苹果蠹蛾细胞色素P450基因CYP332A19和CYP337B19的克隆及表达分析  

Cloning and expression analysis of cytochrome P450 genes CYP332A 19 and CYP337B 19 in the codling moth,Cydia pomonella(Lepidoptera:Tortricidae)

作　　者：陈高满 陈展博 葛辉 杨雪清[1,2] 王小奇 CHEN Gao-Man;CHEN Zhan-Bo;GE Hui;YANG Xue-Qing;WANG Xiao-Qi(College of Plant Protection,Shenyang Agricultural University,Shenyang 110866,China;Key Laboratory of Economical and Applied Entomology of Liaoning Province,Shenyang 110866,China)

机构地区：[1]沈阳农业大学植物保护学院,沈阳110866 [2]辽宁省经济与应用昆虫教育厅重点实验室,沈阳110866

出　　处：《昆虫学报》2020年第8期941-951,共11页Acta Entomologica Sinica

基　　金：中国科协青年人才托举工程项目(YESS20160085);辽宁省高等学校创新人才支持计划(LCR2018024)。

摘　　要：【目的】本研究旨在通过克隆苹果蠹蛾Cydia pomonella细胞色素P450基因CYP332A 19和CYP337B 19,并对其进行序列和表达分析,以更好地了解这两个P450基因在植物次生物质解毒方面的作用,为进一步的功能研究提供依据。【方法】采用本地BLAST搜索苹果蠹蛾转录组数据库获得细胞色素P450基因cDNA序列,采用RT-PCR技术克隆目的基因的编码区。利用生物信息学软件分析目的基因的序列特征及与其他近缘物种的P450基因的系统进化关系。采用RT-qPCR技术测定目的基因在苹果蠹蛾不同发育阶段(卵、1-5龄幼虫、蛹和成虫)、4龄幼虫不同组织(头部、表皮、脂肪体、中肠和马氏管)以及4龄幼虫分别取食添加0.1%香豆素和0.5%槲皮素的人工饲料2 d后的表达水平。【结果】克隆获得苹果蠹蛾细胞色素P450基因CYP332A 19(GenBank登录号:MF574708)和CYP337B 19(GenBank登录号:MF574697)的全长cDNA序列,开放阅读框(ORF)分别长1518和1491 bp,分别编码505和496个氨基酸,其蛋白质分子量分别为58.586和57.734 kD,理论等电点分别为8.99和7.61。结构域分析显示,CYP332A 19和CYP337B 19中均包含包括血色素结合区在内的5个保守的细胞色素P450结构域。系统发育树显示,苹果蠹蛾CYP332A 19与苹淡褐卷蛾Epighyas postvittana CYP332A 9等CYP332A基因聚在一枝,而CYP337B 19与稻纵卷叶螟Cnaphalocrocis medinalis CYP337B 12和六星灯蛾Zygaena filipendulae CYP337B 11等CYP337B基因聚在另一枝。RT-qPCR分析结果表明,CYP332A 19和CYP337B 19在苹果蠹蛾幼虫期的表达水平高于卵期的,分别在4龄幼虫脂肪体和中肠中的表达量最高。取食分别含0.1%香豆素和0.5%槲皮素的人工饲料2 d后,4龄幼虫体内的CYP332A 19和CYP337B 19相对表达量显著高于对照组(取食含2%DMSO的人工饲料)。【结论】CYP332A 19和CYP332B 19分别在苹果蠹蛾幼虫脂肪体和中肠中高表达,且在取食含香豆素和槲皮素的人工饲料的苹果蠹�【Aim】This study aims to clone the sequences of two cytochrome P450 genes CYP332A 19 and CYP337B 19 from the codling moth,Cydia pomonella,and to analyze their structure properties and expression profiles,so as to further understand the roles of the two genes in the detoxification of plant secondary substances and to provide evidence for further functional research.【Methods】The cytochrome P450 gene cDNA sequences were searched from the transcriptome database of C.pomonella by local BLSTA,and the coding regions of the target genes were cloned using RT-PCR.Bioinformatics software was used to analyze the sequence characteristics and phylogenetic relationship of the target genes with P450 genes of other related species.RT-qPCR was employed to determine the expression patterns of the CYP450 genes in different developmental stages(egg,1st-5th instar larva,pupa,and adult),various tissues(head,epidermis,fat body,midgut,and Malpighian tubules)of the 4th instar larvae,and the 4th instar larvae of C.pomonella fed with artificial diets supplemented with 0.1%coumarin and 0.5%quercetin,respectively,for 2 d.【Results】The full-length cDNA sequences of CYP450 genes CYP332A 19(GenBank accession no.:MF574708)and CYP337B 19(GenBank accession no.:MF574697)were cloned,with an open reading frame(ORF)of 1518 bp and 1491 bp in length,respectively,encoding 505 and 496 amino acids,respectively.The molecular mass of CYP332A19 and CYP337B19 is 58.586 and 57.734 kD,respectively,and their theoretical isoelectric points are 8.99 and 7.61,respectively.Domain analysis showed that both CYP332A19 and CYP337B19 contain five conserved regions of CYP450 genes,including the heme binding domain.Phylogenetic analysis indicated that CYP332A 19 and other CYP332A genes such as CYP332A 9 from Epighyas postvittana were clustered together,while CYP337B 19 and CYP337B genes such as CYP337B 12 from Cnaphalocrocis medinalis and CYP337B 11 from Zygaena filipendulae were gathered in another branch.The RT-qPCR results showed that the mRNA levels of CYP332A

关 键 词：苹果蠹蛾 细胞色素P450 植物次生物质 基因表达 解毒代谢 

分 类 号：Q966[生物学—昆虫学]