蝶蛹金小蜂毒液富半胱氨酸结构域蛋白的功能及其作用机理

In this proposal, we willstudy on the functions and the action modes of cysteine-rich domain-containing proteins (CDPs) isolated from the venom of Pteromalus puparum. Post analyses of the tissue specific expression profile of the mRNA in P. puparum venom gland and the proteins composition of the venom, respectively, the venom CDPs has been identified. For our recent researches, we will first clone the full length cDNAs encoding these CDPs mentioned above. After that, we will recombinantly express the cDNA fragments encoding the mature peptides of these CDPs, respectively, in prokaryotic system, following the preparations of the antiserums against these recombinant proteins. We will investigate the mRNA and protein expression profiles of the CDPs, related to the tissue distribution and the developmental dynamics, by real-time PCR and immunoblotting. Additionally, we will express the sequences encoding the mature peptides of the CDPs utilizing the insect cell or yeast system, and study the functions of the recombinant CDPs and their influences on the importantly physiological processes, including immunity, growth and development of host pest. We will use the methods of Co-IP or yeast two-hybrid, RNA-seq, pharmacological investigation, and RNA interference to search the target proteins of CDPs in the host, to analyze alterations of the transcripts levels of the host related genes, to discover the types of the G protein-coupled receptors in the host, and to illustrate the regulating effects of CDPs on Ca 2+ signaling transduction pathways of the host. Following these researches, we are able to explore the mechanisms at physiological and molecular levels. Finally, these results probably obtained here may provide the new theories and methods to further reveal the compositions and functions of the parasitoid venoms and also the new mechanisms of the venom components regulating the host physiological processes.

在采用转录组及蛋白组学方法分别对蝶蛹金小蜂毒腺mRNA特异性表达谱及其毒液蛋白质组成进行分析并鉴定获得毒液富半胱氨酸结构域蛋白（CDPs）基础上，克隆获得编码CDPs的cDNA全序列。分别对其成熟肽编码序列进行原核表达，制备抗体，分析其编码基因的转录与表达的组织特异性及其随寄生蜂发育的动态变化。采用昆虫或酵母表达系统，对CDP编码序列进行表达，研究重组CDPs的功能，及其对寄主菜粉蝶免疫反应及生长发育等重要生理过程的影响。采用免疫共沉淀或酵母双杂交方法、RNA-seq方法、药理学及RNA干扰等系列方法，分别寻找CDPs作用于寄主体内的靶蛋白，分析CDPs处理后寄主相关基因的转录水平变化，明确CDPs作用于寄主体内G蛋白偶联受体类型及其对寄主体内钙离子信号转导途径的调控作用，进而揭示其作用的生理与分子机理。最终，为揭示寄生蜂毒液蛋白组成与其功能，及其调控寄主生理反应的机理提供理论与方法支撑。

寄生蜂是一类重要的天敌资源，其与寄主害虫间的互作机理研究一直是寄生学与昆虫免疫学的研究热点。本项目瞄准蔬菜害虫生物防治之科学问题，以十字花科蔬菜世界性重要害虫菜粉蝶，及其蛹期优势寄生蜂蝶蛹金小蜂为研究系统，综合采用基因组学、转录组学组学、蛋白组学等方法，基因鉴定与克隆、基因表达模式分析、蛋白重组表达与产物纯化、生物活性测定、RNA干扰等等技术与手段，就蝶蛹金小蜂毒液富半胱氨酸蛋白CDPs编码基因的鉴定与筛选，毒液重要的CDPs编码基因cDNA全序列克隆及其表达模式分析，重要CDPs编码基因的重组表达及重组CDPs生物学功能研究，重要CDPs调控寄主害虫生理反应的分子机理研究等方面开展系统工作。取得以下主要成果：1）进一步完善蝶蛹金小蜂基因组组装与基因注释工作，完成蝶蛹金小蜂丝氨酸蛋白酶抑制剂类型CDP编码基因的注释工作；2）筛选并鉴定出蝶蛹金小蜂毒液重要的CDPs（Kazal型蛋白酶抑制剂，PpKazals）编码基因，并完成其cDNA全序列克隆；3）明确6个重要的PpKazal编码基因的时空表达模式；4）建立寄生蜂毒液PpKazal编码基因的重组表达体系，成功获得4个具有生物活性的重组表达产物；5）明确PpdxKazal-1、PpdxKazal-5及Ppthrombin-4均可显著抑制寄主害虫PPO激活级联反应，进而抑制寄主血淋巴黑化过程，探明Ppthrombin-2及Ppthrombin-4抑制水解蛋白酶活性的底物谱，推测PpdxKazal-1和PpdxKazal-5很可能作用于寄主血淋巴中新的蛋白酶靶标；6）解析并明确毒液PpdxKazal-1和PpdxKazal-5重组表达产物在转录水平上调控寄主害虫生理反应的分子机理；7）相关研究成果已发表SCI 期刊论文10篇，申报国内发明专利4项。上述研究结果，一方面为揭示寄生蜂毒液蛋白组成与其功能，及其调控寄主生理反应的机理提供理论与方法支撑；另方面也为建立以害虫免疫系统为靶标的新型杀虫剂－免疫抑制剂的筛选与研发平台，及为基于害虫免疫抑制的新型害虫生物防治策略研究奠定理论基础与技术支撑，进而为“绿色农业”可持续发展提供害虫控制的新途径。