黄萎病菌诱导下陆地棉抗病品种SSH文库的构建

 采用抑制差减杂交技术，构建了陆地棉抗病品种冀棉20经黄萎病菌诱导8 h、12 h、24 h、48 h后的混合SSH文库，文库共包含800个阳性克隆。对质粒的酶切和巢式PCR结果表明，插入片段大小平均为400 bp。随机挑选20个阳性克隆进行测序，利用BLASTn和BLASTx进行序列相似性分析，发现20条EST都可以找到功能已知的同源序列，其中在BLASTn比较结果中，功能已知的有12条，占全部EST的60%；BLASTx的比较结果中有14条功能已知，占全部EST的70%。这些同源序列涉及到10种与抗病防御相关的基因，包括丝氨酸/苏氨酸激酶、谷胱甘肽-S-转移酶、乙醇脱氢酶、细胞色素P450、ACC氧化酶等。将这些序列与GenBank dbEST库进行比对，发现75%的EST都可以找到同源性较高的序列，它们都来自于植物在生物与非生物胁迫条件下特异表达的cDNA文库，这些胁迫包括病原菌诱导、低温处理、紫外线照射、热激处理、盐处理、氧胁迫、水杨酸诱导等。

SSH Library Construction of Upland Cotton Resistant Cultivar under the Stress of Verticillium dahliae

cDNA library of the Upland cotton resistant cultivar, Jimian 20, was constructed using suppression subtractive hybridization (SSH) and pGEM-T Easy vector under the stress of Verticillium dahliae at 8h, 12h, 24h and 48h post inoculation. This SSH library included eight hundred positive clones. The insert sizes ranged from 0.2kb to 1kb, averaged 400bp, based on restriction endoenzyme digestion and nested PCR. Twenty random clones were selected and sequenced. ESTs similarity analysis via BLASTn and BLASTx showed that all of the twenty ESTs were homologous to the sequences with known functions. Twelve were as known sequences in the BLASTn, accounted for 60% of the total ESTs tested. Fourteen, i.e, 70% of the total, were as known sequences in the BLASTx. According to the classification criteria of plant gene function, these homologous sequences were related to ten kinds of disease resistance genes as those reported, including serine/threonine kinase, glutathione S-transferase, alcohol dehydrogenase, cytochrome P450, ACC oxidase,etc. Querying the GenBank dbEST, as much as 75% of these ESTs were of high homology with the cDNAs that were found under the abiotic or biotic stresses in plants. These stress involved pathogen, low temperature, ultraviolet, heat shocking, salt stress, oxygen stress, and salicylic acid etc.