CN110468142B

CN110468142B - 负调控因子AtRTP5基因及其在抗植物疫霉菌上的应用

Info

Publication number: CN110468142B
Application number: CN201910922156.8A
Authority: CN
Inventors: 单卫星; 李卫卫
Original assignee: Northwest A&F University
Current assignee: Northwest A&F University
Priority date: 2019-09-27
Filing date: 2019-09-27
Publication date: 2022-06-07
Anticipated expiration: 2039-09-27
Also published as: US20210095307A1; US11920142B2; CN110468142A

Abstract

本发明公开了一种负调控因子AtRTP5基因在提高抗植物疫霉菌抗性上的应用，AtRTP5基因通过干扰植物激素免疫信号通路，负调控植物对疫霉菌的抗性；AtRTP5基因的核苷酸序列如SEQ ID NO：1所示或与其同源性为50％以上的同源序列。还公开一种由负调控因子AtRTP5基因编码的蛋白在提高抗植物疫霉菌抗性上的应用，通过基因工程减少AtRTP5基因编码的蛋白的表达量，增强植物对疫霉菌的抗性；蛋白的氨基酸序列如SEQ ID NO：2所示或与其同源性为50％以上的同源序列。该负调控因子有潜力被应用于基因工程培育抗病新品种，对于改变现有技术病原菌抗药性增强的情况具有重要的应用价值。

Description

负调控因子AtRTP5基因及其在抗植物疫霉菌上的应用

技术领域

本发明属于生物技术领域，具体涉及一种负调控因子AtRTP5基因及其在抗植物疫霉菌上的应用。

背景技术

卵菌是一种真核病原微生物，包含多种致病菌，寄主范围广泛，能够引发多种农作物、花卉植物病害，造成巨大的经济损失。19世纪五十年代，卵菌中的致病疫霉菌引起的马铃薯晚疫病在欧洲造成了马铃薯大面积死亡，直接导致数十万人因饥饿死亡。烟草黑胫病是世界性的烟草主要病害之一，在中国的各大主要产烟区都有不同程度的发生，对烟草生产造成了毁灭性的损害，严重影响当地烟民的经济收入和生活质量，而烟草黑胫病的致病菌正是卵菌中的寄生疫霉菌。生产上对于马铃薯晚疫病和烟草黑胫病的防治方法大多是采用药剂处理或者抗病品种。但是，由于病原菌抗药性的产生和植物与病原微生物协同进化，目前有效的抗病资源的挖掘和抗性品种的培育仍然是研究工作的重中之重。

抗病基因的鉴定和应用是作物病害防控的首选途径。在植物-病原菌亲和互作过程中，一些植物基因往往被病原菌诱导后上调表达，常被作为病原菌成功侵染植物的“帮手”，被称为植物免疫负调控因子。免疫负调控因子的缺失或功能突变，可使得植物获得抗性，通过抑制病原菌的侵染和定殖而表现抗病，而且这种抗病性具有广谱性。

尽管目前已鉴定有植物的免疫负调控因子，但仍然是少数几个，而且具体的作用机制也并不清晰，免疫负调控因子在抗病育种中的应用更是屈指可数。

发明内容

本发明的一个目的是解决至少上述问题，并提供至少后面将说明的优点。

本发明还有一个目的是提供一种植物免疫负调控因子AtRTP5基因在疫霉菌抗性上的应用。

为了实现本发明的这些目的和其它优点，提供了一种负调控因子AtRTP5基因在提高抗植物疫霉菌抗性上的应用，所述AtRTP5基因通过干扰植物激素免疫信号通路，负调控植物对疫霉菌的抗性；

所述AtRTP5基因的核苷酸序列如SEQ ID NO：1所示或与SEQ ID NO：1所示序列同源性为50％以上的同源序列。

本发明还提供一种由负调控因子AtRTP5基因编码的蛋白在提高抗植物疫霉菌抗性上的应用，通过基因工程减少所述AtRTP5基因编码的蛋白的表达量，增强植物对疫霉菌的抗性；

所述蛋白的氨基酸序列如SEQ ID NO：2所示或与SEQ ID NO：2所示序列同源性为50％以上的同源序列。

优选的是，所述同源序列来源于马铃薯或烟草。

优选的是，所述植物为拟南芥、马铃薯或烟草。

本发明至少包括以下有益效果：

本发明公开的负调控因子AtRTP5基因，首次作为影响植物免疫负调控因子被克隆。本发明通过基因工程构建AtRTP5基因的过表达转基因拟南芥材料，通过离体叶片接菌实验，结果证明AtRTP5基因过表达拟南芥更容易感寄生疫霉菌；同时，通过接菌测试从拟南芥资源中心购买的AtRTP5基因缺失突变体，发现该突变体能够显著增加对寄生疫霉菌的抗性。上述结果充分证明AtRTP5基因作为负调控基因，其过表达可加重疫霉菌的感染，而如果减少该基因的表达量，则能增强植物对于疫霉菌的抗性，因此，本发明负调控因子AtRTP5基因，首次作为影响植物免疫负调控因子使用，为培育抗疫霉菌的植物新品种具有重要的价值和指导意义。

本发明的其它优点、目标和特征将部分通过下面的说明体现，部分还将通过对本发明的研究和实践而为本领域的技术人员所理解。

附图说明

图1为本发明AtRTP5缺陷型拟南芥rtp5-3抗疫霉菌侵染(a)，AtRTP5过表达植株RTP5OE2更感疫霉菌侵染(b)，在烟草上瞬时过表达AtRTP5促进寄生疫霉菌和致病疫霉菌的侵染(c)；

图2为本发明烟草同源基因NbRTP5沉默后的植株抗疫霉菌侵染；

图3为本发明AtRTP5与烟草中同源基因的序列比对结果；

图4为本发明AtRTP5与马铃薯中同源基因的序列比对结果；

图5为本发明pKannibal载体图谱；

图6为本发明pART27载体图谱。

具体实施方式

下面对本发明做进一步的详细说明，以令本领域技术人员参照说明书文字能够据以实施。

应当理解，本文所使用的诸如“具有”、“包含”以及“包括”术语并不排除一个或多个其它元件或其组合的存在或添加。

实施例1拟南芥负调控因子AtRTP5基因的克隆

1、植物材料：野生型拟南芥Col-0(可以通过公开渠道获得)，从其叶片中提取RNA。

2、RNA的抽提

用RNA提取试剂盒(OMGA)抽提RNA，用琼脂糖凝胶电泳鉴定RNA的完整性，然后在分光光度计上测定RNA的纯度和浓度。

3、基因克隆

用反转录试剂盒(TaKaRa)获得Col-0的cDNA，根据拟南芥网站Tair中提供的AtRTP5(At5G43930)的全长编码序列设计上下游引物AtRTP5-F/R，

AtRTP5-F：5’-CCGGAATTCATGACTCAATCTATCTGTTCGTC-3’

AtRTP5-R：5’-GCGCGGATCCTTAGGTGTTTGGTCCTGTG-3’，

以cDNA为模板进行扩增。PCR扩增产物经酶切、连接、菌液PCR验证后构建成载体pKannibal-AtRTP5(pKannibal质粒载体图谱见图5)，然后后送去测序。将测序结果与已公布序列进行比对，正确的质粒用于后续实验。

实施例2拟南芥AtRTP5基因的序列信息与同源性分析

本发明拟南芥AtRTP5基因全长CDS序列为2181bp，详细序列见Tair Gene Locus：At5G43930.1(见本发明SEQ ID NO：1)，氨基酸序列共726个氨基酸，详细序列见TAIRAccession AASequence NO.：1009128620(见本发明SEQ ID NO：2)。

将拟南芥AtRTP5基因编码蛋白的氨基酸序列用BLAST程序进行同源性检索，结果发现它与本氏烟草中的4个基因NbRTP5-1/NbRTP5-2/NbRTP5-3/NbRTP5-4(其核苷酸序列详见SEQ ID NO：3-SEQ ID NO：6)同源性很高，4个基因编码的氨基酸序列与AtRTP5基因的氨基酸序列同源性均大于50％，并与AtRTP5基因具有相似的生物学功能，同源基因编码的氨基酸序列如Sequence ID(根据茄科网站Sol Genomics Network blast工具获得)Niben101Scf00274g03001.1，Niben101Scf05619g00010.1，Niben101Scf11483g00012.1，Niben101Scf02842g01008.1所示(详见本发明SEQ ID NO：7、SEQ ID NO：8、SEQ ID NO：9、SEQ ID NO：10)。

同时，在马铃薯中基因组的检索中也找到3个基因(GenBank Accession Number：NW_006239128.1、NW_006239128.1、NW_006238946.1)同源性也很高，其编码的氨基酸序列如GenBank Accession Number：XP_015167963.1、XP_015167964.1、XP_006340895.1，所示，同源性均在50％以上。由上述内容可推断7个同源基因具有和AtRTP5相同或相似的功能。

本发明的拟南芥AtRTP5基因与本氏烟草中4个同源基因的氨基酸序列的比对结果见图3。

本发明的拟南芥AtRTP5基因编码蛋白的氨基酸序列与马铃薯测序基因组中3个同源基因的氨基酸序列的比对结果见图4。

实施例3 AtRTP5过表达转基因拟南芥的获得

基因表达载体pART27::AtRTP5-Flag的构建

根据拟南芥网站Tair中提供的AtRTP5(At5G43930)的全长编码序列设计上下游引物AtRTP5-F/R，

AtRTP5-F：5’-CCGGAATTCATGACTCAATCTATCTGTTCGTC-3’

AtRTP5-R：5’-GCGCGGATCCTTAGGTGTTTGGTCCTGTG-3’，

以拟南芥Col-0的cDNA为模板进行扩增。PCR产物经核酸内切酶酶切后与载体pKannibal(可从公开渠道获得)连接，然后电击转化入大肠杆菌感受态，在卡那霉素抗性平板上筛选阳性克隆。用载体引物pKAN-F/R,

pKAN-F:5’-CAATCCCACTATCCTTCGCA-3’

pKAN-R:5’-CGGTAAGGATCTGAGCTACA-3’

进行菌液PCR验证正确克隆后提取质粒送测序公司测序。

将测序结果与已公布序列进行比对，正确的质粒用于内切酶NotI酶切后连入载体pART27(可从公开渠道获得，载体图谱见图6)中，然后电击转化入大肠杆菌感受态，在壮观霉素抗性平板上筛选正确克隆，提质粒进行酶切验证。检测正确的质粒被电击转化入农杆菌感受态细胞中，然后在抗性平板上进行阳性克隆筛选。

利用农杆菌介导的蘸花法进行拟南芥转化，具体步骤如下：

1、将基因表达载体pART27::AtRTP5-Flag电击转化入农杆菌感受态细胞，并将转化成功的克隆挑至液体LB培养基中培养24-36小时；

2、将新鲜菌液按照1：100的比例扩摇到200mL LB液体培养基中，过夜培养16小时左右；

3、低转速离心收集菌体，然后用同等体积的含有0.02％的表面活性剂的5％的蔗糖溶液重悬细胞；

4、剪掉野生型拟南芥的果荚，留下未开的花苞，然后将花苞进入重悬菌液中15-30秒，黑暗保湿处理过夜；

5、植物培养间培养1个月左右收获第一代种子，晾干后在抗性平板上筛选转化成功的植株。成功转化的植株能够在抗性平板上正常生长，生长状态和生长速度与正常植株无差异。对成功转化的植株进行目的基因表达量的检测，超过50％的植株都能过量表达目的基因(表达量比野生型高5倍以上)。

实施例4

烟草同源基因NbRTP5沉默植株的获得与接菌

利用病毒介导的基因沉默技术降低目标基因的表达，具体实施如下：

1、构建沉默表达载体TRV2-Nb4RTP5。

用AtRTP5的氨基酸序列在茄科植物基因组网站(https://solgenomics.net/)中进行序列比对，找到本氏烟草中的四个同源基因NbRTP5-1/NbRTP5-2/NbRTP5-3/NbRTP5-4(详见SEQ ID NO：3-SEQ ID NO：6)。我们利用该网站提供的VIGS工具找到能够同时沉默这四个基因的两段特异性序列，然后根据序列设计PCR引物，用本氏烟草的cDNA为模板进行序列扩增，通过融合PCR的方法将两段序列融合成一个序列片段Nb4RTP5，然后通过酶切、纯化后连接入载体pTRV2(可从公开渠道获得)，并通过测序验证其序列正确性。

上述同时沉默四个本氏烟草的同源基因时所选的靶序列如下所示：

TGCATCTGTCCGACTTCTCACTTACTCAACTCCTTCTGGCCAATATGAACTTTTGTTGTCCCCTGTTGAGCCAACTTTATCTCCTGCACAAGCTCAGACTGGTTCTTCTGTTAGGAATATTGAGAATGCATCCGAACCTGTAGTTGATCCTATGGATACTGATGTGCCGGCTGAGGAAAGAAACAATCAATTTTTCCCTTGAAAATTTTGGGGTGAGAACACTAAATGTACTCTTGACTCCTGTGGATTAAAAAGTGAAGTGGCAAGTGATGCTAGACGGGGACTAATATCATGGGTAGAGGCGGAGTCACTGCAACATTTATCGGCCAAGTATTGTTCACTGTTGCCTCCTCCAAGGTCTACCATTGCAGCAGCATTCAGTCCTGATGGGAGGACACTT

扩增上述靶序列所需的引物如下所示：

tNbRTP5-1F:GTTACCGAATTCTCTAGA TGCATCTGTCCGACTTCTCAC

tNbRTP5-1R:CAAAATTTTCAAGGGAAAAATTGATTGTTTCTTTCC

tNbRTP5-2F:TTTTTCCCTTGAAAATTTTGGGGTGAGAACACT

tNbRTP5-2R:GAGCTCGGTACCGGATCC AAGTGTCCTCCCATCAGGACT

2、将沉默载体TRV2-Nb4RTP5电击转化入农杆菌感受态，并将转化成功的克隆，挑至液体LB培养基中培养24-36h；

3、低转速收集菌体，用含有乙酰丁香酮的MES溶液重悬菌体，取部分菌液稀释后用分光光度计检测OD600的浓度；

4、将菌液调整至合适浓度，与TRV1菌液混合注射；

5、选取4-6叶期的本氏烟草幼苗，用1mL去针头的注射器在最大的叶片背面注射，2-3周后选择比注射叶位高两个叶位的叶片进行接菌实验。

6、寄生疫霉菌接种实验操作具体为：将盐培过的寄生疫霉菌用冷水刺激后，在4℃放置半小时，然后转移到23℃培养箱释放1h。游动孢子释放后在显微镜下进行计数，然后将游动孢子浓度稀释至50-80个/μL，在离体叶片背面接种10μL游动孢子悬浮液，接种前用水喷湿叶片后用纸擦干，避免游动孢子悬浮液散落；接种时避开大叶脉，接种2到3天后观察病菌侵染情况，统计病斑直径。如图2所示，结果发现：Nb4RTP5沉默植株叶片上的病斑更小，更抗寄生疫霉菌侵染。

实施例5AtRTP5基因的功能研究

从拟南芥资源中心购买获得AtRTP5缺陷型拟南芥rtp5-3和AtRTP5过表达转基因拟南芥接菌：选取25-28天苗龄的拟南芥莲座叶，叶背面轻轻划伤后接种8-10μL(约3000个)寄生疫霉菌游动孢子悬浮液，23℃培养箱黑暗保湿处理2-3天后观察统计发病情况。

过表达AtRTP5的烟草叶片接菌：在6-8周苗龄的本氏烟草叶片上一侧注射含有质粒pART27::AtRTP5-Flag的农杆菌菌液，另一侧注射含有对照质粒(pART27::GFP，构建方法同pART27::AtRTP5-Flag)的农杆菌菌液，保证两侧注射面积相近，且均不越过中间主叶脉。黑暗保湿处理过夜，然后在植物培养间正常培养2天后剪取注射叶片，在注射孔附近接种8-10μL(约500个)寄生疫霉菌游动孢子悬浮液，23℃培养箱黑暗保湿处理2-3天后，观察统计发病情况或接种10-15μL(约1000个)致病疫霉菌游动孢子悬浮液，18℃培养箱黑暗保湿处理5天后观察统计发病情况。

如图1所示，无论是接种寄生疫霉菌还是接种致病疫霉菌，过表达AtRTP5基因后病斑直径更大，叶片更感病。

以上实施方式仅用于说明本发明，而非对本发明的限制。尽管参照实施例对本发明进行了详细说明，本领域的普通技术人员应当理解，对本发明的技术方案进行各种组合、修改或者等同替换，都不脱离本发明技术方案的精神和范围，均应涵盖在本发明的权利要求范围当中。

序列表

<110> 西北农林科技大学

<120> 负调控因子AtRTP5基因及其在抗植物疫霉菌上的应用

<160> 10

<170> SIPOSequenceListing 1.0

<210> 1

<211> 2181

<212> DNA

<213> 人工序列(Artificial Sequence)

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atgactcaat ctatctgttc gtcgtcacaa gatcaatctt cttctcctcg ctctttgatc 60

acaccacgca aatcggcgac gaagtttcac cgccgcaggg ggaggagtgt atttaacatg 120

ctggttcaga gggaaatgtc accaaaggct aagtttgtgc caaggaaacg atggggtaag 180

agtagatggt atactgattc ttcatgcgga accaacaacg agtctatgaa agagacggga 240

caaagtctta cctcatgggt tgaggcagag tcattgcagc atttatctgc aaaatattgt 300

cctcttggag ctcctccaag gtcaactatt gctgcggctt ttagtactga tggaagaact 360

cttgcttcta cacatggtga tcatactgtg aagattattg attgtgaaac tggaaactgc 420

ttaaaagttt tgactggcca taggaggact ccttgggtag tcagattcca cccgcatcat 480

tcagaaatag ttgcgagtgg aagtttagat ctcgaggtcc gcttatggaa taccacaact 540

tctgaatgca ttagatctca tctattctat cggcctattg cttctatcgc tttccatgct 600

gagggtgaac tacttgctgt tgcttctggt cataagctgc acatgtggca ctacaatagg 660

agaggagagg gatcatcacc aactgttgta ttgaagacga ggcgatcttt gagagctgta 720

cactttcacc ctcatggggc tccattactt ttgaccgcag aggtgaatga gattgattca 780

ttagattctt caatgtctag agcaacatct atgggctact tgaggtatcc gccccctgct 840

attttgttca caagcacgga aagcaatcaa actagtttgg cagcagaaaa tgagaacaga 900

acatcgtctc ctcctcttcc cttggctaca tccagtggtc cttctggtcc aaatagtgtt 960

ccaggaaatt ctcccagcaa catatttctt acccgagctg gagatagaac ttctcctgca 1020

gttgatggca tggatgtaga tgaagctcaa cctgttggaa gaaatggaat ccctagccaa 1080

gtttcaaata gatcagattt ccctgagctt ggacagattc ggcaattatt tcactttagg 1140

gatcgagttt cctgggagtt accttttcta caaggatggt tgatggctca aggtcatggt 1200

gttgctaatc cagtggttac tcctactggc agtagtaatc atggcatctc agctccatcc 1260

tcaacagcca gtctggaggc tgcagtagcg ttattagaaa tcccgagtgg tgttaactta 1320

catgcggtgt ctagaagagg cggggctcag gaacaaactt cacaacccca attctcgaga 1380

actggattac cggaaggtgt atcttctcgt aacactcaac atggaagtga tgctcaacct 1440

gtagtaaata gggtccagtc tgagcttgct acctcaattg ctgcttcggc tgcagcagct 1500

gctgctgcag aattaccttg tactgtcaaa ctcagaatgt ggtcgcatga catcaaagac 1560

ccatatgcac aactgaaatc cgacagatgt ctatttacaa taccgcatgc cgtcctttgc 1620

agtgaaatgg gagctcatta ttcgccatgt gggagatatt tagcggcctg tgttgcgtgt 1680

gtttttcctc atggtgagat agatcctgga ctgcagacac aagctcaaca agattcaggg 1740

cttgcaactt ccccaactcg acaccctgtc acagcacacc aagtcattta cgagcttcgt 1800

gtgtattctc tccagaagga aagttttggt tcagtacttg tgtcacgggc aattagagct 1860

gcacattgct tgacctctat ccagttctca cccacttcgg agcatatact acttgcatat 1920

gggcggcgtc atggttctct tttgaggagc atcgttagtg atggtgaaac aacatcacat 1980

tttttcacag tattggagat atacagagtt tcagatatgg agcttgtgag agtactgcca 2040

agttcagagg atgaagtgaa cgttgcgtgt tttcatcctt ctcctggagg aggtcttgtt 2100

tatgggacaa aggaggggaa actgaggatc ttccagtaca atacagctgc tacctcaaac 2160

ttcacaggac caaacaccta a 2181

<210> 2

<211> 726

<212> PRT

<213> 人工序列(Artificial Sequence)

<400> 2

Met Thr Gln Ser Ile Cys Ser Ser Ser Gln Asp Gln Ser Ser Ser Pro

1 5 10 15

Arg Ser Leu Ile Thr Pro Arg Lys Ser Ala Thr Lys Phe His Arg Arg

20 25 30

Arg Gly Arg Ser Val Phe Asn Met Leu Val Gln Arg Glu Met Ser Pro

35 40 45

Lys Ala Lys Phe Val Pro Arg Lys Arg Trp Gly Lys Ser Arg Trp Tyr

50 55 60

Thr Asp Ser Ser Cys Gly Thr Asn Asn Glu Ser Met Lys Glu Thr Gly

65 70 75 80

Gln Ser Leu Thr Ser Trp Val Glu Ala Glu Ser Leu Gln His Leu Ser

85 90 95

Ala Lys Tyr Cys Pro Leu Gly Ala Pro Pro Arg Ser Thr Ile Ala Ala

100 105 110

Ala Phe Ser Thr Asp Gly Arg Thr Leu Ala Ser Thr His Gly Asp His

115 120 125

Thr Val Lys Ile Ile Asp Cys Glu Thr Gly Asn Cys Leu Lys Val Leu

130 135 140

Thr Gly His Arg Arg Thr Pro Trp Val Val Arg Phe His Pro His His

145 150 155 160

Ser Glu Ile Val Ala Ser Gly Ser Leu Asp Leu Glu Val Arg Leu Trp

165 170 175

Asn Thr Thr Thr Ser Glu Cys Ile Arg Ser His Leu Phe Tyr Arg Pro

180 185 190

Ile Ala Ser Ile Ala Phe His Ala Glu Gly Glu Leu Leu Ala Val Ala

195 200 205

Ser Gly His Lys Leu His Met Trp His Tyr Asn Arg Arg Gly Glu Gly

210 215 220

Ser Ser Pro Thr Val Val Leu Lys Thr Arg Arg Ser Leu Arg Ala Val

225 230 235 240

His Phe His Pro His Gly Ala Pro Leu Leu Leu Thr Ala Glu Val Asn

245 250 255

Glu Ile Asp Ser Leu Asp Ser Ser Met Ser Arg Ala Thr Ser Met Gly

260 265 270

Tyr Leu Arg Tyr Pro Pro Pro Ala Ile Leu Phe Thr Ser Thr Glu Ser

275 280 285

Asn Gln Thr Ser Leu Ala Ala Glu Asn Glu Asn Arg Thr Ser Ser Pro

290 295 300

Pro Leu Pro Leu Ala Thr Ser Ser Gly Pro Ser Gly Pro Asn Ser Val

305 310 315 320

Pro Gly Asn Ser Pro Ser Asn Ile Phe Leu Thr Arg Ala Gly Asp Arg

325 330 335

Thr Ser Pro Ala Val Asp Gly Met Asp Val Asp Glu Ala Gln Pro Val

340 345 350

Gly Arg Asn Gly Ile Pro Ser Gln Val Ser Asn Arg Ser Asp Phe Pro

355 360 365

Glu Leu Gly Gln Ile Arg Gln Leu Phe His Phe Arg Asp Arg Val Ser

370 375 380

Trp Glu Leu Pro Phe Leu Gln Gly Trp Leu Met Ala Gln Gly His Gly

385 390 395 400

Val Ala Asn Pro Val Val Thr Pro Thr Gly Ser Ser Asn His Gly Ile

405 410 415

Ser Ala Pro Ser Ser Thr Ala Ser Leu Glu Ala Ala Val Ala Leu Leu

420 425 430

Glu Ile Pro Ser Gly Val Asn Leu His Ala Val Ser Arg Arg Gly Gly

435 440 445

Ala Gln Glu Gln Thr Ser Gln Pro Gln Phe Ser Arg Thr Gly Leu Pro

450 455 460

Glu Gly Val Ser Ser Arg Asn Thr Gln His Gly Ser Asp Ala Gln Pro

465 470 475 480

Val Val Asn Arg Val Gln Ser Glu Leu Ala Thr Ser Ile Ala Ala Ser

485 490 495

Ala Ala Ala Ala Ala Ala Ala Glu Leu Pro Cys Thr Val Lys Leu Arg

500 505 510

Met Trp Ser His Asp Ile Lys Asp Pro Tyr Ala Gln Leu Lys Ser Asp

515 520 525

Arg Cys Leu Phe Thr Ile Pro His Ala Val Leu Cys Ser Glu Met Gly

530 535 540

Ala His Tyr Ser Pro Cys Gly Arg Tyr Leu Ala Ala Cys Val Ala Cys

545 550 555 560

Val Phe Pro His Gly Glu Ile Asp Pro Gly Leu Gln Thr Gln Ala Gln

565 570 575

Gln Asp Ser Gly Leu Ala Thr Ser Pro Thr Arg His Pro Val Thr Ala

580 585 590

His Gln Val Ile Tyr Glu Leu Arg Val Tyr Ser Leu Gln Lys Glu Ser

595 600 605

Phe Gly Ser Val Leu Val Ser Arg Ala Ile Arg Ala Ala His Cys Leu

610 615 620

Thr Ser Ile Gln Phe Ser Pro Thr Ser Glu His Ile Leu Leu Ala Tyr

625 630 635 640

Gly Arg Arg His Gly Ser Leu Leu Arg Ser Ile Val Ser Asp Gly Glu

645 650 655

Thr Thr Ser His Phe Phe Thr Val Leu Glu Ile Tyr Arg Val Ser Asp

660 665 670

Met Glu Leu Val Arg Val Leu Pro Ser Ser Glu Asp Glu Val Asn Val

675 680 685

Ala Cys Phe His Pro Ser Pro Gly Gly Gly Leu Val Tyr Gly Thr Lys

690 695 700

Glu Gly Lys Leu Arg Ile Phe Gln Tyr Asn Thr Ala Ala Thr Ser Asn

705 710 715 720

Phe Thr Gly Pro Asn Thr

725

<210> 3

<211> 2310

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 3

atgagggcgt cactttggcc ggagaattca agcagtgatt catccacgtc aatttcgcgg 60

cggcggaatt cgccacctca ttccatttcc caaaacccta attgtaaaca cagctgcggt 120

aacgtttttc agttgctaac gaggagggag gtatctcctc gaactaaacg cagttctacg 180

aagttttggg gtgagaactc taaatgttca gtttattcct gtggattaaa aagccaagta 240

gcaagagacc cgagacaggg tcttatatca tgggtagagg cagagtcatt gagacatttt 300

tcagctaagt attgtccact gttgcctccg cccaggtcca ctattgctgc ggcattcagt 360

cctgatggga agacactcgc ttctactcat ggagatcaca cagtaaagat aattgattgc 420

caaagtggaa agtgcttaaa ggtgttaagt ggacatcgca ggactccttg ggtggttcgt 480

ttccatccgt tatactctga tatacttgca agtggaagtt tggaccatga agttcgtcta 540

tgggatgcaa agactgccga gtgtatagga tcacatcgtc ctattgcatc cattgccttc 600

catgcccaag gggaagtttt ggctgttgct tcaggccaca agctgtatat gtggcactac 660

aacagaagag gggaagcatc ttcaccagct atcgtactaa agacaagacg ttcacttcgt 720

gctgtacatt ttcacccaca tggtgcccca tttcttttaa cagctgaggt caatgatttg 780

gactcatcag attcttcaat gacgcttgca acttctccgg gtaacttgcg gtaccctcct 840

cctactgtgt atttgacaga tgctcactcc acttaccgat ctgcttcagc aaatgaattg 900

ccgatcatgt ctctaccatt catgatctgg ccttcaatcg ctagaggtga tcctggaatg 960

cctttgcagc agagcgatgc agacatggct tctgacagca cacagcacag agcggacact 1020

tatgcatctg tccgacttct cacttactca actccttctg gccaatatga acttttgttg 1080

tcccctgttg agccaacttt atctcctgca caagctcaga ctggttcttc tgttaggaat 1140

attgagaatg catccgaacc tgtagttgat cctatggata ctgatgtgcc ggctgaggaa 1200

agaaacaatc aatttttccc tttcagtgac cctgcatact gggaattacc tttcttgcaa 1260

ggatggttga ttggccaaag ccaagctggc cggcgagcga ttcattcaga gcacagtggt 1320

gccacaagta gtgtatcggc ttatggtgaa gtagaacatc ctcctgcagt tccgtcagtc 1380

atttcaaaca gtaatcattc aaggtctgga agatctggtt ctcgtcaccg ttcttcacgc 1440

tctagagcga tacctgttgt tggatctggt gatagcgctg ctcccattaa cgttgtacat 1500

gatgaaaatg attctcaagc ttttatgagt cagttccagt cagagatagc cacttcactg 1560

gctgcagcag cggctcccga attgccttgc actgtgaaac tcagaatatg gccttatgat 1620

gttaaggctc catgtgcacc gcttgatgct gaaaattgtc gcttaatgat accacatgct 1680

gtgctttgta gtgaaatggg agcccacttt tcaccatgtg ggagattttt agcagcttgt 1740

gttgcatgta ttctgcctaa catggaagct gaacctggtt ttcacggaca acttcaccat 1800

gatgccatgg gtgctgcaac ttcacctacc agacatccta ttgcagccca tcgggttatg 1860

tatgagctac gaatatattc cttggaggag gcaacgtttg gtttagtgct tgtatctcgg 1920

gcaattagag ctgcacattg tttgacttca attcagtttt ctccaacttc tgaacatctt 1980

ttacttgcct atgggcgtcg gcacggttca cttctgaaaa gtatagtggt tgatggagat 2040

acaactgtac ccgtttacac gattcttgag gtctacagag tttcagatat ggaacttgtg 2100

agagttcttc ccagtgcaga ggatgaggtt aacgttgctt gtttccatcc tttggttggt 2160

ggcggcctag tctatggaac caaggaagga aaattgagga tcctccaatt tgataaatca 2220

aatgggtgtc atgaaaatat acgtatcagg acaatgactg gtggctgcaa agttgaatgt 2280

gttgctgatg gtgactgtat agttagttga 2310

<210> 4

<211> 2238

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 4

atgagggcgt cactttggcc ggagaattca agcagcgatt catccacgtc aattttgcgg 60

cggcggaatt cgccgcctca ttcgattgcc caaaacccta attgtaaaca tagctgcggt 120

aacgtctttc agctgctaac gaggagggag gtatctcctc gaactaaacg cagttctaag 180

aagttttggg gtgagaactc taaatgttct gttcattcct gtggattaaa aagccaagta 240

gcaagagacc cgaggcaggg tcttatatca tgggtagagg cagagtcatt gagacatttt 300

tcagctaagt attgtccact gttgcgtccg cctaggtcta ctattgctgc ggcattcagt 360

cctgatggga agacactcgc ttctactcat ggagatcaca cagtaaagat aattgattgc 420

caaagtggaa agtgcttaaa ggtgttaagt ggacatcgca ggactccttg ggtggttcgt 480

ttccatccat tatactctga tatacttgca agtggaagtt tggaccatga agttcgttta 540

tgggatgcaa aaactgccga gtgtatagga tcacgtgatt tttatcgtcc tattgcatcc 600

attgccttcc atgcccaagg ggaagttctg gctgttgctt caggccacaa gctgtatatg 660

tggcactaca acagaagagg ggaagcatct tcaccagcta tcgtactaaa gacaagacgt 720

tcacttcgtg ctgtacattt ccacccacat ggtgccccat ttcttttaac agctgaggtc 780

aatgatctgg actcatcaga ttcttcaatg acgcttgcaa cttctccggc aaatgaattg 840

ccgatcatgt ctctaccatt catgatctgg ccttcaatcg ctagaggtga tcctagaatg 900

cctttgcagc tgagcgatgc agacatggct tctgacagca cacagcagag agccgacact 960

tctgcatctg tccgacttct cacttactca actccttctg gccaatatga acttttattg 1020

tcccctgttg agccaacttt atctcctgca caagctcaga ctggttcttc tgttagggat 1080

actgagaatg catccgaacc tgtagttgat cctatggaga ctgatgtgcc ggctgaggaa 1140

agaaacaatc aatttttccc tttcagtgac cctgcatatt gggaattacc tttcttgcaa 1200

ggatggttga ttggccaaag ccaagctggc agacgagcga ttcattcaga gcacagtggt 1260

gccactagta gtgtatcggc ttatggtgaa gtagaacatc ctcctgcagt tccgtcagtc 1320

atttcaaaca gtaatcatcc aaggtctgaa agatctggct ctcgtcaccg ttcttcacgc 1380

tctagagcga tacctgttgt tggatctggt gatagcgctg ctcccattaa cattgtacat 1440

gatgaaaatg attctcatgc ttttatgagt cggttccagt cagagatagc tacttcactg 1500

gctgcagcag ctgctcccga attgccttgc actgtgaaac tcagaatttg gccttatgat 1560

gttaaggctc catgtgcacc gcttgaagct gaaaattgtc gtttaatgat accacatgct 1620

gtgctttgta gtgaaatggg agcccacttt tcaccatgtg ggaggttttt agccgcttgt 1680

gttgcatgta ttctgcctaa catggaagct gatcctggtt ttcaaggaca acttcaccat 1740

gatgccatgg gtgctgcaac ttcacctacc agacatccta ttgcagccca tcgggttatg 1800

tatgagctac gaatatattc cttggaggag gcgacgtttg gtttagtgct tgtatctcgg 1860

gcaattagag ctgcacattg tttgacttca attcagtttt ctccaacttc tgaacatctt 1920

ttacttgcct atgggcgtcg gcacggttca cttctgaaaa gtatagtgat tgatggagat 1980

acaactgttc ctgtttacac gattcttgag gtctacagag tttcagatat ggaacttgtg 2040

agagttcttc ccagtgcaga ggatgaggtt aatgttgctt gtttccatcc tttggttggt 2100

ggcggcctag tctatggaac caaggaagga aaattgagga tcctccaatt tgataaatca 2160

aatggtttgg accgtacagt atcctgtttt cctgatgaag acatgctgga ggttccaaca 2220

tatgctttag aaggctag 2238

<210> 5

<211> 2100

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 5

atgagatcgt ctgtcttgcc ggagaattca ggcagtgcgc cgtcaacttc gcggaattca 60

cctccttctg cgacggcgcc tcgaaactcc aattgtaaac acagtaatgt ctttcaactg 120

ttaacaagaa gggaggtatc ttctcgaact aaacgcgctt ccagaaaatt ttggggtgag 180

aacactaaat gtactcttga ctcctgtgga ttaaaaagtg aagtggcaag tgatgctaga 240

cggggactaa tatcatgggt agaggcggag tcactgcaac atttatcggc caagtattgt 300

tcactgttgc ctcctccaag gtctaccatt gcagcagcat tcagtcctga tgggaggaca 360

cttgcttcta cgcatggaga tcacacagtg aaaataattg actgtcaaac tgggaagtgc 420

ttaaaggttt tgagtggaca ccgcaggaca ccttgggtgg ttcgtttcca tccattgtac 480

cctgaaatac tggcaagtgg aagtttggac cacgaagttc ggctgtggga tgcaaaaact 540

gccgagtgta taggatcgcg agatttttat cgccccatcg catccatagc gttccatgcc 600

caaggggaag ttctagccgt tgcttcaggc cacaagcttt atatatggca ttacaacaga 660

agaggagagg cttctacacc agcaattgta ctgaagacac ggcgttctct tcgtgctgtc 720

catttccacc cacatgctac cccatttctt ttgacagctg aggtcaatga tctggattca 780

tcagattctt caatgacacg tgctacttct ccggcaaatg aactgcctat catgtctcta 840

cctttcctga tctggccgtc aattgcaaga ggtgatccca gaatgactgt gcagcaaact 900

gatatagata tggtaaccga caatgtacag cacagaacag atacttcatc atctgtccgc 960

cttctcacat attcaactcc gtccggccag tatgaacttt tattgtcccc tgttgagcaa 1020

agtgcatctc ccgcacaaga agctcatact ggttcttctg ttggggagaa tgagaatata 1080

ggatggttga ttggccaaag ccaagttgcc caacaagcaa ctcatccaga ccttagtggt 1140

actaccacta atccatcaac ttatgatgaa ctggaaaatc cttctgctgt tccgttggta 1200

atttcaagca atactcatcc aaggtccgga agatctggtt ctcggcaacg ttcttcacgc 1260

tctcgagcga ttcctgttgc tggagctggt gctggtgctg cttcgcttaa cgttatgcat 1320

gatgagagtg attctcaaac ttctattggt cgcatccagt cggagatagc tacttcactg 1380

gctgcagcag cggctgctga attgccttgc actgtgaaac tcagaatatg gcctcatgat 1440

gttaaggttc catgtgcacc ccttcatgct gaaagatgtc gcttaacaat accacatgct 1500

gtactttgca gtgaaatggg agcccatttt tcaccatgtg ggagattttt agcagcttgt 1560

gttgcatgta ttctgccaaa cgtggatgct gatcctggtt ttcatggcca tcttcatcat 1620

gatactatgg cagctggaac ttctccaacc agacatccag ttgctgccca ccaggttatg 1680

tatgagctac ggatatattc cctggaggag gcaacgtttg gttcagtgct tgcatctcga 1740

gcaattagag ctgctcattg tttgacttca attcagtttt ctccggcttc agagcatctg 1800

ttacttgctt atgggcgtcg ccatagttca ctacttaaaa gtgttgttat tgatggagac 1860

acaactatac ccatttacac tattcttgag gtctatagag tttctgatat ggaacttgtg 1920

agagttctcc ccagtgcgga ggatgaggtt aatgtcgctt gcttccatcc ttcggttggt 1980

ggtggccttg tctatggaac caaggaaggg aagttgagga ttctccaata tgacaattca 2040

aatggtttgg gtcgcacaat atcctgttct cctgtcgaaa acattgtcga ggtattataa 2100

<210> 6

<211> 978

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 6

atggagactg atgtgcagcc agaagaaaga aacaatcagt ttttcccttt tagtgaccca 60

gcatactggg aattgccttt tttgcaagga tggttgattg gccaaagcca agttgcccaa 120

caaggaactc atccagacct tagtgatact accactaatc catcaactta tggtgaactg 180

gaaaatcctt ctgctgttcc cttggcaatt tcaagcaata atcgtccaag gtccggaaga 240

tctggttctc ggcaacgttc ttcacgctct cgagcgattc ctgttgctgg aggtggtgct 300

tcttcgctta actttatgca tgatgagagt gattctcaaa cttcgattgc tcgcatccag 360

tcagagatag ctacttcgct ggctgcagca gcggctgctg aattgccttg cactgtgaaa 420

ctcagaatat ggcctcatga tgttaaggtt ccatgtgcac cccttcatgc tgaaagatgt 480

cgcttaacaa taccacatgc tgtactttgc agtgaaatgg gagcccattt ttcaccatgt 540

gggagattct tagcaacttg tgttgcatgt attctgccaa acgtggatgc tgatcctagt 600

tttcatggcc atcttcatca tgatactatg gcagctggaa cttctccaac cagacatcca 660

gttgctgccc accaggttat gtatgagcta cggatatatt ccctggagga ggcagcgttt 720

ggttcagtgc ttgcatctcg agcaattaga gctgctcatt gtttaacttc aattcaggtc 780

tatagagttt ctgatatgga acttgtgaga gttctcccca gtgcggagga tgaggttaat 840

gtcgcttgct tccatccttc ggttggtggt ggccttgtct atggaaccaa ggaagggaag 900

ttgaggattc tccaatatga caattcaaat tggcaaaaag aaattagctc caaatttcca 960

ataactgtat tcttctga 978

<210> 7

<211> 769

<212> PRT

<213> 人工序列(Artificial Sequence)

<400> 7

Met Arg Ala Ser Leu Trp Pro Glu Asn Ser Ser Ser Asp Ser Ser Thr

1 5 10 15

Ser Ile Ser Arg Arg Arg Asn Ser Pro Pro His Ser Ile Ser Gln Asn

20 25 30

Pro Asn Cys Lys His Ser Cys Gly Asn Val Phe Gln Leu Leu Thr Arg

35 40 45

Arg Glu Val Ser Pro Arg Thr Lys Arg Ser Ser Thr Lys Phe Trp Gly

50 55 60

Glu Asn Ser Lys Cys Ser Val Tyr Ser Cys Gly Leu Lys Ser Gln Val

65 70 75 80

Ala Arg Asp Pro Arg Gln Gly Leu Ile Ser Trp Val Glu Ala Glu Ser

85 90 95

Leu Arg His Phe Ser Ala Lys Tyr Cys Pro Leu Leu Pro Pro Pro Arg

100 105 110

Ser Thr Ile Ala Ala Ala Phe Ser Pro Asp Gly Lys Thr Leu Ala Ser

115 120 125

Thr His Gly Asp His Thr Val Lys Ile Ile Asp Cys Gln Ser Gly Lys

130 135 140

Cys Leu Lys Val Leu Ser Gly His Arg Arg Thr Pro Trp Val Val Arg

145 150 155 160

Phe His Pro Leu Tyr Ser Asp Ile Leu Ala Ser Gly Ser Leu Asp His

165 170 175

Glu Val Arg Leu Trp Asp Ala Lys Thr Ala Glu Cys Ile Gly Ser His

180 185 190

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

195 200 205

Val Ala Ser Gly His Lys Leu Tyr Met Trp His Tyr Asn Arg Arg Gly

210 215 220

Glu Ala Ser Ser Pro Ala Ile Val Leu Lys Thr Arg Arg Ser Leu Arg

225 230 235 240

Ala Val His Phe His Pro His Gly Ala Pro Phe Leu Leu Thr Ala Glu

245 250 255

Val Asn Asp Leu Asp Ser Ser Asp Ser Ser Met Thr Leu Ala Thr Ser

260 265 270

Pro Gly Asn Leu Arg Tyr Pro Pro Pro Thr Val Tyr Leu Thr Asp Ala

275 280 285

His Ser Thr Tyr Arg Ser Ala Ser Ala Asn Glu Leu Pro Ile Met Ser

290 295 300

Leu Pro Phe Met Ile Trp Pro Ser Ile Ala Arg Gly Asp Pro Gly Met

305 310 315 320

Pro Leu Gln Gln Ser Asp Ala Asp Met Ala Ser Asp Ser Thr Gln His

325 330 335

Arg Ala Asp Thr Tyr Ala Ser Val Arg Leu Leu Thr Tyr Ser Thr Pro

340 345 350

Ser Gly Gln Tyr Glu Leu Leu Leu Ser Pro Val Glu Pro Thr Leu Ser

355 360 365

Pro Ala Gln Ala Gln Thr Gly Ser Ser Val Arg Asn Ile Glu Asn Ala

370 375 380

Ser Glu Pro Val Val Asp Pro Met Asp Thr Asp Val Pro Ala Glu Glu

385 390 395 400

Arg Asn Asn Gln Phe Phe Pro Phe Ser Asp Pro Ala Tyr Trp Glu Leu

405 410 415

Pro Phe Leu Gln Gly Trp Leu Ile Gly Gln Ser Gln Ala Gly Arg Arg

420 425 430

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

435 440 445

Gly Glu Val Glu His Pro Pro Ala Val Pro Ser Val Ile Ser Asn Ser

450 455 460

Asn His Ser Arg Ser Gly Arg Ser Gly Ser Arg His Arg Ser Ser Arg

465 470 475 480

Ser Arg Ala Ile Pro Val Val Gly Ser Gly Asp Ser Ala Ala Pro Ile

485 490 495

Asn Val Val His Asp Glu Asn Asp Ser Gln Ala Phe Met Ser Gln Phe

500 505 510

Gln Ser Glu Ile Ala Thr Ser Leu Ala Ala Ala Ala Ala Pro Glu Leu

515 520 525

Pro Cys Thr Val Lys Leu Arg Ile Trp Pro Tyr Asp Val Lys Ala Pro

530 535 540

Cys Ala Pro Leu Asp Ala Glu Asn Cys Arg Leu Met Ile Pro His Ala

545 550 555 560

Val Leu Cys Ser Glu Met Gly Ala His Phe Ser Pro Cys Gly Arg Phe

565 570 575

Leu Ala Ala Cys Val Ala Cys Ile Leu Pro Asn Met Glu Ala Glu Pro

580 585 590

Gly Phe His Gly Gln Leu His His Asp Ala Met Gly Ala Ala Thr Ser

595 600 605

Pro Thr Arg His Pro Ile Ala Ala His Arg Val Met Tyr Glu Leu Arg

610 615 620

Ile Tyr Ser Leu Glu Glu Ala Thr Phe Gly Leu Val Leu Val Ser Arg

625 630 635 640

Ala Ile Arg Ala Ala His Cys Leu Thr Ser Ile Gln Phe Ser Pro Thr

645 650 655

Ser Glu His Leu Leu Leu Ala Tyr Gly Arg Arg His Gly Ser Leu Leu

660 665 670

Lys Ser Ile Val Val Asp Gly Asp Thr Thr Val Pro Val Tyr Thr Ile

675 680 685

Leu Glu Val Tyr Arg Val Ser Asp Met Glu Leu Val Arg Val Leu Pro

690 695 700

Ser Ala Glu Asp Glu Val Asn Val Ala Cys Phe His Pro Leu Val Gly

705 710 715 720

Gly Gly Leu Val Tyr Gly Thr Lys Glu Gly Lys Leu Arg Ile Leu Gln

725 730 735

Phe Asp Lys Ser Asn Gly Cys His Glu Asn Ile Arg Ile Arg Thr Met

740 745 750

Thr Gly Gly Cys Lys Val Glu Cys Val Ala Asp Gly Asp Cys Ile Val

755 760 765

Ser

<210> 8

<211> 745

<212> PRT

<213> 人工序列(Artificial Sequence)

<400> 8

Met Arg Ala Ser Leu Trp Pro Glu Asn Ser Ser Ser Asp Ser Ser Thr

1 5 10 15

Ser Ile Leu Arg Arg Arg Asn Ser Pro Pro His Ser Ile Ala Gln Asn

20 25 30

Pro Asn Cys Lys His Ser Cys Gly Asn Val Phe Gln Leu Leu Thr Arg

35 40 45

Arg Glu Val Ser Pro Arg Thr Lys Arg Ser Ser Lys Lys Phe Trp Gly

50 55 60

Glu Asn Ser Lys Cys Ser Val His Ser Cys Gly Leu Lys Ser Gln Val

65 70 75 80

Ala Arg Asp Pro Arg Gln Gly Leu Ile Ser Trp Val Glu Ala Glu Ser

85 90 95

Leu Arg His Phe Ser Ala Lys Tyr Cys Pro Leu Leu Arg Pro Pro Arg

100 105 110

Ser Thr Ile Ala Ala Ala Phe Ser Pro Asp Gly Lys Thr Leu Ala Ser

115 120 125

Thr His Gly Asp His Thr Val Lys Ile Ile Asp Cys Gln Ser Gly Lys

130 135 140

Cys Leu Lys Val Leu Ser Gly His Arg Arg Thr Pro Trp Val Val Arg

145 150 155 160

Phe His Pro Leu Tyr Ser Asp Ile Leu Ala Ser Gly Ser Leu Asp His

165 170 175

Glu Val Arg Leu Trp Asp Ala Lys Thr Ala Glu Cys Ile Gly Ser Arg

180 185 190

Asp Phe Tyr Arg Pro Ile Ala Ser Ile Ala Phe His Ala Gln Gly Glu

195 200 205

Val Leu Ala Val Ala Ser Gly His Lys Leu Tyr Met Trp His Tyr Asn

210 215 220

Arg Arg Gly Glu Ala Ser Ser Pro Ala Ile Val Leu Lys Thr Arg Arg

225 230 235 240

Ser Leu Arg Ala Val His Phe His Pro His Gly Ala Pro Phe Leu Leu

245 250 255

Thr Ala Glu Val Asn Asp Leu Asp Ser Ser Asp Ser Ser Met Thr Leu

260 265 270

Ala Thr Ser Pro Ala Asn Glu Leu Pro Ile Met Ser Leu Pro Phe Met

275 280 285

Ile Trp Pro Ser Ile Ala Arg Gly Asp Pro Arg Met Pro Leu Gln Leu

290 295 300

Ser Asp Ala Asp Met Ala Ser Asp Ser Thr Gln Gln Arg Ala Asp Thr

305 310 315 320

Ser Ala Ser Val Arg Leu Leu Thr Tyr Ser Thr Pro Ser Gly Gln Tyr

325 330 335

Glu Leu Leu Leu Ser Pro Val Glu Pro Thr Leu Ser Pro Ala Gln Ala

340 345 350

Gln Thr Gly Ser Ser Val Arg Asp Thr Glu Asn Ala Ser Glu Pro Val

355 360 365

Val Asp Pro Met Glu Thr Asp Val Pro Ala Glu Glu Arg Asn Asn Gln

370 375 380

Phe Phe Pro Phe Ser Asp Pro Ala Tyr Trp Glu Leu Pro Phe Leu Gln

385 390 395 400

Gly Trp Leu Ile Gly Gln Ser Gln Ala Gly Arg Arg Ala Ile His Ser

405 410 415

Glu His Ser Gly Ala Thr Ser Ser Val Ser Ala Tyr Gly Glu Val Glu

420 425 430

His Pro Pro Ala Val Pro Ser Val Ile Ser Asn Ser Asn His Pro Arg

435 440 445

Ser Glu Arg Ser Gly Ser Arg His Arg Ser Ser Arg Ser Arg Ala Ile

450 455 460

Pro Val Val Gly Ser Gly Asp Ser Ala Ala Pro Ile Asn Ile Val His

465 470 475 480

Asp Glu Asn Asp Ser His Ala Phe Met Ser Arg Phe Gln Ser Glu Ile

485 490 495

Ala Thr Ser Leu Ala Ala Ala Ala Ala Pro Glu Leu Pro Cys Thr Val

500 505 510

Lys Leu Arg Ile Trp Pro Tyr Asp Val Lys Ala Pro Cys Ala Pro Leu

515 520 525

Glu Ala Glu Asn Cys Arg Leu Met Ile Pro His Ala Val Leu Cys Ser

530 535 540

Glu Met Gly Ala His Phe Ser Pro Cys Gly Arg Phe Leu Ala Ala Cys

545 550 555 560

Val Ala Cys Ile Leu Pro Asn Met Glu Ala Asp Pro Gly Phe Gln Gly

565 570 575

Gln Leu His His Asp Ala Met Gly Ala Ala Thr Ser Pro Thr Arg His

580 585 590

Pro Ile Ala Ala His Arg Val Met Tyr Glu Leu Arg Ile Tyr Ser Leu

595 600 605

Glu Glu Ala Thr Phe Gly Leu Val Leu Val Ser Arg Ala Ile Arg Ala

610 615 620

Ala His Cys Leu Thr Ser Ile Gln Phe Ser Pro Thr Ser Glu His Leu

625 630 635 640

Leu Leu Ala Tyr Gly Arg Arg His Gly Ser Leu Leu Lys Ser Ile Val

645 650 655

Ile Asp Gly Asp Thr Thr Val Pro Val Tyr Thr Ile Leu Glu Val Tyr

660 665 670

Arg Val Ser Asp Met Glu Leu Val Arg Val Leu Pro Ser Ala Glu Asp

675 680 685

Glu Val Asn Val Ala Cys Phe His Pro Leu Val Gly Gly Gly Leu Val

690 695 700

Tyr Gly Thr Lys Glu Gly Lys Leu Arg Ile Leu Gln Phe Asp Lys Ser

705 710 715 720

Asn Gly Leu Asp Arg Thr Val Ser Cys Phe Pro Asp Glu Asp Met Leu

725 730 735

Glu Val Pro Thr Tyr Ala Leu Glu Gly

740 745

<210> 9

<211> 699

<212> PRT

<213> 人工序列(Artificial Sequence)

<400> 9

Met Arg Ser Ser Val Leu Pro Glu Asn Ser Gly Ser Ala Pro Ser Thr

1 5 10 15

Ser Arg Asn Ser Pro Pro Ser Ala Thr Ala Pro Arg Asn Ser Asn Cys

20 25 30

Lys His Ser Asn Val Phe Gln Leu Leu Thr Arg Arg Glu Val Ser Ser

35 40 45

Arg Thr Lys Arg Ala Ser Arg Lys Phe Trp Gly Glu Asn Thr Lys Cys

50 55 60

Thr Leu Asp Ser Cys Gly Leu Lys Ser Glu Val Ala Ser Asp Ala Arg

65 70 75 80

Arg Gly Leu Ile Ser Trp Val Glu Ala Glu Ser Leu Gln His Leu Ser

85 90 95

Ala Lys Tyr Cys Ser Leu Leu Pro Pro Pro Arg Ser Thr Ile Ala Ala

100 105 110

Ala Phe Ser Pro Asp Gly Arg Thr Leu Ala Ser Thr His Gly Asp His

115 120 125

Thr Val Lys Ile Ile Asp Cys Gln Thr Gly Lys Cys Leu Lys Val Leu

130 135 140

Ser Gly His Arg Arg Thr Pro Trp Val Val Arg Phe His Pro Leu Tyr

145 150 155 160

Pro Glu Ile Leu Ala Ser Gly Ser Leu Asp His Glu Val Arg Leu Trp

165 170 175

Asp Ala Lys Thr Ala Glu Cys Ile Gly Ser Arg Asp Phe Tyr Arg Pro

180 185 190

Ile Ala Ser Ile Ala Phe His Ala Gln Gly Glu Val Leu Ala Val Ala

195 200 205

Ser Gly His Lys Leu Tyr Ile Trp His Tyr Asn Arg Arg Gly Glu Ala

210 215 220

Ser Thr Pro Ala Ile Val Leu Lys Thr Arg Arg Ser Leu Arg Ala Val

225 230 235 240

His Phe His Pro His Ala Thr Pro Phe Leu Leu Thr Ala Glu Val Asn

245 250 255

Asp Leu Asp Ser Ser Asp Ser Ser Met Thr Arg Ala Thr Ser Pro Ala

260 265 270

Asn Glu Leu Pro Ile Met Ser Leu Pro Phe Leu Ile Trp Pro Ser Ile

275 280 285

Ala Arg Gly Asp Pro Arg Met Thr Val Gln Gln Thr Asp Ile Asp Met

290 295 300

Val Thr Asp Asn Val Gln His Arg Thr Asp Thr Ser Ser Ser Val Arg

305 310 315 320

Leu Leu Thr Tyr Ser Thr Pro Ser Gly Gln Tyr Glu Leu Leu Leu Ser

325 330 335

Pro Val Glu Gln Ser Ala Ser Pro Ala Gln Glu Ala His Thr Gly Ser

340 345 350

Ser Val Gly Glu Asn Glu Asn Ile Gly Trp Leu Ile Gly Gln Ser Gln

355 360 365

Val Ala Gln Gln Ala Thr His Pro Asp Leu Ser Gly Thr Thr Thr Asn

370 375 380

Pro Ser Thr Tyr Asp Glu Leu Glu Asn Pro Ser Ala Val Pro Leu Val

385 390 395 400

Ile Ser Ser Asn Thr His Pro Arg Ser Gly Arg Ser Gly Ser Arg Gln

405 410 415

Arg Ser Ser Arg Ser Arg Ala Ile Pro Val Ala Gly Ala Gly Ala Gly

420 425 430

Ala Ala Ser Leu Asn Val Met His Asp Glu Ser Asp Ser Gln Thr Ser

435 440 445

Ile Gly Arg Ile Gln Ser Glu Ile Ala Thr Ser Leu Ala Ala Ala Ala

450 455 460

Ala Ala Glu Leu Pro Cys Thr Val Lys Leu Arg Ile Trp Pro His Asp

465 470 475 480

Val Lys Val Pro Cys Ala Pro Leu His Ala Glu Arg Cys Arg Leu Thr

485 490 495

Ile Pro His Ala Val Leu Cys Ser Glu Met Gly Ala His Phe Ser Pro

500 505 510

Cys Gly Arg Phe Leu Ala Ala Cys Val Ala Cys Ile Leu Pro Asn Val

515 520 525

Asp Ala Asp Pro Gly Phe His Gly His Leu His His Asp Thr Met Ala

530 535 540

Ala Gly Thr Ser Pro Thr Arg His Pro Val Ala Ala His Gln Val Met

545 550 555 560

Tyr Glu Leu Arg Ile Tyr Ser Leu Glu Glu Ala Thr Phe Gly Ser Val

565 570 575

Leu Ala Ser Arg Ala Ile Arg Ala Ala His Cys Leu Thr Ser Ile Gln

580 585 590

Phe Ser Pro Ala Ser Glu His Leu Leu Leu Ala Tyr Gly Arg Arg His

595 600 605

Ser Ser Leu Leu Lys Ser Val Val Ile Asp Gly Asp Thr Thr Ile Pro

610 615 620

Ile Tyr Thr Ile Leu Glu Val Tyr Arg Val Ser Asp Met Glu Leu Val

625 630 635 640

Arg Val Leu Pro Ser Ala Glu Asp Glu Val Asn Val Ala Cys Phe His

645 650 655

Pro Ser Val Gly Gly Gly Leu Val Tyr Gly Thr Lys Glu Gly Lys Leu

660 665 670

Arg Ile Leu Gln Tyr Asp Asn Ser Asn Gly Leu Gly Arg Thr Ile Ser

675 680 685

Cys Ser Pro Val Glu Asn Ile Val Glu Val Leu

690 695

<210> 10

<211> 325

<212> PRT

<213> 人工序列(Artificial Sequence)

<400> 10

Met Glu Thr Asp Val Gln Pro Glu Glu Arg Asn Asn Gln Phe Phe Pro

1 5 10 15

Phe Ser Asp Pro Ala Tyr Trp Glu Leu Pro Phe Leu Gln Gly Trp Leu

20 25 30

Ile Gly Gln Ser Gln Val Ala Gln Gln Gly Thr His Pro Asp Leu Ser

35 40 45

Asp Thr Thr Thr Asn Pro Ser Thr Tyr Gly Glu Leu Glu Asn Pro Ser

50 55 60

Ala Val Pro Leu Ala Ile Ser Ser Asn Asn Arg Pro Arg Ser Gly Arg

65 70 75 80

Ser Gly Ser Arg Gln Arg Ser Ser Arg Ser Arg Ala Ile Pro Val Ala

85 90 95

Gly Gly Gly Ala Ser Ser Leu Asn Phe Met His Asp Glu Ser Asp Ser

100 105 110

Gln Thr Ser Ile Ala Arg Ile Gln Ser Glu Ile Ala Thr Ser Leu Ala

115 120 125

Ala Ala Ala Ala Ala Glu Leu Pro Cys Thr Val Lys Leu Arg Ile Trp

130 135 140

Pro His Asp Val Lys Val Pro Cys Ala Pro Leu His Ala Glu Arg Cys

145 150 155 160

Arg Leu Thr Ile Pro His Ala Val Leu Cys Ser Glu Met Gly Ala His

165 170 175

Phe Ser Pro Cys Gly Arg Phe Leu Ala Thr Cys Val Ala Cys Ile Leu

180 185 190

Pro Asn Val Asp Ala Asp Pro Ser Phe His Gly His Leu His His Asp

195 200 205

Thr Met Ala Ala Gly Thr Ser Pro Thr Arg His Pro Val Ala Ala His

210 215 220

Gln Val Met Tyr Glu Leu Arg Ile Tyr Ser Leu Glu Glu Ala Ala Phe

225 230 235 240

Gly Ser Val Leu Ala Ser Arg Ala Ile Arg Ala Ala His Cys Leu Thr

245 250 255

Ser Ile Gln Val Tyr Arg Val Ser Asp Met Glu Leu Val Arg Val Leu

260 265 270

Pro Ser Ala Glu Asp Glu Val Asn Val Ala Cys Phe His Pro Ser Val

275 280 285

Gly Gly Gly Leu Val Tyr Gly Thr Lys Glu Gly Lys Leu Arg Ile Leu

290 295 300

Gln Tyr Asp Asn Ser Asn Trp Gln Lys Glu Ile Ser Ser Lys Phe Pro

305 310 315 320

Ile Thr Val Phe Phe

325

Claims

1.一种负调控因子AtRTP5基因在提高植物疫霉菌抗性上的应用，其特征在于，所述AtRTP5基因负调控植物对疫霉菌的抗性；

所述AtRTP5基因的核苷酸序列如SEQ ID NO：1所示。

2.一种由负调控因子AtRTP5基因编码的蛋白在提高植物疫霉菌抗性上的应用，其特征在于，通过基因工程减少所述AtRTP5基因编码的蛋白的表达量，增强植物对疫霉菌的抗性；

所述蛋白的氨基酸序列如SEQ ID NO：2所示。

3.如权利要求1所述的应用，其特征在于，所述植物为拟南芥、马铃薯或烟草。