麦蚜杀虫剂抗性机理的基因调控网络研究

Wheat aphids are notorious and important pests in wheat and have caused huge damages in wheat production in China. The control of them is primarily dependent on the application of insecticides, but the control efficiency is seriously compromised due to dramatic increase of resistance from abuse of insecticides. The mechanism of insecticide resistance in wheat aphids remains unclear according to literatures, which retards the understanding of formation and control of insecticide resistance. In this program, we target two wheat aphids, Macrosiphum avenae and Rhopalosiphum padi. The insecticide resistance status of the two aphids is investigated in fields. Strains with different resistance levels towards organophosphate, pyrethroid, carbamate, and neonicotinoid insecticides, respectively, are screened in the lab. The gene expression profiles of these strains are compared on transcriptomic level with RNAseq sequencing in order to identify gene regulation network and metabolic pathways involved in insecticide resistance. RNAi technique is applied to verify the functions of main genes in the regulation network. Evolutionary characteristics of three detoxification enzyme families, carboxylesterase, cytochrome P450 monooxygenase, and glutathione S-transferase, are analyzed to predict the developmental trend of insecticide resistance in wheat aphids. The innovation of this program is that gene regulation network, in stead of individual genes, is the research target to view the whole picture of resistance mechanism in aphids. The results will promote the progress of research and management of insecticide resistance in aphids.

麦蚜是我国小麦的重要害虫之一，对我国农业生产造成极大危害。多年来对麦蚜的防治仍以化学防治为主，过度使用化学杀虫剂造成麦蚜的抗药性迅速增加，防治难度越来越大。国内外对麦蚜抗药性产生的分子机理研究尚不清晰，制约了对麦蚜抗药性形成的认识以及有效的控制。本项目以小麦上混合发生的麦长管蚜和禾谷缢管蚜为研究对象，在确定麦蚜种群抗药性水平的基础上，筛选出对有机磷、拟除虫菊酯、氨基甲酸酯和新烟碱类杀虫剂不同抗性水平的品系，在转录组水平上比较这些品系基因表达谱的差异，鉴定引起麦蚜抗药性的基因调控网络和代谢通路，利用RNAi技术验证网络上主要基因的功能，分析三大解毒酶基因家族的进化特征，预测麦蚜抗药性发生、发展的进化趋势。本项目的创新点是通过基因网络来阐明抗性机理，突破过去从单基因水平解释抗药性成因的模式，为克服蚜虫抗药性奠定科学基础。研究成果将推进蚜虫抗药性研究和治理的进程。

本项目从河北、山东、黑龙江、河南、陕西采集了不同地理种群的麦长管蚜和禾谷缢管蚜，测定了它们对有机磷（辛硫磷、毒死蜱）、氨基甲酸酯（灭多威）、新烟碱类（吡虫啉）、拟除虫菊酯（溴氰菊酯）5种杀虫剂的抗性。发现麦长管蚜地理种群对所测杀虫剂仅产生了三倍以内的低等抗性，泰安地区的麦长管蚜对5种杀虫剂均产生了低抗，沧州、保定和河南种群对部分杀虫剂具有低抗，而陕西、石家庄和哈尔滨种群对5种杀虫剂均敏感。大部分禾谷缢管蚜种群对所测杀虫剂敏感，仅哈尔滨地区的种群对溴氰菊酯有低水平抗性。为了进一步揭示潜在的抗性机制，我们检测了两种麦蚜各种群三大解毒酶系的活性，即羧酸酯酶（CarE）、细胞色素P450单加氧酶和谷胱甘肽S-转移酶（GST）。结果表明：麦长管蚜泰安种群的CarE、P450与敏感品系相比活性显著升高，杨凌和哈尔滨种群的GST活性显著升高，沧州种群的P450活性显著升高；禾谷缢管蚜哈尔滨和封丘种群的GST与敏感品系相比活性显著升高。利用高通量测序技术，对麦长管蚜和禾谷缢管蚜转录组测序，获得两种麦蚜CarE、GST和P450家族的基因序列，并通过进化分析进行了亚家族分类。为了探讨蚜虫抗药性的基因调控网络和代谢通路，测定了三种蚜虫对吡虫啉、久效磷和灭多威杀虫剂筛选的转录组变化，找到了蚜虫应对化学杀虫剂的基因调控网络。在吡虫啉筛选下，蚜虫的水解酶活性、钙离子结合的基因被上调。在久效磷筛选下，蚜虫的水解酶活性、跨膜转运活性、氧化还原酶活性、蛋白二聚化活性和锌离子结合活性的基因被上调。在灭多威筛选下，蚜虫的DNA结合、RNA结合、ATP结合、GTP结合、水解酶活性、单加氧酶活性、蛋白二聚化活性，转移酶活性和激酶活性的基因被上调。研究成果加深了对麦蚜抗药性形成的分子机制的认识，为研究其它蚜虫抗药性的分子机理提供了科学依据，为将来研制开发快速检测麦蚜种群抗性水平和抗性机理的试剂奠定了坚实的理论基础。