野桑蚕细胞色素P450家族CYP4M5基因的克隆和诱导表达  

Cloning and Induced Expression of CYP4M 5—A Member of Cytochrome P450 Family From Bombyx mandarina

作　　者：王燕红[1] 王东[1] 李兵[1,2] 赵华强[1] 许雅香[1,2] 沈卫德[1,2] 

机构地区：[1]苏州大学基础医学和生物科学学院,江苏苏州215123 [2]苏州大学蚕桑研究所,江苏苏州215123

出　　处：《蚕业科学》2009年第1期78-83,共6页ACTA SERICOLOGICA SINICA

基　　金：国家重点基础研究发展计划"973"项目(编号2005CB1-21005)

摘　　要：细胞色素P450单加氧酶系在生物体对外源化合物的代谢和内源化合物的调节中都起着非常重要的作用,昆虫对杀虫剂产生抗性与单加氧酶系介导的代谢反应有关。以野桑蚕(Bombyxmandarina)中肠为材料,依据家蚕(Bombyxmori)基因组P450基因预测序列设计一对引物,采用RT-PCR方法克隆了一个P450家族基因的cDNA序列,命名为CYP4M5(GenBank登录号:EU273876)。序列分析表明,野桑蚕CYP4M5基因编码区长度为1 512 bp,编码503个氨基酸,相对分子质量约为58 kD,等电点7.8。同源性分析表明该基因与家蚕CYP4M5的同源性最高,达97.42%。分析鉴定了该基因在野桑蚕各组织中的mRNA表达水平以及溴氰菊酯诱导24 h后mRNA的表达水平,结果显示:在正常野桑蚕幼虫体内,CYP4M5在脂肪体中的表达水平最高;野桑蚕经5 ng/μL溴氰菊酯药液处理后,CYP4M5在中肠和脂肪体中表达量分别增加1.1倍和4.4倍。推测该基因可能参与野桑蚕对杀虫剂的解毒代谢作用。The cytochrome P450-dependent monooxygenases paly an extremely important role in metabolic system involved in the catabolism and anabolism of xenobiotics and endogenous compounds. Cytochrome P450 is a key constituent which acts as the terminal oxidase in monooxygenase systems to oxidize diverse substrates. Insecticide resistance has been proven to be correlated with monooxygenase-mediated metabolism. According to the predicted P450 sequences from the genome of Bombyx mori, a pair of primers were de- signed and a novel gene named CYP4M5 of cytochrome P450 was successfully cloned from the midgut mRNA of Bombyx mandarina by RT-PCR （GenBank accession number. EU273876）. Sequence analysis revealed that this gene contains a 1 512 bp ORF, encoding a protein of 503 amino acids. The predicted molecular weight and isoelectric point of this protein was 58 kD and 7.8, respectively. Homology analysis showed that this gene is the homolog of CYP4M5 from Bombyx mori. The results of semi-quantitative RT-PCR showed that this gene was highly expressed in fat body. And the expression level could be increased by induction with cypermethrin. Treatment with 5 ng/uL cypermethrin could increase the expression level in midgut and fat bodv of the Inrvae by 1. 1 fold and 4.4 fold, respectively. It is inferred that CYP4M5 gene may be involved in detoxification to insecticide in Bombyx mandarina.

关 键 词：野桑蚕 细胞色素P450 CYP4M5基因 基因克隆 半定量RT-PCR 

分 类 号：S885.9[农业科学—特种经济动物饲养] Q78[农业科学—畜牧兽医]