【作者】 钱伟；

【导师】 孙日飞；

【作者基本信息】 中国农业科学院 ， 蔬菜学， 2013， 博士

【摘要】 大白菜起源于中国，是我国最重要的蔬菜作物之一。病毒病是影响大白菜生产的主要病害，芜菁花叶病毒（Turnip mosaic virus，TuMV）是引起该病的主要病原。大白菜TuMV抗性基因的定位克隆、功能研究及抗性机制探索对大白菜TuMV高效分子育种具有特殊重要意义。本研究以大白菜抗TuMV高代自交系BP8407和感TuMV高代自交系极早春等为实验材料，对大白菜TuMV抗性基因进行遗传、定位、克隆研究，并对该抗性基因进行功能验证及抗性机制探索，获得了以下研究结果：1、以大白菜抗TuMV高代自交系BP8407和感TuMV高代自交系极早春为亲本，构建F2群体及F2:3家系。对F2群体及F2:3家系进行TuMV C4株系人工机械磨擦接种、ELISA检测。F2群体中共239株，其中抗病植株52株，感病植株187株。经卡方检测，抗感比符合1：3，因此BP8407对TuMV C4抗性受一对隐性基因控制。2、利用混合群体分组分析法（BSA）、SSR和Indel标记技术筛选与TuMV抗性基因连锁的标记。在200对SSR标记中，筛选到一个与TuMV抗性基因连锁的标记BC84。BC84位于白菜A04染色体上的scaffold000048上。进一步在A04染色体上scaffold000048附近的145对Indel标记中筛选到与TuMV抗性基因连锁的17对Indel连锁标记。该抗性基因两侧紧密连锁的分子标记为BrID10694和BrID101309，其与抗病基因连锁距离分别为0.3和0.6cM。该隐性抗性基因位于大白菜A04染色体上的scaffold000060或者scaffold000104上，将其命名为retr02。3、根据白菜参考基因组信息及植物对病毒的隐性抗性机制，对retr02定位目标区域内的基因进行分析，筛选到retr02的侯选基因Bra035393，其编码eIF（iso）4E蛋白。该基因与拟南芥TuMV隐性抗性基因lsp为共线性基因。4、根据白菜参考基因组的Bra035393序列信息，设计引物，对亲本BP8407和极早春的Bra035393的gDNA和cDNA进行克隆分析。对Bra035393的gDNA克隆测序后发现，BP8407的Bra035393与极早春的Bra035393相比存在着3个差异位点，第一个差异位点是在exon1和intron1的连接处多出一个碱基G，第二个差异位点是在intron2中多出一个碱基T，第三个差异位点是在exon3中存在着一个SNP（A/G）。对Bra035393的cDNA克隆测序后发现，极早春的Bra035393正常编码成熟的eIF（iso）4E蛋白。而BP8407的Bra035393由于exon1和intron1的连接处多出一个碱基G发生错误剪切，造成编码提前终止，编码不成熟的eIF（iso）4E蛋白。5、对retr02（BP8407Bra035393）和Retr02（极早春Bra035393）构建原核表达载体（pET-32a），进行蛋白诱导分析，进一步验证出Retr02编码正常的eIF（iso）4E蛋白，而retr02编码的蛋白长度大大缩短，其为不成熟的eIF（iso）4E蛋白。通过酵母双杂互作分析得出，Retr02编码的完整的eIF（iso）4E蛋白可以与TuMV C4VPg蛋白互作，而retr02编码的eIF（iso）4E蛋白不能与TuMV C4VPg蛋白互作。证实retr02编码的不成熟的eIF（iso）4E蛋白，丧失与TuMV C4VPg互作的能力，从而使80122对TuMV C4产生抗性。综上所述，本研究定位、克隆了大白菜TuMV隐性抗性基因retr02，并明确了retr02的抗性机制，为大白菜抗TuMV高效分子育种奠定了基础。更多还原

【Abstract】 Chinese cabbage originated in China, is one of the most important vegetable crops in China. Thevirus disease is the main disease affecting the production of Chinese Cabbage. Turnip mosaic virus(TuMV) is the main pathogen causing virus disease. The researches of the TuMV resistance genecloning, function studies and resistance mechanism were very important to Chinese cabbage TuMVefficient molecular breeding.With the TuMV resistance lines BP8407and susceptible lines Ji Zao Chun, we research theTuMV resitance genetics, the TuMV resitance gene mapping and cloning, the TuMV resitance genefunctional verification and resistance mechanism. And the results are as follows:1. The F2population and F2:3lines, which were deduced from the cross of the TuMV resistancelines BP8407and susceptible lines Ji Zao Chun, were inoculated with TuMV C4isolate. The resistancewas established by ELISA. The segregation data from the TuMV inoculation of the F2generation (52resistant individuals and187susceptible individuals) fitted the expected segregation for a Mendelianmodel based on the action of a single recessive allele.2. The polymorphsic SSR and Indel markers were identified using the BSA method. Among the200pairs of SSR markers, only BC84linked with the resistance gene, which located on the A04chromosome scaffold000048of Brassica rapa. Then, the145Indel markers on A04scaffold000048region were further screened using the BSA method, of which17Indel markers were polymorphsic andlinked to resistance gene. The both sides of the resistance genes closely linked markers were BrID10694and BrID101309, with resistance genes linkage distance respectively0.3and0.6cM. So the resistancegene was located on A04scaffold000060or scaffold000104, and was named retr02.3. Based on the B.rapa reference genome information and plant recessive resistance mechanism tovirus, we analysed the retr02candidate gene in the mapped region. And one candidate gene Bra035393,which coded eIF (iso)4E protein, was identified. This candidate gene was the syntenic gene of theTuMV recessive resistance gene lsp in Arabidopsis thaliana.4. According to Bra035393sequence of B.rapa reference genome information, some primers weredesigned to clone the Bra035393gDNA and cDNA of BP8407and Ji Zao Chun. After the gDNA ofBra035393cloning sequencing, there were three different sites between BP8407and Ji Zao Chun. Thefirst different site was BP8407have one more G at the joint of exon1and intron1. The second differentsite was BP8407have one more A in intron2. And the third different site was the SNP (A/G) in exon3.For Bra035393cDNA cloning sequencing, we found BP8407Bra035393maybe could code earlytermination and code the immature eIF (iso)4E protein, because of the first different site Insert G at thejoint of exon1and intron1. And Ji Zao Chun Bra035393normal encoded mature eIF (iso)4E protein.5. To research the Bra035393expression in transgenic E. coli Rosetta, pET-32a-Bra035393vectorswere constructed using the CDS of BP8407and Ji Zao Chun. And we further verified Retr02normalencoded mature eIF (iso)4E protein, but retr02coded immature eIF (iso)4E protein. Throught yeast two hybrid interaction analysis, we confirmed that the mature eIF (iso)4E protein coded by Retr02could interact with TuMV C4VPg, but the immature eIF (iso)4E protein coded by retr02could notinteract with TuMV C4VPg. So the the immature eIF (iso)4E protein coded by retr02losed its abilityand caused the resistance.To sum up, we mapped and cloned the TuMV recessive resistance gene retr02, and confirmed theresistance mechanism. This work laid a foundation for Chinese cabbage efficient molecular breedingresistance to TuMV.更多还原