紫外线照射辣椒叶片cDNA文库构建及部分防御信号基因cDNA的检测

对抗病的朝天椒地方品种,以紫外线(UV)处理叶片并提取叶片总RNA,采用SMARTTMcDNA文库构建试剂盒构建了一个cDNA文库.该文库含有2.6×106个独立的噬菌斑克隆,扩增后滴度达到1.5×109pfu.μL-1,随机挑取的噬菌斑PCR检测发现重组率达90.0%.在搜索、拼接与植物WRKY、bZIP、AP2和E2等重要调节基因同源的辣椒表达序列标签(ESTs),获得重叠群的基础上,分别设计上述基因ESTs重叠群特异性引物,以cDNA文库为模板进行PCR扩增.结果表明,部分上述基因cDNA存在于文库中.因此,本研究所构建的文库可用于进一步分离响应UV的重要调节基因cDNA的分离.

Construction of cDNA library with RNA isolated from leaves of Capsicum annuum challenged with UV and detection of cDNA of several genes

Total RNA was isolated from leaves of Capsicum annuum challenged with UV and a cDNA library with more than 2.6×106 individual clones was constructed with SMARTTM cDNA construction kit,the tittering of the amplification cDNA library was more than 1.5×109 pfu·μL-1 and the recombinant ratio was more than 90%.ESTs(expressed sequence tag) homology to WRKY,bZIP,AP2 protein,E2 ubiquitin-conjugating enzyme of Arabidopsis thaliana was searched and assembled with DNAMAN,the specific primers were designed according to the sequences of different contigs.The result of PCR with cDNA library as template indicated that a member of the multiple gene family of WRKY,bZIP,AP2 protein,E2 ubiquitin-conjugating enzyme of C.annuum existed in the cDNA library.The cDNA library could be used to isolate the cDNAs of pepper DS genes expressed under the challenge of UV.