飞蝗细胞色素P450基因LmCYP6FD3的表达及其在杀虫剂解毒中的作用  

Expression profiles of the cytochrome P450 gene LmCYP6FD3 in Locusta migratoria(Orthoptera:Acrididae) and its role in insecticide detoxification

作　　者：朱文雅[1,2,3,4] 吴海花[1,2,4] 张学尧[1,2,4] 刘娇[1,2,4] 张建珍[1,2,4] 马恩波[1,2,4] ZHU Wen-Ya WU Hai-Hua ZHANG Xue-Yao LIU Jiao ZHANG Jian-Zhen MA En-Bo(Institute of Applied Biology, Shanxi University, Taiyuan 030006, China College of Life Science, Shanxi University, Taiyuan 030006, China Institute of Plant Protection, Shanxi Academy of Agricultural Sciences, Taiyuan 030031, China Key Laboratory of Integrated Pest Management in Agriculture, Taiyuan 030031, China)

机构地区：[1]山西大学应用生物学研究所,太原030006 [2]山西大学生命科学学院,太原030006 [3]山西省农业科学院植物保护研究所,太原030031 [4]农业有害生物综合治理山西省重点实验室,太原030031

出　　处：《昆虫学报》2017年第7期742-749,共8页Acta Entomologica Sinica

基　　金：国家自然科学基金重大国际合作项目(31320103921);国家自然科学基金青年科学基金项目(31101463);山西省自然科学基金项目(201601D011073)

摘　　要：【目的】研究飞蝗Locusta migratoria细胞色素P450基因的分子特性和生物学功能。【方法】搜索飞蝗转录组数据库,获得细胞色素P450基因cDNA序列,采用RT-PCR技术克隆目的基因cDNA全长序列。采用实时定量PCR(real-time quantitative PCR,RT-qPCR)技术测定其在飞蝗5龄若虫不同组织(胃盲囊、前肠、中肠、后肠、体壁、精巢、卵巢、肌肉、血淋巴、脂肪体和马氏管)中及不同发育阶段(卵、1-5龄若虫及成虫)的表达水平。采用RNA干扰(RNAi)技术沉默飞蝗2龄若虫细胞色素P450基因,检测基因的沉默效率,并研究该基因干扰后,2龄若虫对杀虫剂马拉硫磷、西维因和溴氰菊酯3种杀虫剂的敏感性。【结果】克隆获得飞蝗细胞色素P450基因的cDNA全长序列(Gen Bank登录号:KT316378),将其命名为LmCYP6FD3,其核苷酸序列全长为1 563 bp,编码521个氨基酸。研究发现该基因在飞蝗5龄若虫马氏管中高表达,其次是后肠和脂肪体中,在其他组织中的表达量相对较低;对LmCYP6FD3在飞蝗不同发育阶段的表达进行检测,发现该基因在飞蝗整个发育阶段均有表达,在若虫期表达量较高。RNA干扰结合杀虫剂生物测定结果表明,LmCYP6FD3在RNA干扰24 h时的沉默效率最高;2龄若虫点滴接触西维因,RNAi处理组(dsLmCYP6FD3注射组)与对照组(ds GFP注射组)相比,死亡率提高了32%。【结论】克隆获得飞蝗LmCYP6FD3的cDNA全长序列,该基因在飞蝗马氏管中高表达,并可能参与西维因在飞蝗体内的解毒代谢。【Aim】To study the molecular characterization and biological function of the cytochrome P450 gene LmCYP6FD3 from the migratory locust,Locusta migratoria.【Methods】The cytochrome P450 gene sequences were searched from the transcriptome database of L.migratoria,the cDNA fragment of the target gene was cloned using RT-PCR,and its expression patterns in various tissues（gastric caeca,foregut,midgut,hindgut,integument,testis,ovary,muscles,hemolymph,fat body and Malpighian tubules） of the 5th instar nymphs and different developmental stages（egg,1st-5th instar nymph and adult） were analyzed using real-time quantitative PCR（RT-q PCR）.The target gene was silenced by RNA interference（RNAi）,the silence efficiency was evaluated by injecting dsRNA to the 2nd instar nymphs,and the susceptibility of the nymphs to three insecticides（malathion,carbaryl and deltamethrin） was assessed.【Results 】 The full-length cDNA of cytochrome P450 gene was cloned（Gen Bank accession no.：KT316378） and named LmCYP6FD3.Its length is 1 563 bp,encoding 521 amino acids.The expression level of LmCYP6FD3 was the highest in the Malpighian tubules of the 5th instar nymphs,moderate in the hindgut and fat body,and lower in other tissues.LmCYP6FD3 was expressed during the whole developmental period and had relatively higher expression level in the nymphal stage.Insecticide bioassay in combination with RNAi showed that the highest silence efficiency of LmCYP6FD3 was achieved at 24 h after RNAi,and the mortality of the 2nd instar nymphs of L.migratoria in the RNAi treatment group（injection with ds LmCYP6FD3） increased by 32% as compared with that of the control group（injection with ds GFP） after exposure to carbaryl by dipping method.【Conclusion】In this study,the full-length of cDNA sequence of LmCYP6FD3 was cloned.This gene has the highest expression level in the Malpighian tubules of L.migratoria and might play a significant role in the detoxification of carbaryl in this locust.

关 键 词：细胞色素P450 飞蝗 基因表达 RNA干扰 杀虫剂 

分 类 号：Q966[生物学—昆虫学]