中华按蚊拟除虫菊酯解毒酶基因的鉴定，调控和功能

Mosquito-borne diseases are the main threat to human health, and the control of mosquito-borne diseases mainly depend on mosquito vector control through the use of pyrethroid insecticide. Pyrethroid resistance is considered to be the single biggest obstacle for mosquito vector control; however, the molecular mechanism of pyrethroid resistance is still lack of comprehensive understanding. Anopheles sinensis is a main malaria vector in China and southeastern countries in Asia. In recent years, our research suggests that metabolic detoxification be the main mechanism for pyrethroid resistance. Recently, we have sequenced and annotated the genome of Anopheles sinensis, and also sequenced and preliminarily analyzed the genomes of individuals and transcriptomes of pyrethroid-resistant and -susceptible strains from different geographical populations. Using Anopheles sinensis as a model species, this project aims to: 1) identify and classify the P450, CCE and GST superfamilies of genes and conduct research on their evolution through the analysis of the whole genome; 2) identify the P450, CCE and GST genes responsible for pyrethroid metabolic detoxification, and investigate their molecular characterization through transcriptome and resequencing analyses; 3) elucidate the molecular regulation mechanism of candidate detoxification enzyme genes identified, through genome and resequencing analyses; 4) investigate their expression patterns through quantitative PCR in different development stages, different genders and different tissues; 5) verify pyrethroid metabolic detoxification function of these candidate genes by RNAi technique. This work is of important scientific and application value in understanding pyrethroid resistance mechanism, developing resistance detection molecular techniques, and improving the applied effect of pyrethroid.

蚊媒病是人类健康的主要威胁，蚊媒病的控制主要依赖于拟除虫菊酯对媒介蚊虫的控制。拟除虫菊脂抗性是蚊虫控制的最大单一障碍，其分子机制尚不清楚。中华按蚊是我国的主要传疟媒介。近年来，我们的研究表明代谢解毒是拟除虫菊酯抗性的主要机制。最近，我们测序并注释了中华按蚊基因组，测序并初步分析了不同种群抗药性和敏感品系的个体和表达谱。本项目以中华按蚊为模式种，通过基因组分析鉴定和分类P450、CCE和GST三大解毒酶家族基因；通过表达谱和重测序分析鉴定拟除虫菊酯P450、CCE和GST解毒酶基因并分析它们的分子特征；通过基因组和重测序分析揭示候选P450、CCE和GST解毒酶基因的分子调控机制；通过定量PCR弄清它们在不同发育期、不同性别和不同组织的表达；通过RNAi验证它们的拟除虫菊酯代谢解毒功能。这对于弄清拟除虫菊酯抗性机制，开发抗性检测的分子技术，提高拟除虫菊酯的施用效果具有重要的科学和应用价值。

蚊媒病是人类健康的主要威胁，蚊媒病的控制主要依赖于拟除虫菊酯对媒介蚊虫的控制。拟除虫菊脂抗性是蚊虫控制的最大单一障碍，其分子机制尚不清楚。中华按蚊是我国的主要传疟媒介。本项目以中华按蚊为模式种，主要开展了下列工作：1）蚊虫基因电转化显微注射和高效RNAi技术研究，并据此搭建了相关技术平台。2）通过基因组和转录组生物信息学分析分析，中华按蚊P450、CCE和GST三大解毒酶家族基因共分别有112、57和35个基因。对这些基因开展了系统的分类、命名、特征和进化分析。3）通过表达谱、重测序和RNAi分析，鉴定出参与拟除虫菊酯解毒的主要P450基因5个 (AsCYP6Z2, AsCYP6P3v1, AsCYP6P3v2, AsCYP9J5 和AsCYP306A1)，CCE基因4个（AsAe10，AsAce1，AsBe3 和AsUn5），GST基因2个（GSTe2和GSTd2）。4）通过基因组和重测序分析，初步研究了这些P450、CCE和GST解毒酶基因的分子调控机制；通过RNA-seq和qPCR弄清了它们在不同发育期、不同性别和不同组织的表达。5）利用原核表达系统在体外对AsAe7、GSTE6、GSTe2和GSTd2进行重组表达，获得了这些基因的晶体，为后续解析这些基因的晶体结构以及阐释这些基因参与抗性代谢的分子机制奠定了基础。6）在全基因组水平也鉴定和分析了中华按蚊ABC家族、UGT家族、HSP超家族、线粒体、CP家族，CSP家族和iGluR家族的基因。同时，通过RNA-seq和qPCR等技术初步调查了与拟除虫菊酯抗性相关的ABC、UGT、HSP、蚊线粒体基因。结果表明，P450、CCE和GST家族基因在拟除虫菊酯代谢解毒上具有重要作用，但不同种群，起主要作用的基因具有多塑性。这对于弄清拟除虫菊酯抗性机制，开发抗性检测的分子技术，提高拟除虫菊酯的施用效果具有重要的科学和应用价值。