitated with FLAG antibodies. The results showed that FLAG-RAE1, FLAG-RAE1-1 and FLAG-RAE 1. DELTA.F each co-immunoprecipitated STOP-1-HA (FIG. 5C), and that wild-type RAE1 co-immunoprecipitated STOP-1-HA less, consistent with the Split-LUC test.
RAE1 was shown to be capable of interacting with STOP-1 protein by the in vitro Pull-Down test, tobacco Split-LUC test, and in vivo Co-IP test above.
EXAMPLE 5 investigation of RAE1 modulation of STOP-1 protein levels
To investigate RAE1 regulation of STOP-1 protein levels, pSTOP.sup.1 STOP-1-HA transgenic lines were constructed and pSTOP.sup.1 STOP-1-HA was crossed into the RAE.sup.1-1 mutant and then treated without or with Al to detect STOP-1 protein in the roots. Aluminum treatment allowed for accumulation of STOP-1 protein, and in RAE-1 mutants STOP-1 protein increased over wild-type, both in the absence and presence of aluminum, whereas overexpression of RAE1 promoted degradation of STOP-1 protein (FIG. 6).
To study STOP-1 degradation via the 26S proteasome pathway, pSTOP 1:1-HA transgenic lines were treated with the proteasome inhibitor MG132 for 1h to inhibit STOP-1 degradation and increase STOP-1 protein (FIG. 7A), and MG132 inhibited STOP-1 degradation and increase STOP-1 protein under either aluminum-free or aluminum-free conditions (FIG. 7B). pSTOP 1A STOP1-GUS transgenic line was further constructed, and was subjected to treatment without or with Al and MG132, which indicated that the aluminum treatment allowed accumulation of STOP1 protein, and that MG132 inhibited degradation of STOP1 under either no-aluminum or aluminum-containing conditions to increase STOP1 protein (FIGS. 7C, 7D).
To demonstrate that RAE1 mediates STOP-1 degradation, varying amounts of RAE1/RAE1-1/RAE1 DeltaF and STOP-1 were co-expressed in Arabidopsis protoplasts, with increasing RAE1 expression levels, promoting STOP-1 degradation (FIG. 8A), and conversely increasing RAE-1/RAE 1 DeltaF expression levels, without affecting STOP-1 protein expression levels (FIG. 8A). The protoplast expression system demonstrated that STOP-1 protein was ubiquitinated, whereas mutant proteins RAE-1 and RAE 1. DELTA.F were not able to mediate ubiquitination of STOP-1 (FIGS. 8B, 8C). The above shows that RAE1 mediates STOP-1 protein degradation by ubiquitination modification, whereas RAE-1 muteins and the removal of F-box domain proteins lose the function of mediating STOP-1 ubiquitination and degradation.
Expression of anti-aluminum toxin gene AtALMT1 and the like regulated by STOP-1 was up-regulated due to STOP-1 accumulation in mutant rae1, comparing the secretion of malic acid in wild-type and mutant rae. In the Al-free condition, there was little and no difference in the secretion of both malate (fig. 9A), both increased when Al treatment was performed, and the secretion of malate in mutants rae1-1 and rae1-2 was significantly improved over that in the wild type (fig. 9A). Consistent with this, increased malate secretion in mutant rae1 resulted in less accumulation of Al than the wild type in mutants rae1-1 and rae1-2 (fig. 9B). The resistance of wild type and mutant rae1 to Al toxin was further compared on aluminum-soaked plate medium, with the relative root lengths of mutants rae1-1 and rae1-2 being significantly longer than the wild type under 1mM Al treatment conditions (FIGS. 9C, 9D). The above shows that STOP-1 protein accumulation caused by RAE1 mutation greatly increases the resistance of Arabidopsis to Al toxin, while overexpression of RAE1 reduces the resistance of Arabidopsis to Al toxin.
Example 6 functional conservation of RAE1 homologous Gene
RAE1 has a homologous gene RAL1 (RAE 1 Like 1) in Arabidopsis thaliana, and expression of AtALMT1, pAtALMT1: LUC is up-regulated in RAL 1T-DNA knockout mutant RAL1, but up-regulated to a lower extent than in RAE1-1 (FIGS. 10A, 10B). pAtALMT.LUC expression was higher in RAE-1 RAL1 double mutants than in each single mutant, indicating that RAE1 was functionally redundant with RAL1 (FIG. 10B). Expression of RAL1 was also induced by aluminum toxicity, but the tissue sites where RAL1 was expressed in roots were different from that of RAE1 (fig. 10C, 10D). The tobacco Split-LUC assay demonstrated that RAL1 was able to interact with STOP-1 protein (FIG. 10E). As shown above, RAL1 has the same function as RAE1 for degrading STOP-1 protein, but RAE1 plays an important role in plant resistance to aluminum toxicity because it is not expressed in root tip meristems and elongation regions.
RAE1 and RAL1 have corresponding homologous genes in most monocots (rice, maize, barley, etc.) and dicots (Brassica cabbage, soybean, etc.). The homologous genes of RAE1 in rice are OsRAE1.1 and OsRAE1.2, the amino acid sequences of the two homologous genes are 97.5% similar, the OsRAE1.1 and OsRAE1.2 are expressed in root tips and root base parts, and the expression is induced by Al and regulated by ART1 (the genes homologous to STOP1 in rice) (FIGS. 11A and 11B). Tobacco Split-LUC experiments demonstrated that osrae1.1 was able to interact with ART1 protein (fig. 11C). The above shows that RAE1 is conserved in regulating the stability mechanism of plant anti-aluminum toxin transcription factor STOP1, and that the function deletion mutation of RAE1 homologous gene in crops is possible to improve the anti-aluminum toxin capability of crops.
All documents mentioned in this disclosure are incorporated by reference in this disclosure as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the application as defined in the appended claims.
Sequence listing
<110> Molecular plant science Excellent innovation center of China academy of sciences
<120> Gene for regulating and controlling anti-aluminum toxicity transcription factor STOP-1 protein and application thereof
<130> P2019-0269
<150> 201810339581X
<151> 2018-04-16
<160> 77
<170> PatentIn version 3.5
<210> 1
<211> 51
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 1
ggggacaagt ttgtacaaaa aagcaggctc ctggctcctt ttggttgtct a 51
<210> 2
<211> 54
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 2
ggggaccact ttgtacaaga aagctgggta acaccttttg atggtcactc agct 54
<210> 3
<211> 47
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 3
gacctgcagg catgcaagct ttgtttcagc atatttgcat gtttgat 47
<210> 4
<211> 45
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 4
cacctgtaat tcacacgtgg tgtgctacag aggaaagaag gtttg 45
<210> 5
<211> 45
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 5
ggtacccggg gatcctctag atttagggct tcaaactctt taccc 45
<210> 6
<211> 49
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 6
ggaacatcgt atgggtaaag cttgagacta gtatctgaaa cagactcac 49
<210> 7
<211> 45
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 7
ttggggccca acgttctcga gtttagggct tcaaactctt taccc 45
<210> 8
<211> 48
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 8
cggccgcaaa gtcgacgaat tcgagactag tatctgaaac agactcac 48
<210> 9
<211> 45
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 9
tccatggctg atatcggatc catgaagaag gttaaacaga ttcgt 45
<210> 10
<211> 39
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 10
gtggtggtgg tggtgctcga gaggcgccat ctcttccag 39
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<211> 46
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 11
tccgcgtgga tccccggaat tcatgaagaa ggttaaacag attcgt 46
<210> 12
<211> 42
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 12
gtcacgatgc ggccgctcga gttaaggcgc catctcttcc ag 42
<210> 13
<211> 45
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 13
tccgcgtgga tccccggaat tcatggaaac tgaagacgat ttgtg 45
<210> 14
<211> 46
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 14
gtcacgatgc ggccgctcga gttagagact agtatctgaa acagac 46
<210> 15
<211> 44
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 15
ggtctggtgc cacgcggatc catggaaact gaagacgatt tgtg 44
<210> 16
<211> 46
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 16
acttaagcat tatgcggccg cttagagact agtatctgaa acagac 46
<210> 17
<211> 45
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 17
gtacgcgtcc cggggcggta ccatgaagaa ggttaaacag attcg 45
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<211> 44
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 18
gaacgaaagc tctgcaggtc gacttaaggc gccatctctt ccag 44
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<211> 42
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
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gtacgcgtcc cggggcggta ccctcgatct taccttctgc cc 42
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<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
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gaacgaaagc tctgcaggtc gacttaaggc gccatctctt ccag 44
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<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
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acgagctcgg tacccgggat ccatggaaac tgaagacgat ttgtg 45
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<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
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gacgcgtacg agatctggtc gacgagacta gtatctgaaa cagact 46
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<211> 45
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 23
cttgctccgt ggatcctcta gaatggaaac tgaagacgat ttgtg 45
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<211> 45
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 24
gaacatcgta tgggtatcta gagagactag tatctgaaac agact 45
<210> 25
<211> 42
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 25
cttgctccgt ggatcctcta gaatgaagaa ggttaaacag at 42
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<211> 38
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
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gaacatcgta tgggtatcta gaaggcgcca tctcttcc 38
<210> 27
<211> 45
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
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cttgctccgt ggatcctcta gaatgctcga tcttaccttc tgccc 45
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<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 28
gaacatcgta tgggtatcta gaaggcgcca tctcttcc 38
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cttgctccgt ggatcctcta gaatggaaac tgaagacgat ttgtg 45
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<211> 45
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 30
tgtagtcaga aggcctggta ccgagactag tatctgaaac agact 45
<210> 31
<211> 42
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 31
cttgctccgt ggatcctcta gaatgaagaa ggttaaacag at 42
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<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 32
tgtagtcaga aggcctggta ccaggcgcca tctcttcc 38
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<211> 45
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 33
cttgctccgt ggatcctcta gaatgctcga tcttaccttc tgccc 45
<210> 34
<211> 38
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 34
tgtagtcaga aggcctggta ccaggcgcca tctcttcc 38
<210> 35
<211> 665
<212> PRT
<213> Arabidopsis thaliana (Arabidopsis thaliana)
<400> 35
Met Lys Lys Val Lys Gln Ile Arg Val Leu Lys Pro Phe Asp Leu Leu
1 5 10 15
Ser Glu Glu Leu Val Phe Ile Ile Leu Asp Leu Ile Ser Pro Asn Pro
20 25 30
Ser Asp Leu Lys Ser Phe Ser Leu Thr Cys Lys Ser Phe Tyr Gln Leu
35 40 45
Glu Ser Lys His Arg Gly Ser Leu Lys Pro Leu Arg Ser Asp Tyr Leu
50 55 60
Pro Arg Ile Leu Thr Arg Tyr Arg Asn Thr Thr Asp Leu Asp Leu Thr
65 70 75 80
Phe Cys Pro Arg Val Thr Asp Tyr Ala Leu Ser Val Val Gly Cys Leu
85 90 95
Ser Gly Pro Thr Leu Arg Ser Leu Asp Leu Ser Arg Ser Gly Ser Phe
100 105 110
Ser Ala Ala Gly Leu Leu Arg Leu Ala Leu Lys Cys Val Asn Leu Val
115 120 125
Glu Ile Asp Leu Ser Asn Ala Thr Glu Met Arg Asp Ala Asp Ala Ala
130 135 140
Val Val Ala Glu Ala Arg Ser Leu Glu Arg Leu Lys Leu Gly Arg Cys
145 150 155 160
Lys Met Leu Thr Asp Met Gly Ile Gly Cys Ile Ala Val Gly Cys Lys
165 170 175
Lys Leu Asn Thr Val Ser Leu Lys Trp Cys Val Gly Val Gly Asp Leu
180 185 190
Gly Val Gly Leu Leu Ala Val Lys Cys Lys Asp Ile Arg Thr Leu Asp
195 200 205
Leu Ser Tyr Leu Pro Ile Thr Gly Lys Cys Leu His Asp Ile Leu Lys
210 215 220
Leu Gln His Leu Glu Glu Leu Leu Leu Glu Gly Cys Phe Gly Val Asp
225 230 235 240
Asp Asp Ser Leu Lys Ser Leu Arg His Asp Cys Lys Ser Leu Lys Lys
245 250 255
Leu Asp Ala Ser Ser Cys Gln Asn Leu Thr His Arg Gly Leu Thr Ser
260 265 270
Leu Leu Ser Gly Ala Gly Tyr Leu Gln Arg Leu Asp Leu Ser His Cys
275 280 285
Ser Ser Val Ile Ser Leu Asp Phe Ala Ser Ser Leu Lys Lys Val Ser
290 295 300
Ala Leu Gln Ser Ile Arg Leu Asp Gly Cys Ser Val Thr Pro Asp Gly
305 310 315 320
Leu Lys Ala Ile Gly Thr Leu Cys Asn Ser Leu Lys Glu Val Ser Leu
325 330 335
Ser Lys Cys Val Ser Val Thr Asp Glu Gly Leu Ser Ser Leu Val Met
340 345 350
Lys Leu Lys Asp Leu Arg Lys Leu Asp Ile Thr Cys Cys Arg Lys Leu
355 360 365
Ser Arg Val Ser Ile Thr Gln Ile Ala Asn Ser Cys Pro Leu Leu Val
370 375 380
Ser Leu Lys Met Glu Ser Cys Ser Leu Val Ser Arg Glu Ala Phe Trp
385 390 395 400
Leu Ile Gly Gln Lys Cys Arg Leu Leu Glu Glu Leu Asp Leu Thr Asp
405 410 415
Asn Glu Ile Asp Asp Glu Gly Leu Lys Ser Ile Ser Ser Cys Leu Ser
420 425 430
Leu Ser Ser Leu Lys Leu Gly Ile Cys Leu Asn Ile Thr Asp Lys Gly
435 440 445
Leu Ser Tyr Ile Gly Met Gly Cys Ser Asn Leu Arg Glu Leu Asp Leu
450 455 460
Tyr Arg Ser Val Gly Ile Thr Asp Val Gly Ile Ser Thr Ile Ala Gln
465 470 475 480
Gly Cys Ile His Leu Glu Thr Ile Asn Ile Ser Tyr Cys Gln Asp Ile
485 490 495
Thr Asp Lys Ser Leu Val Ser Leu Ser Lys Cys Ser Leu Leu Gln Thr
500 505 510
Phe Glu Ser Arg Gly Cys Pro Asn Ile Thr Ser Gln Gly Leu Ala Ala
515 520 525
Ile Ala Val Arg Cys Lys Arg Leu Ala Lys Val Asp Leu Lys Lys Cys
530 535 540
Pro Ser Ile Asn Asp Ala Gly Leu Leu Ala Leu Ala His Phe Ser Gln
545 550 555 560
Asn Leu Lys Gln Ile Asn Val Ser Asp Thr Ala Val Thr Glu Val Gly
565 570 575
Leu Leu Ser Leu Ala Asn Ile Gly Cys Leu Gln Asn Ile Ala Val Val
580 585 590
Asn Ser Ser Gly Leu Arg Pro Ser Gly Val Ala Ala Ala Leu Leu Gly
595 600 605
Cys Gly Gly Leu Arg Lys Ala Lys Leu His Ala Ser Leu Arg Ser Leu
610 615 620
Leu Pro Leu Ser Leu Ile His His Leu Glu Ala Arg Gly Cys Ala Phe
625 630 635 640
Leu Trp Lys Asp Asn Thr Leu Gln Ala Glu Leu Asp Pro Lys Tyr Trp
645 650 655
Lys Gln Gln Leu Glu Glu Met Ala Pro
660 665
<210> 36
<211> 3761
<212> DNA
<213> Arabidopsis thaliana (Arabidopsis thaliana)
<400> 36
gggttttttc caaaatcctc aaagagagac tatacttctc tcggatcctt cctgatttca 60
aaatttcttc aacccaccag cagcagcagc agatgaagaa ggttaaacag attcgtgtct 120
taaagccttt cgatcttctc tcggaagagc tcgtctttat catccttgac ctcatctctc 180
ccaacccttc cgatctcaaa tccttctctc ttacttgcaa atctttctac cagctcgagt 240
ccaagcaccg cggatcctta aaacctctcc gctccgacta tctccctcgc atcttgactc 300
gttaccggaa caccaccgat ctcgatctta ccttctgccc gcgtgtcact gactacgcgc 360
ttagcgtcgt tggctgtctc tccggaccta cgcttcgctc cctcgacctc tcgcgctctg 420
gctccttctc cgccgcggga ctactgcgat tggccctcaa atgtgtcaat ttagtcgaga 480
ttgacctgtc caatgcgacg gagatgagag acgccgatgc tgcggtggtg gcggaggcga 540
ggagtctgga gaggctgaag ctgggcagat gcaagatgct gacggacatg ggaatcggat 600
gtatagcagt tggatgtaag aagctcaata cggttagctt gaaatggtgt gttggcgtcg 660
gagatttagg ggttggcttg cttgccgtca aatgcaagga cattcgcacc ttagacctct 720
cctacttgcc ggtaattaac ttctctcttc acattttctt aatccgcaga gccaaatttc 780
gattaatatg gtaagtttgt cgaattggaa ttatttctaa atttgttctt cctgagacct 840
gttggttgat ttttcaagtt aacaacaagt tttggttgaa cttttggtct ttcaacatct 900
gtggatcatt atctatctga attcgacact ttaagtgcca tatgtttgga ttatatcttc 960
agaggagctg gaaattcaaa cctattcttt tgttccgtat tctacagatc acaggaaagt 1020
gtttacatga cattctgaaa cttcaacacc ttgaagaact tcttctagaa gggtgctttg 1080
gagtagatga tgacagtctt aaatcactca gacatgattg caagtcattg aaggtaacct 1140
ctattccaag aaatttgttt gattcttata catgagatca cataatagtt ttggaatcga 1200
cttgacagac aaacttttta gttgatattt tagaacagaa aacttggtat tctcagaata 1260
tggcttggag aagatgttac gcttcttctt gctcgctgca tttattctgg aaacatttaa 1320
cttttatgag agaagatgta taaacaggtt tcatgttttc ttagactcag aaagctattt 1380
atcattgaaa gtctaatgct ggaagactat atagttgatt gttttcatca tcatcttatc 1440
catagacatt tgagatttta cttaccaaaa cctgtgttct gcagaagctt gatgcatcca 1500
gctgccagaa tttaactcat agaggtttaa cctcactttt aagcggggca ggatatcttc 1560
agcgacttga tctatcacac tgttcttctg taagttctcc gttttctcat tatcatcaga 1620
acacatgctt tccttgcttt gttctctggt tatggcctgt atgtaagtat ttgctaaatg 1680
gctgagtcaa cttagtttgg cactaaaact ctttagtagt agcaaaacca gtaattgcac 1740
ataccaccga atagaccatt tgtgtcatgg atcgatagca gtacacagac ccaccaagtt 1800
ctatgatgcc ttcttatatg aggtttattt actgatttag gtgatatcat tggattttgc 1860
gagtagctta aagaaggttt cagcattaca gtcgatcagg ttggatggtt gttctgttac 1920
gcctgatggt ttgaaggcga taggcacatt gtgcaattcc ctgaaagagg ttagcctaag 1980
caaatgcgtg agcgtaactg atgaaggtct ttcttctcta gtaatgaaac tgaaagacct 2040
cagaaaactt gacatcacat gttgccggaa actaagtaga gtttcaatca cccaaatcgc 2100
caattcgtgt cctttactag tctctttgaa gatggagtct tgttctcttg tttccagaga 2160
agccttttgg ttgatcggac aaaagtgtcg gctacttgaa gagcttgact taactgacaa 2220
cgagattgat gatgaaggtt ctttattttc ttcagatcaa ttcttagaac cgtggttttt 2280
ggattatacc ttatcttgtt agtctttcct cgtatgatgc aggactgaaa tccatatcta 2340
gttgtctgag tctttcctcg ttaaagctgg gaatttgtct taacataaca gacaaagggc 2400
tctcgtacat cgggatgggc tgttcaaatc tccgtgaact tgatctctat aggtatctcg 2460
cgtagaaatt tatttttcag tcgggtcaat aataaaattc tccgttctca atctagtctt 2520
tatctactcc aggtcagtgg gaataacaga cgtaggcatc tccacaattg cccaaggctg 2580
cattcatctg gaaacaataa acatttcata ctgccaagac ataacagaca agtccctggt 2640
ttcattgtcc aaatgctcgt tgttacaaac attcgagagc aggggatgtc ctaacatcac 2700
gtcccaagga cttgcagcca ttgctgttcg gtgcaagcga ctcgccaagg ttgacttgaa 2760
gaagtgccca tccatcaatg atgcggggtt gctcgctctg gctcacttct ctcagaatct 2820
caaacaggta ataaaccgtt gaatccttca ctgacagctg aaaaaactac aatatactgg 2880
ttgaatctgt tatctgattg cgatccccat gttgcagata aacgtgtcag acacagctgt 2940
gactgaagtg ggacttctct ccctagccaa catagggtgt ttacagaaca tagcggttgt 3000
gaactcgagt ggtttaagac cgagcggagt agcagcagca ttgctggggt gtggaggatt 3060
aaggaaagcg aaactacatg cgtccctaag atcactgctt cctctttctc taatccacca 3120
cttggaagct cgtggttgtg cgttcctctg gaaagacaat acccttcagg tgaatatata 3180
tacaagtata tgtagattac tacttactac actaaatagc cgcaaaaggt tggtgtagct 3240
ttttatgatt acaaatgaaa atcttagagc gaaggagtct gacatatgga aatgtaaatg 3300
atgtgggaac aggcggagtt agatcccaag tactggaagc aacagctgga agagatggcg 3360
ccttaaatta aaagtgagaa gaaacattct gaaatgggaa agaggagttc ctatgggagc 3420
cagcagacag gccctgtttt gggcccagtg tggattagct ggaacaattt tggtctcttt 3480
tgtgttgttg ttgacggcgc gtgttctaac aaccccacac aaccttacat tataagtcta 3540
gtcacatggt gggtgacatg gtaccgttgt atatgtagtt tttgtttctt tttttttttt 3600
ttttggttgg ggagccgaaa ttaacggacg taacagagtc acaaggggat gccttctctg 3660
tgcctttttg actttttcct cctttctttt ttttctgtaa tcatatgagt tttatgtaat 3720
ttaatgcctc atcaacttgc ctggataggg ccggcctttc c 3761
<210> 37
<211> 677
<212> PRT
<213> Rice (Oryza sativa)
<400> 37
Met Ala Ser Pro Met Pro Thr Pro Thr His Arg Val Lys Arg Arg Arg
1 5 10 15
Leu Asp Leu Ser Pro Pro Pro His Leu Asn Asp Leu Ala Asp Glu Leu
20 25 30
Leu Phe Leu Ile Leu Asp Arg Ala Ala Ala His Asp Pro Arg Ala Leu
35 40 45
Lys Ser Phe Ser Leu Val Ser Arg Ala Cys His Ala Ala Glu Ser Arg
50 55 60
His Arg Arg Val Leu Arg Pro Phe Arg Pro Asp Leu Leu Pro Ala Ala
65 70 75 80
Leu Ala Arg Tyr Pro Ala Leu Ser Arg Leu Asp Leu Ser Leu Cys Pro
85 90 95
Arg Leu Pro Asp Ala Ala Leu Ala Ala Leu Pro Ala Ala Pro Ser Val
100 105 110
Ser Ala Val Asp Leu Ser Arg Ser Arg Gly Phe Gly Ala Ala Gly Leu
115 120 125
Ala Ala Leu Val Ala Ala Cys Pro Asn Leu Thr Asp Leu Asp Leu Ser
130 135 140
Asn Gly Leu Asp Leu Gly Asp Ala Ala Ala Ala Glu Val Ala Lys Ala
145 150 155 160
Arg Arg Leu Gln Arg Leu Ser Leu Ser Arg Cys Lys Arg Ile Thr Asp
165 170 175
Met Gly Leu Gly Cys Ile Ala Val Gly Cys Pro Asp Leu Arg Glu Leu
180 185 190
Ser Leu Lys Trp Cys Ile Gly Val Thr His Leu Gly Leu Asp Leu Leu
195 200 205
Ala Leu Lys Cys Asn Lys Leu Asn Ile Leu Asp Leu Ser Tyr Thr Met
210 215 220
Ile Val Lys Lys Cys Phe Pro Ala Ile Met Lys Leu Gln Ser Leu Gln
225 230 235 240
Val Leu Leu Leu Val Gly Cys Asn Gly Ile Asp Asp Asp Ala Leu Thr
245 250 255
Ser Leu Asp Gln Glu Cys Ser Lys Ser Leu Gln Val Leu Asp Met Ser
260 265 270
Asn Tyr Tyr Asn Val Thr His Val Gly Val Leu Ser Ile Val Lys Ala
275 280 285
Met Pro Asn Leu Leu Glu Leu Asn Leu Ser Tyr Cys Ser Pro Val Thr
290 295 300
Pro Ser Met Ser Ser Ser Phe Glu Met Ile His Lys Leu Gln Thr Leu
305 310 315 320
Lys Leu Asp Gly Cys Gln Phe Met Asp Asp Gly Leu Lys Ser Ile Gly
325 330 335
Lys Ser Cys Val Ser Leu Arg Glu Leu Ser Leu Ser Lys Cys Ser Gly
340 345 350
Val Thr Asp Thr Asp Leu Ser Phe Val Val Pro Arg Leu Lys Asn Leu
355 360 365
Leu Lys Leu Asp Val Thr Cys Cys Arg Lys Ile Thr Asp Val Ser Leu
370 375 380
Ala Ala Ile Thr Thr Ser Cys Pro Ser Leu Ile Ser Leu Arg Met Glu
385 390 395 400
Ser Cys Ser Leu Val Ser Ser Lys Gly Leu Gln Leu Ile Gly Arg Arg
405 410 415
Cys Thr His Leu Glu Glu Leu Asp Leu Thr Asp Thr Asp Leu Asp Asp
420 425 430
Glu Gly Leu Lys Ala Leu Ser Gly Cys Ser Lys Leu Ser Ser Leu Lys
435 440 445
Ile Gly Ile Cys Leu Arg Ile Thr Asp Glu Gly Leu Arg His Val Ser
450 455 460
Lys Ser Cys Pro Asp Leu Arg Asp Ile Asp Leu Tyr Arg Ser Gly Ala
465 470 475 480
Ile Ser Asp Glu Gly Val Thr His Ile Ala Gln Gly Cys Pro Met Leu
485 490 495
Glu Ser Ile Asn Leu Ser Tyr Cys Thr Lys Leu Thr Asp Cys Ser Leu
500 505 510
Arg Ser Leu Ser Lys Cys Ile Lys Leu Asn Thr Leu Glu Ile Arg Gly
515 520 525
Cys Pro Met Val Ser Ser Ala Gly Leu Ser Glu Ile Ala Thr Gly Cys
530 535 540
Arg Leu Leu Ser Lys Leu Asp Ile Lys Lys Cys Phe Glu Ile Asn Asp
545 550 555 560
Met Gly Met Ile Phe Leu Ser Gln Phe Ser His Asn Leu Arg Gln Ile
565 570 575
Asn Leu Ser Tyr Cys Ser Val Thr Asp Ile Gly Leu Ile Ser Leu Ser
580 585 590
Ser Ile Cys Gly Leu Gln Asn Met Thr Ile Val His Leu Ala Gly Val
595 600 605
Thr Pro Asn Gly Leu Ile Ala Ala Leu Met Val Cys Gly Leu Arg Lys
610 615 620
Val Lys Leu His Glu Ala Phe Lys Ser Met Val Pro Ser His Met Leu
625 630 635 640
Lys Val Val Glu Ala Arg Gly Cys Leu Phe Gln Trp Ile Asn Lys Pro
645 650 655
Tyr Gln Val Ala Val Glu Pro Cys Asp Val Trp Lys Gln Gln Ser Gln
660 665 670
Asp Leu Leu Val Gln
675
<210> 38
<211> 677
<212> PRT
<213> Rice (Oryza sativa)
<400> 38
Met Ala Ser Pro Ala Pro Thr His His Ala Lys Arg Arg Arg Leu Ala
1 5 10 15
Leu Pro Pro Pro Pro Pro Pro His Leu Asn Asp Leu Ala Asp Glu Leu
20 25 30
Leu Phe Leu Ile Leu Asp Arg Ala Ala Ala His Asp Pro Arg Ala Leu
35 40 45
Lys Ser Phe Ser Leu Val Ser Arg Ala Cys His Ala Ala Glu Ser Arg
50 55 60
His Arg Arg Val Leu Arg Pro Phe Arg Pro Asp Leu Leu Pro Ala Ala
65 70 75 80
Leu Ala Arg Tyr Pro Ala Ile Ser His Leu Asp Leu Ser Leu Cys Pro
85 90 95
Arg Leu Pro Asp Ala Ala Leu Ala Ala Leu Pro Ala Ala Pro Phe Val
100 105 110
Ser Ala Val Asp Leu Ser Arg Ser Arg Gly Phe Gly Ala Ala Gly Leu
115 120 125
Ala Ala Leu Val Ala Ala Phe Pro Asn Leu Thr Asp Leu Asp Leu Ser
130 135 140
Asn Gly Leu Asp Leu Gly Asp Ala Ala Ala Ala Glu Val Ala Lys Ala
145 150 155 160
Arg Arg Leu Gln Arg Leu Ser Leu Ser Arg Cys Lys Arg Ile Thr Asp
165 170 175
Met Gly Leu Gly Cys Ile Ala Val Gly Cys Pro Asp Leu Arg Glu Leu
180 185 190
Ser Leu Lys Trp Cys Ile Gly Val Thr His Leu Gly Leu Asp Leu Leu
195 200 205
Ala Leu Lys Cys Asn Lys Leu Asn Ile Leu Asp Leu Ser Tyr Thr Met
210 215 220
Ile Val Lys Lys Cys Phe Pro Ala Ile Met Lys Leu Gln Asn Leu Gln
225 230 235 240
Val Leu Leu Leu Val Gly Cys Asn Gly Ile Asp Asp Asp Ala Leu Thr
245 250 255
Ser Leu Asp Gln Glu Cys Ser Lys Ser Leu Gln Val Leu Asp Met Ser
260 265 270
Asn Ser Tyr Asn Val Thr His Val Gly Val Leu Ser Ile Val Lys Ala
275 280 285
Met Pro Asn Leu Leu Glu Leu Asn Leu Ser Tyr Cys Ser Pro Val Thr
290 295 300
Pro Ser Met Ser Ser Ser Phe Glu Met Ile His Lys Leu Gln Lys Leu
305 310 315 320
Lys Leu Asp Gly Cys Gln Phe Met Asp Asp Gly Leu Lys Ser Ile Gly
325 330 335
Lys Ser Cys Val Ser Leu Arg Glu Leu Ser Leu Ser Lys Cys Ser Gly
340 345 350
Val Thr Asp Thr Asp Leu Ser Phe Val Val Pro Arg Leu Lys Asn Leu
355 360 365
Leu Lys Leu Asp Val Thr Cys Cys Arg Lys Ile Thr Asp Val Ser Leu
370 375 380
Ala Ala Ile Thr Thr Ser Cys Pro Ser Leu Ile Ser Leu Arg Met Glu
385 390 395 400
Ser Cys Ser Leu Val Ser Ser Lys Gly Leu Gln Leu Ile Gly Arg Arg
405 410 415
Cys Thr His Leu Glu Glu Leu Asp Leu Thr Asp Thr Asp Leu Asp Asp
420 425 430
Glu Gly Leu Lys Ala Leu Ser Gly Cys Ser Lys Leu Ser Ser Leu Lys
435 440 445
Ile Gly Ile Cys Leu Arg Ile Thr Asp Glu Gly Leu Arg His Val Ser
450 455 460
Lys Ser Cys Pro Asp Leu Arg Asp Ile Asp Leu Tyr Arg Ser Gly Ala
465 470 475 480
Ile Ser Asp Glu Gly Val Thr His Ile Ala Gln Gly Cys Pro Met Leu
485 490 495
Glu Ser Ile Asn Met Ser Tyr Cys Thr Lys Leu Thr Asp Cys Ser Leu
500 505 510
Arg Ser Leu Ser Lys Cys Ile Lys Leu Asn Thr Leu Glu Ile Arg Gly
515 520 525
Cys Pro Met Val Ser Ser Ala Gly Leu Ser Glu Ile Ala Thr Gly Cys
530 535 540
Arg Leu Leu Ser Lys Leu Asp Ile Lys Lys Cys Phe Glu Ile Asn Asp
545 550 555 560
Met Gly Met Ile Phe Leu Ser Gln Phe Ser His Asn Leu Arg Gln Ile
565 570 575
Asn Leu Ser Tyr Cys Ser Val Thr Asp Ile Gly Leu Ile Ser Leu Ser
580 585 590
Ser Ile Cys Gly Leu Gln Asn Met Thr Ile Val His Leu Ala Gly Val
595 600 605
Thr Pro Asn Gly Leu Ile Ala Ala Leu Met Val Cys Gly Leu Arg Lys
610 615 620
Val Lys Leu His Glu Ala Phe Lys Ser Met Val Pro Ser His Met Leu
625 630 635 640
Lys Val Val Glu Ala Arg Gly Cys Leu Phe Gln Trp Ile Asn Lys Pro
645 650 655
Tyr Gln Val Ala Val Glu Pro Cys Asp Val Trp Lys Gln Gln Ser Gln
660 665 670
Asp Leu Leu Val Gln
675
<210> 39
<211> 692
<212> PRT
<213> Corn (Zea mays)
<400> 39
Met Ala Met Ala Ala Gln Gln His Arg His His Lys Arg Arg Arg Ile
1 5 10 15
Ala Leu Ser Pro Ser Pro Ser Pro Ser Leu Ala Pro Ile Pro Gly Ala
20 25 30
Pro Thr Pro Pro Leu Asp Ser Leu Ala Asp Glu Leu Leu Phe Leu Val
35 40 45
Leu Asp Arg Val Ala Gln Ala Asp Pro Arg Ala Leu Lys Ser Phe Ala
50 55 60
Leu Ala Ser Arg Ala Cys His Ala Ala Glu Ser Arg His Arg Arg Thr
65 70 75 80
Leu Arg Pro Leu Arg Ala Asp Leu Leu Pro Ala Ala Leu Ala Arg Tyr
85 90 95
Pro Ser Ala Thr Arg Leu Asp Leu Thr Leu Cys Ala Arg Val Pro Asp
100 105 110
Ala Ala Leu Ala Ser Ala Ala Val Ser Gly Ser Ser Ala Leu Arg Ala
115 120 125
Val Asp Leu Ser Arg Ser Arg Gly Phe Gly Ala Ala Gly Val Ala Ala
130 135 140
Leu Ala Ala Ala Cys Pro Asp Leu Ala Asp Leu Asp Leu Ser Asn Gly
145 150 155 160
Val His Leu Gly Asp Ala Ala Ala Ala Glu Val Ala Arg Ala Arg Ala
165 170 175
Leu Arg Arg Leu Ser Leu Val Arg Trp Lys Pro Leu Thr Asp Met Gly
180 185 190
Leu Gly Cys Val Ala Val Gly Cys Thr Glu Leu Lys Asp Leu Ser Leu
195 200 205
Lys Trp Cys Leu Gly Leu Thr Asp Leu Gly Ile Gln Leu Leu Ala Leu
210 215 220
Lys Cys Arg Lys Leu Thr Ser Leu Asp Leu Ser Tyr Thr Met Ile Thr
225 230 235 240
Lys Asp Ser Leu Pro Ser Ile Met Lys Leu Pro Asn Leu Gln Glu Leu
245 250 255
Thr Leu Val Gly Cys Ile Gly Ile Asp Asp Gly Ala Leu Val Ser Leu
260 265 270
Glu Arg Glu Cys Ser Lys Ser Leu Gln Val Leu Asp Met Ser Gln Cys
275 280 285
Gln Asn Ile Thr Asp Val Gly Val Ser Ser Ile Leu Lys Ser Val Pro
290 295 300
Asn Leu Leu Glu Leu Asp Leu Ser Tyr Cys Cys Pro Val Thr Pro Ser
305 310 315 320
Met Val Arg Asn Phe Gln Lys Leu Pro Lys Leu Gln Ala Leu Lys Leu
325 330 335
Glu Gly Cys Lys Phe Met Ala Asn Gly Leu Lys Ala Ile Gly Thr Ser
340 345 350
Cys Val Ser Leu Arg Glu Leu Ser Leu Ser Lys Ser Ser Gly Val Thr
355 360 365
Asp Thr Glu Leu Ser Phe Val Val Ser Arg Leu Lys Asn Leu Leu Lys
370 375 380
Leu Asp Ile Thr Cys Cys Arg Ser Ile Thr Asp Val Ser Leu Ala Ala
385 390 395 400
Ile Thr Ser Ser Cys Thr Ser Leu Ile Ser Leu Arg Met Glu Ser Cys
405 410 415
Ser His Val Ser Ser Gly Ala Leu Gln Leu Ile Gly Lys His Cys Ser
420 425 430
His Leu Glu Glu Leu Asp Leu Thr Asp Ser Asp Leu Asp Asp Glu Gly
435 440 445
Leu Lys Ala Leu Ala Arg Cys Ser Glu Leu Ser Ser Leu Lys Ile Gly
450 455 460
Ile Cys Leu Lys Ile Ser Asp Glu Gly Leu Ser His Ile Gly Arg Ser
465 470 475 480
Cys Pro Lys Leu Arg Glu Ile Asp Leu Tyr Arg Cys Gly Val Ile Ser
485 490 495
Asp Asp Gly Ile Ile Gln Ile Ala Gln Gly Cys Pro Met Leu Glu Ser
500 505 510
Ile Asn Leu Ser Tyr Cys Thr Glu Ile Thr Asp Arg Ser Leu Ile Ser
515 520 525
Leu Ser Lys Cys Ala Lys Leu Asn Thr Leu Glu Ile Arg Gly Cys Pro
530 535 540
Ser Val Ser Ser Ile Gly Leu Ser Glu Ile Ala Met Gly Cys Arg Leu
545 550 555 560
Leu Ser Lys Leu Asp Ile Lys Lys Cys Phe Gly Ile Asn Asp Val Gly
565 570 575
Met Leu Tyr Leu Ser Gln Phe Ala His Ser Leu Arg Gln Ile Asn Leu
580 585 590
Ser Tyr Cys Ser Val Thr Asp Val Gly Leu Leu Ser Leu Ser Ser Ile
595 600 605
Ser Gly Leu Gln Asn Met Thr Ile Val His Leu Ala Gly Ile Thr Pro
610 615 620
Asn Gly Leu Thr Ala Thr Leu Met Val Cys Gly Gly Leu Thr Lys Val
625 630 635 640
Lys Leu His Glu Ala Phe Arg Ser Met Met Pro Pro His Thr Ile Lys
645 650 655
Asn Val Glu Ala Arg Gly Cys Val Phe Gln Trp Ile Asp Lys Pro Phe
660 665 670
Lys Val Glu Val Glu Pro Cys Asp Val Trp Lys Gln Gln Ser Gln Asp
675 680 685
Val Leu Val Arg
690
<210> 40
<211> 454
<212> PRT
<213> Barley (Hordeum vulgare)
<400> 40
Ile Ser Lys Asp Cys Leu Pro Ala Ile Met Glu Leu Pro Asn Leu Glu
1 5 10 15
Val Leu Ala Leu Val Gly Cys Val Gly Ile Asp Asp Asp Ala Leu Ser
20 25 30
Gly Leu Glu Asn Glu Ser Ser Lys Ser Leu Arg Val Leu Asp Met Ser
35 40 45
Thr Cys Arg Asn Val Thr His Thr Gly Val Ser Ser Val Val Lys Ala
50 55 60
Leu Pro Asn Leu Leu Glu Leu Asn Leu Ser Tyr Cys Cys Asn Val Thr
65 70 75 80
Ala Ser Met Gly Lys Cys Phe Gln Met Leu Pro Lys Leu Gln Thr Leu
85 90 95
Lys Leu Glu Gly Cys Lys Phe Met Ala Asp Gly Leu Lys His Ile Gly
100 105 110
Ile Ser Cys Val Ser Leu Arg Glu Leu Ser Leu Ser Lys Cys Ser Gly
115 120 125
Val Thr Asp Thr Asp Leu Ser Phe Val Val Ser Arg Leu Lys Asn Leu
130 135 140
Leu Lys Leu Asp Ile Thr Cys Asn Arg Asn Ile Thr Asp Val Ser Leu
145 150 155 160
Ala Ala Ile Thr Ser Ser Cys His Ser Leu Ile Ser Leu Arg Ile Glu
165 170 175
Ser Cys Ser His Phe Ser Ser Glu Gly Leu Arg Leu Ile Gly Lys Arg
180 185 190
Cys Cys His Leu Glu Glu Leu Asp Ile Thr Asp Ser Asp Leu Asp Asp
195 200 205
Glu Gly Leu Lys Ala Leu Ser Gly Cys Ser Lys Leu Ser Ser Leu Lys
210 215 220
Ile Gly Ile Cys Met Arg Ile Ser Asp Gln Gly Leu Ile His Ile Gly
225 230 235 240
Lys Ser Cys Pro Glu Leu Arg Asp Ile Asp Leu Tyr Arg Ser Gly Gly
245 250 255
Ile Ser Asp Glu Gly Val Thr Gln Ile Ala Gln Gly Cys Pro Met Leu
260 265 270
Glu Ser Ile Asn Leu Ser Tyr Cys Thr Glu Ile Thr Asp Val Ser Leu
275 280 285
Met Ser Leu Ser Lys Cys Ala Lys Leu Asn Thr Leu Glu Ile Arg Gly
290 295 300
Cys Pro Ser Ile Ser Ser Ala Gly Leu Ser Glu Ile Ala Ile Gly Cys
305 310 315 320
Arg Leu Leu Ala Lys Leu Asp Val Lys Lys Cys Phe Ala Ile Asn Asp
325 330 335
Val Gly Met Phe Phe Leu Ser Gln Phe Ser His Ser Leu Arg Gln Ile
340 345 350
Asn Leu Ser Tyr Cys Ser Val Thr Asp Ile Gly Leu Leu Ser Leu Ser
355 360 365
Ser Ile Cys Gly Leu Gln Asn Met Thr Ile Val His Leu Ala Gly Ile
370 375 380
Thr Pro Asn Gly Leu Leu Ala Ala Leu Met Val Ser Gly Gly Leu Thr
385 390 395 400
Arg Val Lys Leu His Ala Ala Phe Arg Ser Met Met Pro Pro His Met
405 410 415
Leu Lys Val Val Glu Ala Arg Gly Cys Ala Phe Gln Trp Ile Asp Lys
420 425 430
Pro Phe Lys Val Glu Gln Glu Arg Cys Asp Ile Trp Gln Gln Gln Ser
435 440 445
Arg Asp Val Leu Val Arg
450
<210> 41
<211> 668
<212> PRT
<213> Brassica (Brassica rapa)
<400> 41
Met Lys Lys Ala Lys Gln Ile Gln His Met Ile Ser Lys Pro Phe Asp
1 5 10 15
Leu Leu Ser Glu Glu Leu Val Phe Ile Ile Leu Asp Leu Val Ala Gln
20 25 30
Asn Pro Ser Asp Leu Lys Ser Phe Ser Leu Thr Cys Lys Trp Phe Tyr
35 40 45
Gln Val Glu Ala Arg His Arg Arg Ser Leu Lys Pro Leu Arg Ala Glu
50 55 60
Tyr Leu Pro Arg Ile Leu Thr Arg Tyr Arg Asn Thr Ala Asp Leu Asp
65 70 75 80
Leu Ser Phe Cys Pro Arg Val Thr Asp Tyr Ala Leu Ser Val Val Gly
85 90 95
Cys Leu Ser Gly Pro Thr Leu Arg Ser Val Asp Leu Ser Arg Ser Phe
100 105 110
Ser Phe Ser Ala Ala Gly Leu Leu Arg Leu Ala Val Lys Cys Val Ser
115 120 125
Leu Val Glu Ile Asp Leu Ser Asn Ala Thr Glu Met Arg Asp Ala Ala
130 135 140
Ala Ala Val Val Ala Glu Ala Lys Ser Leu Glu Arg Leu Lys Leu Gly
145 150 155 160
Arg Cys Lys Lys Leu Thr Asp Met Gly Ile Gly Cys Ile Ala Val Gly
165 170 175
Cys Arg Lys Leu Lys Arg Val Ser Leu Lys Trp Cys Val Gly Val Gly
180 185 190
Asp Leu Gly Val Gly Leu Leu Ala Val Lys Cys Lys Asp Ile Arg Ser
195 200 205
Leu Asp Leu Ser Tyr Leu Pro Ile Thr Gly Lys Cys Leu His Asp Val
210 215 220
Leu Lys Leu Gln His Leu Glu Glu Leu Leu Leu Gln Gly Cys Phe Gly
225 230 235 240
Val Asp Asp Asp Ser Leu Lys Ser Leu Thr His His Cys Asn Ser Leu
245 250 255
Lys Asn Leu Asp Ala Ser Ser Cys Gln Asn Leu Thr Gln Arg Gly Leu
260 265 270
Thr Ser Leu Leu Ser Gly Ala Gly Cys Leu Glu Arg Leu Asp Leu Ala
275 280 285
His Ser Ser Ser Val Ile Ser Leu Asp Phe Ala Ser Ser Leu Asn Lys
290 295 300
Val Ser Ser Ala Leu Gln Ser Ile Arg Leu Asp Gly Cys Ala Val Thr
305 310 315 320
Cys Asp Gly Leu Lys Ala Ile Gly Thr Leu Cys Ile Ser Leu Arg Glu
325 330 335
Val Ser Leu Ser Lys Cys Val Thr Val Thr Asp Glu Gly Leu Ser Cys
340 345 350
Leu Val Met Lys Leu Lys Asp Leu Arg Lys Leu Asp Ile Thr Cys Cys
355 360 365
Arg Lys Leu Thr Gly Val Ser Ile Thr Gln Val Ala Ser Ser Cys Pro
370 375 380
Leu Leu Val Ser Leu Lys Met Glu Ser Cys Ser Leu Val Ser Arg Asp
385 390 395 400
Ala Phe Trp Leu Ile Gly His Lys Cys Arg Leu Leu Glu Glu Leu Asp
405 410 415
Phe Thr Asp Asn Glu Ile Asp Asp Glu Gly Leu Lys Ser Ile Ser Ser
420 425 430
Cys Arg Ser Leu Ser Ser Leu Lys Leu Gly Ile Cys Leu Asn Ile Thr
435 440 445
Asp Arg Gly Leu Ser Tyr Ile Gly Met Gly Cys Ser Asn Leu Arg Glu
450 455 460
Leu Asp Leu Tyr Arg Ser Val Gly Ile Thr Asp Val Gly Ile Ser Ser
465 470 475 480
Ile Ala Gln Gly Cys Cys His Leu Glu Thr Ile Asn Ile Ser Tyr Cys
485 490 495
Lys Asp Ile Thr Asp Lys Ser Leu Val Ser Leu Ser Lys Cys Ser Met
500 505 510
Leu Gln Thr Phe Glu Ser Arg Gly Cys Pro His Ile Thr Cys Gln Gly
515 520 525
Leu Ala Ala Ile Ala Val Arg Cys Lys Arg Leu Ser Lys Leu Asp Leu
530 535 540
Lys Lys Cys Pro Phe Ile Asn Asp Ser Gly Leu Leu Thr Leu Ala His
545 550 555 560
Phe Ser Gln Gly Leu Lys Gln Ile Ser Val Ser Glu Thr Gly Val Thr
565 570 575
Asp Val Gly Leu Val Ser Leu Ala Asn Ile Gly Cys Leu Gln Asn Ile
580 585 590
Ala Ala Val Asn Thr Arg Gly Leu Ser Pro Ser Gly Val Ala Ala Ala
595 600 605
Leu Val Gly Cys Gly Gly Leu Arg Lys Val Lys Leu His Ala Ser Leu
610 615 620
Arg Ser Leu Leu Pro Ser Ser Leu Ile Asn His Met Glu Ala Arg Gly
625 630 635 640
Cys Ser Phe Leu Trp Lys Asp Tyr Asn Asn His Asn Asn Ser Asn Thr
645 650 655
Leu Gln Ala Glu Leu Asp Pro Lys Tyr Trp Lys Val
660 665
<210> 42
<211> 708
<212> PRT
<213> Soybean (Glycine max)
<400> 42
Met Ser Arg Ile Tyr Ser Ser Asn Ile Glu Thr Lys Gln Thr Asn Pro
1 5 10 15
Ser Leu Leu Ser Pro Gln Leu Phe Ile Ile Asn Leu Gln Asn Asp Gln
20 25 30
Pro Asn Ala Lys Arg Arg Thr Met Lys Lys Gln Lys Leu Ser Glu Pro
35 40 45
Gln Asn Asp Thr Thr Asn Pro Phe Glu Val Leu Ser Glu Glu Leu Met
50 55 60
Phe Val Ile Leu Asp Phe Leu Gln Thr Thr Ser Leu Asp Lys Lys Ser
65 70 75 80
Phe Ser Leu Thr Cys Lys Leu Phe Tyr Ser Val Glu Ala Lys His Arg
85 90 95
Arg Leu Leu Arg Pro Leu Arg Ala Glu His Leu Pro Ala Leu Ala Ala
100 105 110
Arg Tyr Pro Asn Val Thr Glu Leu Asp Leu Ser Leu Cys Pro Arg Val
115 120 125
Gly Asp Gly Ala Leu Gly Leu Val Ala Gly Ala Tyr Ala Ala Thr Leu
130 135 140
Arg Arg Met Asp Leu Ser Arg Ser Arg Arg Phe Thr Ala Thr Gly Leu
145 150 155 160
Leu Ser Leu Gly Ala Arg Cys Glu His Leu Val Glu Leu Asp Leu Ser
165 170 175
Asn Ala Thr Glu Leu Arg Asp Ala Gly Val Ala Ala Val Ala Arg Ala
180 185 190
Arg Asn Leu Arg Lys Leu Trp Leu Ala Arg Cys Lys Met Val Thr Asp
195 200 205
Met Gly Ile Gly Cys Ile Ala Val Gly Cys Arg Lys Leu Arg Leu Leu
210 215 220
Cys Leu Lys Trp Cys Val Gly Ile Gly Asp Leu Gly Val Asp Leu Val
225 230 235 240
Ala Ile Lys Cys Lys Glu Leu Thr Thr Leu Asp Leu Ser Tyr Leu Pro
245 250 255
Ile Thr Glu Lys Cys Leu Pro Ser Ile Phe Lys Leu Gln His Leu Glu
260 265 270
Asp Leu Val Leu Glu Gly Cys Phe Gly Ile Asp Asp Asp Ser Leu Asp
275 280 285
Val Asp Leu Leu Lys Gln Gly Cys Lys Thr Leu Lys Arg Leu Asp Ile
290 295 300
Ser Gly Cys Gln Asn Ile Ser His Val Gly Leu Ser Lys Leu Thr Ser
305 310 315 320
Ile Ser Gly Gly Leu Glu Lys Leu Ile Leu Ala Asp Gly Ser Pro Val
325 330 335
Thr Leu Ser Leu Ala Asp Gly Leu Asn Lys Leu Ser Met Leu Gln Ser
340 345 350
Ile Val Leu Asp Gly Cys Pro Val Thr Ser Glu Gly Leu Arg Ala Ile
355 360 365
Gly Asn Leu Cys Ile Ser Leu Arg Glu Leu Ser Leu Ser Lys Cys Leu
370 375 380
Gly Val Thr Asp Glu Ala Leu Ser Phe Leu Val Ser Lys His Lys Asp
385 390 395 400
Leu Arg Lys Leu Asp Ile Thr Cys Cys Arg Lys Ile Thr Asp Val Ser
405 410 415
Ile Ala Ser Ile Ala Asn Ser Cys Thr Gly Leu Thr Ser Leu Lys Met
420 425 430
Glu Ser Cys Thr Leu Val Pro Ser Glu Ala Phe Val Leu Ile Gly Gln
435 440 445
Lys Cys His Tyr Leu Glu Glu Leu Asp Leu Thr Asp Asn Glu Ile Asp
450 455 460
Asp Glu Gly Leu Met Ser Ile Ser Ser Cys Ser Trp Leu Thr Ser Leu
465 470 475 480
Lys Ile Gly Ile Cys Leu Asn Ile Thr Asp Arg Gly Leu Ala Tyr Val
485 490 495
Gly Met Arg Cys Ser Lys Leu Lys Glu Leu Asp Leu Tyr Arg Ser Thr
500 505 510
Gly Val Asp Asp Leu Gly Ile Ser Ala Ile Ala Gly Gly Cys Pro Gly
515 520 525
Leu Glu Met Ile Asn Thr Ser Tyr Cys Thr Ser Ile Thr Asp Arg Ala
530 535 540
Leu Ile Ala Leu Ser Lys Cys Ser Asn Leu Glu Thr Leu Glu Ile Arg
545 550 555 560
Gly Cys Leu Leu Val Thr Ser Ile Gly Leu Ala Ala Ile Ala Met Asn
565 570 575
Cys Arg Gln Leu Ser Arg Leu Asp Ile Lys Lys Cys Tyr Asn Ile Asp
580 585 590
Asp Ser Gly Met Ile Ala Leu Ala His Phe Ser Gln Asn Leu Arg Gln
595 600 605
Ile Asn Leu Ser Tyr Ser Ser Val Thr Asp Val Gly Leu Leu Ser Leu
610 615 620
Ala Asn Ile Ser Cys Leu Gln Ser Phe Thr Leu Leu His Leu Gln Gly
625 630 635 640
Leu Val Pro Gly Gly Leu Ala Ala Ala Leu Leu Ala Cys Gly Gly Leu
645 650 655
Thr Lys Val Lys Leu His Leu Ser Leu Arg Ser Leu Leu Pro Glu Leu
660 665 670
Leu Ile Arg His Val Glu Ala Arg Gly Cys Val Phe Glu Trp Arg Asp
675 680 685
Lys Glu Phe Gln Ala Glu Leu Asp Pro Lys Cys Trp Lys Leu Gln Leu
690 695 700
Glu Asp Val Ile
705
<210> 43
<211> 2485
<212> DNA
<213> Arabidopsis thaliana (Arabidopsis thaliana)
<400> 43
gggttttttc caaaatcctc aaagagagac tatacttctc tcggatcctt cctgatttca 60
aaatttcttc aacccaccag cagcagcagc agatgaagaa ggttaaacag attcgtgtct 120
taaagccttt cgatcttctc tcggaagagc tcgtctttat catccttgac ctcatctctc 180
ccaacccttc cgatctcaaa tccttctctc ttacttgcaa atctttctac cagctcgagt 240
ccaagcaccg cggatcctta aaacctctcc gctccgacta tctccctcgc atcttgactc 300
gttaccggaa caccaccgat ctcgatctta ccttctgccc gcgtgtcact gactacgcgc 360
ttagcgtcgt tggctgtctc tccggaccta cgcttcgctc cctcgacctc tcgcgctctg 420
gctccttctc cgccgcggga ctactgcgat tggccctcaa atgtgtcaat ttagtcgaga 480
ttgacctgtc caatgcgacg gagatgagag acgccgatgc tgcggtggtg gcggaggcga 540
ggagtctgga gaggctgaag ctgggcagat gcaagatgct gacggacatg ggaatcggat 600
gtatagcagt tggatgtaag aagctcaata cggttagctt gaaatggtgt gttggcgtcg 660
gagatttagg ggttggcttg cttgccgtca aatgcaagga cattcgcacc ttagacctct 720
cctacttgcc gatcacagga aagtgtttac atgacattct gaaacttcaa caccttgaag 780
aacttcttct agaagggtgc tttggagtag atgatgacag tcttaaatca ctcagacatg 840
attgcaagtc attgaagaag cttgatgcat ccagctgcca gaatttaact catagaggtt 900
taacctcact tttaagcggg gcaggatatc ttcagcgact tgatctatca cactgttctt 960
ctgtgatatc attggatttt gcgagtagct taaagaaggt ttcagcatta cagtcgatca 1020
ggttggatgg ttgttctgtt acgcctgatg gtttgaaggc gataggcaca ttgtgcaatt 1080
ccctgaaaga ggttagccta agcaaatgcg tgagcgtaac tgatgaaggt ctttcttctc 1140
tagtaatgaa actgaaagac ctcagaaaac ttgacatcac atgttgccgg aaactaagta 1200
gagtttcaat cacccaaatc gccaattcgt gtcctttact agtctctttg aagatggagt 1260
cttgttctct tgtttccaga gaagcctttt ggttgatcgg acaaaagtgt cggctacttg 1320
aagagcttga cttaactgac aacgagattg atgatgaagg actgaaatcc atatctagtt 1380
gtctgagtct ttcctcgtta aagctgggaa tttgtcttaa cataacagac aaagggctct 1440
cgtacatcgg gatgggctgt tcaaatctcc gtgaacttga tctctatagg tcagtgggaa 1500
taacagacgt aggcatctcc acaattgccc aaggctgcat tcatctggaa acaataaaca 1560
tttcatactg ccaagacata acagacaagt ccctggtttc attgtccaaa tgctcgttgt 1620
tacaaacatt cgagagcagg ggatgtccta acatcacgtc ccaaggactt gcagccattg 1680
ctgttcggtg caagcgactc gccaaggttg acttgaagaa gtgcccatcc atcaatgatg 1740
cggggttgct cgctctggct cacttctctc agaatctcaa acagataaac gtgtcagaca 1800
cagctgtgac tgaagtggga cttctctccc tagccaacat agggtgttta cagaacatag 1860
cggttgtgaa ctcgagtggt ttaagaccga gcggagtagc agcagcattg ctggggtgtg 1920
gaggattaag gaaagcgaaa ctacatgcgt ccctaagatc actgcttcct ctttctctaa 1980
tccaccactt ggaagctcgt ggttgtgcgt tcctctggaa agacaatacc cttcaggcgg 2040
agttagatcc caagtactgg aagcaacagc tggaagagat ggcgccttaa attaaaagtg 2100
agaagaaaca ttctgaaatg ggaaagagga gttcctatgg gagccagcag acaggccctg 2160
ttttgggccc agtgtggatt agctggaaca attttggtct cttttgtgtt gttgttgacg 2220
gcgcgtgttc taacaacccc acacaacctt acattataag tctagtcaca tggtgggtga 2280
catggtaccg ttgtatatgt agtttttgtt tctttttttt ttttttttgg ttggggagcc 2340
gaaattaacg gacgtaacag agtcacaagg ggatgccttc tctgtgcctt tttgactttt 2400
tcctcctttc ttttttttct gtaatcatat gagttttatg taatttaatg cctcatcaac 2460
ttgcctggat agggccggcc tttcc 2485
<210> 44
<211> 2452
<212> DNA
<213> Rice (Oryza sativa)
<400> 44
attattacta ccttcccttc ccttcttctt gttgcagtca acctcgccat ggcctcgcct 60
atgccaacgc ctacccaccg cgtcaagcgc cgccgcctcg acctctcccc gcccccgcac 120
ctcaacgacc tcgccgacga gctcctcttc ctcatcctcg accgtgccgc cgcccatgac 180
ccccgcgccc tcaagtcctt ctccctcgtc tcccgcgcct gccacgccgc cgagtcgcgc 240
caccgccgcg tactccgccc cttccgcccc gacctcctcc ccgccgcgct cgcccgctac 300
cccgccctct cccgcctcga tctctccctc tgcccgcgcc tccccgacgc cgccctcgcc 360
gcgctccccg ccgcgccgtc cgtctccgcc gtcgacctct cccgctcccg ggggttcggc 420
gccgccggcc tcgccgcgct cgtcgccgcg tgccccaatc tcacggacct cgacctctcc 480
aatggcctcg acctcgggga tgccgcggcg gcggaggtgg ccaaggcgcg ccgcctgcag 540
aggctctcgc tgtcgcgatg caagcgcatc actgacatgg ggctcggatg catcgccgtc 600
ggatgccccg acctgcgcga gctctcgctc aagtggtgca tcggggtcac tcatctcgga 660
ctagacctcc tcgccctcaa gtgcaacaag ctcaacatcc tggatctctc ctacaccatg 720
attgtaaaaa aatgctttcc agccatcatg aagctacaaa gtctacaagt gttactactg 780
gtgggatgta atggaattga tgatgatgcc cttactagtc ttgatcaaga atgcagcaaa 840
tcactacagg ttcttgatat gtcaaattat tacaatgtca ctcatgtcgg tgttctgtcc 900
attgtgaagg caatgccgaa tctgttggaa ctcaatctat catactgctc tcctgttact 960
ccttctatgt caagcagctt cgaaatgatt cataaattgc agacactgaa gctggatggt 1020
tgccaattca tggatgatgg attaaaatcc attgggaaat cctgtgtttc tttgagggag 1080
ttaagtctga gcaaatgttc tggagtgaca gacacagatc tttcttttgt cgtgccaaga 1140
ctgaaaaatt tgctgaagct ggatgttact tgttgtcgca aaatcactga tgtttcatta 1200
gctgccatca caacctcatg cccctccctc atctctctga gaatggagtc ctgtagcctt 1260
gtttccagca aaggactaca gctgattgga aggcgctgca ctcacttgga ggaattggat 1320
cttactgaca ctgatttgga tgatgaaggt ttgaaagctc tctctggatg cagcaaactt 1380
tcaagcctaa aaattggcat atgcttgagg ataactgatg agggccttag acacgttagc 1440
aagtcctgtc cagatctccg agatatcgat ttgtacaggt ctggggcgat cagtgatgaa 1500
ggggttactc atatagctca aggatgccca atgttagagt ctatcaattt gtcctactgc 1560
acaaaattaa cagactgttc actgagatca ctttcaaaat gcataaagct gaacacattg 1620
gagattcgtg gctgccccat ggtttcatct gctggtctct cggaaattgc cacaggatgc 1680
aggctacttt ctaagcttga tatcaagaaa tgctttgaga tcaatgacat gggaatgatt 1740
ttcctttccc aattctctca caacctccgg cagataaact tgtcatattg ttcggtcacc 1800
gacattgggc ttatatccct ttcaagcata tgtggcttgc agaacatgac cattgtgcat 1860
ttagcgggtg ttacgcctaa tggactgata gctgctctta tggtctgtgg tttgagaaaa 1920
gtgaagcttc atgaagcatt caaatccatg gtgccatcac atatgctcaa agttgttgaa 1980
gcccgtggtt gtcttttcca gtggattaat aaaccctacc aggttgcggt agaaccgtgt 2040
gatgtatgga agcagcagtc gcaggatttg cttgtacagt gaaatgtttc aaagataaac 2100
gttgtggaaa ctggggcgtg ttttgtggtg ttgaatttat ctagagcaat atctccagtc 2160
ctagagaatg agctccaaaa gttttgtgcc ataactggct gaatagtgta ttgaattgct 2220
gacggtagta ttgtcagaac aacatactag tactggtatt ttgcttgtat gccagtggag 2280
gcgagggagg ttatattctt gctgtgttgt atatagcgca ggtaggaatc aacaatcaaa 2340
gagagatcat tggggtaaag catgttgtaa gtagtgcgga gtatgtgata tgccttgctg 2400
tgcctttttg atgcacaatt tgattaatgg aatggaacat tgcattctca ct 2452
<210> 45
<211> 2463
<212> DNA
<213> Rice (Oryza sativa)
<400> 45
gccaaacgcc caccattatt actaccttcc cttcccttct ctcgtttcag tcaacttcgc 60
catggcctcg cctgcgccca cccaccacgc caagcgccgc cgcctcgccc tgcccccgcc 120
cccgcccccg cacctcaacg acctcgccga cgagctcctc ttcctcatcc tcgaccgtgc 180
cgccgcccat gacccacgcg ccctcaagtc cttctccctc gtctcccgcg cctgccacgc 240
cgccgagtcg cgccaccgcc gcgtcctccg ccccttccgc cccgacctcc tccccgccgc 300
gctcgcccgc taccccgcca tctcccacct cgatctctcc ctctgccccc gcctccccga 360
cgccgccctc gccgcgctcc ccgccgcgcc gttcgtctcc gccgtcgacc tctcccgctc 420
ccgcgggttc ggcgccgccg gcctcgccgc gctcgtcgcc gcgttcccca atctcacgga 480
cctcgacctc tccaatggcc tcgacctcgg ggatgccgcg gcggcggagg tggccaaggc 540
gcgccgcctc cagaggctct cgctgtcgcg atgcaagcgc atcactgaca tggggctcgg 600
atgcatcgcc gtcggatgcc ccgacctgcg cgagctctcg ctcaagtggt gcatcggggt 660
cactcatctg ggactagacc tccttgccct caagtgcaac aagctcaaca tcctggatct 720
ctcctacacc atgatagtaa aaaaatgctt tccagccatc atgaagctac aaaatctaca 780
agtgttacta ctggtgggat gtaatggaat tgatgatgat gcccttacta gtcttgatca 840
agaatgcagc aaatcactac aggttcttga tatgtcaaac tcttacaatg tcactcatgt 900
cggtgttctg tccattgtga aggcaatgcc gaatctgttg gaactcaatc tatcatactg 960
ctctcctgtt actccttcta tgtcaagcag cttcgaaatg attcataaat tgcagaaact 1020
gaagctggat ggttgccaat tcatggatga tggattaaaa tccattggga aatcctgtgt 1080
ttctttgagg gagttaagtc tgagcaaatg ttctggagtg acagacacag acctttcttt 1140
tgtcgtgcca agactgaaaa atttgctgaa gctggatgtt acttgttgtc gcaaaatcac 1200
tgatgtttca ttagctgcca tcacaacctc atgcccatcc ctcatctctc tgagaatgga 1260
gtcctgtagc cttgtttcca gcaaaggact acagctgatt ggaaggcgct gcactcactt 1320
ggaggaattg gatcttactg acactgattt ggatgatgaa ggtttgaaag ctctctctgg 1380
atgcagcaaa ctttcaagcc taaaaattgg catatgcttg aggataactg atgagggcct 1440
tagacacgtt agcaagtcct gtccagatct ccgagatatc gatttgtaca ggtctggggc 1500
gatcagtgat gaaggggtta ctcatatagc tcaaggatgc ccaatgttag agtctatcaa 1560
tatgtcctac tgcacaaaat taacagactg ttcactgaga tcactttcaa aatgcataaa 1620
gctgaacaca ttggagattc gtggctgccc catggtttca tctgctggtc tctcggaaat 1680
tgcaacagga tgcaggctac tttctaagct tgatatcaag aaatgctttg agatcaatga 1740
catgggaatg attttccttt cccaattctc tcacaacctc cggcagataa acttgtcata 1800
ttgttcggtc accgacattg ggcttatatc cctttcaagc atatgtggct tgcagaacat 1860
gaccattgtg catttagcgg gtgttacgcc taatggactg atagctgctc ttatggtctg 1920
tggtttgaga aaagtgaagc ttcatgaagc attcaaatcc atggtgccat cacatatgct 1980
caaagttgtt gaagcccgtg gttgtctttt ccagtggatt aataaaccct accaggttgc 2040
ggtagaaccg tgtgacgtat ggaagcagca gtcgcaggat ttgcttgtac agtgaaatgt 2100
ttcaaagata aacgttgtgg aaactggggc gtgttttgtg gtgttgaatt tatcttagag 2160
caatatctcc agtcctagag aatgagctcc aaaagttttg tgccataact ggctgaatag 2220
tgtattgaat tactgacggt agtattgtca gaacaacata ctagtactgg tattttgctt 2280
gtatgccagt ggaggcgagg gaggttatat tcttgatgtg ttgtatatag cgcaggtagg 2340
aatcaacaat caaagagaga ttattggggt aaagcatgta gtaagtgtgg agtatgtgat 2400
atgccttgtt gtgccttttt gatgcacaat ttgattaatg gaatggaaca ttgcattcgc 2460
act 2463
<210> 46
<211> 2585
<212> DNA
<213> Corn (Zea mays)
<400> 46
ctccatctcc atcgccggcc tccatttctt gcctcccgtg ccagccagtc actctcgtcc 60
gccgcagatc gagaagccac caccaccacc accaccaccg catggccatg gcagcccagc 120
agcaccggca ccacaagcgc cgccgcatcg ccctctcccc ctccccgtcc ccgtccctcg 180
cgcccatccc cggcgccccc acgccgccgc tcgactcgct ggccgacgag ctcctcttcc 240
tggtcctgga ccgcgtggcc caggccgacc cgcgggcgct caagtccttc gcgctggcct 300
cccgcgcctg ccacgccgcg gagtcacggc accgccggac gctccgcccg ctccgcgcgg 360
acctcctgcc cgccgcgctg gcgcggtacc cgtccgcgac ccgcctcgac ctcaccctct 420
gcgcgcgcgt ccccgacgcc gccctcgcct ccgccgccgt ctccggctcc tccgccctcc 480
gcgccgtcga cctctcccgc tcccgcgggt tcggcgccgc gggcgtcgcc gcgctcgccg 540
ccgcgtgccc ggacctcgcc gacctcgacc tctccaatgg ggtccacctc ggggacgccg 600
cggcggccga ggtagcgcgg gccagggcgc tgcggaggct ctcgctggtc cgctggaagc 660
cgctcaccga catgggcctc ggatgcgtcg ccgtcgggtg cacggagctg aaggacctct 720
cgctcaagtg gtgccttgga ctcacggatc tggggatcca gctcctcgcc ctcaagtgca 780
ggaagctcac cagcctggat ctctcctaca ccatgatcac aaaggatagc ttgccttcta 840
tcatgaagct acccaatctt caagagctga cactggtggg gtgtattgga atcgatgatg 900
gtgctcttgt tagtcttgag agagaatgca gtaaatcact acaggtgctt gatatgtctc 960
agtgtcagaa tatcaccgat gtaggagttt catccatcct gaagtcggta cccaatctat 1020
tggaactgga tctttcatac tgctgtcctg ttactccttc tatggtgaga aacttccaga 1080
agcttcctaa actgcaggcc ctgaagctgg aaggctgcaa attcatggcc aatggactaa 1140
aagccattgg gacctcttgt gtttctttaa gggagctaag tcttagcaag tcatctggag 1200
tgacagatac agaactctct tttgttgtgt caaggctaaa gaacctgctg aagctggaca 1260
ttacctgttg tcgcagtatt actgatgttt cactagcggc cataactagt tcgtgcactt 1320
ccctcatctc tctgaggatg gagtcttgta gccatgtttc cagtggagca ctccaactga 1380
ttgggaagca ctgttctcac ttggaagagt tggaccttac tgacagtgat ttggatgatg 1440
aaggattgaa agctcttgcc agatgtagcg aactttcgag cctaaaaatt ggcatttgct 1500
tgaagataag tgatgaaggt cttagccaca ttggaaggtc ttgcccaaaa ctccgcgaga 1560
ttgatttgta caggtgtgga gttattagcg atgatggaat tattcaaatt gcgcagggtt 1620
gtccgatgct agagtctatc aacctatcat actgcacaga aataacagac cgttcactga 1680
tttcactctc aaaatgcgca aagctgaata ctttggagat ccgtggctgc cccagtgttt 1740
catcgattgg gctctcagaa atagcgatgg ggtgcaggct gctttccaag cttgatatta 1800
agaaatgctt cgggattaat gatgttggaa tgctttacct ttcccagttc gctcatagcc 1860
tccgtcagat aaacttgtca tactgttcag tcaccgatgt tgggctcctt tccctttcta 1920
gcatatccgg cctgcagaac atgaccatcg tccatttggc gggtataaca cccaatggct 1980
tgacagcaac tcttatggtt tgcggtgggt tgacgaaagt gaagcttcat gaagcattca 2040
gatccatgat gcctcctcat acgataaaaa atgttgaggc acgtggctgt gttttccagt 2100
ggatcgataa accgttcaag gttgaggtgg agccttgtga tgtatggaag caacagtcac 2160
aagatgtgct tgtgcgatga gaatacagga gacctcgagc gtagccgcta tcgtggaaat 2220
cgtggcatgc atcgtatgga tggatgagtt gttgttggct ggcaatggca tccagaagtg 2280
aagtctgatg gggggagctc caaactcagc gttgtaatca gtgcgattcg gcacaaagat 2340
accagcattt gagcagaggt gtatgtatgt cttgatgttg ttttatactg ctatagatgt 2400
gtagatccca ttgtttggtg aggtgatcat ttgcgaggaa gttgactatt agcatgtatt 2460
aagataaaaa ggaaagagat gagaaaatgt ttttgaaatt tagtacgata tgccttgcag 2520
tgcctgtatc tgttgcattc atatttgtga tcagctggaa tgaagtggct tgcattcatg 2580
tttta 2585
<210> 47
<211> 2458
<212> DNA
<213> Barley (Hordeum vulgare)
<400> 47
gaggttggcc gtggccagcc attgctactc ctccctccct tcttggtgtc gccgcagcgg 60
acaaccaact gccaacttgc cttgcccgtc atggccatgg cgacccacag ccacctcccc 120
aagcgccgac gcgtatgccc tgccgcgggc gcgccgatcg acgagctgcc cgacgagctc 180
ctcttcttgg tcctggaccg ggtggcggcc gccgatccgc gcgcgctcaa gtccttcgcg 240
ctggcctccc gcgcctgcca cgccgccgag tcgcgccacc gccgcgtgct ccgcccgtac 300
cgcgccgacc tactccgcgc cgcgcttgcc cgctacccca ccgccgcccg cctcgacctc 360
accctctgcg cgcgcgtgcc cggcgcggcc ctctcctccg cgcccgtgcc ttccctccgc 420
gccgtcgacc tctcccgctc ccgcggcttc ggggcgcccg gcctcgccgc gctcgtcgcc 480
gcctgccccg ccctggccga cctcgacctc tccaatgggg tcgacctcgg ggacgcggcg 540
gccgcggagc tggcgcgggc gcggggcctg cagaggctct gcctctcgcg ctgcaagccc 600
atcacggaca tgggcctcgg ctgcatcgcc gtcggctgcc cagacctgcg ggacctcacg 660
ctcaactggt gcctcgggat cacggatttg gggatccagc tcctcgccct caagtgcaac 720
aaactcagga acctgcatct ttcctacacc atggttagat ctccaaagac tgccttccag 780
ccatcatgga gctacccaat cttgaggtgt tggcactggt gggatgtgtt ggaatagatg 840
atgatgccct tagtggtctt gagaatgaaa gcagcaaatc actacgggtt ctcgatatgt 900
ctacctgtcg aaatgtcact catacgggag tttcatcagt tgtgaaggca ctgccaaatc 960
tcttggagtt gaatctgtcc tactgctgta atgttactgc atctatggga aaatgcttcc 1020
aaatgcttcc taaattgcag accttgaaat tggaaggctg caagttcatg gctgatggac 1080
taaaacacat tggaatttct tgtgtctctt taagagagtt gagcctgagc aagtgctcag 1140
gagtgacaga tactgatctg tctttcgttg tgtcaagact aaagaatttg ctgaagctgg 1200
acattacttg caatcgcaat atcactgatg tttcgttagc tgccatcact agctcatgcc 1260
attccctcat ctctctaaga atagagtcct gtagccattt ttctagtgaa gggctccgac 1320
ttattgggaa gcgatgttgc catttggaag agttggatat caccgacagt gatttggacg 1380
atgaaggttt gaaagctttg tctggatgca gcaaactgtc aagcttaaaa attggaatat 1440
gcatgaggat aagtgaccaa ggccttatcc acattgggaa gtcttgtcca gaactccgag 1500
atattgattt gtataggtct gggggtatta gtgatgaggg ggttactcaa attgcccaag 1560
gttgtccaat gctagagtct atcaacctgt cgtactgtac agaaataaca gatgtctcgt 1620
tgatgtcgct ctcaaaatgt gcaaagctaa acacactgga gatccgtggt tgccccagta 1680
tttcatctgc tgggctctca gaaatagcaa tcggatgcag gctacttgcc aagcttgatg 1740
tcaagaagtg ctttgcgatc aatgatgtgg ggatgttttt tctttcccag ttctctcata 1800
gcctccgtca gataaacttg tcatactgtt cggtcaccga tattgggctt ctgtccctct 1860
ctagcatatg cgggcttcag aacatgacga ttgtacactt ggcgggtatt acgcctaatg 1920
gcttgctggc tgctctgatg gtctctggtg gtttgacaag ggtgaagctt catgcagcgt 1980
tcagatctat gatgcccccg catatgctca aagtcgttga ggctcgcggc tgtgctttcc 2040
agtggattga taaaccattc aaggtcgagc aagaacgatg cgacatatgg caacaacagt 2100
ctcgagacgt gcttgtacga tgagaaatgt ttgacgtcgt cgtcttggct gtcagcctac 2160
aggatatggt gtagaacgag gctttgcacc aaggtggact tgtgtacaga acagacatta 2220
tcggaacaat gttgtatact catgtttcct tcagtgcact aggaggttct tttgttgtgc 2280
cgtatcctgt atatagagca gcaggagatg gatgaaatca tagaggaatt gctggaggtc 2340
gacgtgtact taatagaaac tcaccgatca atgtgagagt gtgcacagtt ctattacctg 2400
tccaatgcgc tcttgtttcg aatgagtagt agtttagcct gtgtttctcc tggtgtcc 2458
<210> 48
<211> 2465
<212> DNA
<213> Brassica (Brassica rapa)
<400> 48
ctgtcctttt ttattctctg tttcccatag aaactgtgaa tctgtgggtt tttagcaaat 60
cctcatcgaa actgtttctc catttgataa aaaaaaaaat cagtaagtag ttgggttcga 120
atgaagaagg cgaagcagat tcaacacatg atctcaaagc ctttcgatct tctgtcggag 180
gagctggtct tcataatcct agacctcgtc gctcagaacc cttccgatct caaatccttc 240
tctcttacct gcaaatggtt ctaccaagtg gaggccaggc accggagatc cctgaaacct 300
ctcagagcgg agtatcttcc tcgaatcctg acaaggtacc ggaataccgc cgatctcgat 360
cttagcttct gcccgcgcgt cacggactac gcgcttagcg tggtcgggtg cctctccgga 420
ccgacgctga ggtcagtaga cttatcgagg tcattctcct tctcggcggc ggggctgctg 480
agactggccg tgaaatgtgt gagtctggtg gagatagacc tgtcgaacgc gacggagatg 540
agggacgcgg cagcggcggt ggtggcggag gcgaagagcc tggagaggct gaagctgggg 600
agatgcaaga agctgacgga catgggaata ggatgcatag cggtgggatg taggaagctg 660
aagagggtga gcttgaagtg gtgtgtaggc gtcggagatt taggagtcgg tctgcttgcc 720
gtcaaatgca aggacattcg ctccttagac ctctcctact tgccgatcac aggcaagtgt 780
ttgcatgatg ttctcaaact tcaacacctt gaagaacttc ttctacaagg ttgcttcgga 840
gtcgatgatg actctcttaa atcactcact catcattgca actccttaaa gaacctggat 900
gcatcgagct gccagaattt aactcagaga ggtttaacct cacttttaag cggggccgga 960
tgtcttgagc gccttgatct agcacactct tcttctgtga tctcattgga ttttgcaagt 1020
agcctgaaca aggtttcttc agcattacag tcgatcaggt tggatggttg tgcggtaact 1080
tgtgatgggt tgaaggcgat agggacactg tgcatttccc tcagagaggt tagcctaagc 1140
aaatgtgtca ccgtaactga tgaaggtctc tcttgtcttg ttatgaaact caaagacctt 1200
agaaaacttg acatcacctg ttgccggaaa ctaactggag tttcaatcac ccaagtcgcc 1260
agttcttgtc ccttactagt ctccttgaag atggagtctt gttctcttgt ttccagagat 1320
gccttttggt tgatcggaca caagtgtcgc ctacttgaag agcttgactt tactgacaat 1380
gagattgatg atgaaggact aaaatccata tccagttgtc gtagtctttc ctcgttgaag 1440
ttgggaatat gtctgaatat aacagacaga ggactctcgt acattggtat gggctgctca 1500
aatctccgtg aacttgatct ctacaggtcg gtgggaataa cagacgtagg aatctcatcg 1560
atcgctcaag gctgctgtca tctcgaaaca ataaacatat catactgcaa agacatcaca 1620
gacaagtcgt tggtgtcatt gtcaaaatgc tcaatgttac aaacattcga gagccgggga 1680
tgtccacaca ttacttgcca aggacttgcc gccattgctg ttcgatgtaa gcggctcagc 1740
aagctcgact tgaaaaagtg tcctttcatc aatgactccg gtttgctcac tctagctcac 1800
ttttcacagg gcctcaaaca gataagcgtg tccgaaacgg gggtgacaga cgtgggactt 1860
gtgtcgctag caaacatagg gtgtttgcag aacatagcgg cagtgaacac aaggggttta 1920
agcccgagtg gagtagcagc agcgttggtt gggtgtggag gtttaaggaa agtgaaactc 1980
cacgcttccc tcagatcact acttccttcg tctcttataa accacatgga agctcgtggc 2040
tgctccttcc tgtggaaaga ctataacaac cataataata gtaatacact tcaggcggag 2100
ttagatccca agtactggaa ggtatagccg gaagaagata ttgagcttta gaagaaagaa 2160
acatccacaa ggggaaagag acggagccac catacaggcc aaggccccat ccattgtttg 2220
gatttagaaa gaggaactaa agcttggtat ctatctatgt cgatggcgcg tgttgcctca 2280
cacacataca cgtatatcca tccttacgta tgtagtatat ggttgtgaca tggattggtg 2340
gttagtttgt atgtaccctt ttgtaatttc atttcctttt tttggatgcc aaagttaacg 2400
gacgtcacaa gagattgcat ttgtgtcctt tttttgagtt tttcttttct ggatggttct 2460
atcaa 2465
<210> 49
<211> 2682
<212> DNA
<213> Soybean (Glycine max)
<400> 49
actcattcaa tattaataaa tgtctcgcat ttattcctca aacatagaaa ccaaacaaac 60
aaacccttcc cttctctccc cacagttatt catcattaac ctccaaaacg accaaccaaa 120
cgcaaaacgc agaaccatga agaagcagaa gctctccgag cctcaaaacg acaccaccaa 180
ccccttcgag gttctctccg aggagctaat gttcgtcatc ctcgacttcc tccaaacgac 240
gtcgttggac aaaaaatctt tctcgctcac gtgtaagttg ttttactccg tcgaggccaa 300
gcaccgtcgt ttgcttcgcc cgctacgtgc ggaacacctg cccgcgctcg ctgcccgcta 360
cccgaacgta acggaattgg atctttcctt gtgtccgcgc gtgggcgacg gcgcgctggg 420
gctcgtcgct ggcgcgtacg cggcgacgct gcggcgaatg gacctgtcgc ggtcgcggcg 480
gttcacggcg accgggctgc taagcctcgg cgcacggtgc gagcacctgg tggagctgga 540
cttgtcgaac gcgacggagc tgagggacgc cggcgtcgcc gcggtggcgc gtgcgcggaa 600
cttgcggaag ctgtggctgg cgaggtgcaa gatggtgacg gatatgggga ttgggtgtat 660
tgcggtgggg tgcaggaagc tgaggctgct ttgcttgaag tggtgtgtgg gaattgggga 720
tttgggtgtg gatttggttg cgattaagtg taaggagctc acaacattgg atctctctta 780
tttgcctatc acggagaaat gtctaccgtc aatcttcaaa ttgcaacatc ttgaagattt 840
ggtccttgaa ggatgctttg gcattgatga cgacagcctt gatgttgatc tcttaaaaca 900
agggtgcaag acattgaaga gacttgatat ctcaggttgt caaaacataa gtcatgttgg 960
gttatcaaag cttacaagca tttctggagg tttagagaaa ctcattttag cagatggctc 1020
tcctgtcacc ctttctcttg ctgatggttt gaataaactt tccatgttgc aatcaattgt 1080
attagatggc tgccctgtta catctgaagg attacgggcc attggaaatt tgtgcatttc 1140
acttagggag cttagtctaa gcaaatgttt gggagtgaca gatgaggcac tctcatttct 1200
tgtgtcaaaa cacaaagatt taaggaaact tgacatcaca tgctgtcgca agataactga 1260
tgtttccatt gccagcattg caaattcatg cacaggtcta acttctctca aaatggagtc 1320
atgtacacta gttccaagtg aagcatttgt cttgattgga cagaaatgcc attatcttga 1380
ggagcttgac ctaacagata atgaaattga tgatgaaggt cttatgtcca tttcttcttg 1440
ttcttggctt accagcttga aaataggaat atgcctgaac ataactgaca gaggacttgc 1500
ctatgttggc atgcgttgct caaaattaaa ggagctggat ctatacaggt ctactggagt 1560
agatgatttg ggcatttcag caattgctgg tggttgccct ggccttgaga tgataaacac 1620
atcctattgt actagcatta ctgacagggc actaattgcc ttgtcaaaat gttcaaattt 1680
ggagacactt gaaattcgag gatgtcttct cgttacatcc ataggtctgg cagctattgc 1740
aatgaattgc agacaactaa gtcgtctaga cataaaaaag tgttacaaca ttgatgacag 1800
tgggatgatt gctctggctc atttctccca aaatctaaga cagataaatt tgtcatatag 1860
ctcagttaca gatgtggggc ttctgtcact tgctaatatc agttgccttc aaagctttac 1920
cttgcttcac ctgcaaggct tggttccagg aggactggcg gcagccttat tagcttgtgg 1980
agggctaaca aaagtgaagc tccatctttc actaagatct ctgttacctg agctacttat 2040
cagacatgtg gaagcacgtg gctgtgtatt tgaatggaga gataaagagt ttcaggctga 2100
attggacccc aagtgttgga aattacagtt ggaagatgtg atataatagg atttttctca 2160
ggttctttga agttttgata aagacaactc tgctgcatgg ctcatgaaat tcaattcgga 2220
agtcatcatc ttctctctct tctctgttca cctaacctac actacaagaa atgaagaaag 2280
cagcatgaaa aatgccagaa gtattctgtt tagtccattg gaggctacac aggcatttgg 2340
aatggagatt gttgatcatc cttccttaag tctcgctcgc ctaactagga tgagttttgt 2400
ttattctttt ttcttttttt tgctgcaaat agcaggatta gtgaagtact taaagtgtta 2460
caaaccccat ctaacgtctt tcatttattt attttgggag tacaattgga aatgttcaga 2520
gaatggagaa gcagtggcta tcgaatgtat aaaaacacta acttctgttc aatcttttct 2580
tttgacagtg aaaagcaaga ttaagtgtgt aaaacttaga aacctgtttt gtttatatat 2640
gattagagct gttcatggac atccagaata gaagtttcaa aa 2682
<210> 50
<211> 642
<212> PRT
<213> Arabidopsis thaliana (Arabidopsis thaliana)
<400> 50
Met Ser Thr Ser Pro Ser Ile Leu Ser Val Leu Ser Glu Asp Leu Leu
1 5 10 15
Val Arg Val Tyr Glu Cys Leu Asp Pro Pro Cys Arg Lys Thr Trp Arg
20 25 30
Leu Ile Ser Lys Asp Phe Leu Arg Val Asp Ser Leu Thr Arg Thr Thr
35 40 45
Ile Arg Ile Leu Arg Val Glu Phe Leu Pro Thr Leu Leu Phe Lys Tyr
50 55 60
Pro Asn Leu Ser Ser Leu Asp Leu Ser Val Cys Pro Lys Leu Asp Asp
65 70 75 80
Asp Val Val Leu Arg Leu Ala Leu Asp Gly Ala Ile Ser Thr Leu Gly
85 90 95
Ile Lys Ser Leu Asn Leu Ser Arg Ser Thr Ala Val Arg Ala Arg Gly
100 105 110
Leu Glu Thr Leu Ala Arg Met Cys His Ala Leu Glu Arg Val Asp Val
115 120 125
Ser His Cys Trp Gly Phe Gly Asp Arg Glu Ala Ala Ala Leu Ser Ser
130 135 140
Ala Thr Gly Leu Arg Glu Leu Lys Met Asp Lys Cys Leu Ser Leu Ser
145 150 155 160
Asp Val Gly Leu Ala Arg Ile Val Val Gly Cys Ser Asn Leu Asn Lys
165 170 175
Ile Ser Leu Lys Trp Cys Met Glu Ile Ser Asp Leu Gly Ile Asp Leu
180 185 190
Leu Cys Lys Ile Cys Lys Gly Leu Lys Ser Leu Asp Val Ser Tyr Leu
195 200 205
Lys Ile Thr Asn Asp Ser Ile Arg Ser Ile Ala Leu Leu Val Lys Leu
210 215 220
Glu Val Leu Asp Met Val Ser Cys Pro Leu Ile Asp Asp Gly Gly Leu
225 230 235 240
Gln Phe Leu Glu Asn Gly Ser Pro Ser Leu Gln Glu Val Asp Val Thr
245 250 255
Arg Cys Asp Arg Val Ser Leu Ser Gly Leu Ile Ser Ile Val Arg Gly
260 265 270
His Pro Asp Ile Gln Leu Leu Lys Ala Ser His Cys Val Ser Glu Val
275 280 285
Ser Gly Ser Phe Leu Lys Tyr Ile Lys Gly Leu Lys His Leu Lys Thr
290 295 300
Ile Trp Ile Asp Gly Ala His Val Ser Asp Ser Ser Leu Val Ser Leu
305 310 315 320
Ser Ser Ser Cys Arg Ser Leu Met Glu Ile Gly Leu Ser Arg Cys Val
325 330 335
Asp Val Thr Asp Ile Gly Met Ile Ser Leu Ala Arg Asn Cys Leu Asn
340 345 350
Leu Lys Thr Leu Asn Leu Ala Cys Cys Gly Phe Val Thr Asp Val Ala
355 360 365
Ile Ser Ala Val Ala Gln Ser Cys Arg Asn Leu Gly Thr Leu Lys Leu
370 375 380
Glu Ser Cys His Leu Ile Thr Glu Lys Gly Leu Gln Ser Leu Gly Cys
385 390 395 400
Tyr Ser Met Leu Val Gln Glu Leu Asp Leu Thr Asp Cys Tyr Gly Val
405 410 415
Asn Asp Arg Gly Leu Glu Tyr Ile Ser Lys Cys Ser Asn Leu Gln Arg
420 425 430
Leu Lys Leu Gly Leu Cys Thr Asn Ile Ser Asp Lys Gly Ile Phe His
435 440 445
Ile Gly Ser Lys Cys Ser Lys Leu Leu Glu Leu Asp Leu Tyr Arg Cys
450 455 460
Ala Gly Phe Gly Asp Asp Gly Leu Ala Ala Leu Ser Arg Gly Cys Lys
465 470 475 480
Ser Leu Asn Arg Leu Ile Leu Ser Tyr Cys Cys Glu Leu Thr Asp Thr
485 490 495
Gly Val Glu Gln Ile Arg Gln Leu Glu Leu Leu Ser His Leu Glu Leu
500 505 510
Arg Gly Leu Lys Asn Ile Thr Gly Val Gly Leu Ala Ala Ile Ala Ser
515 520 525
Gly Cys Lys Lys Leu Gly Tyr Leu Asp Val Lys Leu Cys Glu Asn Ile
530 535 540
Asp Asp Ser Gly Phe Trp Ala Leu Ala Tyr Phe Ser Lys Asn Leu Arg
545 550 555 560
Gln Ile Asn Leu Cys Asn Cys Ser Val Ser Asp Thr Ala Leu Cys Met
565 570 575
Leu Met Ser Asn Leu Ser Arg Val Gln Asp Val Asp Leu Val His Leu
580 585 590
Ser Arg Val Thr Val Glu Gly Phe Glu Phe Ala Leu Arg Ala Cys Cys
595 600 605
Asn Arg Leu Lys Lys Leu Lys Leu Leu Ala Pro Leu Arg Phe Leu Leu
610 615 620
Ser Ser Glu Leu Leu Glu Thr Leu His Ala Arg Gly Cys Arg Ile Arg
625 630 635 640
Trp Asp
<210> 51
<211> 659
<212> PRT
<213> Rice (Oryza sativa)
<400> 51
Met Ser Glu Glu Val Gln Arg Tyr Gly Gly Gly Gly Gly Gly Gly Gly
1 5 10 15
Gly Gly Val Ala Ala Leu Ser Leu Asp Leu Leu Gly Gln Val Leu Asp
20 25 30
Arg Val Arg Glu Pro Arg Asp Arg Lys Ala Cys Arg Leu Val Ser Arg
35 40 45
Ala Phe Ala Arg Ala Glu Ala Ala His Arg Arg Ala Leu Arg Val Leu
50 55 60
Arg Arg Glu Pro Leu Ala Arg Leu Leu Arg Ala Phe Arg Ala Leu Glu
65 70 75 80
Arg Leu Asp Leu Ser Ala Cys Ala Ser Leu Asp Asp Ala Ser Leu Ala
85 90 95
Ala Ala Leu Ser Gly Ala Asp Leu Ala Gly Val Arg Arg Val Cys Leu
100 105 110
Ala Arg Ala Ser Gly Val Gly Trp Arg Gly Leu Asp Ala Leu Val Ala
115 120 125
Ala Cys Pro Arg Leu Glu Ala Val Asp Leu Ser His Cys Val Gly Ala
130 135 140
Gly Asp Arg Glu Ala Ala Ala Leu Ala Ala Ala Thr Gly Leu Arg Glu
145 150 155 160
Leu Ser Leu Glu Lys Cys Leu Gly Val Thr Asp Met Gly Leu Ala Lys
165 170 175
Val Val Val Gly Cys Pro Arg Leu Glu Lys Leu Ser Leu Lys Trp Cys
180 185 190
Arg Glu Ile Ser Asp Ile Gly Ile Asp Leu Leu Ser Lys Lys Cys His
195 200 205
Glu Leu Arg Ser Leu Asp Ile Ser Tyr Leu Lys Val Gly Asn Glu Ser
210 215 220
Leu Arg Ser Ile Ser Ser Leu Glu Lys Leu Glu Glu Leu Ala Met Val
225 230 235 240
Cys Cys Ser Cys Ile Asp Asp Asp Gly Leu Glu Leu Leu Gly Lys Gly
245 250 255
Ser Asn Ser Leu Gln Ser Val Asp Val Ser Arg Cys Asp His Val Thr
260 265 270
Ser Gln Gly Leu Ala Ser Leu Ile Asp Gly His Asn Phe Leu Gln Lys
275 280 285
Leu Asn Ala Ala Asp Ser Leu His Glu Met Arg Gln Ser Phe Leu Ser
290 295 300
Asn Leu Ala Lys Leu Lys Asp Thr Leu Thr Val Leu Arg Leu Asp Gly
305 310 315 320
Leu Glu Val Ser Ser Ser Val Leu Leu Ala Ile Gly Gly Cys Asn Asn
325 330 335
Leu Val Glu Ile Gly Leu Ser Lys Cys Asn Gly Val Thr Asp Glu Gly
340 345 350
Ile Ser Ser Leu Val Thr Gln Cys Ser His Leu Arg Val Ile Asp Leu
355 360 365
Thr Cys Cys Asn Leu Leu Thr Asn Asn Ala Leu Asp Ser Ile Ala Glu
370 375 380
Asn Cys Lys Met Val Glu His Leu Arg Leu Glu Ser Cys Ser Ser Ile
385 390 395 400
Ser Glu Lys Gly Leu Glu Gln Ile Ala Thr Ser Cys Pro Asn Leu Lys
405 410 415
Glu Ile Asp Leu Thr Asp Cys Gly Val Asn Asp Ala Ala Leu Gln His
420 425 430
Leu Ala Lys Cys Ser Glu Leu Leu Val Leu Lys Leu Gly Leu Cys Ser
435 440 445
Ser Ile Ser Asp Lys Gly Leu Ala Phe Ile Ser Ser Ser Cys Gly Lys
450 455 460
Leu Ile Glu Leu Asp Leu Tyr Arg Cys Asn Ser Ile Thr Asp Asp Gly
465 470 475 480
Leu Ala Ala Leu Ala Asn Gly Cys Lys Lys Ile Lys Met Leu Asn Leu
485 490 495
Cys Tyr Cys Asn Lys Ile Thr Asp Ser Gly Leu Gly His Leu Gly Ser
500 505 510
Leu Glu Glu Leu Thr Asn Leu Glu Leu Arg Cys Leu Val Arg Ile Thr
515 520 525
Gly Ile Gly Ile Ser Ser Val Ala Ile Gly Cys Lys Asn Leu Ile Glu
530 535 540
Ile Asp Leu Lys Arg Cys Tyr Ser Val Asp Asp Ala Gly Leu Trp Ala
545 550 555 560
Leu Ala Arg Tyr Ala Leu Asn Leu Arg Gln Leu Thr Ile Ser Tyr Cys
565 570 575
Gln Val Thr Gly Leu Gly Leu Cys His Leu Leu Ser Ser Leu Arg Cys
580 585 590
Leu Gln Asp Val Lys Met Val His Leu Ser Trp Val Ser Ile Glu Gly
595 600 605
Phe Glu Met Ala Leu Arg Ala Ala Cys Gly Arg Leu Lys Lys Leu Lys
610 615 620
Met Leu Ser Gly Leu Lys Ser Val Leu Ser Pro Glu Leu Leu Gln Met
625 630 635 640
Leu Gln Ala Cys Gly Cys Arg Ile Arg Trp Val Asn Lys Pro Leu Val
645 650 655
Tyr Lys Asp
<210> 52
<211> 660
<212> PRT
<213> Corn (Zea mays)
<400> 52
Met Ser Arg Glu Ala Gln Lys Leu Asp Cys Ala Ala Gly Ala Gly Gly
1 5 10 15
Ile Gly Val Leu Ser Leu Asp Leu Leu Gly Gln Val Leu Glu His Leu
20 25 30
Arg Glu Pro Arg Asp Arg Lys Thr Cys Arg Leu Val Ser Arg Ala Phe
35 40 45
Glu Arg Ala Glu Ala Ala His Arg Arg Ala Leu Arg Val Leu Arg Arg
50 55 60
Glu Pro Leu Pro Arg Leu Leu Arg Ala Phe Pro Ala Leu Glu Arg Leu
65 70 75 80
Asp Leu Ser Ala Cys Ala Ser Leu Asp Asp Ala Ser Leu Ala Ala Ala
85 90 95
Val Ala Asp Ala Gly Gly Gly Leu Ala Gly Leu Arg Ser Val Cys Leu
100 105 110
Ala Arg Ala Asn Gly Val Gly Trp Arg Gly Leu Glu Ala Leu Val Ala
115 120 125
Ala Cys Pro Lys Leu Ala Ala Val Asp Leu Ser His Cys Val Thr Ala
130 135 140
Gly Asp Arg Glu Ala Ala Ala Leu Ala Ala Ala Ser Glu Leu Arg Asp
145 150 155 160
Leu Arg Leu Asp Lys Cys Leu Ala Val Thr Asp Met Gly Leu Ala Lys
165 170 175
Val Ala Val Gly Cys Pro Lys Leu Glu Lys Leu Ser Leu Lys Trp Cys
180 185 190
Arg Glu Ile Ser Asp Ile Gly Ile Asp Leu Leu Ala Lys Lys Cys Pro
195 200 205
Glu Leu Arg Ser Leu Asn Ile Ser Tyr Leu Lys Val Gly Asn Gly Ser
210 215 220
Leu Gly Ser Ile Ser Ser Leu Glu Arg Leu Glu Glu Leu Ala Met Val
225 230 235 240
Cys Cys Ser Gly Ile Asp Asp Glu Gly Leu Glu Leu Leu Ser Lys Gly
245 250 255
Ser Asp Ser Leu Gln Ser Val Asp Val Ser Arg Cys Asp His Val Thr
260 265 270
Ser Glu Gly Leu Ala Ser Leu Ile Asp Gly Arg Asn Phe Leu Gln Lys
275 280 285
Leu Tyr Ala Ala Asp Cys Leu His Glu Ile Gly Gln Arg Phe Leu Ser
290 295 300
Lys Leu Ala Arg Leu Lys Glu Thr Leu Thr Leu Leu Lys Leu Asp Gly
305 310 315 320
Leu Glu Val Ser Asp Ser Leu Leu Gln Ala Ile Gly Glu Ser Cys Asn
325 330 335
Lys Leu Val Glu Ile Gly Leu Ser Lys Cys Ser Gly Val Thr Asp Gly
340 345 350
Gly Ile Ser Ser Leu Val Ala Arg Cys Ser Asp Leu Arg Thr Ile Asp
355 360 365
Leu Thr Cys Cys Asn Leu Ile Thr Asn Asn Ala Leu Asp Ser Ile Ala
370 375 380
Asp Asn Cys Lys Met Leu Glu Cys Leu Arg Leu Glu Ser Cys Ser Leu
385 390 395 400
Ile Asn Glu Lys Gly Leu Glu Arg Ile Thr Thr Cys Cys Pro Asn Leu
405 410 415
Lys Glu Ile Asp Leu Thr Asp Cys Gly Val Asp Asp Ala Ala Leu Gln
420 425 430
His Leu Ala Lys Cys Ser Glu Leu Arg Ile Leu Lys Leu Gly Leu Cys
435 440 445
Ser Ser Ile Ser Asp Arg Gly Ile Ala Phe Ile Ser Ser Asn Cys Gly
450 455 460
Lys Leu Val Glu Leu Asp Leu Tyr Arg Cys Asn Ser Ile Thr Asp Asp
465 470 475 480
Gly Leu Ala Ala Leu Ala Asn Gly Cys Lys Arg Ile Lys Leu Leu Asn
485 490 495
Leu Cys Tyr Cys Asn Lys Ile Thr Asp Thr Gly Leu Gly His Leu Gly
500 505 510
Ser Leu Glu Glu Leu Thr Asn Leu Glu Leu Arg Cys Leu Val Arg Val
515 520 525
Thr Gly Ile Gly Ile Ser Ser Val Ala Ile Gly Cys Lys Asn Leu Ile
530 535 540
Glu Leu Asp Leu Lys Arg Cys Tyr Ser Val Asp Asp Ala Gly Leu Trp
545 550 555 560
Ala Leu Ala Arg Tyr Ala Leu Asn Leu Arg Gln Leu Thr Ile Ser Tyr
565 570 575
Cys Gln Val Thr Gly Leu Gly Leu Cys His Leu Leu Ser Ser Leu Arg
580 585 590
Cys Leu Gln Asp Ile Lys Met Val His Leu Ser Trp Val Ser Ile Glu
595 600 605
Gly Phe Glu Met Ala Leu Arg Ala Ala Cys Gly Arg Leu Lys Lys Leu
610 615 620
Lys Met Leu Cys Gly Leu Lys Thr Val Leu Ser Pro Glu Leu Leu Gln
625 630 635 640
Met Leu Gln Ala Cys Gly Cys Arg Ile Arg Trp Val Asn Lys Pro Leu
645 650 655
Val Tyr Lys Asp
660
<210> 53
<211> 661
<212> PRT
<213> Barley (Hordeum vulgare)
<400> 53
Met Ser Glu Glu Glu Ala Gln Arg Tyr Gly Gly Gly Thr Gly Gly Gly
1 5 10 15
Gly Val Gly Ala Leu Ser Val Asp Leu Leu Gly Gln Val Leu Asp Arg
20 25 30
Val Leu Glu Arg Arg Asp Arg Lys Ala Cys Arg Leu Val Ser Arg Ala
35 40 45
Phe Ala Arg Ala Glu Ala Ala His Arg Arg Ala Leu Arg Val Leu Arg
50 55 60
Arg Glu Pro Leu Pro Arg Leu Leu Arg Ala Phe Pro Ala Leu Glu Arg
65 70 75 80
Leu Asp Leu Ser Ala Cys Ala Ser Leu Asp Asp Ala Ser Leu Ala Ala
85 90 95
Ala Leu Ala Gly Ala Asp Leu Gly Thr Val Arg Gln Val Cys Leu Ala
100 105 110
Arg Ala Ser Gly Val Gly Trp Arg Gly Leu Glu Ala Leu Val Ala Ala
115 120 125
Cys Pro Arg Leu Glu Ala Val Asp Leu Ser His Cys Val Gly Ala Gly
130 135 140
Asp Arg Glu Ala Ala Ala Leu Ala Ala Ala Ser Gly Leu Arg Glu Leu
145 150 155 160
Asn Leu Glu Lys Cys Leu Gly Val Thr Asp Met Gly Leu Ala Lys Val
165 170 175
Ala Val Gly Cys Pro Arg Leu Glu Thr Leu Ser Phe Lys Trp Cys Arg
180 185 190
Glu Ile Ser Asp Ile Gly Val Asp Leu Leu Val Lys Lys Cys Arg Asp
195 200 205
Leu Arg Ser Leu Asp Ile Ser Tyr Leu Lys Val Ser Asn Glu Ser Leu
210 215 220
Arg Ser Ile Ser Thr Leu Glu Lys Leu Glu Glu Leu Ala Met Val Ala
225 230 235 240
Cys Ser Cys Ile Asp Asp Glu Gly Leu Glu Leu Leu Ser Arg Gly Ser
245 250 255
Asn Ser Leu Gln Ser Val Asp Val Ser Arg Cys Asn His Val Thr Ser
260 265 270
Gln Gly Leu Ala Ser Leu Ile Asp Gly His Ser Phe Leu Gln Lys Leu
275 280 285
Asn Ala Ala Asp Ser Leu His Glu Ile Gly Gln Asn Phe Leu Ser Lys
290 295 300
Leu Val Thr Leu Lys Ala Thr Leu Thr Val Leu Arg Leu Asp Gly Phe
305 310 315 320
Glu Val Ser Ser Ser Leu Leu Ser Ala Ile Gly Glu Gly Cys Thr Asn
325 330 335
Leu Val Glu Ile Gly Leu Ser Lys Cys Asn Gly Val Thr Asp Glu Gly
340 345 350
Ile Ser Ser Leu Val Ala Arg Cys Ser Tyr Leu Arg Lys Ile Asp Leu
355 360 365
Thr Cys Cys Asn Leu Val Thr Asn Asp Ser Leu Asp Ser Ile Ala Asp
370 375 380
Asn Cys Lys Met Leu Glu Cys Leu Arg Leu Glu Ser Cys Ser Ser Ile
385 390 395 400
Asn Glu Lys Gly Leu Glu Arg Ile Ala Ser Cys Cys Pro Asn Leu Lys
405 410 415
Glu Ile Asp Leu Thr Asp Cys Gly Val Asn Asp Glu Ala Leu His His
420 425 430
Leu Ala Lys Cys Ser Glu Leu Leu Ile Leu Lys Leu Gly Leu Ser Ser
435 440 445
Ser Ile Ser Asp Lys Gly Leu Gly Phe Ile Ser Ser Lys Cys Gly Lys
450 455 460
Leu Ile Glu Leu Asp Leu Tyr Arg Cys Ser Ser Ile Thr Asp Asp Gly
465 470 475 480
Leu Ala Ala Leu Ala Asn Gly Cys Lys Lys Ile Lys Leu Leu Asn Leu
485 490 495
Cys Tyr Cys Asn Lys Ile Thr Asp Ser Gly Leu Ser His Leu Gly Ala
500 505 510
Leu Glu Glu Leu Thr Asn Leu Glu Leu Arg Cys Leu Val Arg Ile Thr
515 520 525
Gly Ile Gly Ile Ser Ser Val Val Ile Gly Cys Lys Ser Leu Val Glu
530 535 540
Leu Asp Leu Lys Arg Cys Tyr Ser Val Asn Asp Ser Gly Leu Trp Ala
545 550 555 560
Leu Ala Arg Tyr Ala Leu Asn Leu Arg Gln Leu Thr Ile Ser Tyr Cys
565 570 575
Gln Val Thr Gly Leu Gly Leu Cys His Leu Leu Ser Ser Leu Arg Cys
580 585 590
Leu Gln Asp Val Lys Met Val His Leu Ser Trp Val Ser Ile Glu Gly
595 600 605
Phe Glu Met Ala Leu Arg Ala Ala Cys Gly Arg Leu Lys Lys Leu Lys
610 615 620
Ile Leu Gly Gly Leu Lys Ser Val Leu Ser Pro Asp Leu Leu Gln Leu
625 630 635 640
Leu Gln Ala Cys Gly Cys Arg Ile Arg Trp Val Asn Lys Pro Leu Val
645 650 655
Tyr Lys Asp Ala Ile
660
<210> 54
<211> 639
<212> PRT
<213> Brassica (Brassica rapa)
<400> 54
Met Pro Leu Ser Pro Ser Ile Leu Ser Val Leu Ser Glu Asp Leu Leu
1 5 10 15
Val Arg Val Tyr Gly Phe Leu Asp Pro Pro Cys Arg Lys Lys Trp Arg
20 25 30
Leu Val Ser Lys Glu Phe His Arg Val Asp Ser Leu Ser Arg Thr Ser
35 40 45
Ile Arg Ile Leu Arg Val Glu Phe Leu Pro Ala Leu Leu Ser Asn Tyr
50 55 60
Pro His Leu Ser Ser Leu Asp Leu Ser Val Cys Pro Lys Leu Asp Asp
65 70 75 80
Asp Val Val Leu Arg Leu Ala Ser Tyr Gly Ala Val Ser Ile Lys Ser
85 90 95
Leu Asn Leu Ser Arg Ala Thr Ala Leu Arg Ala Arg Gly Leu Glu Thr
100 105 110
Leu Ala Arg Leu Cys Arg Gly Leu Glu Arg Val Asp Val Ser His Cys
115 120 125
Trp Gly Phe Gly Asp Arg Glu Ala Ala Ala Leu Ser Val Ala Ala Gly
130 135 140
Leu Arg Glu Val Arg Leu Asp Lys Cys Leu Ser Leu Ser Asp Val Gly
145 150 155 160
Leu Ala Arg Ile Val Leu Gly Cys Ser Asn Leu Ser Lys Ile Ser Leu
165 170 175
Lys Trp Cys Met Glu Ile Ser Asp Leu Gly Ile Asp Leu Leu Cys Lys
180 185 190
Lys Cys Lys Asp Leu Lys Ser Leu Asp Val Ser Tyr Leu Lys Ile Thr
195 200 205
Asn Asp Ser Ile Arg Ser Ile Ala Leu Leu Pro Lys Leu Glu Val Leu
210 215 220
Glu Met Val Asn Cys Pro Leu Val Asp Asp Asp Gly Leu Gln Tyr Leu
225 230 235 240
Glu Asn Gly Cys Pro Ser Leu Gln Glu Ile Asp Val Thr Arg Cys Glu
245 250 255
Arg Val Ser Leu Ser Gly Val Val Ser Ile Val Arg Gly His Pro Asp
260 265 270
Leu Gln His Leu Lys Ala Ser His Cys Val Ser Glu Val Ser Leu Ser
275 280 285
Phe Leu His Asn Ile Lys Ala Leu Lys His Leu Lys Thr Leu Trp Ile
290 295 300
Asp Gly Ala Arg Val Ser Asp Ser Ser Leu Leu Thr Leu Ser Ser Ser
305 310 315 320
Cys Arg Pro Leu Thr Asp Ile Gly Val Ser Lys Cys Val Gly Val Thr
325 330 335
Asp Ile Gly Ile Thr Gly Leu Ala Arg Asn Cys Ile Asn Leu Lys Thr
340 345 350
Leu Asn Leu Ala Cys Cys Gly Phe Val Thr Asp Ala Ala Ile Ser Ala
355 360 365
Val Ala Gln Ser Cys Arg Asn Leu Glu Thr Leu Lys Leu Glu Ser Cys
370 375 380
His Met Ile Thr Glu Lys Gly Leu Gln Ser Leu Gly Cys Tyr Ser Lys
385 390 395 400
His Leu Gln Glu Leu Asp Leu Thr Asp Cys Tyr Gly Val Asn Asp Arg
405 410 415
Gly Leu Glu Tyr Ile Ser Lys Cys Ser Asn Leu Leu Arg Leu Lys Leu
420 425 430
Gly Leu Cys Thr Asn Ile Ser Asp Lys Gly Met Phe His Ile Gly Ser
435 440 445
Lys Cys Ser Lys Leu Leu Glu Leu Asp Leu Tyr Arg Cys Gly Gly Phe
450 455 460
Gly Asp Asp Gly Leu Ala Ala Ile Ser Arg Gly Cys Lys Ser Leu Asn
465 470 475 480
Arg Leu Ile Ile Ser Tyr Cys Gly Glu Leu Thr Asp Thr Gly Val Glu
485 490 495
Gln Ile Arg Gln Leu Glu His Leu Ser His Leu Glu Leu Arg Gly Leu
500 505 510
Lys Asn Ile Thr Gly Ala Gly Leu Ala Ala Val Ala Cys Gly Cys Lys
515 520 525
Lys Leu Asp Tyr Leu Asp Leu Lys Lys Cys Glu Asn Ile Asp Asp Ser
530 535 540
Gly Phe Trp Ala Leu Ala Tyr Phe Ala Arg Asn Leu Arg Gln Ile Asn
545 550 555 560
Leu Cys Tyr Cys Ser Val Ser Asp Thr Ala Leu Cys Met Leu Met Ser
565 570 575
Asn Leu Ser Arg Val Gln Asp Val Asp Leu Val Asn Leu Asn Arg Val
580 585 590
Thr Val Glu Gly Ser Glu Phe Ala Leu Arg Ala Cys Cys Asn Arg Leu
595 600 605
Lys Lys Leu Lys Leu Phe Ala Pro Leu Arg Phe Leu Leu Ser Ser Glu
610 615 620
Leu Leu Glu Met Leu His Ala Arg Gly Cys Arg Ile Arg Trp Asp
625 630 635
<210> 55
<211> 641
<212> PRT
<213> Soybean (Glycine max)
<400> 55
Met Leu Ser Glu Ser Val Phe Cys Leu Leu Thr Glu Asp Leu Leu Ile
1 5 10 15
Arg Val Leu Glu Lys Leu Gly Pro Asp Arg Lys Pro Trp Arg Leu Val
20 25 30
Cys Lys Glu Phe Leu Arg Val Glu Ser Ser Thr Arg Lys Lys Ile Arg
35 40 45
Ile Leu Arg Ile Glu Phe Leu Leu Gly Leu Leu Glu Lys Phe Cys Asn
50 55 60
Ile Glu Thr Leu Asp Leu Ser Met Cys Pro Arg Ile Glu Asp Gly Ala
65 70 75 80
Val Ser Val Val Leu Ser Gln Gly Ser Ala Ser Trp Thr Arg Gly Leu
85 90 95
Arg Arg Leu Val Leu Ser Arg Ala Thr Gly Leu Gly His Val Gly Leu
100 105 110
Glu Met Leu Ile Arg Ala Cys Pro Met Leu Glu Ala Val Asp Val Ser
115 120 125
His Cys Trp Gly Tyr Gly Asp Arg Glu Ala Ala Ala Leu Ser Cys Ala
130 135 140
Ala Arg Leu Arg Glu Leu Asn Met Asp Lys Cys Leu Gly Val Thr Asp
145 150 155 160
Ile Gly Leu Ala Lys Ile Ala Val Gly Cys Gly Lys Leu Glu Arg Leu
165 170 175
Ser Leu Lys Trp Cys Leu Glu Ile Ser Asp Leu Gly Ile Asp Leu Leu
180 185 190
Cys Lys Lys Cys Leu Asp Leu Lys Phe Leu Asp Val Ser Tyr Leu Lys
195 200 205
Val Thr Ser Glu Ser Leu Arg Ser Ile Ala Ser Leu Leu Lys Leu Glu
210 215 220
Val Phe Val Met Val Gly Cys Ser Leu Val Asp Asp Val Gly Leu Arg
225 230 235 240
Phe Leu Glu Lys Gly Cys Pro Leu Leu Lys Ala Ile Asp Val Ser Arg
245 250 255
Cys Asp Cys Val Ser Ser Ser Gly Leu Ile Ser Val Ile Ser Gly His
260 265 270
Gly Gly Leu Glu Gln Leu Asp Ala Gly Tyr Cys Leu Ser Glu Leu Ser
275 280 285
Ala Pro Leu Val Lys Cys Leu Glu Asn Leu Lys Gln Leu Arg Ile Ile
290 295 300
Arg Ile Asp Gly Val Arg Val Ser Asp Phe Ile Leu Gln Thr Ile Gly
305 310 315 320
Thr Asn Cys Lys Ser Leu Val Glu Leu Gly Leu Ser Lys Cys Val Gly
325 330 335
Val Thr Asn Lys Gly Ile Val Gln Leu Val Ser Gly Cys Gly Tyr Leu
340 345 350
Lys Ile Leu Asp Leu Thr Cys Cys Arg Phe Ile Ser Asp Ala Ala Ile
355 360 365
Ser Thr Ile Ala Asp Ser Cys Pro Asp Leu Val Cys Leu Lys Leu Glu
370 375 380
Ser Cys Asp Met Val Thr Glu Asn Cys Leu Tyr Gln Leu Gly Leu Asn
385 390 395 400
Cys Ser Leu Leu Lys Glu Leu Asp Leu Thr Asp Cys Ser Gly Val Asp
405 410 415
Asp Ile Ala Leu Arg Tyr Leu Ser Arg Cys Ser Glu Leu Val Arg Leu
420 425 430
Lys Leu Gly Leu Cys Thr Asn Ile Ser Asp Ile Gly Leu Ala His Ile
435 440 445
Ala Cys Asn Cys Pro Lys Met Thr Glu Leu Asp Leu Tyr Arg Cys Val
450 455 460
Arg Ile Gly Asp Asp Gly Leu Ala Ala Leu Thr Ser Gly Cys Lys Gly
465 470 475 480
Leu Thr Asn Leu Asn Leu Ser Tyr Cys Asn Arg Ile Thr Asp Arg Gly
485 490 495
Leu Glu Tyr Ile Ser His Leu Gly Glu Leu Ser Asp Leu Glu Leu Arg
500 505 510
Gly Leu Ser Asn Ile Thr Ser Ile Gly Ile Lys Ala Val Ala Ile Ser
515 520 525
Cys Lys Arg Leu Ala Asp Leu Asp Leu Lys His Cys Glu Lys Ile Asp
530 535 540
Asp Ser Gly Phe Trp Ala Leu Ala Phe Tyr Ser Gln Asn Leu Arg Gln
545 550 555 560
Ile Asn Met Ser Tyr Cys Ile Val Ser Asp Met Val Leu Cys Met Leu
565 570 575
Met Gly Asn Leu Lys Arg Leu Gln Asp Ala Lys Leu Val Cys Leu Ser
580 585 590
Lys Val Ser Val Lys Gly Leu Glu Val Ala Leu Arg Ala Cys Cys Gly
595 600 605
Arg Ile Lys Lys Val Lys Leu Gln Arg Ser Leu Arg Phe Ser Leu Ser
610 615 620
Ser Glu Met Leu Glu Thr Met His Ala Arg Gly Cys Lys Ile Arg Trp
625 630 635 640
Asp
<210> 56
<211> 3093
<212> DNA
<213> Arabidopsis thaliana (Arabidopsis thaliana)
<400> 56
ctttctttat tttatttacc ttttaccaaa gttacacttc tacccctatt tttcttcaat 60
aagagatgga agagcaaggg caattaagta atttgacaat atccttctcc ttcaagactc 120
tctctctccc acagaaaaca aactgtttgt tgttaataac tgtgggtttt ttccaaaatc 180
ctcaaagaga gactatactt ctctcggatc cttcctgatt tcaaaatttc ttcaacccac 240
cagcagcagc agcagatgaa gaaggttaaa cagattcgtg tcttaaagcc tttcgatctt 300
ctctcggaag agctcgtctt tatcatcctt gacctcatct ctcccaaccc ttccgatctc 360
aaatccttct ctcttacttg caaatctttc taccagctcg agtccaagca ccgcggatcc 420
ttaaaacctc tccgctccga ctatctccct cgcatcttga ctcgttaccg gaacaccacc 480
gatctcgatc ttaccttctg cccgcgtgtc actgactacg cgcttagcgt cgttggctgt 540
ctctccggac ctacgcttcg ctccctcgac ctctcgcgct ctggctcctt ctccgccgcg 600
ggactactgc gattggccct caaatgtgtc aatttagtcg agattgacct gtccaatgcg 660
acggagatga gagacgccga tgctgcggtg gtggcggagg cgaggagtct ggagaggctg 720
aagctgggca gatgcaagat gctgacggac atgggaatcg gatgtatagc agttggatgt 780
aagaagctca atacggttag cttgaaatgg tgtgttggcg tcggagattt aggggttggc 840
ttgcttgccg tcaaatgcaa ggacattcgc accttagacc tctcctactt gccgatcaca 900
ggaaagtgtt tacatgacat tctgaaactt caacaccttg aagaacttct tctagaaggg 960
tgctttggag tagatgatga cagtcttaaa tcactcagac atgattgcaa gtcattgaag 1020
aagcttgatg catccagctg ccagaattta actcatagag gtttaacctc acttttaagc 1080
ggggcaggat atcttcagcg acttgatcta tcacactgtt cttctgtgat atcattggat 1140
tttgcgagta gcttaaagaa ggtttcagca ttacagtcga tcaggttgga tggttgttct 1200
gttacgcctg atggtttgaa ggcgataggc acattgtgca attccctgaa agaggttagc 1260
ctaagcaaat gcgtgagcgt aactgatgaa ggtctttctt ctctagtaat gaaactgaaa 1320
gacctcagaa aacttgacat cacatgttgc cggaaactaa gtagagtttc aatcacccaa 1380
atcgccaatt cgtgtccttt actagtctct ttgaagatgg agtcttgttc tcttgtttcc 1440
agagaagcct tttggttgat cggacaaaag tgtcggctac ttgaagagct tgacttaact 1500
gacaacgaga ttgatgatga aggactgaaa tccatatcta gttgtctgag tctttcctcg 1560
ttaaagctgg gaatttgtct taacataaca gacaaagggc tctcgtacat cgggatgggc 1620
tgttcaaatc tccgtgaact tgatctctat aggtcagtgg gaataacaga cgtaggcatc 1680
tccacaattg cccaaggctg cattcatctg gaaacaataa acatttcata ctgccaagac 1740
ataacagaca agtccctggt ttcattgtcc aaatgctcgt tgttacaaac attcgagagc 1800
aggggatgtc ctaacatcac gtcccaagga cttgcagcca ttgctgttcg gtgcaagcga 1860
ctcgccaagg ttgacttgaa gaagtgccca tccatcaatg atgcggggtt gctcgctctg 1920
gctcacttct ctcagaatct caaacagata aacgtgtcag acacagctgt gactgaagtg 1980
ggacttctct ccctagccaa catagggtgt ttacagaaca tagcggttgt gaactcgagt 2040
ggtttaagac cgagcggagt agcagcagca ttgctggggt gtggaggatt aaggaaagcg 2100
aaactacatg cgtccctaag atcactgctt cctctttctc taatccacca cttggaagct 2160
cgtggttgtg cgttcctctg gaaagacaat acccttcagg cggagttaga tcccaagtac 2220
tggaagcaac agctggaaga gatggcgcct taaattaaaa gtgagaagaa acattctgaa 2280
atgggaaaga ggagttccta tgggagccag cagacaggcc ctgttttggg cccagtgtgg 2340
attagctgga acaattttgg tctcttttgt gttgttgttg acggcgcgtg ttctaacaac 2400
cccacacaac cttacattat aagtctagtc acatggtggg tgacatggta ccgttgtata 2460
tgtagttttt gtttcttttt tttttttttt tggttgggga gccgaaatta acggacgtaa 2520
cagagtcaca aggggatgcc ttctctgtgc ctttttgact ttttcctcct ttcttttttt 2580
tctgtaatca tatgagtttt atgtaattta atgcctcatc aacttgcctg gatagggccg 2640
gcctttccac gtcaccttag tcgcgttttg cggtaaaaaa agtaaaaaga aaggccgtga 2700
ctcagtaggg cccattactg gtccagtcca ggctctcacc gtctgtacaa aaatagcgag 2760
tcagcgactc ggtgtgacag caacaaccct ctctcgttga aaaacggcta atgcgcccga 2820
cacttccatc ctcatattcc attacaaaag aaatccagct atgtagtatc aaactaccga 2880
aaacatacac atgtcaatca agtccaaaaa gtatcaccgt caaatgagtg attcagattt 2940
tgtttgctca tgaaagataa accaaatgat tacttcctta atcacctgcc ccttgttttc 3000
cattttttaa ttacataatg gaacccccca ttattaaaac atgatcattt tctttcgtaa 3060
taattaaact tcccaactaa atgcctctta ata 3093
<210> 57
<211> 2563
<212> DNA
<213> Rice (Oryza sativa)
<400> 57
atgagcgagg aggtgcagag gtatggaggt ggtgggggtg ggggaggagg tggggtggcg 60
gcgctgtcgc tggatttgct cggccaggtg ctcgaccggg tgcgggagcc gcgggaccgc 120
aaggcgtgca ggctcgtgag ccgcgccttc gcccgcgccg aggccgcgca ccgccgcgcg 180
ctgcgggtgc tccgccgcga gcccctggcg cgcctcctcc gcgcgttccg ggcgctggag 240
cggctcgacc tctccgcctg cgcctccctc gacgacgcct ccctcgccgc cgcgctctcc 300
ggcgcggatc tcgccggggt gcgccgggtc tgcctcgccc gcgccagcgg cgtcgggtgg 360
cgcggcctcg acgcgctcgt ggcggcgtgc ccgaggctgg aggccgtcga cctgtcgcac 420
tgcgtcggcg ccggcgaccg cgaggccgcc gcgctggccg cggcgacggg gctgagggaa 480
ttgagcctcg agaagtgcct cggcgtcacg gacatggggc tcgccaaggt ggtggtcggg 540
tgcccgaggc tggagaagct gagcctcaag tggtgccgcg agatctccga catcggcatc 600
gatttgctct ccaagaagtg ccacgagctc cggagcctcg acatctccta cctcaaggtt 660
ggaaatgaat cccttagatc aatatcctca cttgagaaac ttgaggagtt ggcaatggtt 720
tgttgctcat gcatagatga tgatggcctg gaattactag gcaaggggag caactcactg 780
cagagcgttg atgtttcaag atgtgatcat gtaacctccc agggattagc ttcactcata 840
gatggtcaca attttctcca gaagttaaat gctgctgata gtttgcatga gatgagacag 900
tcgtttctgt ccaacttggc aaaactgaag gataccttga cagtgcttag acttgatggt 960
cttgaagtct catcctctgt tcttctggcc attggtggtt gtaataactt ggtcgagatt 1020
ggccttagca aatgcaatgg cgttacagat gaaggaatct cttcacttgt aactcaatgc 1080
agccacttaa gggttattga tctcacatgc tgtaacctcc ttaccaacaa tgcccttgat 1140
tcaatagctg agaactgtaa gatggttgaa catctccgtt tggaatcctg ttcttccata 1200
agcgaaaagg gactggagca gattgcaacc tcctgcccca atctaaagga gatagacctc 1260
actgactgtg gagtgaatga tgcagcattg cagcacttgg ccaagtgctc tgaactgctt 1320
gtactgaaat taggcctgtg ctcaagtatt tctgacaaag gtcttgcttt tatcagttca 1380
agctgtggaa agctgattga gcttgatctc tatcgctgca attctattac cgatgatggg 1440
ctggcagctt tagctaatgg ctgcaagaag attaagatgc taaacctatg ctactgcaac 1500
aagattaccg acagtggttt gggccaccta ggctctctag aggagctcac aaaccttgaa 1560
ctgaggtgct tggtccgtat aacaggtatt ggaatctcat cagttgccat cggctgcaag 1620
aacctgatag agatagactt gaagcgttgc tattctgtcg atgatgctgg cttgtgggct 1680
cttgcccgat atgcactaaa ccttagacag cttactatat catactgcca agtcactggc 1740
ctgggcctgt gtcacctgtt aagctccctg aggtgcctcc aggatgttaa gatggtacac 1800
ctctcatggg tctctataga agggttcgag atggcactga gagcggcttg cgggaggctg 1860
aagaaactga agatgctgag tgggctgaag tctgtgctgt ctcctgagct gctccagatg 1920
ctgcaggcct gcggctgccg catccgttgg gtcaacaagc ctcttgtcta caaggactaa 1980
acctgctctc atatcatcat ctgtgcagtg ttaatatcct cggatcatac accatggagc 2040
ccttaccaag tctagcatgc ttacaagcca tgttagatac ccgcaataat cgtcaatttt 2100
catacccaag atgtactgac gatggtgtct ctgcattgct gcagtgccat ccttatgttt 2160
ggcaactcct agaccaatgt tgtcatcttc agattaaaat ccaacatttg caaacatgag 2220
gcatcgaaac agttatgttc tgagcaagtg atccttgtac cagactgcaa tgaaatgctc 2280
tacactttca attggtatat gcaaagaaca atggcatgtt tgcctttgtc attgctccaa 2340
ctccaggctt gctgaccgat gcatctttta gagtttaaga acagctttag atcgcagaaa 2400
aactcttcct tgatgctcca ggatctgggc tgggacactg gtttcatggt tgtctgtatt 2460
gtatgactgg tggacccgtc cttttttttt taccttgatt acaataatct cgatgtactc 2520
attcaggcag agatttgaag taatgtattg aatcagtttt tat 2563
<210> 58
<211> 2844
<212> DNA
<213> Corn (Zea mays)
<400> 58
cggcatcacc tatccacatc tttctccctc tctctctctc tcctcttccg tccctcctct 60
ctccctctct acgccacgcc tccacctcca cacctgcacg cctctcgctc tctctcctca 120
cctgtcgagc tgcccacagc ccgtacgtac ctgcccccag tcccagtccc cgccgccccg 180
cctgcccgac ccgcggctta tccaccacgt ccgccgctgg gctggatcga tcgggaccgg 240
tatcgcgccg ccgccgctgc cctcggcact cgcgggatcc tgttcctacc atgcagcagc 300
gctgcgccct tgcccgaccg cgccggcccg ccgaaccgcg cgttccgctc cccgacgatc 360
actccctcgt cgccgtcttc tccttcctct tcatccccac ctaaatcaac ctcctcgccg 420
cgaaattctg ctacaatttc ttcatcttcc cttcccgccg ggagcggacg gatcgccgct 480
tcctcggccg gcgggatgag ccgggaggcg cagaaactcg actgcgccgc cggcgccggg 540
gggatcgggg tcctgtcgct ggacctactg ggccaggtgc tggaacacct gcgggagccc 600
cgggaccgca agacgtgccg cctcgtcagc cgcgccttcg agcgcgccga ggccgcgcac 660
cgccgcgcgc ttcgggtgct ccgacgcgag ccgctcccgc gcctgctccg cgcgttcccg 720
gcgctcgagc ggctcgacct ctccgcctgc gcctcgctcg acgacgcctc cctcgccgcg 780
gccgtcgccg acgccggcgg agggctcgcc ggcctccgca gcgtgtgcct cgcgcgggcc 840
aatggtgtcg gctggcgcgg cctcgaggcg ctcgtcgcgg cctgccccaa gctcgcggcc 900
gtcgacctgt cgcactgcgt caccgccggg gaccgcgagg ccgccgcgtt ggcggcggcg 960
tccgagctca gggacctgag gctggacaaa tgccttgccg tcaccgacat ggggctcgcc 1020
aaggtggctg ttgggtgccc caagctggag aagctcagcc tcaagtggtg ccgtgagatc 1080
tctgacattg gaattgatct gctggccaag aagtgccctg agctccgcag cctcaacata 1140
tcctacctca aggtgggcaa tggatccctt ggatcaattt cgtcacttga gaggcttgag 1200
gaattggcaa tggtttgttg ttcaggtata gatgacgaag gcttggaatt gctgagcaag 1260
gggagcgatt cgctgcagag tgttgatgtg tcaagatgtg atcatgtgac ttccgagggg 1320
ttagcttcac tcatagatgg tcgcaatttt cttcagaagt tatatgctgc agattgtttg 1380
catgagatag gacagcgttt tctatccaag ttggcaagac tgaaggaaac cttgacattg 1440
ctgaaactcg acggtcttga ggtctcggac tctcttcttc aagccattgg tgaaagctgt 1500
aacaaattgg ttgagattgg gcttagcaaa tgcagtggtg ttactgatgg aggaatctca 1560
tctcttgtag ctcggtgtag tgacctaaga acaattgatc tcacatgctg caatctcatt 1620
accaacaatg cgcttgattc aatagctgac aactgtaaga tgcttgaatg tttgcggttg 1680
gaatcatgct ctttgataaa tgagaaggga ctagagcgaa ttacaacttg ttgccccaac 1740
cttaaggaga tagacctcac tgactgcgga gtagatgatg cagcattgca gcacttggct 1800
aaatgctctg aattgcgaat attgaaatta ggcctatgct caagtatttc tgacagaggc 1860
attgcattta tcagttcgaa ttgtggaaag ctcgtggagc ttgatctcta ccggtgcaac 1920
tctattactg atgatgggtt ggcagcttta gcaaatgggt gcaagaggat taagttactg 1980
aacttgtgtt actgcaacaa gatcactgat actggtttgg gtcacctagg ctccctggag 2040
gagctcacaa accttgaact gaggtgcttg gtccgcgtaa caggcattgg gatctcctca 2100
gttgcaatcg gatgcaagaa cctgatagag ctggacttga aacgatgcta ttcggttgat 2160
gacgctggcc tgtgggccct tgctcgttat gctttgaacc ttagacagct tacgatatcg 2220
tactgccaag tcacggggtt gggcctgtgc cacctgctga gctccctgcg atgcctccag 2280
gacattaaga tggtgcacct ctcatgggtc tcaatagaag ggtttgagat ggctctgcga 2340
gcagcctgcg ggaggctgaa gaagctgaag atgctctgcg ggctgaagac cgtgctctcc 2400
cctgagctcc tccagatgct gcaggcttgt ggctgccgaa taagatgggt caacaagcct 2460
cttgtctaca aggactagac agactcgtgt tcctcattgt gtgtgcagtg tcagcatcag 2520
catgtattgt caagatcttg caggtttgcc ctggctcacg ttgtgagtcc gcaataatcg 2580
ttgggggcgt aagctcccgt cccccagatg tggttatggt gcctttgggg cacttcttgc 2640
gccatttgtc actctctgct ccggtgtatc tgtgtatgca aacggaggca ccggtaatgt 2700
tcatgtgtat atagcgtgca gtagaatgat cccattttat gggattagtt agtgtgtaat 2760
tgttacctca tttcgcatga ttcgaatctt gagatgaaag aatcataagg ggtatgatcg 2820
atgcattgca ttcaccggtt ctta 2844
<210> 59
<211> 2642
<212> DNA
<213> Barley (Hordeum vulgare)
<400> 59
ggggtgctac ctatccacat ctttctcctc ccatcctccc tccctctccc cactccccac 60
ccatcgaccc accttcctcc ctccgtacct tcgccaccac cggccgtcgc cttgccgctc 120
ctccgccgcc cggatcagga acgatcgcac cgtgcggccg cctcccgccc tgcgcgcgga 180
tcctaccatg ctgaggcgct gcgccccata ccgaccccgc agggccgccg ccgtaccgcg 240
cctcccgatc cccggcggcc acccctacct cgtcgccctc tccttcctct tcatcccgac 300
ctaagctcca cgcctcgccg acaggccggc cctcgccgcc ggagctgccc ggtggaatcg 360
tcagccgcga tgagcgagga ggaggcgcag aggtacggcg gcgggaccgg cggcggcggc 420
gtcggtgcgc tgtccgtgga tctgctcggc caggtgctcg atcgcgtgct ggagcggcgg 480
gaccgcaagg cctgccgcct cgtcagccgc gccttcgcgc gcgctgaggc cgcgcaccgc 540
cgggccctcc gggtgctccg ccgggagccg ctccctcgcc tgctccgcgc cttcccggcg 600
ctcgagcgcc tcgatctctc agcctgtgcc tcgctcgacg acgcgtctct tgcagccgct 660
ttagccggcg cggacctcgg caccgtccga caggtctgcc tggcgcgggc cagcggagtg 720
ggctggcgcg ggctggaagc cctcgtggcc gcctgcccca ggctcgaggc tgtcgacctg 780
tcgcactgcg tcggtgctgg ggatagggag gctgccgccc tggccgccgc ctctgggctg 840
agggagctaa atctggaaaa gtgccttggg gtcactgata tggggctcgc caaggtagcc 900
gtgggctgcc ccagactgga gactctgagc ttcaagtggt gccgtgaaat ctctgacatc 960
ggcgtcgatc tgcttgtcaa aaagtgccgc gacctccgca gccttgacat ctcctaccta 1020
aaggtgagca atgagtccct tagatcaata tcgactcttg agaagctaga ggagttggcc 1080
atggttgctt gctcatgtat agatgatgaa ggcctggaat tgcttagcag aggaagcaat 1140
tcattgcaga gtgttgatgt ctcaagatgc aatcacgtga cttcccaggg gttagcttca 1200
ctgatagatg gtcacagttt tctccagaag ttaaatgccg cagatagttt gcatgagatt 1260
ggacagaatt ttctatccaa gttggtaaca ctgaaggcaa ccttgaccgt gttgagactt 1320
gacggctttg aagtgtcatc ctctcttctt tcagcgattg gtgaaggttg taccaacttg 1380
gttgagattg gactaagcaa atgcaacggt gttacagatg aaggcatctc ttcgcttgta 1440
gctcgctgta gctacctaag gaaaattgat ctcacatgct gcaatctagt cacaaacgat 1500
tcccttgatt caatagctga caactgtaag atgcttgaat gcctccggtt ggagtcctgc 1560
tcttctataa acgagaaagg actagagaga attgcaagct gttgccccaa tctaaaggag 1620
atagatctca ctgattgtgg agtgaacgac gaagcgttgc atcatttggc gaagtgctct 1680
gaactgctga tattgaaatt aggcctgagc tcaagtattt cggacaaagg ccttggtttt 1740
attagttcaa agtgtgggaa gctcattgaa cttgacctct atcgctgcag ttctatcact 1800
gatgatgggc tggcagcctt agccaacggc tgcaagaaaa ttaagctgct gaacctttgt 1860
tactgcaaca agataactga tagtggtttg agccacctgg gcgctcttga ggagctcaca 1920
aaccttgagc tgaggtgcct cgttcgcatt acaggcatcg gaatttcttc cgttgtcatt 1980
ggctgtaaga gcctggtaga acttgacttg aagcgctgct attctgtcaa tgattctggg 2040
ctatgggctc ttgcccgata tgctctaaac cttagacagc tcaccatatc atactgccaa 2100
gttactggcc taggcttgtg ccacctgctt agctccttga ggtgcctcca ggacgtgaag 2160
atggtgcacc tgtcatgggt ttccatagaa gggttcgaga tggctctgcg agccgcttgc 2220
gggaggctga agaagctgaa gatactcggc ggtttgaagt ccgtgctatc ccctgacctg 2280
ctccagcttc tgcaagcctg cggctgccgc atcagatggg tcaacaagcc tcttgtctac 2340
aaggatgcca tctgatctga gaagtaccat attgttcatc ctttcggcaa gtttgtcatc 2400
gctgcgtagc cctgttagaa aattcacaat aactacacgc ctcgtcaatg ccatgcctaa 2460
ggtgtggtca agatcatgtc tgtttgagat gtcgagtaaa catggcagct gctgtcgaat 2520
atatgtcatg tacagtgaag cctttttttt tggacgggat gtacagtgaa gcccttttgc 2580
atagcagagt tgtgcaactg gagttctcta cttgatgtga aggaacctga aaaatatgct 2640
gt 2642
<210> 60
<211> 2389
<212> DNA
<213> Brassica (Brassica rapa)
<400> 60
gggaaacaat tgaagtaaag agagagaggt tcggttttgt gtttccaacc ggaaaaaaaa 60
ccccgaaggt ttgacgatga acccgaatct aataacccgt aacccaattc ggatcctgga 120
tcaaccggtt caccactctc tcctctatct ccgagctgtc tttctcttca tcccgactta 180
aaagatccgt actttttttt ttttgaaact ttccgaccaa actaaaagta aacacatact 240
tgtctttttt tttcttgttc tgtcgcgaaa tgccgctgtc tccatccatt ctatccgttc 300
tgtcggaaga tcttctagtt cgtgtctacg ggttcctaga cccgccctgt cggaaaaaat 360
ggcgactcgt cagcaaagag tttcaccgag tcgactcact gagccgcaca tcgatccgaa 420
tcctccgagt cgagttcctc cccgcgcttc tctccaacta ccctcacctc tcctccctcg 480
acctctccgt ctgccccaaa ctcgacgacg acgtcgttct gcgcctagcg tcctacggcg 540
ccgtttcgat aaagtcgctg aacctgagcc gcgccaccgc gctccgagcg aggggactgg 600
agacgctggc tcgcttgtgc cgaggcctcg agcgagtcga cgtgtctcac tgctgggggt 660
tcggagacag ggaagcggcg gcgctctccg tcgcggcggg gctgagggag gtgaggttgg 720
acaagtgctt gagcctaagc gacgtcggat tggcgaggat cgtcctcggg tgtagtaatc 780
tgagcaagat tagtttgaag tggtgtatgg agatctctga tctagggatc gatcttctct 840
gtaagaaatg caaagacttg aagtctctcg acgtctctta tcttaagatc acgaatgatt 900
cgatccggtc catagctttg ttgccaaagc tcgaggtttt agagatggtg aactgtccgt 960
tggtagatga tgatgggtta cagtatcttg agaatggttg tccttcgtta caggagattg 1020
atgtcacaag gtgtgagcgt gtgagtttgt ctggcgttgt ctccattgtc agaggccatc 1080
ctgatcttca acacctgaaa gccagtcact gtgtatcaga agtatctctg agcttcttac 1140
ataacatcaa agctttgaag catctcaaga ctctatggat cgatggagct cgtgtctctg 1200
actcctctct cttaacccta agctccagct gtagaccctt aacagatatc ggagtgagca 1260
aatgtgtggg tgtgacggat attggcatca caggactagc acgcaactgc ataaacctga 1320
aaaccctaaa cctagcgtgc tgcgggtttg tgactgatgc agccatctct gcggtagctc 1380
agtcttgccg caatctggag actcttaagt tagagtcttg tcatatgata accgagaaag 1440
gtcttcaatc actcggatgt tactccaagc atcttcaaga actcgatctt accgactgtt 1500
atggcgtcaa tgacagaggg ctagaatata tctcaaagtg ttcgaatctt ctaaggttga 1560
aacttggcct ctgcacaaat atctcagaca aagggatgtt tcatatcggt tccaaatgtt 1620
ccaagcttct agaacttgat ctataccgct gtggtggttt tggagatgac ggtttagcag 1680
ctatatcccg aggttgcaag agcttgaacc ggctcattat atcgtactgt ggtgagctaa 1740
cagacacagg ggttgaacaa atccgccagc ttgaacatct aagccatctt gaacttcgag 1800
ggctaaagaa tataaccggt gctggtctag ctgcagttgc gtgcggctgc aagaaattgg 1860
attacttgga cctcaagaag tgcgagaata tagatgactc aggcttctgg gcgcttgctt 1920
actttgcaag aaacctaaga cagataaact tgtgctattg ctcggtttct gatacggctc 1980
tatgcatgtt aatgagcaat ctaagtcggg ttcaagacgt tgacttagtc aacctgaacc 2040
gtgtgacagt ggaagggtct gagtttgctc taagagcctg ttgcaatagg ctcaagaagc 2100
ttaaactttt cgctcctctc agattcttgc tctcatctga attgcttgag atgcttcatg 2160
ctcgtggttg ccgcattaga tgggactgaa agacgaaact ttcttcaatg gaactttact 2220
agtaacactt taacgtgttc atatgtctac ttgttgtatc tataaaaaat tcgactatgt 2280
ttttaaattc ggtttaccaa ttatatgtct accgctttgg tattatcttc caacataaac 2340
aatagtagtt ttagaaaaca aaaatattat tttggtattc tatgcaaaa 2389
<210> 61
<211> 2687
<212> DNA
<213> Soybean (Glycine max)
<400> 61
tttcaacaat ttttctctca tgtccaagtt ttgtcctgag caggacaact taattggcgg 60
ttcccttctc gcccttggat ctttaattgg ttgttccctc cgctccaaga aaaaaaaggg 120
catttatggc aaaaaagagc agcagcattc aacaagttgc gagaggaaca caggactcaa 180
ttcacatggc cgtacccgaa gaaagcgaaa agtgtgaaac acgcaccctc tgaaatctaa 240
aattcaattt tcccttccct gaccctgttt cttctattca cattgtattc tccttgtttt 300
ttcgcttttc aattttccat tgaactcaga caaaaacaga aagaaagggg aaaaaaatga 360
acccagagaa atctcgttgc aggttgccac ttcgcacgct ctctaaacac acgcaccacc 420
ctcttctcca cctccgcctc cttttcctct tcgttcccac ctaaactgtt tcccttcagt 480
tccacgtcac attaacaaac cttttttttt cttaaaaaaa aaactttagt aattacgaaa 540
tttctgatac cttaatgttg tctgaatccg ttttctgcct cttgaccgag gacctgctca 600
tccgggtcct cgaaaagctc gggccggatc ggaaaccgtg gcggctggtg tgcaaggagt 660
ttctccgggt cgaatcgtcg acccggaaga agattcggat cctccgaatc gagtttctgc 720
ttgggttgtt ggagaagttc tgcaacattg agacgctgga cctgtcgatg tgtccgcgga 780
tcgaggacgg agctgtgtcg gttgtgctga gtcagggatc ggcgagttgg actcggggac 840
tgaggagact cgtgctgagt cgcgccaccg ggttggggca tgtgggcttg gagatgctga 900
ttcgggcgtg tcccatgttg gaggccgtgg atgtgtccca ttgttggggg tatggcgaca 960
gagaggctgc ggcgctatcg tgcgccgcga ggttgaggga actcaacatg gataagtgtt 1020
tgggagttac tgatattggg ttggccaaga ttgctgtcgg gtgtgggaaa ttggagaggc 1080
tgagtttgaa gtggtgcttg gagatttctg atctggggat tgatcttctt tgcaaaaagt 1140
gcttggattt gaaatttctc gacgtgtcat atctcaaggt aacaagtgaa tctttgagat 1200
caatagcttc tctgttaaag cttgaggttt ttgttatggt tggctgctct ttagtggatg 1260
acgttggatt gcggtttctt gaaaaagggt gtccactgct taaggcaatt gatgtatcaa 1320
ggtgtgattg tgttagctct tccggtttaa tatctgtaat tagtggacat ggaggtcttg 1380
agcagttgga tgcaggatat tgcctctctg agctttcagc acctcttgtt aaatgcttgg 1440
agaatttaaa gcagctgaga ataattagaa ttgatggtgt tcgagtttct gactttatcc 1500
tccagacaat tggcaccaat tgcaagtctt tagtggaact tggtttaagc aaatgcgttg 1560
gagtgaccaa caagggaatt gtgcagctag tatctggctg tggctatttg aagatacttg 1620
atttgacttg ttgtcggttc atatctgatg cagcaatctc tactatagca gactcttgtc 1680
cagaccttgt ctgtctgaag ctagaatctt gtgatatggt gactgagaat tgtctttatc 1740
aacttggatt aaattgctcg cttctcaaag agcttgatct tactgattgc tctggtgttg 1800
atgacatagc tctaagatat ctatcaagat gttcagaact tgtaagattg aaattaggat 1860
tatgcacaaa tatatcagac ataggattgg cacacattgc ttgtaactgc ccaaaaatga 1920
ctgaacttga tctctatcga tgtgtacgta ttggagatga tgggctagcg gcactaacga 1980
gtggatgcaa ggggttgaca aacctcaact tgtcatattg caatagaatt acagacagag 2040
ggttggagta tatcagccat cttggtgaac tatctgatct ggagttgcgt gggctttcta 2100
atatcacaag cattggtata aaagcagttg caataagttg caagagattg gcagatttag 2160
atttgaaaca ttgcgaaaaa attgatgatt caggtttctg ggcccttgct ttttattcgc 2220
aaaacctgcg gcagataaat atgagctact gtatcgtgtc agatatggtg ttgtgcatgc 2280
ttatgggtaa cctgaaacgc ctgcaagatg ccaaactggt ttgtctttct aaagtgagtg 2340
taaaaggatt ggaagttgcc cttagagctt gctgtggtcg gattaaaaag gttaaactgc 2400
agaggtccct caggttctcg ctttcctctg aaatgctcga gacaatgcat gcacgagggt 2460
gcaagatcag atgggattag ccaattgtca cgctattaat ctgttatact ttcttttctt 2520
ggaagggtgc tgcttcatta agatcttttt tattcaaaat tttatatttc tctagcattt 2580
tcttttcttt aaactctgtt accagatttc tacggaaggt atttttttca ccttcttaag 2640
ttacatcatc tccaataaag catgaggtta cttatcttcg aattaaa 2687
<210> 62
<211> 46
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 62
ttggggccca acgttctcga gtgtttcagc atatttgcat gtttga 46
<210> 63
<211> 42
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 63
cggccgcaaa gtcgacgaat tctgctgctg ctgctggtgg gt 42
<210> 64
<211> 52
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 64
cgacctgcag gcatgcaagc ttaaagtctg tatatatgac acagaagaaa cc 52
<210> 65
<211> 43
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 65
cacctgtaat tcacacgtgg tgttaaggcg ccatctcttc cag 43
<210> 66
<211> 41
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 66
ttggggccca acgttctcga gtgacctgag gctgaaaatc g 41
<210> 67
<211> 47
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 67
cggccgcaaa gtcgacgaat tctttgcaac agaaccagaa ataaaca 47
<210> 68
<211> 47
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 68
gcctgcaggc tctagaggat cctgtttcag catatttgca tgtttga 47
<210> 69
<211> 42
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 69
atgtttttgg cgtcttccat ggtgctgctg ctgctggtgg gt 42
<210> 70
<211> 47
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 70
gcctgcaggc tctagaggat cctgtttcag catatttgca tgtttga 47
<210> 71
<211> 57
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 71
agactatctc ggttaaagac tgcctcgatt aacagattcg agtgcggaca gtacgat 57
<210> 72
<211> 51
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 72
taatcgaggc agtctttaac cgagatagtc ttctactacc aatggcctaa c 51
<210> 73
<211> 42
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 73
atgtttttgg cgtcttccat ggtgctgctg ctgctggtgg gt 42
<210> 74
<211> 96
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 74
tcaatctatc gtactgtccg cactcgaatc tgtccttcct cgcatgctta ctccaccata 60
tacttctact accaatggcc taacttgttg attgcg 96
<210> 75
<211> 96
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 75
cgcaatcaac aagttaggcc attggtagta gaagtatatg gtggagtaag catgcgagga 60
aggacagatt cgagtgcgga cagtacgata gattga 96
<210> 76
<211> 96
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 76
tcaatctatc gtactgtccg cactcgaatc tgttaatcga ggcagtcttt aaccgagata 60
gtcttctact accaatggcc taacttgttg attgcg 96
<210> 77
<211> 96
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 77
cgcaatcaac aagttaggcc attggtagta gaagactatc tcggttaaag actgcctcga 60
ttaacagatt cgagtgcgga cagtacgata gattga 96

Claims (4)

1. An isolated mutant RAE1 protein, wherein the mutant RAE1 protein is a non-native protein and the mutant RAE1 protein is used for one or more purposes selected from the group consisting of:
(1) Increasing the expression level of STOP-1 protein;
(2) Enhancing the aluminum toxicity resistance of plants; and/or
(3) Increasing or up-regulating expression of a gene selected from the group consisting of: atALMT1, atm mate, ALS3, or a combination thereof;
The RAE1 mutant protein has the following mutation in the RAE1 protein corresponding to the wild type: G167R, G439R, R466K, Q524STOP, S568L, G116R, G193R or W400STOP, and the amino acid sequence of the wild-type RAE1 protein is shown as SEQ ID NO. 35.
2. The protein of claim 1, wherein said RAE1 mutant protein has the following mutations in the corresponding wild-type RAE1 protein: G167R and G439R, the amino acid sequence of the wild-type RAE1 protein is shown as SEQ ID NO. 35.
3. The protein of claim 1, wherein said plant is selected from the group consisting of: arabidopsis, tobacco, rice, wheat, corn, sorghum, barley, brassica, soybean, or a combination thereof.
4. A polynucleotide encoding the mutant RAE1 protein of claim 1.
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Tabata,S.等上传.GenBank: CP002688.1，Arabidopsis thaliana chromosome 5, partial sequence.《NCBI Genbank》.2017,第1-8页的网页正文及其链接内容. *
Tabata,S.等上传.NCBI Reference Sequence: NM_120250.4，Arabidopsis thaliana RNI-like superfamily protein mRNA.《NCBI Genbank》.2017,第1-7页的网页正文及其链接内容. *

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