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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 P450 is a family of genes that play an important role in the metabolism of organisms and carries out many important physiological functions including metabolism of plant secondary metabolites and detoxification of insecticides.In this study, Total RNA was used as a template to design and synthesize the degenerate primers according to the conserved amino acid sequences of different insect P450 gene families. The cDNA fragment of the cytochrome P450 gene of Ostrinia furnacalis was amplified by RT-PCR and cloned into pMD19-T vector Was sequenced to obtain a 641 bp DNA fragment encoding 213 amino acid residues, named OfP450 (GenBank accession number: EU807990); compared with the published cotton bollworm, silkworm, parsnip caterpillar, diamondback moth and black belly Drosophila and other cytochrome P450 amino acid sequences were compared, the consistency of 55%, 50%, 49%, 44% and 31% respectively.The plant ginseng ginseng, Dingbu added to artificial diet, corn The results showed that the transcript level of P450 gene in corn borer of Corn borer (Gossypiella collina) fed gossypol and sophoridine was detected by RT-PCR using 18S rRNA as internal standard and optimized semi-quantitative RT- Was gossypol, cloth Ding Suggesting that the cloning of the experimental cloning cytosolic cytochrome P450 of plant secondary metabolites and the P450 expression of a positive correlation.The study for the next step plant-mediated RNA interference technology applied to the biological control of pests provide a solid foundation .