桑螟细胞色素P450基因的鉴定及在不同来源样本和杀虫剂诱导下的表达分析  

Identification of Cytochrome P450 Genes in Glyphodes pyloalis and Their Expression in Sam ples from Different Areas and After Insecticide Induction

作　　者：苏航[1] 占鹏飞 董久鸣 钱秋杰 刘云财 徐豫松[1] 王华兵[1] Su Hang;Zhan Pengfei;Dong Jiuming;Qian Qiujie;Liu Yuncai;Xu Yusong;Wang Huabing(College of Animal Science, Zhejiang University, Hangzhou 310058, China;Huzhou Academy of Agricultural Sciences, Huzhou Zhejiang 313000, China;Agricultural Technology Extension Center of Zhejiang, Hangzhou 310000, China;Haining Sericulture Technology Extension Station, Haining Zhejiang 314400, China)

机构地区：[1]浙江大学动物科学学院,杭州310058 [2]湖州市农业科学研究院,浙江湖州313000 [3]浙江省农业技术推广中心,杭州310000 [4]海宁蚕业技术服务站,浙江海宁314400

出　　处：《蚕业科学》2018年第2期196-204,共9页ACTA SERICOLOGICA SINICA

基　　金：浙江省公益技术应用研究项目(No.2016C32005);湖州市科技项目(No.2015GZ06)

摘　　要：桑螟（Glyphodes pyloalis）是桑树的主要害虫,因其对专用杀虫剂产生了一定抗性,导致防治效果下降。从分子水平研究桑螟对杀虫剂的抗性机制,有利于制定新的防治策略。通过对桑螟中肠转录组数据分析,鉴定了2个细胞色素P450基因CYP9A20和CYP324A19。系统进化分析表明,桑螟CYP9A20和CYP324A19基因均属于CYP3家族,其中CYP324A19基因编码氨基酸序列与同属鳞翅目的稻纵卷叶螟（Cnaphalocrocis medinalis）的同源序列亲缘关系比较近,序列相似度为63%;桑螟CYP9A20基因编码氨基酸序列同样与稻纵卷叶螟的同源序列亲缘关系较近,序列相似度高达80%。以浙江省杭州、湖州和海宁3个蚕区桑园采集的桑螟样本提取DNA,经qRT-PCR分析幼虫中肠的CYP9A20基因表达水平没有明显差异,而CYP324A19基因的表达水平存在显著差异,其中来自湖州、海宁的桑螟样本中该基因的表达量分别为来自杭州的桑螟样本的3.2倍、3.0倍。用15μg/m L辛硫磷和15μg/m L残杀威处理来自湖州的桑螟幼虫24 h后,qRT-PCR检测CYP9A20基因在幼虫中肠中的表达水平无明显变化,而CYP324A19基因的表达显著上调,辛硫磷、残杀威处理后该基因的表达量分别上调4.0倍、7.8倍。研究结果表明,桑螟的2个细胞色素P450基因在进化上出现了不同分歧,从而导致二者在功能上可能存在明显差异,CYP324A19基因可能通过上调表达参与桑螟对杀虫剂胁迫的响应过程。Glyphodes pyloalis is a major pest of mulberry. G. pyloalis has certain resistance to special insecticides that led to decrease of control effect against it. Studying the resistance mechanism of G. pyloalis to pesticides at the molecular level will help to develop new control strategies. In this study,two cytochrome P450 genes（ CYP9 A20 and CYP324 A19）were identified based on analysis to transcriptome data of G. pyloalis midgut. Phylogenetic analysis showed that both CYP9 A20 and CYP324 A19 belonged to CYP3 family. The amino acid sequence encoded by CYP324 A19 gene had closer relationship with its homologous sequence from Cnaphalocrocis medinalis which is also from Lepidoptera.Their amino acid sequences shared 63% identity. The amino acid sequence encoded by CYP9 A20 gene also had closer relationship with its homologous sequence from C. medinalis,and their amino acid sequences shared 80% identity. qRT-PCR analysis showed that there was no significant difference in CYP9 A20 gene expression levels between the midgut samples of G. pyloalis collected in mulberry fields of Hangzhou,Huzhou and Haining of Zhejiang Province,but the expression level of CYP324 A19 gene was significantly different. The expression level of CYP324 A19 gene in G. pyloalis collected from Hangzhou and Haining was 3. 2 and 3. 0 fold to that from Hangzhou. After G. pyloalis larvae from Huzhou were treated with 15μg/m L phoxim and 15 μg/m L propoxur for 24 h,t he expression level of CYP9 A20 gene in larval midgut had no obvious variation,but that of CYP324 A19 gene was significantly increased. After treatment with phoxim and propoxur,the expression level of CYP324 A19 gene was increased by 4. 0-fold and 7. 8-fold respectively. Our study indicated that CYP9 A20 and CYP324 A19 have varied evolutionary pathway,which can be the cause of obvious functional difference between them.CYP324 A19 gene may be involved in response to insecticide stress through upexpression.

关 键 词：桑螟 细胞色素P450 基因鉴定 表达分析 耐药性 

分 类 号：S888.722[农业科学—特种经济动物饲养] Q786[农业科学—畜牧兽医]