利用SSH技术鉴定黄瓜抗霜霉病相关基因

Using SSH Technology to Identify Relevant Genes Resistant to Cucumber Downy Mildew

采用SSH技术对黄瓜抗霜霉病自交系M801-3-1侵染霜霉病菌前后的差异表达基因进行了研究。利用SSH技术构建抗病黄瓜材料接种霜霉病菌和未接种差异表达的差减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。通过SSH-EST代表基因功能的分析,说明抗氧化胁迫能力和叶绿体的重建和保护机制对抗病品种抗病有重要作用。同时,提出SSH-EST代表的clpb、HSP70、HSP22和肽脯氨酰顺反异构酶还可能参与了R基因介导的防御反应,smHSP有可能就是R蛋白的"保卫靶",负责监测细胞内的异常,这一发现为揭示活性氧机制和R基因介导的防卫机制之间的关系提供了关键证据。

Adopting SSH technology,this paper studied on the difference expressive genes of downy mildew resistant cucumber inbred line ‘M801-3-1’ before and after the infection of downy mildew.Disease-resistant cucumber material vaccinated downy mildew and unvaccinated cDNA library was constructed using SSH technology.Forty-eight positive clones were obtained by reverse Northern blot hybridization detection, and 14 UniESTs were obtained including 8 singletons and 6 contigs via molecular biology software.The SSH-EST genes function analysis indicated that oxidative stress resistance and chloroplast reconstruction and protection mechanisms had very important role on disease resistance.At the same time,the paper put forward that Clpb,HSP70,HSP22 and peptidyl-prolyl cis-trans isomerase might be involved in the R gene-mediated defense reaction,and smHSP might be the ‘defending target’ of R protein,responsible for monitoring intracellular exception.This study has provided a key evidence for revealing the relations between active oxygen mechanism and R gene mediate protection mechanism.