基于靶标酶线粒体ATPase的朱砂叶螨抗性分子毒理机制研究

The carmine spider mite,Tetranychus cinnabarinus (Boisduval),is the most serious crop mite pests and widely distributed in China.Due to its short life cycle,abundant progeny and arrhenotokous reproduction,it is able to develop resistance to acricides very rapidly.Resistance mechanisms elucidated in mites to date are similar to those found in insects and consist chiefly of reduced penetration,enhanced degradation,and insensitive target,either separately or together.ATPase as a modified target site has been reported in a number of studies.Acaricides affect the function of this protein,but it is not clear that this is what leads to biological activity.Through the studies in the project,we seek to elucidate that the molecular mechanisms of ATPase as a potential target-site resistance in the carmine spider mite.The contents as follows:(1)Resistance selection and biochemical characterization of ATPase;(2)Cloning and sequence analysis of the ATPase genes from susceptible and resistant spider mite.The molecular basis of target-site resistance was examined by comparing sequences of different strains;（3）Amplification of ATPase gene with RT-PCR and its expression influenced by acaricide in different strains of Tetranychus cinnabarinus.Purification and partial characterization of ATPase ;（4）Expression of ATPase gene from susceptible and acaricide-resistant in Sf9 and properties of ATPase protein..The aim of this study is to detect the mutation point and expressio level of these important genes in susceptible and acaricide-resistant in order to clarify role of these genes mutation point in acaricide resistance.

线粒体ATPase是能量合成过程中的关键酶，自然界最精妙的纳米机器之一，是目前分子生物学、生物化学、生物物理学等领域的研究热点，该酶也是杀螨剂的作用靶标之一，在害螨抗药性形成中起着不可忽视的作用。本项目以重要农林害螨朱砂叶螨为对象，运用昆虫生理生化和分子生物学等学科知识，比较朱砂叶螨敏感和抗性品系对炔螨特等常用杀螨剂的敏感性及ATPase生化特性差异；应用RT-PCR和 RACE技术克隆朱砂叶螨ATPase基因全长序列，解析杀螨剂处理后朱砂叶螨不同发育阶段、不同品系中ATPase基因表达模式，找出与抗性相关的突变位点；通过ATPase的异源表达并分析表达产物的酶学特性，从而阐明基于线粒体ATPase靶标的朱砂叶螨抗性机制。研究结果不仅能在理论上阐明线粒体ATPase靶标抗性的分子毒理学机理，在生产实践中对害螨抗药性的综合治理具有重要参考价值。

线粒体 ATPase 是能量合成过程中的关键酶，也是杀螨剂的作用靶标之一，在害螨抗药性形成中起着不可忽视的作用。本项目以重要农林害螨朱砂叶螨为对象，比较敏感和抗性品系对炔螨特等常用杀螨剂敏感性及ATPase生化特性差异，解析朱砂叶螨不同品系、不同发育阶段和药剂诱导其ATPase基因表达模式，寻找与抗性相关的基因，阐明基于线粒体ATPase靶标的朱砂叶螨抗性机制。通过研究，课题组建立了朱砂叶螨敏感和抗炔螨特品系，明确了朱砂叶螨对炔螨特抗药性发生发展规律；明确了GSTs与朱砂叶螨对炔螨特解毒代谢抗性密切相关，并首次报道GSTs介导的朱砂叶螨炔螨特抗药性研究；克隆获得朱砂叶螨线粒体ATPase基因全长序列，发现朱砂叶螨TcATPase7，TcATPase9 和 TcATPase11基因在不同发育阶段、不同品系和药剂诱导中表达显著差异；构建了TcATPase11在真核细胞表达体系，纯化获得PR-TcATPase11 与 SS-PR-TcATPase11表达蛋白样品并解析其特性。依托项目资助，课题组发表研究论文4篇，其中SCI收录2篇，中文核心期刊2篇。1人获得博士学位，1人获得硕士学位，1人正在攻读硕士研究生，2人晋升职称。本研究对于深入解析以线粒体ATPase 为靶标的螨类抗性机制具有重要意义，以此为基础可进一步研究螨类线粒体ATPase靶点、结构与功能特征，为杀螨剂研发提供精准靶标。