Identification Of Cucumber Downy Mildew Resistance Genes And Expression Analysis

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.