陆地棉黄萎病菌诱导抑制消减杂交cDNA文库的构建与分析

Construction and Analysis of A SSH Library of Upland Cotton Upon Infection with Verticillium dahliae

为探明棉花黄萎病抗性的相关基因,对本实验室的一个高抗黄萎病陆地棉材料进行抑制消减杂交(SSH),以黄萎病菌诱导后48 h和未诱导的植株分别作为Tester和D river,提取总RNA并分离mRNA,通过合成cDNA双链及两次PCR扩增,富集诱导后植株中差异表达的片段。对两次PCR后的产物纯化,连接并转化到大肠杆菌中完成文库构建,共获得了434个阳性克隆。对插入片段进行PCR扩增后发现大小从0.45 kb到0.70 kb不等。指纹图谱分析将所有克隆分为20类,于每一类中取1个克隆进行测序分析。BLAST分析表明大部分克隆片段与其他病原菌诱导缩减库中的EST相似。缩减片段编码的蛋白与拟南芥的P450单加氧酶、病程相关蛋白、类甜蛋白以及几丁质酶等相似。此cDNA文库的建立为进一步分析基因的差异表达及分离抗黄萎病基因奠定了基础。

To explore disease resistant-related candidate genes of Verticillium wilt from upland cotton, a high Verticillium wilt resistant cotton cuhivar was used for construction of a suppression substrative hybridization （SSH）library. The cDNAs from the inoculated seedlings 48 h after disease inoculation were used as the tester and those from the control seedlings as the driver. The total RNA and mRNA were successfully isolated using a method of modified CTAB, followed by the synthesis of double strand cDNA. After two rounds of PCR, the up-regulated fragments responding for induction of Verticillium wilt were accumulated in the SSH library. The purified PCR products were ligated to the vector and transferred into E. coli. 434 clones with 0. 45 kb to 0. 70 kb inserts were obtained. All the clones have been grouped to 20 kinds by fingerprinting and each clone of every kind was chosen to be sequenced. Sequence were blasted to NCBI database. Most of them had high similarity to genes or ESTs from the SSH libraries induced by other pathogens. Their amino acid sequences showed homology to some resistant-related genes, such as cytochrome P450 mono-oxygenase, Thaumatin-like protein, chitinase and PR proteins. Information of this study could be used to isolate the Verticillium wilt resistant-related genes and understand the molecular mechanisms of disease response in cotton.