CN108424438A - 一种小麦白粉病抗性相关蛋白TaWRKY49及其编码基因与应用 - Google Patents
一种小麦白粉病抗性相关蛋白TaWRKY49及其编码基因与应用 Download PDFInfo
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- CN108424438A CN108424438A CN201810472232.5A CN201810472232A CN108424438A CN 108424438 A CN108424438 A CN 108424438A CN 201810472232 A CN201810472232 A CN 201810472232A CN 108424438 A CN108424438 A CN 108424438A
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- tawrky49
- powdery mildew
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Abstract
本发明提供一种小麦白粉病抗性相关蛋白TaWRKY49及其编码基因与应用。该蛋白为如下(a)或(b)的蛋白质:(a)SEQ ID No.2所示的氨基酸序列组成的蛋白质;(b)SEQ ID No.2所示的氨基酸序列经过一个或几个氨基酸残基的取代、缺失或添加且与抗病相关或具有转录激活活性的由(a)衍生的蛋白质。本发明发现小麦蛋白TaWRKY49,将其编码基因瞬时过量表达,发现其可抗小麦白粉菌生理小种E09引起的小麦白粉病,进一步研究发现该蛋白还具有转录激活活性,其可以作为转录激活因子。本发明提供的该蛋白可以为培育具有抗小麦白粉病的转基因小麦研究奠定基础。
Description
技术领域
本发明涉及生物技术领域,尤其涉及一种小麦白粉病抗性相关蛋白TaWRKY49及其编码基因与应用。
背景技术
小麦白粉病是由专性寄生真菌禾布氏白粉菌Bgt(Blumeria graminis fsp.tritici)引起的一种真菌性病害,属世界性分布的病害,严重影响了世界小麦的产量。近年来,由于作物种植密度的增高,氮肥施用量的增大,以及作物种植的单一,使小麦白粉病造成的危害日益严重。该病可侵害小麦植株地上部各器官,但以叶片和叶鞘为主,发病重时颖壳和芒也可受害,一般可减产10%~40%,重病田减产达50%以上。由于小麦白粉病菌具有群体大、适应范围广、生理小种众多,且变异速度快,使得许多有效抗病基因丧失抗性。目前育种工作者一直以选育和推广抗病品种作为病害的主要防治手段,多年来颇见成效,但抗性丧失一直是未能解决的问题,抗源多样化是实现抗病性持久的有效途径。为此通过生物学的方法克隆新的抗病基因,利用转基因的方法提高小麦对白粉病的抗性是今后防治白粉病的有效策略之一。但是,目前小麦白粉病抗性基因资源相对匮乏,亟需发掘小麦抗病基因资源。
发明内容
本发明的目的在于提供一种小麦白粉病抗性相关蛋白TaWRKY49及其编码基因与应用。
本发明一方面提供一种小麦白粉病抗性相关蛋白,所述蛋白为如下(a)或(b)的蛋白质:
(a)SEQ ID No.2所示的氨基酸序列组成的蛋白质;
(b)SEQ ID No.2所示的氨基酸序列经过一个或几个氨基酸残基的取代、缺失或添加且与抗病相关或具有转录激活活性的由(a)衍生的蛋白质。
上述(a)或(b)中的蛋白可人工合成,也可先合成其编码基因,再进行生物表达得到。上述(b)中的蛋白的编码基因可通过将序列表中SEQ ID No.1所示的DNA序列缺失一个或几个氨基酸残基的密码子,和/或进行一个或几个碱基对的错义突变,和/或在其5′端和/或3′端连上表2所示的标签的编码序列得到。
上述蛋白的氨基酸序列中一个或几个氨基酸残基的取代、替换和/或添加,有的是由于自然发生的变异引起的,有的是由人工诱变处理引起的。
本发明还提供如下1)-3)中任一种的DNA分子:
1)SEQ ID No.1所示的DNA分子;
2)在严格条件下与1)限定的DNA序列杂交且编码与抗病相关或具有转录激活活性蛋白的DNA分子;
3)与1)限定的DNA序列至少具有70%、至少具有75%、至少具有80%、至少具有85%、至少具有90%、至少具有95%、至少具有96%、至少具有97%、至少具有98%或至少具有99%同源性且编码与抗病相关或具有转录激活活性蛋白的DNA分子。
上述严格条件为:在6×SSC,0.5%SDS的溶液中,在65℃下杂交,然后用2×SSC,0.1%SDS和1×SSC,0.1%SDS各洗膜一次。
本发明还提供上述基因重组载体、表达盒、转基因细胞系或重组菌。其中,重组载体可以为将SEQ ID No.1插入pUbi-Adaptor-NOS载体中得到的载体;也可以为将SEQ IDNo.1插入35S-BD载体的BamHⅠ与XhoⅠ酶切位点间得到的载体。
本发明还提供上述蛋白在培育具有小麦白粉病抗病性的转基因植物中的应用。
进一步将上述编码基因导入植物小麦中,得到小麦白粉病抗病转基因小麦。
本发明还提供上述蛋白作为转录激活因子的应用。上述蛋白作为转录激活因子体现在激活基因表达。
本发明发现了一种小麦蛋白TaWRKY49,将其编码基因瞬时过量表达,发现其可抗小麦白粉菌生理小种E09引起的小麦白粉病,进一步研究发现该蛋白还具有转录激活活性,其可以作为转录激活因子。本发明提供的该蛋白可以为培育具有抗小麦白粉病的转基因小麦研究奠定基础。
附图说明
图1为实施例2 TaWRKY49瞬时过量表达后对小麦白粉菌吸器形成指数的影响结果。
图2为实施例2 TaWRKY49转录激活报告基因表达结果图。
图3为实施例3 TaWRKY49定位于小麦细胞核内的结果图。
图4为实施例3 TaWRKY49受小麦白粉菌诱导的表达图。
具体实施方式
下面结合具体实施例及附图对本发明做进一步详细说明。
下述实施例中所使用的实验方法如无特殊说明,均为常规方法。
下述实施例中所用的材料,均可从青岛大学得到。
下述实施例中的部分材料如下:
小麦品种山农20记载在研究论文小麦新品种“山农20”抗病基因的分子检测。作物学报,2014,40:611-621。公众可从青岛大学获得;
小麦品种京411记载在研究论文利用京411为骨干亲本培育高产小麦新品种。作物学报,2009,4:1-5。公众可从青岛大学获得;
小麦品种科农199记载在研究论文高产广适小麦新品种科农199。麦类作物学报,2007,27(2):368-370。公众可从青岛大学获得;
小麦品种豫麦66记载在研究论文普通小麦品种“豫麦66”抗白粉病基因的鉴定与分子标记。作物学报,2008,34(4):545-550。公众可从青岛大学获得;
小麦白粉菌生理小种E09记载在研究论文利用人工合成小麦培育的大穗抗白粉小麦基因资源GB4。植物遗传资源学报,2007,8:378。公众可从青岛大学获得;
Columbia生态型拟南芥(col-0)记载在研究论文Molecular characterizationof the submergence response of the Arabidopsis thaliana ecotype Columbia.NewPhytologist.2011,190:457-471.公众可从青岛大学获得;
载体pUbi-GUS记载在研究论文A transient assay system for the functionalassessment of defense-related genes in wheat.Molecμlar Plant-MicrobeInteractions,1999,12:647-654.公众可从青岛大学获得;
载体pUbi-Adaptor-NOS记载在研究论文Recognition specificity and RAR1/SGT1dependence in barley MLA disease resistance genes to the powdery mildewfungus.The Plant Cell,2003,15:732-744.公众可从青岛大学获得;
载体35S-BD记载在研究论文Soybean GmPHD-type transcription regulatorsimprove stress tolerance in transgenic Arabidopsis plants.PLoS One,2009,4(9):e7209.公众可从青岛大学获得;
载体35S-BD-VP16记载在研究论文Soybean GmPHD-type transcriptionregulators improve stress tolerance in transgenic Arabidopsis plants.PLoSOne,2009,4(9):e7209.公众可从青岛大学获得;
载体5×GAL4-LUC记载研究论文在Soybean GmPHD-type transcriptionregulators improve stress tolerance in transgenic Arabidopsis plants.PLoSOne,2009,4(9):e7209.公众可从青岛大学获得;
载体Pptrl记载在研究论文Soybean GmPHD-type transcription regulatorsimprove stress tolerance in transgenic Arabidopsis plants.PLoS One,2009,4(9):e7209.公众可从青岛大学获得;
载体pUbi-Gateway-mYFP载体记载在研究论文The CC-NB-LRR-type Rdg2aresistance gene confers immunity to the seed-borne balrey leaf stripepathogen in the absence of hypersensitive cell death.PLoS One,2010,5:e12599.公众可从青岛大学获得。
实施例1、TaWRKY49基因的获得
1、RNA的获得
小麦品种山农20的7天幼苗接菌小麦白粉菌生理小种E09,48小时取材,提RNA。
2、反转录获得cDNA
反转录体系:
RNA 2.5μL,Oligo-dT引物1μL,DEPC水6.5μL,离心管混合上述溶液,65℃保温5分钟,冰上再放置5分钟。
再在上述体系中加入5×反转录缓冲溶液5μL,dNTP mix 1.25μL,抑制剂0.625μL,M-MLV反转录酶1μL,DEPC水7.125μL,42℃温浴1小时,95℃5分钟,得到cDNA。
3、克隆TaWRKY49
将上述cDNA加入25μL双蒸水,取1μL进行后续PCR反应为模板,用下述引物进行PCR扩增,
正向引物:GTGAGCACGCCCTACTCTC(SEQ ID No.3)
反向引物:CCCAATATACATGCATACAC(SEQ ID No.4)
PCR体系如下:KOD缓冲液5μL,MgSO4 2μL,dNTP 5μL,模板100ng,正向引物2μL,反向引物2μL,KOD plus 1μL,水32μL。
PCR程序如下:94℃5min,然后94℃30s,60℃30s,68℃90s,共29个循环,最后,68℃10min,16℃10min。
将得到的PCR产物送去测序,结果为该PCR产物具有序列表中SEQ ID No.1所示的核苷酸,该序列所示的基因命名为TaWRKY49,编码区为序列表中SEQ ID No.1自5’末端第1-1143位核苷酸;该基因编码的蛋白命名为TaWRKY49,该蛋白的氨基酸序列为序列表中的SEQID No.2。
实施例2、TaWRKY49基因在抗白粉病中的应用
一、基因枪瞬间超表达技术鉴定TaWRKY49对小麦白粉病抗性的调控作用
1、载体构建
1)以实施例1得到的PCR产物(或者人工合成序列1)为模板,用下述引物进行PCR扩增,得到约1.2Kb的PCR产物。
正向引物:GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGGCATCTTGTGGCGGC(SEQ IDNo.5)
反向引物:GGGGACCACTTTGTACAAGAAAGCTGGGTCTCATAAAAAAATATTACTAC(SEQ IDNo.6)
2)用步骤1)得到的PCR产物与pDNOR201载体(购自invitrogen公司)进行BP反应,得到ENTRY载体。
上述BP反应体系:PCR产物75ng,pDNOR201 75ng,BP酶0.5μL,25℃过夜。
3)将上述ENTRY载体与pUbi-Adaptor-NOS载体进行LR反应,生成pUbi-TaMYB1载体。
LR反应体系:ENTRY载体75ng,pUbi-Adaptor-NOS载体75ng,LR酶0.5μL,25℃过夜。
载体pUbi-TaWRKY49通过测序鉴定,该载体为将序列表中的SEQ ID No.1插入pUbi-Adaptor-NOS载体中得到的载体。
2、基因枪瞬时超表达试验
1)金粉的制备
(1)称取9mg(w)金粉放在离心管中,65℃放置4小时以上;
(2)加入70%乙醇,漩涡振荡8分钟,静止15分钟;
(3)2000r/min离心2s,去掉上清液;
(4)加1mL消毒蒸馏水,旋涡振荡2分钟,静止1分钟,2000rpm/min离心2s,弃掉上清液;
(5)重复步骤(4)三次;
(6)在沉淀的金粉中加入50%甘油,漩涡振荡。
2)DNA子弹的制作
(1)振荡50%甘油中的金粉5分钟,使之成为悬浮液;
(2)取50μl的悬浮液于离心管中;
(3)加入5μl的质粒DNA(2μg),边振荡便加入50μl CaCl2水溶液(2.5M),振荡中再在小管加入20μl亚精胺(0.1M),振荡3分钟,之后静置1min,2000rpm/min离心2s,去掉上清液;
(4)加入140μl 70%的乙醇,漩涡振荡,使沉淀均匀散开,则离心(2000rpm/min2s),弃掉上清液;
(5)加入140μl 100%的乙醇,漩涡振荡,使沉淀均匀散开,则离心(2000rpm/min2s),弃掉上清液;
(6)加入12μl 100%的乙醇,漩涡振荡,使沉淀均匀散开。
3)基因枪轰击转化方法
基因枪介导的方法:分别将小麦山农20、京411、科农199和豫麦66的种子经保湿吹芽种植后,待长至一周左右,剪下第一片叶,叶面朝上,放置于含有培养基(1%的琼脂,100mg/L苯丙咪唑)的培养皿上,恢复4小时,得到小麦山农20、京411、科农199和豫麦66的靶材料,备用基因枪轰击;
将目的质粒pUbi-TaWRKY49和质粒pUbi-GUS按1:1体积充分混合,包裹在直径为1μm金粉微粒上,采用PDS-1000/He(美国Bio-Rad)分别按照上述方法轰击小麦山农20、京411、科农199和豫麦66的靶材料,基因枪轰击参数为:阻挡网与轰击材料之间的距离为5.5cm,可裂膜压力为1100Pa)。
基因枪轰击完毕后,将轰击后的小麦山农20、京411、科农199和豫麦66的靶材料叶片分别放入培养箱中恢复培养4小时(培养条件22℃、16h光照/18℃8h黑暗),得到转入pUbi-TaWRKY49和pUbi-GUS的小麦山农20、京411、科农199和豫麦66叶片;静置15小时后接种小麦白粉菌生理小种E09的孢子;
上述接种的方法均如下:将对应小种的孢子抖落在对应的小麦叶片上,保证2个孢子/mm2。
将上述接种后的小麦山农20、京411、科农199和豫麦66叶片静置培养48小时(培养条件22℃、16h光照/18℃8h黑暗),进行GUS染色,37℃过夜,染色完毕后进行脱色,两天后利用显微镜进行细胞观察。
统计表达GUS的细胞中感病细胞的比例来计算吸器指数,根据吸器指数来判断基因TaWRKY49的功能。吸器指数越低,说明抗性越高。
抗病细胞的判断标准:细胞内表达GUS,且细胞内不含有吸器,而细胞表面附着有分生孢子的细胞为抗病细胞。
感病细胞的判断标准:细胞内表达GUS,且细胞内含有吸器的细胞为感病细胞。
吸器指数的计算公式:吸器指数=感病细胞的个数/(抗病细胞的个数+感病细胞的个数)×100%。
以转入pUbi-Adaptor-NOS和pUbi-GUS的小麦山农20叶片细胞(OE-EV)、转入pUbi-Adaptor-NOS和pUbi-GUS的小麦京411叶片细胞(OE-EV)、转入pUbi-Adaptor-NOS和pUbi-GUS的小麦科农199(KN199)叶片细胞(OE-EV)、转入pUbi-Adaptor-NOS和pUbi-GUS的小麦豫麦66叶片细胞(OE-EV)作为空对照。实验设3次重复,结果取平均值。
结果如图1所示:
转入pUbi-Adaptor-NOS和pUbi-GUS的小麦山农20叶片细胞的吸器指数为48.39%,转入pUbi-TaWRKY49和pUbi-GUS的小麦山农20叶片细胞的吸器指数为26.63%;
转入pUbi-Adaptor-NOS和pUbi-GUS的小麦京411叶片细胞的吸器指数为53.75%,转入pUbi-TaWRKY49和pUbi-GUS的小麦京411叶片细胞的吸器指数为31.36%;
转入pUbi-Adaptor-NOS和pUbi-GUS的小麦科农199叶片细胞的吸器指数为50.41%,转入pUbi-TaWRKY49和pUbi-GUS的小麦科农199叶片细胞的吸器指数为28.09%;
转入pUbi-Adaptor-NOS和pUbi-GUS的小麦豫麦66叶片细胞的吸器指数为48.24%,转入pUbi-TaWRKY49和pUbi-GUS的小麦豫麦66叶片细胞的吸器指数为28.45%。
结果显示,在小麦叶片中过表达TaWRKY49后,吸器指数均明显下降,说明TaWRKY49正向调控小麦的白粉病抗性。
二、TaWRKY49作为转录激活因子的转录激活活性分析
1、35S-BD-TaWRKY49载体构建
1)以实施例1得到的PCR产物(或者人工合成序列1)为模板,用下述引物进行PCR扩增,得到约1.2Kb的PCR产物。
正向引物:AAATTTGGATCCATGGCATCTTGTGGCGGCG(SEQ ID No.7)
反向引物:GGGCCTCTCGAGTCATAAAAAAATATTACTAC(SEQ ID No.8)
将制得的PCR产物经过BamHⅠ与XhoⅠ双酶切后与经过同样酶切的35S-BD载体连接,得到载体35S-BD-TaWRKY49。
35S-BD-TaWRKY49载体通过测序鉴定,该载体为将序列表中的序列1插入35S-BD载体的BamHⅠ与XhoⅠ酶切位点间得到的载体。
2、原生质体制备
三周Columbia型拟南芥幼嫩叶片若干,用刀片切成1毫米细条,黑暗下25度酶解4小时,经过滤、离心等一系列操作后用细胞计数板在显微镜下计数,一般每100μL中有2×105个原生质体。
3、质粒转化及荧光值测定
分装100μL原生质体到2mL的EP管中,分别加入下列质粒:Effectors:35S-BD-TaWRKY49;CK-:35S-BD;CK+:35S-BD-VP16;Reporter:5×GAL4-LUC;Internal control:Pptrl。25℃暗培养16小时后检测报告基因LUC表达情况。
检测萤火虫萤光素酶和海肾萤光素酶的荧光值,得到两者比率(萤火虫萤光素酶荧光值/海肾萤光素酶荧光值),即为报告基因相对活性。(详细检测方法见Promega试剂盒Dual-Reporter Assay System货号E1910)
结果如图2所示,转入35S-BD-TaWRKY49和5×GAL4-LUC的原生质体中LUC的相对活性为5.32;
转入35S-BD和5×GAL4-LUC的原生质体中LUC的相对活性为1;
转入35S-BD-VP16和5×GAL4-LUC的原生质体中LUC的相对活性为12.98;
上述结果表明,TaWRKY49能够激活报告基因的表达,荧光值是对照质粒35S-BD的5.32倍,由此可以确定TaWRKY49具有转录激活活性,是转录激活因子。
实施例3、TaWRKY49的亚细胞定位和受白粉菌诱导的表达模式分析
一、TaWRKY49的亚细胞定位
1、载体构建
1)以实施例1得到的PCR产物(或者人工合成序列1)为模板,用下述引物进行PCR扩增,得到约1.2Kb的PCR产物。
正向引物:GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGGCATCTTGTGGCGGCG(SEQ IDNo.9)
反向引物:GGGGACCACTTTGTACAAGAAAGCTGGGTCTAAAAAAATATTACTACTAC(SEQ IDNo.10)
2)用步骤1)制得的PCR产物与pDNOR201载体(购自invitrogen公司)进行BP反应,得到ENTRY-TaWRKY49载体。
上述BP反应体系:PCR产物75ng,pDNOR201 75ng,BP酶0.5μL,25℃过夜。
3)将上述ENTRY-TaWRKY49载体与pUbi-Gateway-mYFP载体LR反应,生成pUbi-TaWRKY49-mYFP载体。
LR反应体系:
中间载体75ng,pUbi-Gateway-mYFP载75n,LR酶0.5μL,25℃过夜。
pUbi-TaWRKY49-mYFP载体通过测序鉴定,该载体为将序列表中的序列1插入pUbi-Gateway-mYFP载体中得到的载体。
2、基因枪介导的单细胞瞬时转化技术鉴定TaWRKY49的亚细胞定位情况
小麦山农20种子经保湿吹芽种植后,待长至一周左右,剪下第一片叶,叶面朝上,放置于含有培养基(1%的琼脂,100mg/L苯丙咪唑)的培养皿上,恢复4小时,进行基因枪轰击;将目的质粒pUbi-TaWRKY49-mYFP包裹在直径为1μm金粉微粒上,采用PDS-1000/He(美国Bio-Rad)轰击靶材料,基因枪轰击参数为:阻挡网与轰击材料之间的距离为5.5cm,可裂膜压力为1100Pa。
基因枪轰击后叶片放置于植物培养室正常生长36小时后进行DAPI染色以标记细胞核,共聚焦显微镜不同荧光通道观察TaWRKY49的亚细胞定位情况。
结果如图3所示,TaWRKY49-mYFP的荧光与DAPI的荧光完全重叠,故TaWRKY49定位于细胞核内。
二、TaWRKY49受白粉菌诱导的表达模式分析
小麦山农20种子经保湿吹芽种植后,待长至一周左右,剪下第一片叶,叶面朝上,放置于含有培养基(1%的琼脂,100mg/L苯丙咪唑)的培养皿上,恢复24小时,接种小麦白粉病菌生理小种E09,分不同时间点取材,提RNA,反转录得到cDNA,将cDNA稀释10倍,取2μL进行Real time PCR。Real time PCR体系及程序参照promega公司试剂盒GoTaq-qPCR MasterMix(目录号A6001)操作说明。以未接菌处理为对照。
Real time PCR的引物如下:
正向引物:CGTGGAGACCGTGCAGGAG(SEQ ID No.11)
反向引物:CCGGACCAGCTGGCAGAG(SEQ ID No.12)
Real time PCR体系如下:
qPCR Master Mix 5μL,template 2μL,正向引物0.2μL,反向引物0.2μL,CXR 1μL,水1.6μL。
Real time PCR程序如下:第一阶段96℃2min,第二阶段95℃15s,61℃1min,共45个循环,第三阶段溶解曲线制作。
结果如图4所示,白粉菌生理小种E09接菌处理能够强烈诱导TaWRKY49的表达,接菌3小时后表达量,提高约1.9倍,而接菌24小时TaWRKY49的表达出现高峰,达到未接菌时的7.8倍,而后表达量降低,接菌36小时TaWRKY49的表达量为未接菌时的4.9倍。
序列表
<110> 青岛大学
<120> 一种小麦白粉病抗性相关蛋白TaWRKY49及其编码基因与应用
<160> 12
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1143
<212> DNA
<213> 小麦(Triticum aestivum)
<400> 1
atggcatctt gtggcggcgc gggccatgac ggcgggcgct cacggccgac ggtggtctac 60
gacgacctgg tggaggtgcg cgagcacgcg gcgacgctgc agaccatcct gcaggggtcg 120
ccgagcgtgt cggccgtgga cgcgagagag ctcgtgaagg ggatgatggc caagctgtcc 180
agcgctatgt cggtgctggg cgccaccagc ggcagcggtg tggagtcgtc tttggggaca 240
ggccgaggac caggtccagg tgggaggagg aagaaacccg gcacggcgtt gtccgggccg 300
caccgccgga gcagctccag gagaaggtcg aagagccctt tcatcaacat ggtcactgct 360
aggacgctca acgatggcaa gacatggagg aagtacggcc aaaaatatat tcatgcctct 420
actagcccga ggagctacta caggtgctct cataagccag accaaggctg ccaggccacg 480
aggcaggtcc aggaatccga ctccaacccg tcggagtaca tcatcagcta ctacggccag 540
cacacctgca aagacccctc cacgttccgg tcactcctca tccaaggcgc cgccgacgct 600
gccccgccag cagactgctc aaacctcatc agcttccagt cgatcaatgg cgcggctgcg 660
agcacgagca cgagcgcttt tgctcatcat gtcgtgaaag aagcggttga tcttcatccg 720
gtaccttact cccgcttctt caactacagc tcctggccgc cggtgcagga gggtatgtcc 780
agcggctcgc tgtcgccggc tggccacggg aagttcatgc agtacgccgg tgggcagctc 840
gtcaacgtta ttggccgaag gacgttgccg ttgaccgtgg gatcagcgcc ggcggagtac 900
tggccggtcg tgggagccgc cagcgtcgac acggacgctg gcgcaggcat ggacagcttc 960
ccttcctcgc cgagcagcct cgggctcatg tcgggatcgt taggggggtc atttggcaat 1020
aacatttacg acgacgacct gttcgacttg attcctgacc aaggtcgtgt acccatgcat 1080
atgcatgcat gcaacacacg gccgactttg catgcatggg gtagtagtaa tattttttta 1140
tga 1143
<210> 2
<211> 380
<212> PRT
<213> 小麦(Triticum aestivum)
<400> 2
Met Ala Ser Cys Gly Gly Ala Gly His Asp Gly Gly Arg Ser Arg Pro
1 5 10 15
Thr Val Val Tyr Asp Asp Leu Val Glu Val Arg Glu His Ala Ala Thr
20 25 30
Leu Gln Thr Ile Leu Gln Gly Ser Pro Ser Val Ser Ala Val Asp Ala
35 40 45
Arg Glu Leu Val Lys Gly Met Met Ala Lys Leu Ser Ser Ala Met Ser
50 55 60
Val Leu Gly Ala Thr Ser Gly Ser Gly Val Glu Ser Ser Leu Gly Thr
65 70 75 80
Gly Arg Gly Pro Gly Pro Gly Gly Arg Arg Lys Lys Pro Gly Thr Ala
85 90 95
Leu Ser Gly Pro His Arg Arg Ser Ser Ser Arg Arg Arg Ser Lys Ser
100 105 110
Pro Phe Ile Asn Met Val Thr Ala Arg Thr Leu Asn Asp Gly Lys Thr
115 120 125
Trp Arg Lys Tyr Gly Gln Lys Tyr Ile His Ala Ser Thr Ser Pro Arg
130 135 140
Ser Tyr Tyr Arg Cys Ser His Lys Pro Asp Gln Gly Cys Gln Ala Thr
145 150 155 160
Arg Gln Val Gln Glu Ser Asp Ser Asn Pro Ser Glu Tyr Ile Ile Ser
165 170 175
Tyr Tyr Gly Gln His Thr Cys Lys Asp Pro Ser Thr Phe Arg Ser Leu
180 185 190
Leu Ile Gln Gly Ala Ala Asp Ala Ala Pro Pro Ala Asp Cys Ser Asn
195 200 205
Leu Ile Ser Phe Gln Ser Ile Asn Gly Ala Ala Ala Ser Thr Ser Thr
210 215 220
Ser Ala Phe Ala His His Val Val Lys Glu Ala Val Asp Leu His Pro
225 230 235 240
Val Pro Tyr Ser Arg Phe Phe Asn Tyr Ser Ser Trp Pro Pro Val Gln
245 250 255
Glu Gly Met Ser Ser Gly Ser Leu Ser Pro Ala Gly His Gly Lys Phe
260 265 270
Met Gln Tyr Ala Gly Gly Gln Leu Val Asn Val Ile Gly Arg Arg Thr
275 280 285
Leu Pro Leu Thr Val Gly Ser Ala Pro Ala Glu Tyr Trp Pro Val Val
290 295 300
Gly Ala Ala Ser Val Asp Thr Asp Ala Gly Ala Gly Met Asp Ser Phe
305 310 315 320
Pro Ser Ser Pro Ser Ser Leu Gly Leu Met Ser Gly Ser Leu Gly Gly
325 330 335
Ser Phe Gly Asn Asn Ile Tyr Asp Asp Asp Leu Phe Asp Leu Ile Pro
340 345 350
Asp Gln Gly Arg Val Pro Met His Met His Ala Cys Asn Thr Arg Pro
355 360 365
Thr Leu His Ala Trp Gly Ser Ser Asn Ile Phe Leu
370 375 380
<210> 3
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
gtgagcacgc cctactctc 19
<210> 4
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
cccaatatac atgcatacac 20
<210> 5
<211> 49
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
ggggacaagt ttgtacaaaa aagcaggctt catggcatct tgtggcggc 49
<210> 6
<211> 50
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
ggggaccact ttgtacaaga aagctgggtc tcataaaaaa atattactac 50
<210> 7
<211> 31
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
aaatttggat ccatggcatc ttgtggcggc g 31
<210> 8
<211> 32
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
gggcctctcg agtcataaaa aaatattact ac 32
<210> 9
<211> 50
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
ggggacaagt ttgtacaaaa aagcaggctt catggcatct tgtggcggcg 50
<210> 10
<211> 50
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
ggggaccact ttgtacaaga aagctgggtc taaaaaaata ttactactac 50
<210> 11
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
cgtggagacc gtgcaggag 19
<210> 12
<211> 18
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
ccggaccagc tggcagag 18
Claims (8)
1.一种小麦白粉病抗性相关蛋白,其特征在于,所述蛋白为如下(a)或(b)的蛋白质:
(a)SEQ ID No.2所示的氨基酸序列组成的蛋白质;
(b)SEQ ID No.2所示的氨基酸序列经过一个或几个氨基酸残基的取代、缺失或添加且与抗病相关或具有转录激活活性的由(a)衍生的蛋白质。
2.权利要求1所示的蛋白的编码基因。
3.含有权利要求2所述的基因的重组载体、表达盒、转基因细胞系或重组菌。
4.权利要求1所述的蛋白在培育具有小麦白粉病抗病性的转基因植物中的应用。
5.根据权利要求4所述的应用,其特征在于,将权利要求2所述的编码基因导入植物小麦中,得到抗白粉病的转基因小麦。
6.根据权利要求5所述的应用,其特征在于,将权利要求2所述的编码基因通过重组表达载体导入,该重组表达载体插入pUbi-Adaptor-NOS载体中得到的。
7.权利要求1所述的蛋白在作为转录激活因子中的应用。
8.根据权利要求7所述的应用,其特征在于,将权利要求2所述的编码基因插入35S-BD载体的BamHⅠ与XhoⅠ酶切位点间得到重组载体。
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