【作者】 康厚祥；

【导师】 谢丙炎；

【作者基本信息】 中国农业科学院 ， 植物病理学， 2011， 博士

【摘要】 黄瓜黑星病是黄瓜的主要病害之一，针对黄瓜抗黑星病基因的研究对明确黄瓜抗病机制和抗病育种都具有重要的意义。本研究以高感黑星病黄瓜材料9930 (中国华北保护地栽培品种)、高抗黑星病材料9110gt (欧洲温室品种)、Gy14 (美国加工品种)、Hardwickii (印度野生黄瓜)等为试材，对黄瓜全基因组水平NBS-encoding (Nucleotide banding site encoding)基因进行分析，对黄瓜抗黑星病基因进行遗传、定位和克隆研究，并对黄瓜与黑星病菌的亲和与不亲和互作进行研究，获得如下研究成果：1、全基因组水平分析发现9930、Gy14、Hardwikii中分别含有NBS-encoding基因58、60、60个，在基因组上的密度为20个/100 Mb基因组，为拟南芥、水稻、杨树等平均密度的1/5，且NBS结构内Kinase-2、Kinase-3、GLPL等保守域与其它几个物种相比，具明显的氨基酸不一致现象，LRR区域则高度保守。通过手工注释的方法发现三个黄瓜基因组中7个直系同源基因发生了移码突变、1个基因发生提前终止密码子突变、1个基因缺失突变，说明不同黄瓜品种中抗病基因差异大。2、精细定位了黄瓜抗黑星病基因Ccu，发现Ccu区域存在一个紧密连锁的抗病基因簇。利用148个株系的F9-RILs群体和2000个单株的F2群体为试材，采用分子标记、遗传连锁作图等技术将Ccu定位到黄瓜2号染色体短臂末端一个长度为140 kb的DNA区域，此区域包含一个紧密连锁的抗病基因簇，抗病基因簇中有三个基因呈串联相邻排列(Csa006756、Csa006757、Csa006758)。通过比较基因组学发现该抗病簇与甜瓜、拟南芥、杨树等同源区域具有共线性(图4-6、4-7),表明此同源抗病基因簇由一个古老的抗病区域进化而来。3、图位克隆了Ccu候选基因。通过构建黄瓜抗黑星病品种9110gt BAC文库，筛选出包含目的抗病簇的BAC克隆H77-J7和H74-D1，进一步构建了长度为11 kb的亚克隆文库，筛选出包含候选基因起始密码子上游到终止密码子下游各约2.5 kb的目的亚克隆Csa00672756-1、Csa006757-1、Csa006758-1，可用于后续转基因验证其功能。4、通过黄瓜与黑星菌亲和与不亲和互作的表达谱分析，揭示了黄瓜抗黑星病的机制。以黄瓜高抗黑星病品种Gy14和高感黑星病品种9930为试材，利用高通量的测序技术对接种病菌材料及其对照材料进行表达谱分析，发现抗病品种在接种24小时后已全面上调了细胞内的2条HR(Hypersensitive Reaction)反应程序和1条PR (Pathogenesis related)基因相关的增强防御程序(图5-11、表5-5)中的编码关键因子基因。与抗病材料相比，感病材料中只部分启动了第三条WRKY转录因子参与的PR基因防御程序，其中WRKY转录因子表达水平显著上调，但其下游的PR基因却没有上调表达，植物未进入对病害的防御状态(图5-11、表5-5)。综上所述，本研究精细定位了黄瓜抗黑星病基因Ccu，并图位克隆了Ccu的候选基因，为黄瓜抗黑星病分子育种奠定了基础；表达谱分析明确了黄瓜抗黑星病的抗病机制，为黄瓜抗病研究提供了参考模式。更多还原

【Abstract】 Scab, is a very important disease of cucumber worldwide, and it is meaningful for usto further study of this disease.Cucumber types of“9930”,“9110gt”,“Gy14”,“Hardwickii”were employed in thepresent study. 9930 is a Northern china fresh market type that is susceptible to scab, 9110gtis a European green house type cucumber with scab resistance conferred by Ccu gene,Gy14 is a US processing type cucumber, and Hardwikii is a wild type originating fromIndia. Whole genomes of 9930、Gy14 and Hardwikii have been sequenced. Based on thesebackground, genome-wide analysis methods were used for studying cucumberNBS-encoding genes. The molecular markers and gene cloning technologies were used forfine mapping and map based cloning of Ccu candidates. The compatible and incompatibleinteractions between cucumber and the scab pathogen were studied using expressionprofiling supported by Illunima GA sequencing technology. The main conclusions are asfollows:1 Genome-wide analysis of cucumbers revealed that there were 58、60 and 60NBS-encoding genes in 9930, Gy14 and Hardwikii respectively, and the averagedistribution in genome was about 20 genes / 100 Mb genome, which was one-fifth thanthose in Arabidopsis, rice and poplar. In Kinase-2, Kinase-3, and GLPL domains,differences were found between different species. Seven frameshift mutations were foundamong the three cucumbers’NBS-encoding orthologouses.2 Fine mapping localizes Ccu into an R gene cluster. A F9 recombinant inbred lines(RILs 148 lines) population and a F2 population (2000 plants) were developed in this study.Ccu locus was delimit into a 140 kb genomic DNA region in the terminal of chromosome 2,it contained four nucleotide binding site encoding resistance gene analogs (RGAs), three ofthem were tandem repeats in this region. Comparative genomics analysis revealed that Ccugene region have syntenic regions in melon, Arabidopsis, poplar and grape (Figure 4-6 &4-7). It implied that Ccu homologous regions were originating from an old R gene cluster.3 Ccu candidates have been cloned. A 9110gt BAC library consists of 4100 individualclones was constructed, and BAC clones of H77-J7 and H74-D1 which contain Ccu regionwere selected, The H77-J7 clone was used for 11 kb library construction, and the targetclones which contain the Ccu candidate genes were screened.4 Expression profiling revealed the mechanism of cucumber scab diseases resistance.Gy14 (Scab Resistance) and 9930 (Scab Susceptible) were employed in this study.Twenty-four hours after inoculation, two HR pathways and a PR gene related diseaseresistance pathway (Figure 5-11 table 5-5) have been up-regulated in Gy14. On thecontrary, in 9930, the WRKY genes were up-regulated, but no PR genes were up-regulated, the plant did not detected the pathogen.In conclusion, Ccu has been fine mapped to a 140 kb DNA region. The Ccu candidateswere cloned by a map-based approach. It provided a platform for MAS of cucumber scabdisease resistance breeding and function analysis of Ccu gene. Expression profilingrevealed the mechanism of cucumber scab diseases resistance, which provides a model forcucumber diseases study.更多还原