玉米螟P450基因cDNA的克隆及植物次生物质对其诱导表达

Cloning of Ostrinia furnacalis P450 Gene cDNA and Inducible Expression of Secondary Plant Metabolite

昆虫细胞色素P450是一类在生物代谢中起重要作用的基因家族,承担着许多重要的生理功能,包括对植物次生物质代谢和杀虫剂解毒等.本研究以玉米螟5龄幼虫中肠的总RNA为模板,根据不同昆虫P450基因家族保守氨基酸序列,设计并合成简并引物,利用RT-PCR扩增出了玉米螟细胞色素P450基因cDNA片段,将其克隆到pMD19-T载体进行序列测定.测序获得了编码213个氨基酸残基的641 bp的DNA片段,命名为OfP450(GenBank登录号:EU807990);与已公布的棉铃虫、家蚕、欧洲防风草结网毛虫、小菜蛾和黑腹果蝇等的细胞色素P450氨基酸序列进行比较,其一致性分别为55%、50%、49%、44%和31%.将植物次生物质棉酚、丁布添加入人工饲料中,对玉米螟进行了饲喂实验,采用优化半定量RT-PCR,以18SrRNA为内标,RT-PCR检测进食棉酚、丁布植物化合物的玉米螟中肠P450基因的转录.结果表明,其转录水平能被棉酚、丁布显著诱导.提示本实验克隆的玉米螟细胞色素P450对植物次生物质的代谢作用与P450表达量呈正相关.该研究为下一步将植物介导的RNA干扰技术应用于害虫生物防治提供坚实的基础.

Insect cytochrome P450s constitute one protein family that plays important roles in biological metabolism and performs many important physiological functions, including degrading secondary plant metabolites and insecticides. In this study, total RNA sample obtained from the midgut of fifth Jnstar larva of Ostriniafurnacalis was served as a template and a pair of degenerate primers were designed based on the reported sequences of conservative amino acid regions of P450 protein family in insects. The cDNA encoding the cytochrome P450 protein was isolated from the midgut of fifth instar larvae of Ostrinia furnacalis by reverse transcription polymerase chain reaction （RT-PCR） and the cDNA fragment was cloned into vector pMD19-T. The sequnence analysis showed that the DNA fragment of Ostrinia furnaealis P450 （GenBank accession No. EU807990） was 641 bp, eneoding 213 amino acid residues. The resulting amino aeid sequence showed high identity to that of reported sequences of P450 family from other inseets sueh as Helicoverpa armigera （55%）, Bombyx mori （50%）, Depressaria pastinacella （49%）, Plutella xylosteUa （44%）, Drosophila melanogaster （31%）. By 18S rRNA as an intenaal eontrol, an optimal semiquantitative RT-PCR method was sueeessfully established. The transeription of Ostrinia furnaealis P450 midgut gene was studied, when Ostrinia furnacalis larvae were fed with seeondary plant metabolite like gossypol or DIMBOA. It is showed that the gene transcription was significantly induced by gossypol or DIMBOA. These results suggested that insect cytoehrome P450 could affect secondary plant metabolite and may be related to the high expression of Ostrinia furnacalis P450 gene after treatment. This work layed a foundation for application of plant mediated RNAi technology in bio-control of pest insects.