棉铃虫中肠cDNA文库的构建及EST分析

cDNA library construction and EST analysis of the larval midgut of Helicoverpa armigera (Lepidoptera: Noctuidae)

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。

Midgut is the main target for Bacillus thuringiensis （Bt） action, and a number of insect midgut proteins have been proposed as putative Bt toxin receptors. In order to study the resistance mechanism of the cotton bollworm, Helicoverpa armigera to Bt, we constructed a larval midgut cDNA library of the cotton bollworm using the Switching Mechanism at 5′ end of the RNA Transcript （SMART）technique. The total RNA of 5th instar larval midgut was extracted and the double-stranded cDNA synthesized. After the normalization treatment, cDNAs were digested and ligated into vector, and then the recombinants were transformed into competent cells. The titer was tested and the cDNA library was amplified and sequenced. The quality evaluation showed that the library had a complexity of 2×106 pfu/mL, and the recombination rate was 100%. The average length of inserted cDNA fragments was over 1 000 bp, and 50% fragments were in the full-length form. A total of 1 098 expressed sequence tags （ESTs） were generated successfully after editing and trimming the vector and ambiguous sequences, and 789 unigene sequences were identified, including 132 contigs and 657 singlets. The assembled 789 ESTs were analyzed with Blast in NT, NR and SWISSPORT database of NCBI. The Blast analysis showed that 218 ESTs （27.62%） had no comparable sequences in databases, 119 ESTs （15.08%） had no definite annotations, and the rest 452 ESTs （57.29%） had high homologies with the available sequences, which had definite annotation with over 300 protein products. Through this study, a high-quality cDNA library of the larval midgut of H. armigera has been constructed, which will be a useful tool for studing gene functions in H. armigera midgut.