烟夜蛾几丁质酶基因的克隆与表达
Cloning and expression of a chitinase gene from Helicoverpa assulta
涂洪涛1,2,付晓伟1,郭线茹1*, 李为争1 ,罗梅浩1 原国辉1
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作者单位：1. 河南农业大学植物保护学院 郑州 450002； 2. 中国农业科学院郑州果树研究所 郑州 450009
中文关键词：烟夜蛾； 几丁质酶； 基因克隆， 原核表达
英文关键词：Helicoverpa assulta, chitinase, gene cloning, prokaryotic expression
中文摘要：运用RT-PCR技术扩增编码烟夜蛾Helicoverpa assulta(Guenée)幼虫几丁质酶基因的cDNA片段，将其克隆至pMD18-T载体，获得该基因的成熟蛋白阅读框序列。将该基因重组到表达型质粒pGEX-4T-2中，并转化入原核细胞中表达，序列测定结果表明，烟夜蛾幼虫几丁质酶基因的成熟蛋白阅读框全长1 338 bp，编码445个氨基酸残基，预测分子量和等电点分别为50.1 kDa和9.26；推导的氨基酸序列与其近缘种棉铃虫几丁质酶氨基酸序列的一致性达99％，与其他6种昆虫几丁质酶的氨基酸序列也高度一致(65％～76％)，并具有几丁质酶的典型特征。将该基因克隆到原核表达载体pGEX-4T-2上并转化BL21，SDS-PAGE和Western印迹分析表明，经IPTG诱导，76 kDa附近没有特异蛋白条带出现，表明烟夜蛾几丁质酶基因不能在原核表达载体pGEX-4-2中表达。
英文摘要：The cDNA encoding the chitianse (named as Has-Chit) was isolated from adipose tissue and epidermis of Helicoverpa assulta(Guenee) larvae by reverse transcription polymerase chain reaction (RT-PCR). The cDNA fragment was further cloned into pMD18-T vector, and then constructed into pGEX-4T-2 for expression in prokaryotic cells. Sequence analyses showed that the open reading frame of Has-Chit was 1 338 bp in length, encoding 162 amino acid residues; its predicted molecular weight and isoelectric point were 501 kDa and 9.26, respectively. The deduced amino acid sequence showed a high identity with the reported sequences of chitinases from its sibling species H. armigera (99%) and other 6 species of insects (65%～76%), all of which shared the typical structural features of chitinases from other insects. The fragment containing Has-Chit gene was inserted into pGEX-4T-2 for expression in prokaryotic cells (BL21), and induced by IPTG. The results showedthat the specific protein of about 76 kDa was not observed by SDS polyacrylamide gel electrophoresis, which indicated that extrinsic gene, Has-Chit gene, can not expressed in E. coli.