用RNA-seq和QTL定位筛选中华按蚊抗药性基因

Anopheles sinensis is an important vector of malaria in China, pyrethroid insecticides indoor-residual spraying (IRS) is the main measures to eliminate malaria. In recent years, the monitoring results on insecticide resistance to Anopheles sinensis indicate that, due to the rapid development of resistance to deltamethrin, high resistance groups have appeared in some areas. Therefore，the intensive usage of deltamethrin has resulted in the severe insecticide resistance in this vector and it has leaded to significant impediment of progress in malaria elimination program in China. We will apply high-through out sequencing in next generation system (RNA-seq), combined with quantitative trait loci (QTL) mapping techniques in this study. Firstly, the gene expression between deltamethrin-susceptible and resistant An. sinensis populations will be analyzed by high-through out sequencing. The specifically and the differentially expressed genes related to insecticide resistance will be identified by transcriptome analysis. Secondly, the genetic linkage map will then be constructed based on the bulked segregant analysis (BSA) method, combined with random amplified fragment length polymorphism (AFLP) technique. The quantitative trait locus (QTL) mapping will be conducted according to the phenotype of insecticide and genotype of molecular markers to identify the gene loci conferring the insecticide resistance of An. sinensis. The RNA-seq results will be combined with QTL mapping outcome to demonstrate the explicitly insecticide resistant genes in An. sinensis. This study considerably improves the understanding of insecticide resistances in An. sinenis, provides the critical references in genomics study in An. sinensis, lays a sold foundation for insecticide resistance of An. sinensis in molecular mechanism, and can support project of malaria elimination in China.

中华按蚊是我国疟疾的主要传播媒介，拟除虫菊酯类杀虫剂室内滞留喷洒(IRS)是我国疟疾消除阶段的重要措施。近年来，中华按蚊对溴氰菊酯杀虫剂的抗性发展迅速，在某些地区已成为高抗性群体。这不仅影响到现有媒介控制措施的效果，还可能延缓我国消除疟疾行动的进程。 本项目拟结合转录组测序(RNA-seq)和数量性状基因座(QTL)定位技术，利用高通量测序技术平台，对中华按蚊溴氰菊酯杀虫剂抗性不同表型在转录组水平上进行深度测序，分析抗性差异表达基因和特异表达基因；同时利用分群分析（BSA）法结合AFLP分子标记技术，构建中华按蚊杀虫剂抗性分子连锁遗传图谱，结合杀虫剂抗性表型进行数量性状基因定位分析，筛选中华按蚊溴氰菊酯杀虫剂抗性相关基因和主效基因，并进行交互验证,以进一步确定抗性基因。本课题将为深入研究中华按蚊抗性机理提供基础，也为我国疟疾媒介控制措施的制订提供科学依据。

中华按蚊是我国疟疾的主要传播媒介，拟除虫菊酯类杀虫剂室内滞留喷洒(IRS)是我国疟疾消除阶段的重要措施。近年来，中华按蚊对溴氰菊酯杀虫剂的抗性发展迅速，在某些地区已成为高抗性群体。这不仅影响到现有媒介控制措施的效果，还可能延缓我国消除疟疾行动的进程。本项目拟结合转录组测序(RNA-seq)和数量性状基因座(QTL)定位技术，利用高通量测序技术平台，对中华按蚊溴氰菊酯杀虫剂抗性不同表型在转录组水平上进行深度测序，分析抗性差异表达基因和特异表达基因；同时利用分群分析（BSA）法结合AFLP分子标记技术，构建中华按蚊杀虫剂抗性分子连锁遗传图谱，结合杀虫剂抗性表型进行数量性状基因定位分析，筛选中华按蚊溴氰菊酯杀虫剂抗性相关基因和主效基因，并进行交互验证,以进一步确定抗性基因。结果表明：通过蚊敏感品系与抗性品系转录组测序比较，发现共计167个表达上调基因，145个表达下调基因。通过部分目的基因实时荧光定量PCR验证后，目的基因XM_001863852和XM_001845881在蚊实验室抗性品系中的表达量低于实验室敏感品系，表达量变化倍数分别为：0.177,0.548；XM_001845883.1在抗性品系中的表达量是其在敏感品系中的表达量的2.281倍，提示该表皮蛋白基因参与蚊虫抗药性的形成。建立了蚊溴氰菊酯抗性、敏感品系杂交F2代群体，采用BSA法，筛选到溴氰菊酯抗性性状紧密连锁的AFLP分子标记108个，并分别在F2代溴氰菊酯抗性、敏感群体中进行验证，为建立淡色库蚊溴氰菊酯抗性遗传图谱和数量性状分析（QTL）提供了作图群体和分子标记。筛选出的两个表皮蛋白抗性相关基因XM_001863852和XM_001845881，并经相互验证后应用于现场检测蚊虫溴氰菊酯杀虫剂抗性，也取得理想的实验结果。本研究将为深入研究中华按蚊抗性机理提供基础，也为我国疟疾媒介控制措施的制订提供科学依据。