害虫天敌拟环纹豹蛛全部乙酰胆碱酯酶的特征与生理功能

Acetylcholinesterases (AChEs) and acetylcholine receptors (AChRs) are the key components for the transmission of acetylcholine (ACh), the most important transmitter, in invertebrate central nervous system, which makes it the targets for several classes of insecticides. Spiders are important predator enemies against insect pests in farmlands. However, some spiders are also sensitive to insecticides, which reminds us that the insecticide selectivity between insect pests and spider enemies is highly important. In previous studies in our laboratory, four AChEs had been identified in Pardosa pseudoannulata, the spider enemy against rice planthoppers, and another three candidate AChEs were also found by sequence analysis, among which only two AChE-encoding genes were highly expressed in the brain. These results indicated the function differentiation of multiple AChEs. In this study, with data from transcriptome and genome sequencing, all candidate AChEs in P. pseudoannulata will be figured out, with the analyses on protein structure modeling and tissue expression profile. Using heterologous systems, the catalytic activity, biochemical properties and insecticide sensitivity will be tested on different recombinant AChEs. Combining cell lines from P. pseudoannulata and RNAi technique, the biological roles of different AChEs in cells will be explored. Taking data together, the biological importance of each AChE in P. pseudoannulata will be elucidated. By comparing to insect AChEs, the selectivity mechanisms of insecticides targeting AChEs between insect pests and spider enemies will be clarified in terms of the number, structure, function and property of AChEs. The results will provide theoretical support for the design of selective insecticides and rational use of the existing insecticides.

乙酰胆碱酯酶是无脊椎动物神经系统胆碱能传递的重要组成，是多类杀虫剂的分子靶标。农田蜘蛛是很多害虫的捕食性天敌，与昆虫同属于无脊椎动物，对部分杀虫剂敏感，因此杀虫剂在害虫和蜘蛛天敌之间的选择性十分重要。本实验室前期在水稻害虫稻飞虱天敌拟环纹豹蛛中鉴定了4个乙酰胆碱酯酶（AChE），并发现了另外3个潜在的AChE，但仅2个AChE在脑中高表达，说明多个AChE存在明显的功能分化。结合转录组、基因组数据，本项目将鉴定拟环纹豹蛛全部AChE，分析不同AChE的序列特征、组织表达谱，通过外源细胞表达分析重组酶的生化特征和杀虫剂敏感性，结合细胞培育和RNAi技术探索不同AChE的细胞生理功能，最终阐明AChE在拟环纹豹蛛中的生理功能。对比害虫与蜘蛛AChE，从数量、结构、功能和特征等方面阐明害虫和蜘蛛对作用于AChE的杀虫剂敏感性差异的机制，为杀虫剂的开发和合理使用提供理论支撑。

蜘蛛被认为是乙酰胆碱酯酶（AChE）数量最多的动物物种，但是确证的数量一直没有定论，多个乙酰胆碱酯酶发挥什么样的生理功能、如何发挥功能不清楚，多个乙酰胆碱酯酶是否能够提升天敌类蜘蛛对杀虫剂的抵抗能力，同样未知。为了解决这些关键科学问题，在国家自然科学基金面上项目的资助下，开展了害虫天敌拟环纹豹蛛中乙酰胆碱酯酶鉴定、组织特异性、酶动力学特征、胆碱能经典功能与非神经系统的其他功能、多个乙酰胆碱酯酶的分工协作等方面的研究。项目实施按照计划书内容顺利开展，已经取得较好的研究成果，主要包括一下几个方面。1、鉴定和验证了一个新的乙酰胆碱酯酶PpAChE5，至此发现了5个AChEs，是目前所有生物中被证实的乙酰胆碱酯酶数量最多的物种，这5个AChEs均具有该类蛋白的特征结构。2、明确了5个乙酰胆碱酯酶基因的表达谱，ace1，ace2和ace5在中枢神经系统（脑）中高表达，ace3和ace4在脂肪体和消化道中高表达。3、对包括PpAChE5在内的5个AChEs均进行了体外重组表达，分析了重组酶的酶活动力学特征和对有机磷、氨基甲酸酯类杀虫剂敏感性的差异。4、发现PpAChE1、PpAChE2和PpAChE5协同完成中枢神经系统中乙酰胆碱浓度的调控，当有机磷杀虫剂使用后，通过ace5的上调表达，挽救PpAChE1、PpAChE2和PpAChE5酶活被抑制的影响，提升拟环纹豹蛛对有机磷杀虫剂胁迫的抵御能力。5、建立了拟环纹豹蛛脂肪体细胞系（有限细胞系），发现PpAChE4的编码基因ace4在脂肪体（细胞）中高表达，具有促进细胞凋亡的生理功能。6、建立了通过喂食表达了dsRNA的大肠杆菌干扰拟环纹豹蛛基因的高通量RNAi方法。发表第一标注该项目编号的SCI论文共4篇，第二标注发表SCI论文1篇，授权国家发明专利1项。明确多个乙酰胆碱酯酶协同调控拟环纹豹蛛神经系统中乙酰胆碱浓度，为该类天敌保护与利用、天敌安全性杀虫剂的创制提供了理论基础和技术支撑。