关键词: 中国野生葡萄, 抑制消减杂交, 霜霉病, EST序列

Abstract:

【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.

Key words: Chinese wild Vitis, suppression subtractive hybridization(SSH)library, downy mildew, express sequence tag