CN101955945A

CN101955945A - 长穗偃麦草TeWKS1基因的克隆、改造和应用

Info

Publication number: CN101955945A
Application number: CN2010100115276A
Authority: CN
Inventors: 付道林; 李新征; 王洪刚
Original assignee: Shandong Agricultural University
Current assignee: Shandong Agricultural University
Priority date: 2010-01-07
Filing date: 2010-01-07
Publication date: 2011-01-26

Abstract

本发明涉及长穗偃麦草TeWKS1基因的克隆、改造和应用，属分子生物学和生物技术领域。本发明利用同源克隆的方法从十倍体长穗偃麦草(Thinopyrumelongatum)获得小麦条锈病候选抗性基因TeWKS1，即TeWKS1-1和TeWKS1-2W。并将TeWKS1-2W基因第1294位胸腺嘧啶(T)置换为鸟嘌呤(G)，其第1294-1296位核苷酸序列由“TAG”(终止密码子)变为“GAG”(谷氨酸密码子)，改造后命名为TeWKS1-2M。TeWKS1-1和TeWKS1-2M基因转录产物mRNA开放阅读框部分有1935bp，可编码具有644个氨基酸的蛋白，含有Kinase和START两个保守序列。两个基因在小麦中的应用，可提高小麦对条锈病菌的广谱抗性。

Description

长穗偃麦草TeWKS1基因的克隆、改造和应用

(一)发明领域

本发明涉及长穗偃麦草TeWKS1基因的克隆、改造和应用，属于分子生物学和生物技术领域。

(二)技术背景

小麦族(Triticeae)是禾本科植物中一个重要的种群，包括小麦、大麦和黑麦等麦类作物和许多重要牧草植物。小麦占全球栽培面积的16％，提供全球可食用干物质的23％左右(FAOSTATdata 2005)。在我国，小麦是仅次于水稻的第二大粮食作物，播种面积超过3.5亿亩，保障小麦的稳产、高产对保障粮食供应、确保粮食生产安全具有重要意义。

小麦条锈病，俗称″黄疸″，是由条锈病菌(Puccinia striiformis Westend.f.sp.tritici)引起。条锈病菌借助空气传播，具有传播远、流行快、危害大和大区流行等特点。一般流行年份，条锈病造成30％左右减产，严重年份时减产高达50-75％(Conner and Kuzyk 1988；Roelfset al.1992)，严重危害世界小麦粮食生产。在我国，小麦条锈病普遍发生，常年危害面积6000-8000万亩，并呈逐年加重趋势。

近年来，条锈病菌优势小种毒性变宽，导致条锈病控制难度加大。目前，多数栽培品种对优势小种，Su-14和CYR32，表现高度感染(Wan et al.2007)。在生产上，使用杀菌剂控制条锈病虽然有一定效果，但经济效益低，造成药物残留、环境污染，危害人类健康。发掘或利用潜在抗条锈病基因，通过常规育种手段与生物技术相结合，培育具有高抗条锈性的小麦新品种，成为控制条锈病害的最为经济实效的手段，并成为现代小麦育种的一个重要目标。

目前，已经定名的小麦条锈病抗性基因41个(McIntosh et al.2008)，其中Yr5、Yr9、Yr15和Yr26等基因在生产上已得到应用(李峰奇等2008)。最近，Yr18和Yr36两个广谱高抗条锈病基因得以克隆(Fu et al.2009；Krattinger et al.2009)。Yr36基因最初在野生二粒小麦(Triticum turgidum ssp.dicoccoides)中发现，对小麦条锈病具有广谱抗性，能够有效降低条锈病危害，减少产量损失(Fu et al.2009；Uauy et al.2005)。该基因编码一类新型抗病蛋白，携带2个同防御反应相关的Kinase和START保守区，因此被命名为WHEATKINASE-START 1(WKS1)基因(Fu et al.2009)。研究证明，WKS1基因只存在于部分野生麦族种质，而在栽培小麦品种中丧失。为此，挖掘其它麦族种质的优异WKS1基因，用以提高麦类作物的条锈病抗性，是本项发明创造的重要目的。

长穗偃麦草(Thinopyrum elongatum，Syn：Agropyron elongatum)，又名彭梯卡偃麦草、彭梯卡薄冰草等，是小麦族的重要多年生牧草植物。抗病性鉴定证明，长穗偃麦草对小麦条锈病高抗到免疫，对小麦叶锈病、杆锈病、白粉病等病完全免疫，是麦类作物遗传改良的重要种质资源。我国在利用偃麦草资源方面取得了世界瞩目的成就。自上世纪50年代起，李振声等开展了普通小麦和偃麦草的远缘杂交工作，将十倍体长穗偃麦草中的优异基因转入小麦中，育成了以小偃6号为代表的优质、高产、抗病小偃系列小麦新品种(Li et al.2008)。小偃6号在陕西、山西、河南等省份累计推广面积达1.5亿亩，增产40亿公斤。近二十年来，小偃***衍生良种70多个，累计推广面积3亿亩以上，增产小麦超过了75亿公斤。因此，长穗偃麦草是潜在的克隆广谱高抗条锈病基因(WKS1基因)，用以提高麦类作物条锈病抗性的理想基因资源，也是本研究的目的。

(三)发明内容

本发明利用同源克隆的方法，从我国育种上广泛使用的十倍体长穗偃麦草(2n＝10X＝70，基因组StStStStEeEeEbEbExEx)中克隆到了两个TeWKS1类似基因，即TeWKS1-1和TeWKS1-2W。序列分析显示，TeWKS1-1正常通读；而最初克隆的TeWKS1-2W基因携带早熟终止子，其翻译产物同TeWKS1-1相比，缺少TeWKS1-1蛋白碳端的213个氨基酸，导致START保守区缺失，序列分析显示，早熟终止子系点突变(G→T)所致，TeWKS1-2W第1294-1296位核酸序列为TAG终止子，为保证TeWKS1-2W的基因活性，本发明对早熟终止子进行点突变修复，将第1294位胸腺嘧啶(T)置换为鸟嘌呤(G)，1294-1296位由终止子信号TAG突变为谷氨酸密码子GAG，经突变修复改造后的TeWKS1-2W基因，命名为TeWKS1-2M，该修复使得TeWKS1-2M基因编码644个氨基酸。

其中TeWKS1-1基因核苷酸序列如SEQ ID NO 1所示，氨基酸序列如SEQ ID NO 2所示；TeWKS1-2M基因核苷酸序列如SEQ ID NO 3所示，氨基酸序列如SEQ ID NO 4所示。

TeWKS1-1和TeWKS1-2M两基因转录产物mRNA开放阅读框部分各1935bp，由此推得可编码具有644个氨基酸的蛋白，各自携带两个保守序列，Kinase和START。TeWKS1-1和TeWKS1-2M基因在核酸和编码产物蛋白氨基酸水平上都具有99％的同源性。生物信息学分析表明，长穗偃麦草TeWKS1-1和TeWKS1-2M两基因与已发表的小麦条锈病高温成株抗性基因Yr36的核酸序列同源性是97％；氨基酸同源性95％，具有潜在的广谱高抗条锈病功能。

本发明的另一个目的在于上述两个基因在小麦抗条锈病育种的应用，如上所述，TeWKS1-1和TeWKS1-2M两基因具有潜在的广谱高抗条锈病功能，通过基因转化或远缘杂交等技术手段转化到栽培小麦中，可如Yr36基因(Fu et al.2009；Uauy et al.2005)一样，提高小麦对条锈病的广谱抗性，有效降低条锈病危害，减少产量损失。而来源于长穗偃麦草的基因正如技术背景所述，已在小麦品种改良中广泛应用，不存在技术上等问题。

本发明基因序列如下：

(1)SEQ ID NO 1的信息

(a)序列特征

*长度：1935碱基对

*类型：核酸

*链型：双链

*拓扑结构：线性

(b)分子类型：cDNA

(c)假设：否

(d)反义：否

(e)最初来源：长穗偃麦草

(f)序列描述：SEQ IN NO.1

1 ATGGAGCTCC CACGAAACAA ACTTGCAGAT TTGAACCAAG GAAATGAAAA TTTAAAGGCA 60

61 AAGGCAAAGT GGACACCCAA TGCCAGGAGG TTCACAGAAC ATCAGATTAA AAGAATTACT 120

121 AAGAACTATA GAACTCATGT TGGCAAAGGT GCCTTTGGTG AGGTTTTCCG AGGTTTTCTT 180

181 GACGATGGCA GTCCAGTTGC AGTGAAGAAA TACATCAACC AGAATATGAA AGAAGGGTTT 240

241 GACAAAGAGA TAACTATCCA TTGCCAAGTC AACCACAAGA ACATAGTCAA GCTTTTGGGT 300

301 TATTGTTCAG AGGGAAATGC CTTGACGATG GTCACTGAGT ACATTCCTAG AGGAAACCTC 360

361 AAAGACCTCC TTCATGGAAG TGATGATCCC ATTTCTTTTG AGGCAAGATT GTGTATTGCT 420

421 ATAGATTGTG CAGAGGCATT AGCTTTCATG CATTCAAAGG ATCCGCCAAT CATTCACGGT 480

481 GACATCAAAC CTGACAATAT ACTCTTGGAT GATAACTTGG GTGCAAAATT ATCTGACTTT 540

541 GGAATATCAA GGTTGCTTTC TATGGAGAAT AGTTATTTTA CTAATAATGT AATAGGAAGC 600

601 AGAGGTTACA TGGATCCAGA ACACATTCAG ACTGGCCGCG TTGATCCTAA GAATGATGTT 660

661 TACAGTTTTG GGGTTGTTTT GGTAGAACTA GTTACCAGAG CTATGGCAGC TGAGAATGGG 720

721 ACATGTAGCG ACCTTGCAAA AAAATTCATT GAAGCTTTCC TCCAAAAAAA TATTTTTTTG 780

781 AAAGTTTTTG GAAAGCAAAA AAAGGCAAGA GAGATGTTTG ATACCCAGAT AGCAAATGCG 840

841 AGCAACATGG AGGTTCTAGA AAAAATTGGA GAGCTGGCAA TTGAGTGTCT TAGAAGGGAT 900

901 ATCAAGAAAC GTCCTGAAAT GAATCATGTT GTAGAACGTC TTCGAATGCT TGGTAAAGAT 960

961 CATGAAAAAA GACAAAATAG AAAAGCAGAA AAGCAACATG GAGTGGTGCC TCCTGATGGT 1020

1021 AATACAACCT TTAGCTCTTC GTGGCCAAAG GGTAGATTAG AAAATGGAAG GAAATCTTCT 1080

1081 TCCTCTGATG CCGAGAGTTT GTTCAGTCAT AGGGAGGTGG AAGCAGTAGA CGAAGAAAAT 1140

1141 CAACATCCAT TATTGCGAAG AAAATCAATT GGAAATGGTC CTCCGGAATC ATTTCATGAT 1200

1201 TGGACCTATG GGAACGACAT AGGAGGCCCT GACGAAGTAT TCTCTAGAGG ATACTGGCAT 1260

1261 CTTCTCGGAT GCCAGAATGG CCTCCACATT TTTGAGCCCC TCGAGTATGG TGATTACCTT 1320

1321 GTAAGAGCCG TTGGCAAAGT GATGAAGGCC GTTGGTGTGA TTGAGGCACC TTGTGAGGCT 1380

1381 ATATTTCAGC TTCTCATGAG CATTGACGCT AGCCGTTATG AGTGGGACTG CAGCTTCATG 1440

1441 TATGGTAGTC TAGTGGAGGA GCTAGATGGC CACACTGCAA TACTATACCA TAGGCTACAC 1500

1501 CTAGATTGGT TCTTGACGTT TGTTTGGCCT CGTGATCTTT GTTATGTACG ATATTGGCAG 1560

1561 CGTAATGACG ACGGAGGTTA CGTGGTGTTG TTCCAATCCA GAGAGCACCC AAACTGTGGT 1620

1621 CCACAGCCAG GATTTGTGAG GGCACACATT GAGATTGGCG GGTTCAAAAT TTCTCCACTG 1680

1681 AAAACCCGTA ATGGAAGAAC TCGAACACAA GTACAATACC TTATGAAGAT GGATTTGAAG 1740

1741 GGTTGGGGCG TTGGTTACTT ATCCTCGTTT CAGCAGCATT GCGTCCTCCG CATGCTGAAC 1800

1801 ACTATTGCTG GGCTCAGGGA ATGGTTTTCA CGAAGTGATG AAATTCCAAC TTCGATGGAT 1860

1861 CAAAGTAGAT ATTCTACAAT GCTTGAAGAG GAGTCAGATG AAGACGAGTT GTCTTCAGGA 1920

1921 AGTGATGAAA GTTGA 1935

(2)SEQ IN NO.2的信息

(a)序列特征

*长度：644氨基酸

*类型：氨基酸

*链型：单链

*拓扑结构：线性

(b)分子类型：蛋白质

(c)序列描述：SEQ IN NO.2

MET Glu Leu Pro Arg Asn Lys Leu Ala Asp Leu Asn Gln Gly Asn

1 5 10 15

Glu Asn Leu Lys Ala Lys Ala Lys Trp Thr Pro Asn Ala Arg Arg

16 20 25 30

Phe Thr Glu His Gln Ile Lys Arg Ile Thr Lys Asn Tyr Arg Thr

31 35 40 45

His Val Gly Lys Gly Ala Phe Gly Glu Val Phe Arg Gly Phe Leu

46 50 55 60

Asp Asp Gly Ser Pro Val Ala Val Lys Lys Tyr Ile Asn Gln Asn

61 65 70 75

MET Lys Glu Gly Phe Asp Lys Glu Ile Thr Ile His Cys Gln Val

76 80 85 90

Asn His Lys Asn Ile Val Lys Leu Leu Gly Tyr Cys Ser Glu Gly

91 95 100 105

Asn Ala Leu Thr MET Val Thr Glu Tyr Ile Pro Arg Gly Asn Leu

106 110 115 120

Lys Asp Leu Leu His Gly Ser Asp Asp Pro Ile Ser Phe Glu Ala

121 125 130 135

Arg Leu Cys Ile Ala Ile Asp Cys Ala Glu Ala Leu Ala Phe MET

136 140 145 150

His Ser Lys Asp Pro Pro Ile Ile His Gly Asp Ile Lys Pro Asp

151 155 160 165

Asn Ile Leu Leu Asp Asp Asn Leu Gly Ala Lys Leu Ser Asp Phe

166 170 175 180

Gly Ile Ser Arg Leu Leu Ser MET Glu Asn Ser Tyr Phe Thr Asn

181 185 190 195

Asn Val Ile Gly Ser Arg Gly Tyr MET Asp Pro Glu His Ile Gln

196 200 205 210

Thr Gly Arg Val Asp Pro Lys Asn Asp Val Tyr Ser Phe Gly Val

211 215 220 225

Val Leu Val Glu Leu Val Thr Arg Ala MET Ala Ala Glu Asn Gly

226 230 235 240

Thr Cys Ser Asp Leu Ala Lys Lys Phe Ile Glu Ala Phe Leu Gln

241 245 250 255

Lys Asn Ile Phe Leu Lys Val Phe Gly Lys Gln Lys Lys Ala Arg

256 260 265 270

Glu MET Phe Asp Thr Gln Ile Ala Asn Ala Ser Asn MET Glu Val

271 275 280 285

Leu Glu Lys Ile Gly Glu Leu Ala Ile Glu Cys Leu Arg Arg Asp

286 290 295 300

Ile Lys Lys Arg Pro Glu MET Asn His Val Val Glu Arg Leu Arg

301 305 310 315

MET Leu Gly Lys Asp His Glu Lys Arg Gln Asn Arg Lys Ala Glu

316 320 325 330

Lys Gln His Gly Val Val Pro Pro Asp Gly Asn Thr Thr Phe Ser

331 335 340 345

Ser Ser Trp Pro Lys Gly Arg Leu Glu Asn Gly Arg Lys Ser Ser

346 350 355 360

Ser Ser Asp Ala Glu Ser Leu Phe Ser His Arg Glu Val Glu Ala

361 365 370 375

Val Asp Glu Glu Asn Gln His Pro Leu Leu Arg Arg Lys Ser Ile

376 380 385 390

Gly Asn Gly Pro Pro Glu Ser Phe His Asp Trp Thr Tyr Gly Asn

391 395 400 405

Asp Ile Gly Gly Pro Asp Glu Val Phe Ser Arg Gly Tyr Trp His

406 410 415 420

Leu Leu Gly Cys Gln Asn Gly Leu His Ile Phe Glu Pro Leu Glu

421 425 430 435

Tyr Gly Asp Tyr Leu Val Arg Ala Val Gly Lys Val MET Lys Ala

436 440 445 450

Val Gly Val Ile Glu Ala Pro Cys Glu Ala Ile Phe Gln Leu Leu

451 455 460 465

MET Ser Ile Asp Ala Ser Arg Tyr Glu Trp Asp Cys Ser Phe MET

466 470 475 480

Tyr Gly Ser Leu Val Glu Glu Leu Asp Gly His Thr Ala Ile Leu

481 485 490 495

Tyr His Arg Leu His Leu Asp Trp Phe Leu Thr Phe Val Trp Pro

496 500 505 510

Arg Asp Leu Cys Tyr Val Arg Tyr Trp Gln Arg Asn Asp Asp Gly

511 515 520 525

Gly Tyr Val Val Leu Phe Gln Ser Arg Glu His Pro Asn Cys Gly

526 530 535 540

Pro Gln Pro Gly Phe Val Arg Ala His Ile Glu Ile Gly Gly Phe

541 545 550 555

Lys Ile Ser Pro Leu Lys Thr Arg Asn Gly Arg Thr Arg Thr Gln

556 560 565 570

Val Gln Tyr Leu MET Lys MET Asp Leu Lys Gly Trp Gly Val Gly

571 575 580 585

Tyr Leu Ser Ser Phe Gln Gln His Cys Val Leu Arg MET Leu Asn

586 590 595 600

Thr Ile Ala Gly Leu Arg Glu Trp Phe Ser Arg Ser Asp Glu Ile

601 605 610 615

Pro Thr Ser MET Asp Gln Ser Arg Tyr Ser Thr MET Leu Glu Glu

616 620 625 630

Glu Ser Asp Glu Asp Glu Leu Ser Ser Gly Ser Asp Glu Ser

631 635 640 644

(3)SEQ ID NO 3的信息

(a)序列特征

*长度：1935碱基对

*类型：核酸

*链型：双链

*拓扑结构：线性

(b)分子类型：cDNA

(c)假设：否

(d)反义：否

(e)最初来源：长穗偃麦草

(f)序列描述：SEQ IN NO.3

1 ATGGAGCTCC CACGAAACAA ACTTGCAGAT TTGAACCAAG GAAATGAAAA TTTAAAGGCA 60

61 AAGGCAAAGT GGACACCCAA TGCCAGGAGG TTCACAGAAC ATCAGATTAA AAGAATTACT 120

121 AAGAACTATA GAACTCATGT TGGCAAAGGT GCCTTTGGTG AGGTTTTCCG AGGTTTTCTT 180

181 GACGATGGCA GTCCAGTTGC AGTGAAGAAA TACATCAACC AGAATATGAA AGAAGGGTTT 240

241 GACAAAGAGA TAACTATCCA TTGCCAAGTC AACCACAAGA ACATAGTCAA GCTTTTGGGT 300

301 TATTGTTCAG AGGAAAATGC CTTGACGATG GTCACTGAGT ACATTCCTAG AGGAAACCTC 360

361 AAAGACCTCC TTCATGGAAG TGATGATCCC ATTTCTTTTG AGGCAAGATT GTGTATTGCT 420

421 ATAGATTGTG CAGAGGCATT AGCTTTCATG CATTCAAAGG ATCCGCCAAT CATTCACGGT 480

481 GACATCAAAC CTGACAATAT ACTCTTGGAT GATAACTTGG GTGCAAAATT ATCTGACTTT 540

541 GGAATATCAA GGTTGCTTTC TATGGAGAAT AGTTATTTTA CTAATAATGT AATAGGAAGC 600

601 AGAGGTTACA TGGATCCAGA ACACATTCAG ACTGGCCGCG TTGATCCTAA GAATGATGTT 660

661 TACAGTTTTG GGGTTGTTTT GGTAGAACTA GTTACCAGAG CTATGGCAGC TCAGAATGGG 720

721 ACATGTAGCG ACCTTGCAAA AAAATTCATT GAAGCTTTCC TCCAAAAGAA TATTTTTTTG 780

781 AAAGTTTTTG GAAAGCAAAA AAAGGCAAGA GAGATGTTTG ATACCCAGAT AGCAAATGCG 840

841 AGCAACATGG AGGTTCTAGA AAAAATTGGA GAGCTGGCAA TTGAGTGTCT TAGAAGGGAT 900

901 ATCAAGAAAC GTCCTGAAAT GAATCATGTT GTAGAACGTC TTCGAATGCT TGGTAAAGAT 960

961 CATGAAAAAA GACAAAATAG AAAAGCAGAA AAGCAACATG GAGTGGTGCC TCCTGATGGT 1020

1021 AATACAACCT TTAGCTCTTC GTGGCCAAAG GGTAGATTAG AAAATGGAAG GAAATCTTCT 1080

1081 TCCTCTGATG CCGAGAGTCT GTTCAGTCAT AGGGAGGTGG AAGCAGTAGA CGAAGAAAAT 1140

1141 CAACATCCAT TATTGCGAAG AAAATCAATT GGAAATGGTC CTCCGGAATC ATTTCATGAT 1200

1201 TGGACCTATG GGAACGACAT AGGAGGCCCT GACGAAGTAT TCTCTAGAGG ATACTGGCAT 1260

1261 CTTCTCGGAT GCCAGAATGG CCTCCACATT TTTGAGCCCC TCGAGTATGG TGATTACCTT 1320

1321 GTAAGAGCCG TTGGCAAAGT GATGAAGGCC GTTGGTGTGA TTGAGGCACC TTGTGAGGCT 1380

1381 ATATTTCAGC TTCTCATGAG CATGGACGCT AGCCGTTATG AGTGGGACTG CAGCTTCATG 1440

1441 TATGGTAGTC TAGTGGAGGA GCTAGATGGC CACACTGCAA TACTATACCA TAGGCTACAC 1500

1501 CTAGATTGGT TCTTGACGTT TGTTTGGCCT CGTGATCTTT GTTATGTACG ATATTGGCAG 1560

1561 CGTAATGACG ACGGAGGTTA CGTGGCGTTG TTCCAATCCA GAGAGCACCC AAACTGTGGT 1620

1621 CCACAGCCAG GATTTGTGAG GGCACACATT GAGATTGGCG GGTTCAAAAT TTCTCCACTG 1680

1681 AAAATCCGTA ATGGAAGAAC TCGAACACAA GTACAATACC TTATGAAGAT GGATTTGAAG 1740

1741 GGTTGGGGCG TTGGTTACTT ATCCTCGTTT CAGCAGCATT GCGTCCTCCG CATGCTGAAC 1800

1801 ACTATTGCTG GGCTCAGGGA ATGGTTTTCA CGAAGTGATG AAATTCCAAC TTCGATGGAT 1860

1861 CAAAGTAGAT ATTCTACAAT GCTTGAAGAG GAGTCAGATG AAGACGAGTT GTCTTCAGGA 1920

1921 AGTGATGAAA GTTGA 1935

(4)SEQ IN NO.4的信息

(a)序列特征

*长度：644氨基酸

*类型：氨基酸

*链型：单链

*拓扑结构：线性

(b)分子类型：蛋白质

(c)序列描述：SEQ IN NO.4

MET Glu Leu Pro Arg Asn Lys Leu Ala Asp Leu Asn Gln Gly Asn

1 5 10 15

Glu Asn Leu Lys Ala Lys Ala Lys Trp Thr Pro Asn Ala Arg Arg

16 20 25 30

Phe Thr Glu His Gln Ile Lys Arg Ile Thr Lys Asn Tyr Arg Thr

31 35 40 45

His Val Gly Lys Gly Ala Phe Gly Glu Val Phe Arg Gly Phe Leu

46 50 55 60

Asp Asp Gly Ser Pro Val Ala Val Lys Lys Tyr Ile Asn Gln Asn

61 65 70 75

MET Lys Glu Gly Phe Asp Lys Glu Ile Thr Ile His Cys Gln Val

76 80 85 90

Asn His Lys Asn Ile Val Lys Leu Leu Gly Tyr Cys Ser Glu Glu

91 95 100 105

Asn Ala Leu Thr MET Val Thr Glu Tyr Ile Pro Arg Gly Asn Leu

106 110 115 120

Lys Asp Leu Leu His Gly Ser Asp Asp Pro Ile Ser Phe Glu Ala

121 125 130 135

Arg Leu Cys Ile Ala Ile Asp Cys Ala Glu Ala Leu Ala Phe MET

136 140 145 150

His Ser Lys Asp Pro Pro Ile Ile His Gly Asp Ile Lys Pro Asp

151 155 160 165

Asn Ile Leu Leu Asp Asp Asn Leu Gly Ala Lys Leu Ser Asp Phe

166 170 175 180

Gly Ile Ser Arg Leu Leu Ser MET Glu Asn Ser Tyr Phe Thr Asn

181 185 190 195

Asn Val Ile Gly Ser Arg Gly Tyr MET Asp Pro Glu His Ile Gln

196 200 205 210

Thr Gly Arg Val Asp Pro Lys Asn Asp Val Tyr Ser Phe Gly Val

211 215 220 225

Val Leu Val Glu Leu Val Thr Arg Ala MET Ala Ala Gln Asn Gly

226 230 235 240

Thr Cys Ser Asp Leu Ala Lys Lys Phe Ile Glu Ala Phe Leu Gln

241 245 250 255

Lys Asn Ile Phe Leu Lys Val Phe Gly Lys Gln Lys Lys Ala Arg

256 260 265 270

Glu MET Phe Asp Thr Gln Ile Ala Asn Ala Ser Asn MET Glu Val

271 275 280 285

Leu Glu Lys Ile Gly Glu Leu Ala Ile Glu Cys Leu Arg Arg Asp

286 290 295 300

Ile Lys Lys Arg Pro Glu MET Asn His Val Val Glu Arg Leu Arg

301 305 310 315

MET Leu Gly Lys Asp His Glu Lys Arg Gln Asn Arg Lys Ala Glu

316 320 325 330

Lys Gln His Gly Val Val Pro Pro Asp Gly Asn Thr Thr Phe Ser

331 335 340 345

Ser Ser Trp Pro Lys Gly Arg Leu Glu Asn Gly Arg Lys Ser Ser

346 350 355 360

Ser Ser Asp Ala Glu Ser Leu Phe Ser His Arg Glu Val Glu Ala

361 365 370 375

Val Asp Glu Glu Asn Gln His Pro Leu Leu Arg Arg Lys Ser Ile

376 380 385 390

Gly Asn Gly Pro Pro Glu Ser Phe His Asp Trp Thr Tyr Gly Asn

391 395 400 405

Asp Ile Gly Gly Pro Asp Glu Val Phe Ser Arg Gly Tyr Trp His

406 410 415 420

Leu Leu Gly Cys Gln Asn Gly Leu His Ile Phe Glu Pro Leu Glu

421 425 430 435

Tyr Gly Asp Tyr Leu Val Arg Ala Val Gly Lys Val MET Lys Ala

436 440 445 450

Val Gly Val Ile Glu Ala Pro Cys Glu Ala Ile Phe Gln Leu Leu

451 455 460 465

MET Ser MET Asp Ala Ser Arg Tyr Glu Trp Asp Cys Ser Phe MET

466 470 475 480

Tyr Gly Ser Leu Val Glu Glu Leu Asp Gly His Thr Ala Ile Leu

481 485 490 495

Tyr His Arg Leu His Leu Asp Trp Phe Leu Thr Phe Val Trp Pro

496 500 505 510

Arg Asp Leu Cys Tyr Val Arg Tyr Trp Gln Arg Asn Asp Asp Gly

511 515 520 525

Gly Tyr Val Ala Leu Phe Gln Ser Arg Glu His Pro Asn Cys Gly

526 530 535 540

Pro Gln Pro Gly Phe Val Arg Ala His Ile Glu Ile Gly Gly Phe

541 545 550 555

Lys Ile Ser Pro Leu Lys Ile Arg Asn Gly Arg Thr Arg Thr Gln

556 560 565 570

Val Gln Tyr Leu MET Lys MET Asp Leu Lys Gly Trp Gly Val Gly

571 575 580 585

Tyr Leu Ser Ser Phe Gln Gln His Cys Val Leu Arg MET Leu Asn

586 590 595 600

Thr Ile Ala Gly Leu Arg Glu Trp Phe Ser Arg Ser Asp Glu Ile

601 605 610 615

Pro Thr Ser MET Asp Gln Ser Arg Tyr Ser Thr MET Leu Glu Glu

616 620 625 630

Glu Ser Asp Glu Asp Glu Leu Ser Ser Gly Ser Asp Glu Ser

631 635 640 644

(四)附图说明

图1：TeWKS1-1编码蛋白的保守区示意图

图2：TeWKS1-2基因改造示意图

图1中：TeWKS1-1和TeWKS1-2M基因编码的蛋白携带Kinase和START保守区，这是最近克隆的小麦条锈病抗性基因Yr36的典型特征(Fu et al.2009)。初步推测，Kinase区域涉及信号转导，而START区域同脂类分子结合以及蛋白的构象变化有关。由于TeWKS1基因编码的蛋白与小麦条锈病抗性基因YR36编码产物有95％的同源性，TeWKS1基因能提供优异的条锈病抗性。

图2中：TeWKS1-1和TeWKS1-2基因分别由不同背景图案表示。两基因有99％的同源性，而在BspEI和XhoI两酶切位点之间的124bp范围内，两者唯一的差异在于导致TeWKS1-2W提前终止的点突变(红线所示)。用BspEI和XhoI双酶切TeWKS1-1的相应片段替代TeWKS1-1W的对应区段，进而获得TeWKS1-2M基因。

(五)具体发明实施方式

实施例1：长穗偃麦草TeWKS1基因cDNA的获得

本发明使用Trizol试剂(Invitrogen，CA，USA)提取长穗偃麦草总RNA，使用SuperScript

II逆转录试剂盒(Invitrogen)合成cDNA。RNA提取和cDNA合成方法依照该公司试剂盒配套技术。本发明设计了正向特异引物：5’-CACCATGGAGCTCCCACGAAACAAAC-3’和反向特异引物：5’-AGTACTTCGATGAAACAGATGGAA-3’扩增长穗偃麦草TeWKS1基因。扩增反应使用高保真DNA聚合酶Phusion^TM(New EnglandBiolabs，MA，USA)，反应体系参照产品说明书；反应条件为：98度预变性30秒，98度变性10秒，58度复性30秒，72度延伸60秒(30个循环)，72度终延伸10分钟，4度待机。扩增产物经0.8％琼脂糖凝胶电泳分离，使用DNA胶回收试剂盒(Qiagen，CA，USA)回收。回收产物克隆到载体pENTR/D-TOPO

(Invitrogen)上，由上海生工生物工程技术服务有限公司进行序列测定。

通过对22个cDNA克隆进行测序，发现其中19个属于TeWKS1-1类型，3个属于TeWKS1-2类型。利用克隆载体上的通用测序引物(M13正反向引物)以及TeWKS1基因引物，对代表性克隆进行全序测定，获得1985bp的cDNA片断，其中1935bp为编码序列。利用BLAST软件将分离出的cDNA序列和翻译产物氨基酸序列与基因库中的相应序列进行比较，确定他们是与小麦条锈病高温成株抗性基因Yr36具有高度同源性的基因。

实施例2：长穗偃麦草TeWKS1-2M基因的改造

TeWKS1-1和TeWKS1-2基因有99％的同源性，而在BspEI和XhoI两酶切位点之间的124bp范围内，两者唯一的差异在于导致TeWKS1-2W提前终止的点突变(图2.红线所示)。由于pENTR/D-TOPO

(Invitrogen)克隆质粒上BspEI和XhoI都为单一酶切位点，因此用BspEI和XhoI双酶切TeWKS1-1的相应片段替代TeWKS1-1W的对应区段，改造前TeWKS1-2W基因第1294位核苷酸为胸腺嘧啶(T)，以其起始的三连密码子(TAG)为终止子；改造后的长穗偃麦草TeWKS1-2M基因第1294位核苷酸为鸟嘌呤(G)，以其起始的三连密码子(GAG)编码谷氨酸(Glu)，该修复使得TeWKS1-2M基因编码644个氨基酸。进而获得TeWKS1-2M基因。改造后的TeWKS1-2M基因送交上海生工生物工程技术服务有限公司进行测序，测序结果表明序列无误，改造成功。

实施例3：TeWKS1-1和TeWKS1-2M两个基因在小麦中的应用

通过基因转化技术，将上述两个基因转入栽培小麦品种中，可提高小麦对条锈病的广谱抗性，有效降低条锈病危害。

<110>山东农业大学

<120>长穗偃麦草TeWKS1基因的克隆、改造及应用

<160>4

<210>1

<211>1935

<212>DNA

<213>长穗偃麦草(Thinopyrum elongatum，Syn：Agropyron elongatum)

<400>1

1 atggagctcc cacgaaacaa acttgcagat ttgaaccaag gaaatgaaaa tttaaaggca 60

61 aaggcaaagt ggacacccaa tgccaggagg ttcacagaac atcagattaa aagaattact 120

121 aagaactata gaactcatgt tggcaaaggt gcctttggtg aggttttccg aggttttctt 180

181 gacgatggca gtccagttgc agtgaagaaa tacatcaacc agaatatgaa agaagggttt 240

241 gacaaagaga taactatcca ttgccaagtc aaccacaaga acatagtcaa gcttttgggt 300

301 tattgttcag agggaaatgc cttgacgatg gtcactgagt acattcctag aggaaacctc 360

361 aaagacctcc ttcatggaag tgatgatccc atttcttttg aggcaagatt gtgtattgct 420

421 atagattgtg cagaggcatt agctttcatg cattcaaagg atccgccaat cattcacggt 480

481 gacatcaaac ctgacaatat actcttggat gataacttgg gtgcaaaatt atctgacttt 540

541 ggaatatcaa ggttgctttc tatggagaat agttatttta ctaataatgt aataggaagc 600

601 agaggttaca tggatccaga acacattcag actggccgcg ttgatcctaa gaatgatgtt 660

661 tacagttttg gggttgtttt ggtagaacta gttaccagag ctatggcagc tgagaatggg 720

721 acatgtagcg accttgcaaa aaaattcatt gaagctttcc tccaaaaaaa tatttttttg 780

781 aaagtttttg gaaagcaaaa aaaggcaaga gagatgtttg atacccagat agcaaatgcg 840

841 agcaacatgg aggttctaga aaaaattgga gagctggcaa ttgagtgtct tagaagggat 900

901 atcaagaaac gtcctgaaat gaatcatgtt gtagaacgtc ttcgaatgct tggtaaagat 960

961 catgaaaaaa gacaaaatag aaaagcagaa aagcaacatg gagtggtgcc tcctgatggt 1020

1021 aatacaacct ttagctcttc gtggccaaag ggtagattag aaaatggaag gaaatcttct 1080

1081 tcctctgatg ccgagagttt gttcagtcat agggaggtgg aagcagtaga cgaagaaaat 1140

1141 caacatccat tattgcgaag aaaatcaatt ggaaatggtc ctccggaatc atttcatgat 1200

1201 tggacctatg ggaacgacat aggaggccct gacgaagtat tctcta9agg atactggcat 1260

1261 cttctcggat gccagaatgg cctccacatt tttgagcccc tcgagtatgg tgattacctt 1320

1321 gtaagagccg ttggcaaagt gatgaaggcc gttggtgtga ttgaggcacc ttgtgaggct 1380

1381 atatttcagc ttctcatgag cattgacgct agccgttatg agtgggactg cagcttcatg 1440

1441 tatggtagtc tagtggagga gctagatggc cacactgcaa tactatacca taggctacac 1500

1501 ctagattggt tcttgacgtt tgtttggcct cgtgatcttt gttatgtacg atattggcag 1560

1561 cgtaatgacg acggaggtta cgtggtgttg ttccaatcca gagagcaccc aaactgtggt 1620

1621 ccacagccag gatttgtgag ggcacacatt gagattggcg ggttcaaaat ttctccactg 1680

1681 aaaacccgta atggaagaac tcgaacacaa gtacaatacc ttatgaagat ggatttgaag 1740

1741 ggttggggcg ttggttactt atcctcgttt cagcagcatt gcgtcctccg catgctgaac 1800

1801 actattgctg ggctcaggga atggttttca cgaagtgatg aaattccaac ttcgatggat 1860

1861 caaagtagat attctacaat gcttgaagag gagtcagatg aagacgagtt gtcttcagga 1920

1921 agtgatgaaa gttga 1935

<110>山东农业大学

<120>长穗偃麦草TeWKS1基因的克隆、改造及应用

<210>2

<211>644

<212>PRT

<213>长穗偃麦草(Thinopyrum elongatum，Syn：Agropyron elongatum)

<400>2

MET Glu Leu Pro Arg Asn Lys Leu Ala Asp Leu Asn Gln Gly Asn

1 5 10 15

Glu Asn Leu Lys Ala Lys Ala Lys Trp Thr Pro Asn Ala Arg Arg

16 20 25 30

Phe Thr Glu His Gln Ile Lys Arg Ile Thr Lys Asn Tyr Arg Thr

31 35 40 45

His Val Gly Lys Gly Ala Phe Gly Glu Val Phe Arg Gly Phe Leu

46 50 55 60

Asp Asp Gly Ser Pro Val Ala Val Lys Lys Tyr Ile Asn Gln Asn

61 65 70 75

MET Lys Glu Gly Phe Asp Lys Glu Ile Thr Ile His Cys Gln Val

76 80 85 90

Asn His Lys Asn Ile Val Lys Leu Leu Gly Tyr Cys Ser Glu Gly

91 95 100 105

Asn Ala Leu Thr MET Val Thr Glu Tyr Ile Pro Arg Gly Asn Leu

106 110 115 120

Lys Asp Leu Leu His Gly Ser Asp Asp Pro Ile Ser Phe Glu Ala

121 125 130 135

Arg Leu Cys Ile Ala Ile Asp Cys Ala Glu Ala Leu Ala Phe MET

136 140 145 150

His Ser Lys Asp Pro Pro Ile Ile His Gly Asp Ile Lys Pro Asp

151 155 160 165

Asn Ile Leu Leu Asp Asp Asn Leu Gly Ala Lys Leu Ser Asp Phe

166 170 175 180

Gly Ile Ser Arg Leu Leu Ser MET Glu Asn Ser Tyr Phe Thr Asn

181 185 190 195

Asn Val Ile Gly Ser Arg Gly Tyr MET Asp Pro Glu His Ile Gln

196 200 205 210

Thr Gly Arg Val Asp Pro Lys Asn Asp Val Tyr Ser Phe Gly Val

211 215 220 225

Val Leu Val Glu Leu Val Thr Arg Ala MET Ala Ala Glu Asn Gly

226 230 235 240

Thr Cys Ser Asp Leu Ala Lys Lys Phe Ile Glu Ala Phe Leu Gln

241 245 250 255

Lys Asn Ile Phe Leu Lys Val Phe Gly Lys Gln Lys Lys Ala Arg

256 260 265 270

Glu MET Phe Asp Thr Gln Ile Ala Asn Ala Ser Asn MET Glu Val

271 275 280 285

Leu Glu Lys Ile Gly Glu Leu Ala Ile Glu Cys Leu Arg Arg Asp

286 290 295 300

Ile Lys Lys Arg Pro Glu MET Asn His Val Val Glu Arg Leu Arg

301 305 310 315

MET Leu Gly Lys Asp His Glu Lys Arg Gln Asn Arg Lys Ala Glu

316 320 325 330

Lys Gln His Gly Val Val Pro Pro Asp Gly Asn Thr Thr Phe Ser

331 335 340 345

Ser Ser Trp Pro Lys Gly Arg Leu Glu Asn Gly Arg Lys Ser Ser

346 350 355 360

Ser Ser Asp Ala Glu Ser Leu Phe Ser His Arg Glu Val Glu Ala

361 365 370 375

Val Asp Glu Glu Asn Gln His Pro Leu Leu Arg Arg Lys Ser Ile

376 380 385 390

Gly Asn Gly Pro Pro Glu Ser Phe His Asp Trp Thr Tyr Gly Asn

391 395 400 405

Asp Ile Gly Gly Pro Asp Glu Val Phe Ser Arg Gly Tyr Trp His

406 410 415 420

Leu Leu Gly Cys Gln Asn Gly Leu His Ile Phe Glu Pro Leu Glu

421 425 430 435

Tyr Gly Asp Tyr Leu Val Arg Ala Val Gly Lys Val MET Lys Ala

436 440 445 450

Val Gly Val Ile Glu Ala Pro Cys Glu Ala Ile Phe Gln Leu Leu

451 455 460 465

MET Ser Ile Asp Ala Ser Arg Tyr Glu Trp Asp Cys Ser Phe MET

466 470 475 480

Tyr Gly Ser Leu Val Glu Glu Leu Asp Gly His Thr Ala Ile Leu

481 485 490 495

Tyr His Arg Leu His Leu Asp Trp Phe Leu Thr Phe Val Trp Pro

496 500 505 510

Arg Asp Leu Cys Tyr Val Arg Tyr Trp Gln Arg Asn Asp Asp Gly

511 515 520 525

Gly Tyr Val Val Leu Phe Gln Ser Arg Glu His Pro Asn Cys Gly

526 530 535 540

Pro Gln Pro Gly Phe Val Arg Ala His Ile Glu Ile Gly Gly Phe

541 545 550 555

Lys Ile Ser Pro Leu Lys Thr Arg Asn Gly Arg Thr Arg Thr Gln

556 560 565 570

Val Gln Tyr Leu MET Lys MET Asp Leu Lys Gly Trp Gly Val Gly

571 575 580 585

Tyr Leu Ser Ser Phe Gln Gln His Cys Val Leu Arg MET Leu Asn

586 590 595 600

Thr Ile Ala Gly Leu Arg Glu Trp Phe Ser Arg Ser Asp Glu Ile

601 605 610 615

Pro Thr Ser MET Asp Gln Ser Arg Tyr Ser Thr MET Leu Glu Glu

616 620 625 630

Glu Ser Asp Glu Asp Glu Leu Ser Ser Gly Ser Asp Glu Ser

631 635 640 644

<110>山东农业大学

<120>长穗偃麦草TeWKS1基因的克隆、改造及应用

<210>3

<211>1935

<212>DNA

<213>长穗偃麦草(Thinopyrum elongatum，Syn：Agropyron elongatum)

<400>3

1 atggagctcc cacgaaacaa acttgcagat ttgaaccaag gaaatgaaaa tttaaaggca 60

61 aaggcaaagt ggacacccaa tgccaggagg ttcacagaac atcagattaa aagaattact 120

121 aagaactata gaactcatgt tggcaaaggt gcctttggtg aggttttccg aggttttctt 180

181 gacgatggca gtccagttgc agtgaagaaa tacatcaacc agaatatgaa agaagggttt 240

241 gacaaagaga taactatcca ttgccaagtc aaccacaaga acatagtcaa gcttttgggt 300

301 tattgttcag aggaaaatgc cttgacgatg gtcactgagt acattcctag aggaaacctc 360

361 aaagacctcc ttcatggaag tgatgatccc atttcttttg aggcaagatt gtgtattgct 420

421 atagattgtg cagaggcatt agctttcatg cattcaaagg atccgccaat cattcacggt 480

481 gacatcaaac ctgacaatat actcttggat gataacttgg gtgcaaaatt atctgacttt 540

541 ggaatatcaa ggttgctttc tatggagaat agttatttta ctaataatgt aataggaagc 600

601 agaggttaca tggatccaga acacattcag actggccgcg ttgatcctaa gaatgatgtt 660

661 tacagttttg gggttgtttt ggtagaacta gttaccagag ctatggcagc tcagaatggg 720

721 acatgtagcg accttgcaaa aaaattcatt gaagctttcc tccaaaagaa tatttttttg 780

781 aaagtttttg gaaagcaaaa aaaggcaaga gagatgtttg atacccagat agcaaatgcg 840

841 agcaacatgg aggttctaga aaaaattgga gagctggcaa ttgagtgtct tagaagggat 900

901 atcaagaaac gtcctgaaat gaatcatgtt gtagaacgtc ttcgaatgct tggtaaagat 960

961 catgaaaaaa gacaaaatag aaaagcagaa aagcaacatg gagtggtgcc tcctgatggt 1020

1021 aatacaacct ttagctcttc gtggccaaag ggtagattag aaaatggaag gaaatcttct 1080

1081 tcctctgatg ccgagagtct gttcagtcat agggaggtgg aagcagtaga cgaagaaaat 1140

1141 caacatccat tattgcgaag aaaatcaatt ggaaatggtc ctccggaatc atttcatgat 1200

1201 tggacctatg ggaacgacat aggaggccct gacgaagtat tctctagagg atactggcat 1260

1261 cttctcggat gccagaatgg cctccacatt tttgagcccc tcgagtatgg tgattacctt 1320

1321 gtaagagccg ttggcaaagt gatgaaggcc gttggtgtga ttgaggcacc ttgtgaggct 1380

1381 atatttcagc ttctcatgag catggacgct agccgttatg agtgggactg cagcttcatg 1440

1441 tatggtagtc tagtggagga gctagatggc cacactgcaa tactatacca taggctacac 1500

1501 ctagattggt tcttgacgtt tgtttggcct cgtgatcttt gttatgtacg atattggcag 1560

1561 cgtaatgacg acggaggtta cgtggcgttg ttccaatcca gagagcaccc aaactgtggt 1620

1621 ccacagccag gatttgtgag ggcacacatt gagattggcg ggttcaaaat ttctccactg 1680

1681 aaaatccgta atggaagaac tcgaacacaa gtacaatacc ttatgaagat ggatttgaag 1740

1741 ggttggggcg ttggttactt atcctcgttt cagcagcatt gcgtcctccg catgctgaac 1800

1801 actattgctg ggctcaggga atggttttca cgaagtgatg aaattccaac ttcgatggat 1860

1861 caaagtagat attctacaat gcttgaagag gagtcagatg aagacgagtt gtcttcagga 1920

1921 agtgatgaaa gttga 1935

<110>山东农业大学

<120>长穗偃麦草TeWKS1基因的克隆、改造及应用

<210>4

<211>644

<212>PRT

<213>长穗偃麦草(Thinopyrum elongatum，Syn：Agropyron elongatum)

<400>4

MET Glu Leu Pro Arg Asn Lys Leu Ala Asp Leu Asn Gln Gly Asn

1 5 10 15

Glu Asn Leu Lys Ala Lys Ala Lys Trp Thr Pro Asn Ala Arg Arg

16 20 25 30

Phe Thr Glu His Gln Ile Lys Arg Ile Thr Lys Asn Tyr Arg Thr

31 35 40 45

His Val Gly Lys Gly Ala Phe Gly Glu Val Phe Arg Gly Phe Leu

46 50 55 60

Asp Asp Gly Ser Pro Val Ala Val Lys Lys Tyr Ile Asn Gln Asn

61 65 70 75

MET Lys Glu Gly Phe Asp Lys Glu Ile Thr Ile His Cys Gln Val

76 80 85 90

Asn His Lys Asn Ile Val Lys Leu Leu Gly Tyr Cys Ser Glu Glu

91 95 100 105

Asn Ala Leu Thr MET Val Thr Glu Tyr Ile Pro Arg Gly Asn Leu

106 110 115 120

Lys Asp Leu Leu His Gly Ser Asp Asp Pro Ile Ser Phe Glu Ala

121 125 130 135

Arg Leu Cys Ile Ala Ile Asp Cys Ala Glu Ala Leu Ala Phe MET

136 140 145 150

His Ser Lys Asp Pro Pro Ile Ile His Gly Asp Ile Lys Pro Asp

151 155 160 165

Asn Ile Leu Leu Asp Asp Asn Leu Gly Ala Lys Leu Ser Asp Phe

166 170 175 180

Gly Ile Ser Arg Leu Leu Ser MET Glu Asn Ser Tyr Phe Thr Asn

181 185 190 195

Asn Val Ile Gly Ser Arg Gly Tyr MET Asp Pro Glu His Ile Gln

196 200 205 210

Thr Gly Arg Val Asp Pro Lys Asn Asp Val Tyr Ser Phe Gly Val

211 215 220 225

Val Leu Val Glu Leu Val Thr Arg Ala MET Ala Ala Gln Asn Gly

226 230 235 240

Thr Cys Ser Asp Leu Ala Lys Lys Phe Ile Glu Ala Phe Leu Gln

241 245 250 255

Lys Asn Ile Phe Leu Lys Val Phe Gly Lys Gln Lys Lys Ala Arg

256 260 265 270

Glu MET Phe Asp Thr Gln Ile Ala Asn Ala Ser Asn MET Glu Val

271 275 280 285

Leu Glu Lys Ile Gly Glu Leu Ala Ile Glu Cys Leu Arg Arg Asp

286 290 295 300

Ile Lys Lys Arg Pro Glu MET Asn His Val Val Glu Arg Leu Arg

301 305 310 315

MET Leu Gly Lys Asp His Glu Lys Arg Gln Asn Arg Lys Ala Glu

316 320 325 330

Lys Gln His Gly Val Val Pro Pro Asp Gly Asn Thr Thr Phe Ser

331 335 340 345

Ser Ser Trp Pro Lys Gly Arg Leu Glu Asn Gly Arg Lys Ser Ser

346 350 355 360

Ser Ser Asp Ala Glu Ser Leu Phe Ser His Arg Glu Val Glu Ala

361 365 370 375

Val Asp Glu Glu Asn Gln His Pro Leu Leu Arg Arg Lys Ser Ile

376 380 385 390

Gly Asn Gly Pro Pro Glu Ser Phe His Asp Trp Thr Tyr Gly Asn

391 395 400 405

Asp Ile Gly Gly Pro Asp Glu Val Phe Ser Arg Gly Tyr Trp His

406 410 415 420

Leu Leu Gly Cys Gln Asn Gly Leu His Ile Phe Glu Pro Leu Glu

421 425 430 435

Tyr Gly Asp Tyr Leu Val Arg Ala Val Gly Lys Val MET Lys Ala

436 440 445 450

Val Gly Val Ile Glu Ala Pro Cys Glu Ala Ile Phe Gln Leu Leu

451 455 460 465

MET Ser MET Asp Ala Ser Arg Tyr Glu Trp Asp Cys Ser Phe MET

466 470 475 480

Tyr Gly Ser Leu Val Glu Glu Leu Asp Gly His Thr Ala Ile Leu

481 485 490 495

Tyr His Arg Leu His Leu Asp Trp Phe Leu Thr Phe Val Trp Pro

496 500 505 510

Arg Asp Leu Cys Tyr Val Arg Tyr Trp Gln Arg Asn Asp Asp Gly

511 515 520 525

Gly Tyr Val Ala Leu Phe Gln Ser Arg Glu His Pro Asn Cys Gly

526 530 535 540

Pro Gln Pro Gly Phe Val Arg Ala His Ile Glu Ile Gly Gly Phe

541 545 550 555

Lys Ile Ser Pro Leu Lys Ile Arg Asn Gly Arg Thr Arg Thr Gln

556 560 565 570

Val Gln Tyr Leu MET Lys MET Asp Leu Lys Gly Trp Gly Val Gly

571 575 580 585

Tyr Leu Ser Ser Phe Gln Gln His Cys Val Leu Arg MET Leu Asn

586 590 595 600

Thr Ile Ala Gly Leu Arg Glu Trp Phe Ser Arg Ser Asp Glu Ile

601 605 610 615

Pro Thr Ser MET Asp Gln Ser Arg Tyr Ser Thr MET Leu Glu Glu

616 620 625 630

Glu Ser Asp Glu Asp Glu Leu Ser Ser Gly Ser Asp Glu Ser

631 635 640 644

Claims

1.一种长穗偃麦草TeWKS1基因，其特征在于所述的TeWKS1基因包括TeWKS1-1和TeWKS1-2M两个基因；TeWKS1-1基因核苷酸序列如SEQ ID NO 1所示，氨基酸序列如SEQ ID NO2所示；TeWKS1-2M基因核苷酸序列如SEQ ID NO 3所示，氨基酸序列如SEQ ID NO 4所示。

2.根据权利要求1所述的核苷酸序列，其特征在于该基因克隆于长穗偃麦草。

3.一种长穗偃麦草TeWKS1基因的应用，其特征在于TeWKS1-1和TeWKS1-2M两个基因在小麦中的应用，可提高小麦对条锈病菌的广谱抗性。