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中肠是苏云金芽孢杆菌Bacillus thuringiensis(Bt)发挥作用的主要部位,中肠上很多蛋白被认为是Bt毒素的结合蛋白。为了探索棉铃虫Helicoverpa armigera对Bt的抗性机制,我们运用RNA转录过程中的5’末端转换机制(Switching Mechanism at5’end of the RNA Transcript,SMART)技术构建了棉铃虫中肠的cDNA文库。先提取棉铃虫5龄幼虫中肠组织的总RNA,合成双链cDNA,经过均一化处理后,酶切、连接载体、转化到大肠杆菌Escherichia coli感受态细胞,最后进行滴度测试、文库扩增并进行EST序列测定。对该文库质量分析表明:文库滴度为2×106pfu/mL,重组率为100%,平均插入片段大于1kb,全长率为50%。最终成功得到1098条表达序列标签(expressed sequence tags,ESTs)序列,经Phrap程序聚类拼接后得到789条单基因簇(unigene),包括132个重叠群(congtigs)和657个单拷贝(singlets)。将聚类拼接后的789条ESTs序列在NT,NR和SWISSPORT库中进行本地化搜索,比对后发现:218条序列(27.62%)没有注解;119条序列(15.08%)注解不明确;452条序列(57.29%)有功能注解,并表达300多种基因产物。构建的文库各项指标均达到要求,该文库的构建为棉铃虫中肠各类基因的克隆及功能分析奠定了基础。
The midgut is the main site where Bacillus thuringiensis (Bt) plays its role. Many proteins in the midgut are thought to bind to Bt toxins. In order to explore the resistance mechanism of Helicoverpa armigera against Bt, we constructed a cDNA library of midgut of Helicoverpa armigera by using the Switching Mechanism at5’end of the RNA Transcript (SMART) technique. The total RNA was extracted from the midgut tissues of the 5th instar larvae of Helicoverpa armigera and double-stranded cDNA was synthesized. After homogenization, the cDNA was digested and ligated into Escherichia coli E.coli competent cells. Finally, the titer was tested and the library was amplified And EST sequence determination. The library quality analysis showed that the library titer was 2 × 106pfu / mL, the recombination rate was 100%, the average inserted fragment was more than 1kb, the full length rate was 50%. Finally, 1098 expressed sequence tags (ESTs) sequences were successfully obtained, and 789 unigene were obtained by Phrap clustering, including 132 congtigs and 657 singlets, . After clustering, 789 ESTs were localized in NT, NR and SWISSPORT libraries. The comparison showed that 218 sequences (27.62%) did not annotate, 119 sequences (15.08%) were not clear, and 452 The sequence (57.29%) was functionally annotated and expressed more than 300 gene products. All the indexes of the constructed library met the requirements. The construction of the library laid the foundation for cloning and functional analysis of various genes in the midgut of Helicoverpa armigera.