中华按蚊保幼激素受体基因AsMet分子特性、表达模式及在生殖中的作用分析  

Molecular characterization and expression profiling of juvenile hormone receptor gene AsMet in Anopheles sinensis(Diptera:Culicidae)and an analysis of its roles in reproduction

作　　者：幸晓清 陈斌[1] 司风玲[1] XING Xiao-Qing;CHEN Bin;SI Feng-Ling(Chongqing Key Laboratory of Vector Control and Utilization,Institute of Entomology and Molecular Biology,College of Life Sciences,Chongqing Normal University,Chongqing 401331,China)

机构地区：[1]重庆师范大学生命科学学院昆虫与分子生物学研究所,媒介生物控制和利用重庆市重点实验室,重庆401331

出　　处：《昆虫学报》2024年第11期1475-1483,共9页Acta Entomologica Sinica

基　　金：重庆市教委科学技术委员会青年项目(KJQN202000535);重庆市科学技术委员会基础与前沿研究计划(面上)项目(cstc2019jcyj-msxmX0595);重庆师范大学基金项目(20XLB016)。

摘　　要：【目的】为明确保幼激素受体Methoprene-tolerant基因AsMet在中华按蚊Anopheles sinensis体内的时空表达模式,并探究其对中华按蚊生殖调控和发育的影响。【方法】基于中华按蚊转录组数据,采用RACE克隆AsMet全长cDNA序列并进行分子特征分析;利用qPCR分析AsMet在不同发育阶段(蛹和雌成蚊)、3日龄雌成蚊不同组织[头、胸、腹部前端(腹部前3节)、腹部后端(腹部剩余部分)、中肠、马氏管、脂肪体、卵巢和体壁]中的表达量;通过向中华按蚊末期雌蛹显微注射ds AsMet进行RNAi后,测定AsMet,AsKr-h1和AsVg的表达量,并观测雌成蚊卵巢发育情况、羽化率、产卵量和卵孵化率。【结果】中华按蚊AsMet(GenBank登录号:OR783325)的cDNA序列全长6841 bp,开放阅读框(open reading frame,ORF)长3159 bp,编码1052个氨基酸,预测其分子质量为114.46 kD,等电点为6.63。AsMet有4个保守结构域,包括1个螺旋-环-螺旋结构域、2个PAS结合域和1个C末端保守基序。AsMet与冈比亚按蚊An.gambiae、埃及伊蚊Aedes aegypti和尖音库蚊Culex pipiens的Met聚为一支。AsMet在化蛹后30 h和成蚊大部分阶段显著高表达,且在雌成蚊头和胸部中显著高表达,在中肠、马氏管和卵巢中呈现低表达。显微注射ds AsMet到末期雌蛹后24,48和72 h时,AsMet的表达量较显微注射ds EGFP的对照组分别下调70.05%,41.05%和68.64%,羽化率低于显微注射ds EGFP且在雌蚊交配吸血后卵巢发育不良,产卵量比正常注射ds EGFP的低67.58%,卵孵化率比正常注射ds EGFP的下降93.10%。【结论】AsMet表达被降低后影响中华按蚊卵巢的正常发育且使产卵量和孵化率显著降低。研究结果为深入研究JH调控中华按蚊生殖发育机理奠定基础,对保幼激素信号通路和昆虫生殖调控的分子机制的认识提供理论依据。【Aim】To clarify the spatiotemporal expression pattern of the juvenile hormone receptor methoprene-tolerant gene AsMet in Anopheles sinensis and explore its influence on the reproductive regulation and development of An.sinensis.【Methods】Based on the transcriptome data of An.sinensis,the full-length cDNA sequence of AsMet was cloned by RACE and its molecular characteristics were analyzed.qPCR was used to analyze the expression levels of AsMet in different developmental stages(pupa and female adult)and different tissues[head,throax,anterior part of abdomen(the first 3 segments of abdomen),posterior part of abdomen(the remaining part of abdomen),midgut,Malpighian tubules,fat body,ovary and integument]of the 3-day-old female adults.ds AsMet was microinjected into the last instar female pupa for RNAi,and the expression levels of AsMet,AsKr-h1 and AsVg,the development of ovaries of female adults,emergence rate,number of eggs laid and egg hatching rate were observed and detected.【Results】The full-length cDNA sequence of AsMet of An.sinensis(GenBank accession no.:OR783325)was 6841 bp with the open reading frame(ORF)of 3159 bp in length,encoding 1052 amino acids with the predicted molecular weight of 114.46 kD and the isoelectric point of 6.63.AsMet had four conserved domains,including one helix-loop-helix domain,two PAS-binding domains,and one C-terminal conserved motif.AsMet clustered with Mets of An.gambiae,Aedes aegypti and Culex pipiens.AsMet was significantly highly expressed at 30 h after pupation and at most stages of adults,significantly highly expressed in the head and thorax of female adults,and lowly expressed in the midgut,Malpighian tubules and ovary.The expression levels of AsMet were reduced by 70.05%,41.05%and 68.64%,respectively,at 24,48 and 72 h after ds AsMet microinjection into the last instar female pupa as compared with those in the control group microinjected with ds EGFP.The emergence rate in microinjection group with ds AsMet was lower than that in microinjection group with ds EGFP,an

关 键 词：中华按蚊 保幼激素受体基因 分子特性 表达模式 生殖调控 

分 类 号：Q966[生物学—昆虫学]