CN116410282B - 来源欧洲芜菁ECD04的BraA03g008044E基因在根肿菌抗性改良中的应用 - Google Patents
来源欧洲芜菁ECD04的BraA03g008044E基因在根肿菌抗性改良中的应用 Download PDFInfo
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Abstract
本发明公开了来源欧洲芜菁ECD04的BraA03g008044E基因在根肿菌抗性改良中的应用,属于基因克隆技术领域。所述BraA03g008044E基因的核苷酸序列如SEQ ID No.1或SEQ IDNo.2所示,其编码蛋白的氨基酸序列如SEQ ID No.3所示;该目的基因位于欧洲芜菁ECD04的A03染色体上,其物理位置为3918065‑3922651。本发明公开了该基因对根肿菌的抗病功能,为通过基因工程手段或分子标记辅助选择技术选育芸薹属抗根肿病植物新品种提供科学和技术依据。
Description
技术领域
本发明涉及基因克隆技术领域,特别是涉及一种来源欧洲芜菁ECD04的BraA03g008044E基因在根肿菌抗性改良中的应用。
背景技术
根肿病是由芸薹根肿菌(P.brassicae)侵染引起的一种世界性土传病害(DixonGR.The occurrence and economic impact ofPlasmodiophora brassicae and clubrootdisease.JPlant Growth Regul,2009,28:194-202)。该病原菌专性侵染十字花科植物,包括油菜、大白菜、小白菜、甘蓝、萝卜、花椰菜、芥菜等在内的多种栽培和野生种(Howard RJ,Strelkov SE,Harding MW.Clubroot ofcruciferous crops-new perspectives on anold disease.Can JPlantPathol,2010,32(1):43-57)。其病原菌可以通过带病菌的种子、被侵染的植物材料、带有病原菌的土壤、洪水、动物的饲料和粪便等媒介进行传播(ChaiAL,Xie XW,Shi YX,Li BJ.Research status of clubroot(Plasmodiophora brassicae)oncruciferous crops in China.Can JPlantPathol,2014,36:142-153)。据报道,全球已有超过60个国家有根肿病的发生。近年来根肿病在我国快速蔓延,已成为影响中国十字花科作物生产的严重的病害之一。
根肿菌的侵染对植物的生长发育造成了严重危害,植物在感染早期根部形态与未被感染植物的根部并无明显差别,但随着感染时间的延长,易感植物的根系表现出肿胀的特征,导致根系水分和养分输送功能受阻,最终整株植物枯萎死亡(AhmedA,Munir S,HePF,Li YM,He PB,Wu YX,He YQ.Biocontrol arsenals ofbacterial endophyte:Animminent triumph against clubroot disease.MicrobiolRes,2020,241:126565)。在全球,很多国家和地区种植了十字花科作物,甚至还是一些国家的主要经济作物,但近年来根肿病对十字花科作物的危害逐渐加重,造成了巨大的经济损失(Dixon GR.The occurrenceand economic impact ofPlasmodiophora brassicae and clubroot disease.JPlantGrowth Regul,2009,28:194-202)。目前根肿病侵染的区域呈现扩大的趋势,已经严重威胁到了十字花科作物的生产。
培育抗根肿病的抗性品种被认为是最经济有效和环境友好的措施之一。发根农杆菌又称发根根瘤菌,是根瘤菌科的一种可以诱导植物伤口产生毛状根的土壤杆菌。发根农杆菌的Ti质粒携带具有主导诱根性的rol基因,可通过侵染植物伤口处快速将目的基因插入并整合到宿主植物染色体上(Sevon,N.and Oksman-Caldentey,K.M.(2002)Agrobacterium rhizogenes-mediated transformation:Root cultures as a sourceofalkaloids.PlantaMed,68,859-868)。由于发根转化系统具有可靠、重复性好、高效等特点,因此近年以发根农杆菌为介导的转化体系被广泛用于基因的功能研究(Meng,D.,Yang,Q.,Dong,B.Y.,Song,Z.H.,Niu,L.L.,Wang,L.T.,etal.(2019)Development of anefficient root transgenic system for pigeon pea and its application to otherimportant economically plants.Plant Biotechnology Journal,17,1804-1813)。
当前的抗根肿病工作主要通过将抗性基因导入对根肿病敏感的材料中从而获得优良抗病品种,因此鉴定抗根肿病的抗性基因(clubroot resistance gene,CR)尤为重要。Yang等将含多个抗病位点的抗源材料ECD04进行了从头测序和组装,并将已报道的28个抗根肿病位点整合为15个位点,并预测出NBS-LRR\RLK\RLP类型的抗病候选基因62个(Yang,Z.,Jiang,Y.,Gong,J.,Li,Q.,Dun,B.,Liu,D.,et al.(2022)R gene triplicationconfers European fodder turnip with improved clubroot resistance.PlantBiotechnol J,20,1502-1517),这为抗根肿病基因克隆和功能验证奠定良好的基础。
发明内容
本发明的目的是提供一种来源欧洲芜菁ECD04的BraA03g008044E基因在根肿菌抗性改良中的应用,以解决上述现有技术存在的问题,本发明以甘蓝型油菜丙409为转化受体,克隆并验证了一个对中国根肿菌4号优势生理小种具备有效抗性的基因BraA03g008044E,提供了一个依托该抗性基因为通过分子标记辅助选择和基因工程方法改良油菜、白菜和甘蓝等芸薹属植物的根肿病抗性的育种方法。
为实现上述目的,本发明提供了如下方案:
本发明提供BraA03g008044E基因在赋予根肿菌抗性中的应用,所述BraA03g008044E基因的核苷酸序列如SEQ ID No.1或SEQ ID No.2所示,其编码蛋白的氨基酸序列如SEQ ID No.3所示。
进一步地,所述BraA03g008044E基因位于欧洲芜菁ECD04的A03染色体上,其物理位置为3918065-3922651。
本发明还提供BraA03g008044E基因在根肿病抗病育种中的应用,所述BraA03g008044E基因通过转基因或有性杂交转育途径用于芸薹属植物根肿病抗病育种中的应用,所述芸薹属植物包括油菜、白菜、甘蓝或萝卜,所述BraA03g008044E基因的核苷酸序列如SEQ ID No.1或SEQ ID No.2所示。
本发明还提供BraA08g039497E基因及根据其所开发的标记在芸薹属植物分子标记辅助选择中的应用,所述芸薹属植物包括油菜、白菜、甘蓝或萝卜,所述BraA08g039497E基因的核苷酸序列如SEQ ID No.1或SEQ ID No.2所示。
本发明还提供一种用于扩增BraA03g008044E基因全长的引物,包括如SEQ IDNo.4所示的正向引物和SEQ ID No.5所示的反向引物。
本发明还提供一种用于构建BraA03g008044E基因遗传转化载体的引物,包括如SEQ ID No.6所示的正向引物和SEQ ID No.7所示的反向引物。
本发明还提供一种用于BraA03g008044E基因阳性检测的引物,包括SEQ ID No.8所示的正向引物和SEQ ID No.9所示的反向引物。
本发明还提供一种用于检测BraA03g008044E基因Quantitative Real-time PCR(Q-PCR)的引物,包括SEQ ID No.10所示的正向引物和SEQ ID No.11所示的反向引物。
本发明公开了以下技术效果:
本发明公开了来源于欧洲芜菁ECD04的BraA03g008044E基因对中国根肿菌4号生理小种具有抗性功能,并通过发根农杆菌介导的转化体系,以甘蓝型油菜丙409为背景对其进行根部超表达验证,从而验证BraA03g008044E基因对根肿菌的抗病功能。本发明可以提供一种BraA03g008044E基因对抗根肿菌病基础研究、抗病育种和分子标记辅助选择中的应用,通过该基因所在的定位区间去选育抗根肿菌的品种,从而缩短育种进程。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案,下面将对实施例中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图仅仅是本发明的一些实施例,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他的附图。
图1:本发明实施例中BraA03g008044E基因在pCAMBIA3301植物表达载体上克隆模式图;
图2:本发明实施例中BraA03g008044E基因全长PCR扩增后的琼脂糖凝胶图,M为DL5000DNALadder,1-3为扩增目的条带,条带大小为4067bp;
图3:BraA03g008044E阳性克隆检测琼脂糖凝胶图,M为DL 2000DNAMarker,1-13为菌落PCR阳性检测条带,条带大小为502bp;
图4:本发明实施例中BraA03g008044E基因在甘蓝型油菜受体材料丙409中超量表达后接中国根肿菌4号优势生理小种表型图,以丙409作为感病对照,409R作为抗病对照,从左至右分别为409R、0级、1级、3级、丙409;
图5:本发明实施例中BraA03g008044E转化丙409植株根系目的基因检测琼脂糖凝胶图,M为DL 2000DNAMarker,N为阴性对照,P为阳性对照,目的片段大小为502bp;
图6:本发明实施例中BraA03g008044E转化植株根系目的基因相对表达量Q-PCR统计图,R表示为抗病单株,S1表示为感病1级单株,S3表示为感病3级单株,ECD04和409R为抗病对照,409S为感病对照;
图7:BraA03g008044E以及其在拟南芥、感病白菜、感病甘蓝型油菜中同源基因的基因结构比对图;BraA03g008044E,BraA02g029240E,BraA10g046894E来源于ECD04(AA);AT5G18350,AT5G18360,AT5G18370来源于拟南芥;BraA-03g008470.3C,BraA03t10201Z,A03p07970.1_BraMIZ,A03p08230.1_BraCCB,A03p08800.1_BraPCB,A03p09280.1_BraCCA来源于不同基因型的感病白菜A3染色体(AA);A03p08770.1_BnaDAR,A03p007940.1_BnaEXP,BnaA03T006430-0TA,ZS11A03G007920,BnaA03T0098400ZY分别来源于甘蓝型油菜darmor,Express,Tapidor,ZS11,Zheyou(AACC)。
具体实施方式
现详细说明本发明的多种示例性实施方式,该详细说明不应认为是对本发明的限制,而应理解为是对本发明的某些方面、特性和实施方案的更详细的描述。
应理解本发明中所述的术语仅仅是为描述特别的实施方式,并非用于限制本发明。另外,对于本发明中的数值范围,应理解为还具体公开了该范围的上限和下限之间的每个中间值。在任何陈述值或陈述范围内的中间值,以及任何其他陈述值或在所述范围内的中间值之间的每个较小的范围也包括在本发明内。这些较小范围的上限和下限可独立地包括或排除在范围内。
除非另有说明,否则本文使用的所有技术和科学术语具有本发明所述领域的常规技术人员通常理解的相同含义。虽然本发明仅描述了优选的方法和材料,但是在本发明的实施或测试中也可以使用与本文所述相似或等同的任何方法和材料。本说明书中提到的所有文献通过引用并入,用以公开和描述与所述文献相关的方法和/或材料。在与任何并入的文献冲突时,以本说明书的内容为准。
在不背离本发明的范围或精神的情况下,可对本发明说明书的具体实施方式做多种改进和变化,这对本领域技术人员而言是显而易见的。由本发明的说明书得到的其他实施方式对技术人员而言是显而易见得的。本发明说明书和实施例仅是示例性的。
关于本文中所使用的“包含”、“包括”、“具有”、“含有”等等,均为开放性的用语,即意指包含但不限于。
实施例1
1、克隆BraA03g008044E基因,将目的基因PCR扩增出来,用于扩增BraA03g008044E基因组引物序列为:
正向引物(SEQ ID No.4):5’-ctgtaggaggacgctgagaag-3’;
反向引物(SEQ ID No.5):5’-acatcacaacacttacacaaggc-3’;
以改造过的pCAMBIA3301(将Basta抗性改为Hyg抗性)植物表达载体为骨架,构建由CaMV35S驱动目的基因的表达载体(图1),最后转入K599农杆菌感受态细胞,详细说明如下:
以供体材料欧洲芜菁ECD04基因组为模板对BraA03g008044E基因进行扩增。
PCR反应扩增引物由pCAMBIA3301载体同源臂、酶切位点、目的基因同源序列三部分组成,便于后续基因克隆操作。
正向引物序列(SEQ ID No.6):
5’-acgggggactcttgaccatggctgtaggaggacgctgagaag-3’;
反向引物序列(SEQ ID No.7):
5’-ggggaaattcgagctggtcaccacatcacaacacttacacaaggc-3’;
采取CTAB法提取欧洲芜菁ECD04的DNA,使用Phanta Max Super-Fidelity DNAPolymerase(南京诺唯赞生物科技公司)进行目的片段扩增。
50μl反应体系:2×PhantaMax Buffer 25μl、Forward Primer(10μM)2μl、ReversePrimer(10μM)2μl、dNTP Mix(10mM each)1μl、DNA模板2μl、ddH2O 18μl。
PCR反应条件:预变性95℃3min;变性95℃15s,复性58℃15s,延伸72℃4min,34个循环;72℃延伸5min;25℃5min。
扩增产物用1%的琼脂糖凝胶电泳检测,条带大小预期为4067bp(图2)。
对扩增出来的目的片段使用DNAPurification Kit(天根生化科技有限公司)试剂盒进行PCR原液回收并测量纯化回收后目的片段浓度。
酶切改造过的pCAMBIA3301(Hyg抗性)植物表达载体。
酶切体系:buffer 6μl,质粒DNA 50μl,NcoI FastDigest enzyme(s)1μl,BstEII FastDigest enzyme(s)1μl,ddH2O 2μl。
将上述反应液离心混匀,置于37℃水浴锅中反应30min。反应结束后进行1%琼脂糖凝胶电泳检测,并切胶用DNAPurification Kit(天根生化科技有限公司)试剂盒回收并测量浓度。
使用ClonExpress II One Step Cloning Kit(南京诺唯赞生物科技公司)将目的基因片段和酶切好的植物表达载体进行连接。
将连接好的产物转入DH5α大肠感受态细胞:
a.在冰上解冻克隆感受态细胞;
b.取10μl重组产物加入到100μl感受态细胞中,轻弹管壁混匀(请勿振荡混匀),冰上静置30min;
c.42℃水浴热激45sec后,立即置于冰上冷却5min;
d.加入700μl LB培养基(不添加抗生素),37℃摇菌1h(转速200r/min);
e.将Kana抗性的LB平板固体培养基在37℃培养箱中预热;
f.5000r/min离心5min,弃掉600μl上清,将剩余菌体重悬,用无菌涂布棒在含有Kana抗性的平板上轻轻涂匀;
g.37℃培养箱中倒置培养12-16h。
单克隆阳性鉴定,在转化平板上挑取单克隆,进行菌液PCR阳性鉴定。
阳性检测正向引物序列(SEQ ID No.8):5’-actatccttcgcaagaccctt-3’;
阳性检测反向引物序列(SEQ ID No.9):5’-gctcgcctccattcatgta-3’;
菌液PCR 12μl反应体系:2×Taq Master Mix(Dye Plus)6μl、Forward Primer(10μM)1μl、Reverse Primer(10μM)1μl、菌液2μl、ddH2O 2μl。
菌液PCR反应条件:预变性95℃3min;变性95℃15s,复性58℃15s,延伸72℃30s,34个循环;72℃延伸5min;25℃5min。
将PCR反应产物用1%琼脂糖凝胶电泳检测,条带在502bp为阳性单克隆(图3),对于阳性克隆,直接将其转化好的DH5α菌液送往武汉擎科生物技术有限公司进行一代测序。
选取测序结果与基因组序列完全一致的克隆子扩大培养,使用TIANprep RapidMini PlasmidKit(天根生化科技有限公司)提取质粒。将质粒体转化到发根农杆菌K599中,具体步骤如下:
取K599感受态细胞于冰上融化。向100μl感受态细胞中加入0.01-1μg重组质粒。冰上放置30min,转至液氮中放置5min,37℃水浴5min,冰上5min,加入400μl无抗LB,28℃摇床孵育1小时。取100μl转化产物涂布于带有Kana和Str的抗性LB板上。28℃培养36-48h。挑取单克隆进行检测,所用引物和试验步骤与以上大肠杆菌阳性鉴定一致。
2、发根农杆菌介导的转化体系
将转化成功的发根农杆菌进行扩摇,挑取单克隆菌斑,加入400μl含有Kana和Str抗性的LB,放置于28℃摇床200r/min转速中过夜。小摇后的400μl菌液取100-150μl均匀涂布至含有Kana和Str抗生素的平板上,放置于28℃恒温箱中培养24h至平板上长满菌。将菌刮取至100ml MS液体培养基中,加入AS,并调整OD值至0.4-0.6,配置成侵染液。将光照下生长7d的材料在子叶节点下约0.5cm处进行剪切,保留带柄子叶,将剪切后的材料放入侵染液中,并放置于28℃摇床90r/min转速中侵染45min后拿出,用滤纸吸干残留菌液,移至皿中(放入两层MS浸湿的滤纸),放置于黑暗的温室条件下进行暗培养2-3d,再移至土中。
3、甘蓝型油菜株系(丙409)接种体系
室内接种使用的根肿菌,是中国根肿菌4号生理小种(季海雯,任莉,陈坤荣,徐理,刘凡,孙超超,李俊,刘胜毅,方小平.油菜根肿病病原主要生理小种和品种抗病性鉴定[J].中国油料作物学报,2013,35(03):301-306.)。根肿病抗病性室内接菌鉴定采用的温室菌土法接种(战宗祥,江莹芬,朱紫媛等.与位点PbBa8.1紧密连锁分子标记的开发及甘蓝型油菜根肿病抗性育种[J].中国油料作物学报,2015,37(6):766-771.)。首先将冻存于-20℃的菌根置于室温下解冻后,用匀浆机将其磨碎,将菌根和基质土按1:20的比例混匀,混匀后的菌土放置于25℃黑暗环境下封存48h。温室培养条件16L/8D,温度24℃,湿度60%-80%。培养30-35d后即可调查病情。
4、丙409超表达BraA03g008044E后接种病情调查
把接菌后的株系表型分为三个等级,分别为0级、1级、2级、3级。0级表现为根部无肿瘤,根系发育正常;1级表现为侧根着生少许肿瘤或主根少有澎粗;2级表现为主根着生肿瘤,有明显的较大结节状或球状肿块;3级表现为主根现大的肿瘤成纺锤形,植株矮小生长停止。
发病率和病情相关指数计算方式:
株系发病率=(感病株数/调查总株数)×100%;
株系病情指数=[∑(各级病株数×相应级数)/调查总株数×最高级别值]×100。
经调查,丙409超表达BraA03g008044E后,发病率降为50%,病情指数降为28.3(见图4和表1)。
表1丙409超表达BraA03g008044E后接中国根肿菌4号生理小种鉴定结果
注:丙409作为感病对照,409R作为抗病对照。
5、将接种过根肿菌4号生理小种的BraA03g008044E植株根系进行阳性鉴定。
将接种根肿菌的植株根系洗净,取一定量样品用CTAB法提取DNA,用阳性检测引物(SEQ ID No.8-9)进行检测。
20μl PCR扩增体系,2×Taq Master Mix(Dye Plus)10μl、Forward Primer(10μM)1μl、Reverse Primer(10μM)1μl、DNA模板2μl、ddH2O 6μl。
PCR反应条件:预变性95℃3min;变性95℃15s,复性58℃15s,延伸72℃30s,34个循环;72℃延伸30s;25℃5min,最后用1%琼脂糖凝胶电泳检测,502bp位置为目的条带(图5)。
6、BraA03g008044E转化至丙409后其表达情况
使用Super总RNA提取试剂盒(普洛麦格(北京)生物技术有限公司)对丙409转化BraA03g008044E植株接菌后根部样品的总RNA进行提取并进行RNA浓度测量。
根据样品RNA的浓度,用RNase-free ddH2O调整样品浓度一致,使得RNA进样总体积为12μl,加入4×gDNAWiper Mix 4μl,用移液器轻轻吹打混匀,42℃2min。
在上述反应管中直接加入5×HiScript II qRT SuperMix II 4μl,用移液器轻轻吹打混匀,然后进行逆转录反应:50℃15min,85℃5s。
Quantitative Real-time PCR分析BraA03g008044E基因的表达量,利用CEDesign设计BraA03g008044E基因的定量引物,定量引物序列如下:
正向定量引物(SEQ ID No.10):5’-gctccgagaattagagggagag-3’;
反向定量引物(SEQ ID No.11):5’-gtcgggaagacagttgagg-3’;
qPCR反应体系:qPCR SYBR Green Master Mix 11.8μl,F/R Primer 0.6μl,模板cDNA2μl,ddH2O to 15μl。
qPCR反应程序:95℃5min,95℃10s,56℃20s,72℃20s,40个循环,溶解曲线阶段默认设置1。
BraA03g008044E在转基因植株中的表达情况,该基因在丙409中超量表达并且表现为完全抗病的材料相对表达量均比丙409以及感病的转化植株高(图6)。
7、抗根肿菌基因BraA03g008044E其在A03染色体上的物理位置为3918065-3922651。BraA03g008044E基因在根部超量表达后能发挥其抗病功能,赋予感病材料丙409抗根肿菌的能力。BraA03g008044E基因的核苷酸序列如SEQ ID No.1和SEQ ID No.2所示,其编码蛋白氨基酸序列如SEQ ID No.3所示,具有典型的NBS-LRR结构域(图7),因此该基因可能具有抗根肿菌的功能。
SEQ ID No.1:
CTGTAGGAGGACGCTGAGAAGTTTCTTGCGTTCATGATACTTCCTCTCCACTTGACAAGTCAATTGTAAGAGGACCCCCCAGAAGTTTACTGTTTCCTTATCCCTTTACGCGTTCTCACCTTTTTTTTTTATGTGGCTACGAGCAACACACACATATTTACAAAGTCTCTTTTTGATTATTTATTCTTCAAATCTAAAAACTTTGTCATTCTTTTCAGAATTCCTTCGTGCTTGTTATTCATGGCTTCTTCATCATCTGTGTCTTGCGGTTCGTTGTATGACGTCTTCCTGAGCTTCCGTGGAGAAGACGTGCGCAAAGGGTTTCTTAGCCACGTTGTGAAAGAGTTCAAAAGCAAGGGGATCGAACCGTTCATTGATAACGAGATGGAGCGAGGCAAATCTGTCGGTCCCACCCTTGTAGGAGCGATCAGACAATCAAGGGTCGCCATTGTTTTGCTCTCCCGTAACTACGCTTCTTCAAGCTGGTGTTTGGATGAGTTAGTAGAGATCATGAAGTGCAGGGAAGATGATCAAAAAAGAGTGATAACCGTTTTCTACGAAGTGGATCCATCTGATGTTAGGAAGCAGATCGGAGAT
TTCGGAAAAGCCTTTGAGAAAACCTGTGTGGAAAAAACTGAAGAGGTGATACATGAAT
GGAGGCGAGCTTTGAAGGAAGTCGCGGGTATAGCTGGTTATGCCTCTAGCAACTGGTTC
GTTCACTAGTTTTCTTTTTAATGAATGCTCAACTAATTTAGTTTTGCTTAGGAAAGAAAGA
TTAAATATGAATATTTATCGATACTAGAACCATGTTTCCACGGCTTTCTTTTTTTGCTGTGT
GTAGCTTCTTTTCATGATGTATGTTCTAATGGATATCCTCTGTTTTTGTTAGGGAGAGTGA
AGCTGATTTGATCAACGAACTTGCCACAAATATTATGGGTGTGTTGCCTTTGACACCATC
AAATGATTTCGATGACTTTGTTGGCATAGAAGATCGTGTCATGGAGATGAAAACGATGTT
GAATCTACAAACAAAAGAAGTTAAGGTGATTGGGATTTGGGGTACTGCCGGGATTGGTA
AGACGACGGCCGCCAGAGTTTTGTATAACCAAATATCTCCTGGATTTTCATTCAGCACAT
TTTTGGAGAATATCAAAGGATGTTTTGAGAGGTCCTGTGGTAACGATCATCAGTTGAAGT
TGCGTTTTCAGGGAAAGTTGCTGTCTCAAATTTTTAACCAAAAGGATATTGTGGTTGGTC
ACTTGGGATGGGCACCACAAAAGCTGAGTGACAAAAAAGTGTTGGTTGTTCTTGATGA
AGTGGATAGCTGGTGGCAACTAGAGGAAATGGCAAAAAGAGCATGGTTTGGTCCCGGA
AGTATGATTATCATTACAACCGAAGATAGAAAACTTTTCAAGACACTCGGACTCGAGGC
CAATCAAATCTACGAGATGAAACTTCCAATTGGAGATGAGGCTCTTCAGATCTTCTGTCT
ATATGCCTTTGGTCAAAAGTTTCCAGATAATGGTTTCGAAAGTCTTGCTTGGGAAGTTAC
GAAACTTGCGGGTAAACTTCCTCTAGGCTTGAGAGTTATGGGGTCGTATCTACGAGGAAT
GTCCAAGGACGAGTGGATAGACGCACTACCAAGCCTCAGGTCTAGCCTTGACAGCGAA
ATTGAATCAACTCTGAGATTGAGCTACAACGTCTTAAGTGTTAAAGAAAAAGCACTTTT
CCTGCATATTGCATGTTTCTTTGTTGGTTTCAAGGATGATAGGGTCAAGAGTATTCTTGAA
AAAAGCGATTTGAACGTCATCCATGGGCTGCAAACCTTAGCTTACAGAAGTCTTATATAT
GTAGAGCACAGATTAATAAGGATGCATAGTTTACTGCAACAAATGGGTGAAGAAATTGT
CTATGGACAGTCTGCCGAGCCTGGAAAGCGACAGTTCCTGACGAAAGCCACTGAGATTT
CTGAAATTTTTGAAGAAAATACTGTAAGTTCCTTTTTTCTTCTGTTTTCTTTATTTTTTAAG
CTGAGATTTTTTTTCTTTTTGAATAATCTTTTAAAGGGTACTGGAACGGTTCTAGGCATAA
AGCTCCGAGAATTAGAGGGAGAGGAAATCCAAATGAGTGAAAGCGCTTTCCAAGGGAT
GAACCATCTCCAGTTTCTAGATGTCGATTGTATCTTATGCTTACCCGAAGGCCTCAACTGT
CTTCCCGACAAACTTAGACTGCTAGATTGGAAGCAGTGTCCATTGAGATTTTGGCCTTCC
AAGTTCTCTGGAAAGTTTCTTGTTGAACTAATCATGCCCAACAACAAATTTGAGAAACT
TTGGGAGGGAATCAAAGTAAAAAATAGTAGGCCTTTTTCTAATTATATATAATTGCGTGTA
AAGCGCGGTTAGTCATTTCTCTAAAACTTTTTTTTTTGTATAGCCTCTCCCATGTCTCAAG
CTGATGGATTTGAGCTACTCTGACTATCTGAAAGAGATTCCAGATCTCTCGAAAGCAACC
AGTATCGAGATACTAAAACTCCATGACTGCAGAAGTTTGTTAGAGCTCCCCTTTTCTATT
GGTAGGTTAATCAATCTCGAGGAATTATACCTCAGCTTTTGCGGGAGTTTGGAAAAAATC
GGTGGTTGCTCGAGTTTGGAAAAACTCAGTGATTGCTCGAGTTTGAAAGAACTCGATCT
GAGCTATTCTGGGATAGGAGCATTGGAATTGCCATTATCAGTTCGTACTTGGTCTAGCTTT
TATAGATTGGATATGTCAGGGTTGAGTGACCTCAAGAAATTCCCAAATGTTCCTTACAGC
ATCGTAGAGTTGGATCTGAGCAACACAGGGATAGAGGAAATTCCTCCATGCATTGAGAA
TCTATATTGTCTGGAGAAACTAAAAATGATGGGATGCAGGAAGCTGGAAATTGTATCTCC
AAACATTTCTAAGTTGGAGAAACTTAAGACTATTGCACTGTGTAAAAATGGCGCTGAGG
CGCTTGGTGTGGATGGACCTTTTAAAGCAACGATCGATGGGTTTGATAAATGGTGTCACC
AGTGGCTTCTCATATCAGACTTAAACATTCACTACATTTTACCGAGATGTCTAACGGAGA
AGGTTCTTACGCCTCCATTTTCATCATTACGTTTAATCGGTCCTGAATTAAACACTATTCC
AGATTGCATCAGACGTCTCTCCGGACTAGGTCAGCTTGATATCTCAGCTTGTCGAAAGCT
CACAGCACTGCCACCGCTTCCAGCTTCCCTTGAAGCACTAAATGCATGCGGGTGTGCCT
ACTTGGAGAGTATAGACTCTTCTTTTCAAAATCCAATTCGCCTAAACTTTGCTGACTGCT
ACAACCTGAATCAAAAAGCGAGAAAGCTCATCCAGACATCAGCTTGCGAATATGCGCTC
TTACCGGGTGAAGAAGTGCCTGCACATTTCACTCACCAAGCTATTTCTAGTTCACTAACG
ATCGATCTGACTCCAAGACATCTTCCTTCATCCTTCAGATTCAAAGCTTGCATCTTACTGT
CAAGACTCCTTGGTTCCCTTAATGTGTCTTGTTGCGTCAGAGGTAAACAGACTGGCCTC
ACTCTCCCATATGGATCAACCCAGCTTCGTTATATGTCAGATCTATACTCACCTCGAAACC
ATCTTTATATATTTGAAGATTCTGTCTCTCTAAGCCAGGATTTCCCTGAAGCCAAAGACGC
CACTTTCAGCGAGCTTTCGTTTCTGTTCACAGTGAGGGATGATGAATCATCCTTGGTGGT
GGTTAAGGGCTGTGGTGTACGAATTTTGGAGGCAAACAATGAAAATGCAGATAATGATG
ATGATGATGAAGGTGGTAATGATAGTAAAAGTGCAGACGATGATGATGATGATACTGATG
ACGTTGATGAGGATATAGAGGATGATATCGATGATAATGACGCTGGCTTTTCTGATTCAGT
AATTTGACATAAACATAGAGGCAAACAAAAGAAGGAAAAGAATCAGGACGTGAGGACA
GTGCAGAGACGAGGAGCTGGAAACGAATGAGGTTAAGCCTTGTGTAAGTGTTGTGATGT。
SEQ ID No.2:
ATGGCTTCTTCATCATCTGTGTCTTGCGGTTCGTTGTATGACGTCTTCCTGAGCTTCCGTGGAGAAGACGTGCGCAAAGGGTTTCTTAGCCACGTTGTGAAAGAGTTCAAAAGCAA
GGGGATCGAACCGTTCATTGATAACGAGATGGAGCGAGGCAAATCTGTCGGTCCCACCC
TTGTAGGAGCGATCAGACAATCAAGGGTCGCCATTGTTTTGCTCTCCCGTAACTACGCTT
CTTCAAGCTGGTGTTTGGATGAGTTAGTAGAGATCATGAAGTGCAGGGAAGATGATCAA
AAAAGAGTGATAACCGTTTTCTACGAAGTGGATCCATCTGATGTTAGGAAGCAGATCGG
AGATTTCGGAAAAGCCTTTGAGAAAACCTGTGTGGAAAAAACTGAAGAGGTGATACAT
GAATGGAGGCGAGCTTTGAAGGAAGTCGCGGGTATAGCTGGTTATGCCTCTAGCAACTG
GGAGAGTGAAGCTGATTTGATCAACGAACTTGCCACAAATATTATGGGTGTGTTGCCTTT
GACACCATCAAATGATTTCGATGACTTTGTTGGCATAGAAGATCGTGTCATGGAGATGAA
AACGATGTTGAATCTACAAACAAAAGAAGTTAAGGTGATTGGGATTTGGGGTACTGCCG
GGATTGGTAAGACGACGGCCGCCAGAGTTTTGTATAACCAAATATCTCCTGGATTTTCAT
TCAGCACATTTTTGGAGAATATCAAAGGATGTTTTGAGAGGTCCTGTGGTAACGATCATC
AGTTGAAGTTGCGTTTTCAGGGAAAGTTGCTGTCTCAAATTTTTAACCAAAAGGATATTG
TGGTTGGTCACTTGGGATGGGCACCACAAAAGCTGAGTGACAAAAAAGTGTTGGTTGT
TCTTGATGAAGTGGATAGCTGGTGGCAACTAGAGGAAATGGCAAAAAGAGCATGGTTTG
GTCCCGGAAGTATGATTATCATTACAACCGAAGATAGAAAACTTTTCAAGACACTCGGA
CTCGAGGCCAATCAAATCTACGAGATGAAACTTCCAATTGGAGATGAGGCTCTTCAGAT
CTTCTGTCTATATGCCTTTGGTCAAAAGTTTCCAGATAATGGTTTCGAAAGTCTTGCTTGG
GAAGTTACGAAACTTGCGGGTAAACTTCCTCTAGGCTTGAGAGTTATGGGGTCGTATCTA
CGAGGAATGTCCAAGGACGAGTGGATAGACGCACTACCAAGCCTCAGGTCTAGCCTTG
ACAGCGAAATTGAATCAACTCTGAGATTGAGCTACAACGTCTTAAGTGTTAAAGAAAAA
GCACTTTTCCTGCATATTGCATGTTTCTTTGTTGGTTTCAAGGATGATAGGGTCAAGAGTA
TTCTTGAAAAAAGCGATTTGAACGTCATCCATGGGCTGCAAACCTTAGCTTACAGAAGT
CTTATATATGTAGAGCACAGATTAATAAGGATGCATAGTTTACTGCAACAAATGGGTGAA
GAAATTGTCTATGGACAGTCTGCCGAGCCTGGAAAGCGACAGTTCCTGACGAAAGCCA
CTGAGATTTCTGAAATTTTTGAAGAAAATACTGGTACTGGAACGGTTCTAGGCATAAAGC
TCCGAGAATTAGAGGGAGAGGAAATCCAAATGAGTGAAAGCGCTTTCCAAGGGATGAA
CCATCTCCAGTTTCTAGATGTCGATTGTATCTTATGCTTACCCGAAGGCCTCAACTGTCTT
CCCGACAAACTTAGACTGCTAGATTGGAAGCAGTGTCCATTGAGATTTTGGCCTTCCAA
GTTCTCTGGAAAGTTTCTTGTTGAACTAATCATGCCCAACAACAAATTTGAGAAACTTTG
GGAGGGAATCAAACCTCTCCCATGTCTCAAGCTGATGGATTTGAGCTACTCTGACTATCT
GAAAGAGATTCCAGATCTCTCGAAAGCAACCAGTATCGAGATACTAAAACTCCATGACT
GCAGAAGTTTGTTAGAGCTCCCCTTTTCTATTGGTAGGTTAATCAATCTCGAGGAATTATA
CCTCAGCTTTTGCGGGAGTTTGGAAAAAATCGGTGGTTGCTCGAGTTTGGAAAAACTCA
GTGATTGCTCGAGTTTGAAAGAACTCGATCTGAGCTATTCTGGGATAGGAGCATTGGAAT
TGCCATTATCAGTTCGTACTTGGTCTAGCTTTTATAGATTGGATATGTCAGGGTTGAGTGA
CCTCAAGAAATTCCCAAATGTTCCTTACAGCATCGTAGAGTTGGATCTGAGCAACACAG
GGATAGAGGAAATTCCTCCATGCATTGAGAATCTATATTGTCTGGAGAAACTAAAAATGA
TGGGATGCAGGAAGCTGGAAATTGTATCTCCAAACATTTCTAAGTTGGAGAAACTTAAG
ACTATTGCACTGTGTAAAAATGGCGCTGAGGCGCTTGGTGTGGATGGACCTTTTAAAGC
AACGATCGATGGGTTTGATAAATGGTGTCACCAGTGGCTTCTCATATCAGACTTAAACAT
TCACTACATTTTACCGAGATGTCTAACGGAGAAGGTTCTTACGCCTCCATTTTCATCATTA
CGTTTAATCGGTCCTGAATTAAACACTATTCCAGATTGCATCAGACGTCTCTCCGGACTA
GGTCAGCTTGATATCTCAGCTTGTCGAAAGCTCACAGCACTGCCACCGCTTCCAGCTTC
CCTTGAAGCACTAAATGCATGCGGGTGTGCCTACTTGGAGAGTATAGACTCTTCTTTTCA
AAATCCAATTCGCCTAAACTTTGCTGACTGCTACAACCTGAATCAAAAAGCGAGAAAGC
TCATCCAGACATCAGCTTGCGAATATGCGCTCTTACCGGGTGAAGAAGTGCCTGCACATT
TCACTCACCAAGCTATTTCTAGTTCACTAACGATCGATCTGACTCCAAGACATCTTCCTTC
ATCCTTCAGATTCAAAGCTTGCATCTTACTGTCAAGACTCCTTGGTTCCCTTAATGTGTCT
TGTTGCGTCAGAGGTAAACAGACTGGCCTCACTCTCCCATATGGATCAACCCAGCTTCG
TTATATGTCAGATCTATACTCACCTCGAAACCATCTTTATATATTTGAAGATTCTGTCTCTC
TAAGCCAGGATTTCCCTGAAGCCAAAGACGCCACTTTCAGCGAGCTTTCGTTTCTGTTC
ACAGTGAGGGATGATGAATCATCCTTGGTGGTGGTTAAGGGCTGTGGTGTACGAATTTT
GGAGGCAAACAATGAAAATGCAGATAATGATGATGATGATGAAGGTGGTAATGATAGTA
AAAGTGCAGACGATGATGATGATGATACTGATGACGTTGATGAGGATATAGAGGATGATATCGATGATAATGACGCTGGCTTTTCTGATTCAGTAATTTGA。
SEQ ID No.3:
MASSSSVSCGSLYDVFLSFRGEDVRKGFLSHVVKEFKSKGIEPFIDNEMERGKSVGPTLVGAIRQSRVAIVLLSRNYASSSWCLDELVEIMKCREDDQKRVITVFYEVDPSDVRKQIGDFGKAFEKTCVEKTEEVIHEWRRALKEVAGIAGYASSNWESEADLINELATNIMGVLPLTPSNDFDDFVGIEDRVMEMKTMLNLQTKEVKVIGIWGTAGIGKTTAARVLYNQISPGFSFSTFLENIKGCFERSCGNDHQLKLRFQGKLLSQIFNQKDIVVGHLGWAPQKLSDKKVLVVLDEVDSWWQLEEMAKRAWFGPGSMIIITTEDRKLFKTLGLEANQIYEMKLPIGDEALQIFCLYAFGQKFPD NGFESLAWEVTKLAGKLPLGLRVMGSYLRGMSKDEWIDALPSLRSSLDSEIESTLRLSYNVLSVKEKALFLHIACFFVGFKDDRVKSILEKSDLNVIHGLQTLAYRSLIYVEHRLIRMHSLLQQMGEEIVYGQSAEPGKRQFLTKATEISEIFEENTGTGTVLGIKLRELEGEEIQMSESAFQGMNHLQFLDVDCILCLPEGLNCLPDKLRLLDWKQCPLRFWPSKFSGKFLVELIMPNNKFEKLWEGIKPLPCLKLMDLSYSDYLKEIPDLSKATSIEILKLHDCRSLLELPFSIGRLINLEELYLSFCGSLEKIGGCSSLEKLSDCSSLKELDLSYSGIGALELPLSVRTWSSFYRLDMSGLSDLKKFPNVPYSIVELDLSNTGIEEIPPCIENLYCLEKLKMMGCRKLEIVSPNISKLEKLKTIALCKNGAEALGVDGPFKATIDGFDKWCHQWLLISDLNIHYILPRCLTEKVLTPPFSSLRLIGPELNTIPDCIRRLSGLGQLDISACRKLTALPPLPASLEALNACGCAYLESIDSSFQNPIRLNFADCYNLNQKARKLIQTSACEYALLPGEEVPAHFTHQAISSSLTIDLTPRHLPSSFRFKACILLSRLLGSLNVSCCVRGKQTGLTLPYGSTQLRYMSDLYSPRNHLYIFEDSVSLSQDFPEAKDATFSELSFLFTVRDDESSLVVVKGCGVRILEANNENADNDDDDEGGNDSKSADDDDDDTDDVDEDIEDDIDDNDAGFSDSVI*。
8、BraA03g008044E基因在根肿病抗性育种和分子标记辅助选择中的应用
BraA03g008044E基因作为NBS-LRR基因,使得油菜转基因材料获得更好的根肿病抗性,可以抵御中国根肿菌4号生理小种的侵染,有效保护根肿菌污染田块中油菜的产量。BraA03g008044E除了对甘蓝型油菜有抗根肿效应,对其他农艺性状影响不大,表明该基因可以作为抗性基因通过基因工程手段进行油菜、白菜、甘蓝和萝卜等芸薹属植物的抗性育种。根据基因全长序列(包含上游序列和编码区)及与本基因连锁的其它序列所开发的标记进行的分子标记辅助育种,也可以大大加速根肿病抗性育种进程。
以上所述的实施例仅是对本发明的优选方式进行描述,并非对本发明的范围进行限定,在不脱离本发明设计精神的前提下,本领域普通技术人员对本发明的技术方案做出的各种变形和改进,均应落入本发明权利要求书确定的保护范围内。
Claims (3)
1.BraA03g008044E基因在赋予芸薹属植物根肿菌抗性中的应用,其特征在于,所述BraA03g008044E基因的核苷酸序列如SEQ ID No.1所示;所述根肿菌为中国根肿菌4号生理小种。
2.BraA03g008044E基因在根肿病抗病育种中的应用,其特征在于,所述BraA03g008044E基因通过转基因或有性杂交转育途径用于芸薹属植物根肿病抗病育种中的应用,所述芸薹属植物包括油菜、白菜或甘蓝,所述BraA03g008044E基因的核苷酸序列如SEQ ID No.1所示;所述根肿病的病原菌为中国根肿菌4号生理小种。
3.BraA03g008044E基因在芸薹属植物根肿菌抗性分子标记辅助选择中的应用,其特征在于,所述芸薹属植物包括油菜、白菜或甘蓝,所述BraA03g008044E基因的核苷酸序列如SEQ ID No.1所示;所述根肿菌为中国根肿菌4号生理小种。
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