致倦库蚊几丁质合成酶基因CqCHS1和CqCHS2的表达与分析

Expression and Analysis of Chitin Synthase Gene CqCHS1 and CqCHS2 in Culex quinquefasciatus

利用生物信息学方法比较分析致倦库蚊几丁质合成酶CqCHS1、CqCHS2的基因结构、氨基酸序列、蛋白理化特性,预测蛋白跨膜结构和空间结构,并基于6种昆虫绘制分子进化树,随后使用Real-time PCR方法探测CqCHS1、CqCHS2基因在致倦库蚊不同发育时期的表达谱。结果显示:CqCHS1、CqCHS2基因长度分别为39 206 bp和17 920 bp,分别包含10个和9个外显子;CqCHS1、CqCHS2氨基酸序列长度分别为1 579 aa和1 214 aa;理论分子量分别为179.581 k D和138.816 k D,等电点分别分别为6.21和8.28,亲水性平均系数分别为-0.076和0.101;预测跨膜次数分别为17次和13次;二者具有相近的二级结构组成和三级结构。进化树显示,6种昆虫CHS1和CHS2分别聚类,形成两大分支。它们CHS的同源性与物种的亲缘关系基本一致。Real-time PCR结果显示,CqCHS1在卵期呈低表达,幼虫时期表达增高,到蛹期达到峰值,成虫期表达量降低。CqCSH2在幼虫期表达量最低,卵和蛹期表达量较低且差异不大,成虫期表达量最大。本研究的结果可为深入了解致倦库蚊的发育情况提供有价值的信息,并为围绕几丁质代谢为基础的杀虫剂研制,提供潜在的药物靶点。

The gene structures, amino acid sequences, protein physicochemical characteristics, protein transmembrane helices and senior structures of CqCHS1, CqCHS2 in Culex quinquefasciatus were compared and analyzed via bioinformatics methods, following with the phylogenetic tree of CHS proteins based on 6 insects; Then the relative expression of CqCHS1, CqCHS2 in different development stages of C. quinquefaseiatus was determined by Real-time PCR. Real-time PCR results indicated that the gene Cq CHS1 lowly expressed in eggs, then increased in larva until the maximum expression in pupal stage, and deduced in adult at last; the gene CqCHS2 presented the minimum expression in larva but the maximum that in adult. There was no significant difference in expression between larva and pupal stage, during which both expressed at lower level. The results displayed that genes CqCHS1 and CqCHS2 had the length of 39 206 bp and 17 920 bp, containing with 10 and 9 exons. CqCHS1 and CqCHS2 proteins had the length of 1 579 and 1 214 amino acid, the molecular weight of 179.581 kD and 138.816 kD, the pI of 6.21 and 8.28, the GRAVY of-0.076 and 0.101. There were 17 transmembrane helices predicted in CqCHS1, while as 13 in CqCHS2. Both proteins contained similar components of secondary structures and tertiary structures. The phylogenetic tree showed a positive correlation between the homology of the CHS proteins and genetic relationship of species for the and form two branches. Our study would provide 6 insects, in which CHS 1 and CHS2 clustered separately valuable information for development research in C. quinquefasciatus, and offer potential drug targets for insecticides based on the chitin metabolism.