中国野生华东葡萄抗霜霉病抑制消减文库构建及初步分析

Construction and Preliminary Analysis of the SSH Library of Chinese Wild Vitis pseudoretioulata Resistance to Downy Mildew

【目的】分离和获得中国野生华东葡萄抗霜霉病相关基因片段的EST序列。【方法】以高抗霜霉病的中国野生华东葡萄(V.pseudoreticulata)白河-35-1株系为材料,以田间接种葡萄霜霉菌的幼嫩叶片为处理,以田间自然生长的未接种幼嫩叶片为对照,分别提取RNA,采用抑制消减杂交技术构建抗霜霉病基因的消减正交和反交两个文库,在文库中,选择3个EST序列,用半定量PT-PCR检验构建文库中EST片段的表达情况。【结果】构建的文库中EST片段的大小在150～900bp,经筛选文库,得到正交库有效的ESTs85条,反交库ESTs29条,所获序列经过BLASTn和BLASTx网上序列比对,其中有78条ESTs与GenBank中其它植物相关基因序列有较高的同源性,31条为未知功能序列,已登录GenBank114条ESTs,登录号:FG106789-FG106902。进一步利用半定量RT-PCR对文库中3个基因的表达情况进行了分析,证明这些EST序列是可以表达的。【结论】经初步分析这些序列的功能,涉及抗病信号传导、能量代谢、蛋白质代谢、核酸代谢、光合作用及膜运输和代谢等方面,其中获得与抗病直接相关的ESTs23条,下一步是通过对获得的EST序列进行末端快速分析(RACE)技术,获得抗霜霉病基因全长序列,并进行原核和真核表达研究验证基因全长序列功能,为研究葡萄抗霜霉病的基因提供依据。

[Objective] Isolating disease-resistance ESTs against downy mildew from Chinese wild Vitis pseudoretioulata Baihe-35-1. [Method] To obtain a differentially expressed genes library, a forward-suppression subtractive hybridization library and a reverse-suppression subtractive hybridization library were constructed. The young grape leaves was inoculated with Plasmopara viticola as the treated materials, and the untreated leaves were used as control materials. ESTs from the library were sequenced. Three ESTs was analized with the Semi-Quantitative RT-PCR. [Result] The inserted segments were 150-900 bp in length. Altogether 85 forward SSH library ESTs and 29 reverse SSH library ESTs were obtained. BLASTn and BLASTx analysis revealed that 78 ESTs have high homology with known genes in GenBank, and 31 ESTs are unknown genes. 114 ESTs of them had been submitted to GenBank, and the accession number is FG106789-FG106902. Three genes were selected and proved that these ESTs from the library could express in the plant by reverse transcription semi-quantitative. [Conclusion] The differentially expressed genes are involved in signal transduction, energy metabolism, protein and nucleic acid metabolism, photosynthesis and transmembrane transport, and 23 ESTs of them are probably related to disease resistance. The next step is to obtain downy mildew resistance gene span sequence through rapid amplification of cDNA ends （RACE） technology. And carries out researches on the nucleus and the eukaryon expression confirmation gene for providing a basis for further study of the grape genes resistance to downy mildew.