【作者】 刘大军；

【导师】 秦智伟；

【作者基本信息】 东北农业大学 ， 蔬菜学， 2013， 博士

【摘要】 黄瓜霜霉病是由卵菌纲的古巴假霜霉病菌(Pseudoperonospora cubensis)引起的。在黄瓜生产中,霜霉病是具有毁灭性的叶部病害。近年来随着病原菌的变异,有加速流行的趋势。利用药剂防治已经可以在一定程度上控制该病的发生。但是,药剂的使用存在食品安全问题和环境问题,也存在霜霉病菌的抗药性问题。培育并使用抗病品种是减轻该病危害的最有效措施。因此,黄瓜抗霜霉病基因的发掘和黄瓜抗霜霉病机制的深入研究对于黄瓜抗霜霉病育种和黄瓜生产实践具有重要意义。本论文主要进行了以下几方面的研究：(1)对不同类型黄瓜材料进行苗期接种鉴定和田间评价鉴定,评价霜霉病抗性；(3)测序并分析“M801-3-1”和“M302-3”侵染霜霉病菌后的转录组。(3)采用SSH技术对黄瓜抗霜霉病品种“M801-3-1”侵染霜霉病菌前后的差异表达基因进行分析；(4)从植物抗性基因数据库网站(http://prgdb.crg.eu/old.php)下载拟南芥和甜瓜抗霜霉病家族的蛋白质氨基酸序列,用其分别检索“瓜类基因组数据库”(http://www.icugi.org/)中的“黄瓜基因组数据库”,通过BLAST比对查找出在“黄瓜基因组数据库”中的黄瓜抗霜霉病候选基因,并利用DNAMAN6.0软件对这些基因进行聚类分析,通过RT-PCR试验和实时定量RT-PCR在抗霜霉病品种“M801-3-1”和感病材料“M302-3”分析候选基因的表达情况；(5)根据Csa002921扩增406bp片段产物构建干扰载体pRNA002921,通过农杆菌介导法转化“M801-3-1”和“M302-3”。本研究主要结果如下：(1)对不同类型黄瓜材料通过苗期接种鉴定和田间评价鉴定,鉴定出抗霜霉病材料5份,中抗材料3份,感病材料3份。确定抗霜霉病品种“M801-3-1”和感病材料“M302-3”。(2)通过测序感病黄瓜品种和抗病黄瓜品种受霜霉病菌侵染后的转录组,证实在抗感品种间霜霉病菌侵染后的转录组存在较大差别。抗霜霉病品种M8O1-3-1中3599个蛋白表达量较高,生长素相关蛋白,钙结合蛋白,热激蛋白,谷胱甘肽氧化酶,谷胱甘肽还原酶,谷胱甘肽硫转移酶,肽脯氨酰顺反异构酶,叶绿体结合蛋白,泛素连接酶,蛋白酶体组成蛋白等在抗病品种中表达量较高,其中47个蛋白仅在M801-3-1中表达,包括DNA损伤诱导蛋白和抗坏血酸过氧化物酶等。证实抗病品种中存在1个表达量较高的CC-NBS-LRR类R基因,可能为抗病R基因。感病品种M302-3蛋白表达量较高的有1727个,乙烯氧化酶,乙烯相应转录因子,水杨酸羧基甲基转移酶,WRKY转录因子蛋白等在感病品种中表达量较高,其中有168个蛋白仅在感病品种M302-3中表达,包括TIR-NBS-LRR疾病抗性蛋白,核糖体组成蛋白,乙烯合成酶,泛素蛋白和效应因子蛋白。证实在感病品种中存在2个特异性表达的TIR-NBS-LRR类R基因和1个高表达的TIR-NBS-LRR,可能为感病R基因。(3)利用SSH技术构建抗病黄瓜材料霜霉病菌接种和未接种差异表达的消减cDNA文库。经反向Northern blot杂交检测,共得到48个阳性克隆。利用分子生物学软件对测序后的序列进行质量检测和聚类、拼接,共得到14个UniESTs,其中包括8个singletons和6个contigs。将上述EST序列在“瓜类基因组数据库”中的“黄瓜基因组数据库”进行BLAST比对,有10个EST片段找到了与之匹配的同源序列,有4个EST片段未找到同源序列,推测可能是新基因。(4)在“黄瓜基因组数据库”进行BLAST比对,确定黄瓜基因组中存在185个和拟南芥抗霜霉病基因同源的基因,2个和甜瓜抗霜霉病基因同源的基因。RT-PCR试验和实时定量RT-PCR表明Csa001907和Csa002921在抗病品种中特异下调表达有可能是黄瓜抗霜霉病的R基因。RT-PCR试验和实时定量RT-PCR证明了大部分R基因是组成型表达的,本研究中未发现R基因存在上调表达的现象。RT-PCR试验和实时定量RT-PCR都证实了Csa001907和Csa002921基因在抗病品种中存在下调表达现象,实时定量RT-PCR试验表明Csa001907和Csa002921在感病品种中不存在下调表达现象。(5)本试验还构建了Csa002921的RNAi干扰表达载体,通过转基因实验能够进一步确定Csa002921在黄瓜抗霜霉病中的作用。更多还原

【Abstract】 Cucumber downy mildew caused by Pseudoperonospora cubensis. Pseudoperonospora cubensis is a destructive leaf disease in recent years. The spread of disease is accelerated with the variability of the pathogen. The application of the pesticides can be controlled to some degree cucumber downy mildew. Food safety and environmental issues become serious with the pesticide application. Also Pseudoperonospora cubensis produce pesticide resistance. Cultivate and use of resistant varieties is the most effective measures to reduce disease. Therefore, study of the mechanisms of resistance to downy mildew and resistant gene exploration in cucumber to downy mildew is important.This research can be divided into the following aspects:(1) Different types of cucumber material was identified by inoculation of seedling and field evaluation identified.(2) Cucumber Downy Mildew Resistant variety "M801-3-1" differentially expressed before and after the infection downy mildew been studied using SSH technology.(3) Arabidopsis genes resistant to downy mildew and melon genes resistance to downy mildew homologous genes protein sequence was downloaded from Plant Resistance Gene database (http://prgdb.crg.eu/old.php). Respectively retrieve Cucurbit Genomics Database (http://www.icugi.org/). BLAST was used to find out cucumber downy mildew resistance candidate genes in Cucurbit Genomics Database. And cluster analysis of these genes use DNAMAN6.0software.Downy mildew resistant varieties "M801-3-1" and susceptible varieties "M302-3" by RT-PCR and real-time quantitative RT-PCR analysis of the expression of candidate genes.(4) Candidate R gene RNAi interference vector pRNA002921was constructed.(5) Sequencing and analysis transcriptome of "M801-3-1" and "M302-3" infected by downy mildew.The main results of this study are as follows.(1) Different types of cucumber were identified using the seedling stage vaccination and field evaluation. Five disease-resistant varieties, three resistant varieties, three susceptible varieties identified. Downy Mildew Resistant varieties "M801-3-1" and susceptible "M302-3" were Select the research material.(2) By sequencing the susceptible cucumber varieties and resistant cucumber varieties transcriptome infected by downy mildew, that was obvious difference in between resistant and susceptible varieties transcriptome of downy mildew infection. The expression of the3599proteins were higher in downy mildew resistance variety M801-3-1, of which47protein only expressed in M801-3-1. The expression of the1727proteins were higher in the susceptible cultivar M302-3, of which168proteins expressed only in the susceptible cultivar M302-3. It was Confirmed the presence of specific expression of susceptible R gene in susceptible cultivars.(3) Disease-resistant cucumber varieties downy mildew vaccinated and unvaccinated suppression subtractive hybridization (SSH) cDNA library was constructed using SSH technology.48positive clones were obtained by reverse Northern blot hybridization detection.14UniESTs sequencing were obtained the, including eight singletons and six contigs via molecular biology software. The EST were BLAST in9930cucumber genome database,10EST fragments were found in homologous sequences matching, four EST fragments did not find the same source sequence, presumably new genes.(4) They have oxidoreductase activity.185Arabidopsis resistance to downy mildew gene homologous genes, the two melon resistant to downy mildew gene homologous gene, were identified by BLAST in Cucurbit Genomics Database. RT-PCR test and real-time quantitative RT-PCR showed that Csa001907, and Csa002921their specific down-regulated expression in the disease-resistant varieties may cucumber R gene resistant to downy mildew. RT-PCR test and real-time quantitative RT-PCR proved that most of the R gene is constitutively expressed. R genes upregulated were not found in SSH test. RT-PCR testing and real-time quantitative RT-PCR confirmed Csa001907, and Csa002921genes down-regulated expression in resistant varieties. Real-time quantitative RT-PCR test showed that there is no down-regulated expression the Csa001907and Csa002921in susceptible varieties.(5) Csa002921RNAi expression vector,used in transgenic experiments, was constructed in this study, to further clarify the role Csa002921resistant to downy mildew in cucumber.更多还原