七星瓢虫保幼激素环氧水解酶基因克隆及表达分析

Cloning and expression analysis of juvenile hormone epoxide hydrolase in Coccinella septempunctata

保幼激素(juvenile hormone, JH)可以调控昆虫滞育,保幼激素环氧水解酶(juvenile hormone epoxide hydrolase, JHEH)是调节保幼激素代谢的关键酶之一。为探索JHEH在七星瓢虫Coccinella septempunctata L.滞育中的调控作用,利用RT-PCR和RACE技术克隆获得七星瓢虫JHEH全长基因,命名为Csjheh(GenBank登录号:MH932586),该基因cDNA全长2 077 bp,开放阅读框(ORF)1 380 bp,编码459个氨基酸,预测蛋白质分子量为51.39 kD,理论等电点(pI)为8.79。疏水性分析结果显示该基因具有典型环氧水解酶的N末端疏水结构。氨基酸序列比对结果表明,Csjheh与中欧山松大小蠹、赤拟谷盗、丽蝇蛹集金小蜂、内华达古白蚁保幼激素环氧水解酶同源性达到64.24%。利用实时荧光定量PCR技术研究其时空表达模式,结果表明Csjheh基因在七星瓢虫成虫初羽化阶段表达量较高,滞育诱导条件下表达量呈先下降后上升的趋势,滞育60 d时与初羽化阶段接近。本研究结果对揭示JHEH参与JH的调控作用,进而调控昆虫滞育提供了理论参考。

Juvenile hormone epoxide hydrolase(JHEH) is one of the most important enzymes in regulating juvenile hormone(JH) metabolism, which is of great significance in insect diapause regulation. In order to explore the regulation of JHEH in diapause of Coccinella septempunctata L., the full-length cDNA of the gene encoding JHEH in C.septempunctata(Csjheh) was cloned by using RT-PCR and RACE. The full-length sequence was 2 077 bp, which consisted of a 1 380 bp open reading frame(ORF) encoding 459 amino acid residues with a molecular mass of 51.39 kD and a theoretical isoelectric point of 8.79. The results of amino acid sequence alignment showed that Csjheh was homologous to those of Dendroctonus ponderosae, Tribolium castaneum, Nasonia vitripennis and Zootermopsis nevadensis, with an amino acid identity of 64.24%. The expression profile of Csjheh was detected by using real-time fluorescent quantitative PCR at different stages of diapause. The results showed that the expression level of Csjheh gene was the highest in the initial emergence stage of the adult ladybug, and then it decreased firstly but increased in the later time of diapause, at which stage the expression level was close to that at the initial emergence stage. The results of this study have important implications for revealing the role of JHEH in the regulation of JH, which plays an important role in the regulation of diapause.