大白菜霜霉菌诱导抑制性消减杂交cDNA文库的构建和分析

Construction and Analysis of Suppression Subtractive Hybridization cDNA Library in Chinese Cabbage(Brassica rapa ssp.pekinensis) Leaves Induced by Peronospora parasitica

以抗霜霉病大白菜双单倍体系(DH)‘T12-19’为材料,构建了霜霉病诱导表达的正向抑制性消减文库,并利用反向Northern斑点杂交技术对768个阳性克隆进行了筛选,共获得57个病原菌诱导上调表达的克隆。测序后得到55条通读表达序列标签(ESTs),对这些ESTs序列进行聚类和拼接分析,共获得50个unigenes。Blast分析表明,37个unigenes与已知基因高度同源,占全部非重复序列的67.3%。对已知功能基因按MIPS的分类方法进行功能分类,发现这些基因的功能主要涉及物质与能量代谢、转录调控、蛋白质合成与代谢、膜及转运、信号转导、抗病防御等。为了验证文库筛选结果的可靠性,采用实时荧光定量PCR技术分析了其中2个克隆BFCH10和BFIA7的表达谱。结果表明,这2个克隆在接种病菌6h后明显上调表达,与反向Northern斑点杂交结果基本一致。

A forward suppression subtractive hybridization（SSH） library was constructed from leaves of Chinese cabbage（Brassica rapa ssp.pekinensis） double haploid（DH） line ＇T12-19＇ resistant to downy mildew which was induced by Peronospora parasitica.By reverse Northern blot analysis,57 up-regulated expressed sequence tags（ESTs） were identified from 768 clones of the SSH library and were sequenced to generate 55 high-quality ESTs.These sequences clustered into 50 unigenes.Blast analyses showed that 37 unigenes shared high identity with genes of known functions involved in energy metabolism,transcription regulation,protein synthesis and fating,membrane transportation,signal transduction,defense,etc.To verify the screening efficiency,the expression profiling of two clones,BFCH10 and BFIA7,was analyzed by real-time PCR.The significant upregulation was observed in leaves of 6 h post-inoculation for these two clones.These results matched the study by reverse Northern blot analysis.