冈比亚按蚊性别决定基因doublesex的克隆、序列分析及表达谱

【目的】doublesex是控制昆虫性别分化的关键基因,决定了昆虫体细胞与生殖细胞的性别。本研究旨在克隆、鉴定重要疟疾媒介冈比亚按蚊Anopheles gambiae性别决定基因doublesex(Angdsx),分析其在雌雄个体内的剪切体及在不同发育时期的表达模式。【方法】基于冈比亚按蚊转录组数据库,比对到Angdsx相关片段,分别以雌雄成蚊c DNA为模板,采用RT-PCR与RACE方法克隆分别获得雌雄个体内Angdsx全长基因,利用生物信息软件对所得序列进行结构域预测、氨基酸序列比对和进化树分析。根据Angdsx特异性表达引物,利用RT-PCR方法研究其在冈比亚按蚊雌雄个体及不同发育时期的表达谱。【结果】分别从冈比亚按蚊雌雄成虫中克隆获得Angdsx c DNA全长序列,分别命名为AngdsxF(Gen Bank登录号:KM978937)和AngdsxM(Gen Bank登录号:KM978938)。Angdsx位于2号常染色体右臂,基因横跨接近80 kb基因组长度。AngdsxF长度为4 874 nt,编码长度为265个氨基酸的雌性特异性蛋白DSXF;AngdsxM长度为3 183 nt,编码长度为633个氨基酸的雄性特异性蛋白DSXM。结构域分析发现Angdsx包括doublesex保守的TRA/TRA-2结合位点、dsx重复序列、富含精氨酸/丝氨酸双肽区、多聚嘌呤增强子序列和RNA结合蛋白结合序列,以及连续的双核苷酸GT为主的重复序列。与AngdsxF相比,AngdsxM具有一个雌性特异性的外显子。AngdsxM在0-2 h卵中高表达,随后逐渐减少,在12-24 h卵中降至最低,之后再次升高;AngdsxF则在6-8 h卵中开始表达。【结论】本研究获得了冈比亚按蚊性别决定基因Angdsx在雌雄个体内的全长序列,Angdsx具有保守的结构域与表达特征。本研究结果为蚊虫性别分化的分子机制及将其最终应用于显性致死昆虫施放技术进行蚊媒的防制提供了理论基础。

Molecular cloning,characterization and expression analysis of sex determiantion gene doublesex from Anonpheles gambiae (Diptera: Culicidae)

【Aim】 Doublesex (dsx) is a key gene involved in the sex determination system, which controls sex determination in the germ line and soma of insects. This study aims to obtain the full-length cDNA sequence of  dsx  from major malaria mosquito  Anopheles gambiae , to reveal the sex-specific differences in splicing patterns of dsx pre-mRNA, and to determine the expression profiles of  dsx  in different developmental stages. 【Methods】 The full-length cDNA of doublesex (Angdsx) were obtained from A. gambiae using RT-PCR and RACE methods. The motif prediction, multiple alignments of amino acid sequences and-phylogenetic analysis were conducted by corresponding bioinformatics softwares. RT-PCR analysis was performed to analyze its sex-specific and development-specific expression profiles. 【Results】 The full-length cDNA sequences of Angdsx  were cloned from female and male adults of A. gambiae, and named Angdsx^M (GenBank accession no. KM978938) and Angdsx^F (GenBank accession no. KM978937), respectively. Angdsx  covers an 80 kb genome region on chromosome 2R. Angdsx^F and Angdsx^M are 4 874 nt and 3 183 nt in length, encoding 265 and 633 amino acids, respectively. Besides the conserved motifs including TRA/TRA-2 binding site, dsx  repeat elements (dsx REs), arginine-serine rich protein, purine-rich enhancer and RNA-binding proteins target sequences, the GT repeat motifs were also indentified in Angdsx by meme program. Compared with Angdsx^M, Angdsx^F contains an extra female specific exon. RT-qPCR analysis displayed that the expression level of Angdsx^M was higher in 0-2 h  embryo and then declined, but increased from 12-24 h embryo again, while that of Angdsx^F raised from 6-8 h. 【Conclusion】 The full-length Angdsx gene was isolated and identified from A. gambiae, which shows conservation of functional domains and sex-specific splicing. This study contributes to understanding the sex differentiation in mosquitoes and may facilitate the application of transgenic mosquitoes based release of insects carrying a dominant lethal (RIDL) technology on vector control.