蝶蛹金小蜂毒液免疫抑制小肽的鉴定、结构与功能研究

For the experiments that will be carried out in this project, series of technological methods will be used, including peptide purification and identification, gene cloning and recombinant expression, biological activity assay, and protein structural analysis. Based on the composition of small immunosuppressive peptides from the venom of a pupal stage endoparasitoid, Pteromalus puparum, of its host Peris rapae, the genes encoding small immunosuppressive peptides will be first screened and cloned. Second, the transcripts and expression levels of different peptide genes in different tissues and developmental stages of the parasitoid will be investigated. Third, the selective small peptides will be expressed in yeast system (or be chemically synthesized) and the biological functions of their recombinant or synthesized products will be assayed. Forth, the structures of the small peptides will be analyzed and the relationship between structure and function will also be studied. Fifth, the small peptides will be recombinantly expressed with a fusion protein, Galanthus nivalis agglutinin (GNA), and the influences of the fusion peptides on the physiologicail aspects of host and no-host insects will be determined. Finally, the structural and functional compositions of small immunosuppressive peptides from parasitoid venom and the application prospects of these peptides will be considered. These results obtained can provide the new theories and methods to further reveal the composition and function of parasitoid venom and also the new mechanism of the venom components regulating the host innate immune responses. On the other hand, these results also can establish the platform for the developing and screening of immunosuppressive agents, which will be a new kind of pesticides targeting to the innate immune system of pest insects. At the same time, these results will also contribute to the newly biological control strategies, based on the immunosuppression of pest immune responses. In the end, the completing of this project will supply the newly pest control strategies and approaches for the production and sustainable development of the green agriculture in China.

采用小肽分离与鉴定、基因克隆与表达、生物活性测定及小肽结构解析等技术，在明确菜粉蝶重要寄生蜂蝶蛹金小蜂毒液中免疫抑制小肽组成的基础上，筛选并克隆免疫抑制小肽的编码基因，探明免疫抑制小肽编码基因转录与表达水平的时空变化；进而采用酵母表达系统对免疫抑制小肽进行表达（或采用化学法对其进行合成），测定免疫抑制小肽生物学功能，并解析其结构，研究结构与功能间关系；最后对免疫抑制小肽与凝集素GNA的融合蛋白进行表达，测定其对寄主与非寄主生理功能的影响。最终，明确该蜂毒液免疫抑制小肽的结构与功能，并探明其在害虫生防中的应用前景。一方面为深入揭示寄生蜂毒液组成与功能，及功能组分调控寄主先天免疫反应的机理提供新思路与新方法；另方面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，及基于抑制免疫反应的新型害虫生防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。

寄生蜂是一类重要的天敌资源，是自然界中调控害虫种群数量的重要因子。寄生蜂与寄主昆虫之间的互作机理研究一直以来都是寄生学与昆虫免疫学的研究热点。本项目以内寄生蜂蝶蛹金小蜂及其寄主害虫菜粉蝶蛹为研究系统，采用基因组学、肽组学、小肽分离与鉴定、基因克隆、基因表达模式分析、小肽重组表达与产物纯化、小肽结构解析、小肽功能研究等实验技术与手段，就蝶蛹金小蜂毒液小肽鉴定与组成分析、小肽编码基因克隆及其表达模式分析、小肽结构解析、小肽重组表达及其产物功能分析，以及小肽融合重组及其产物用于害虫控制初探等方面开展系统研究。取得以下主要结果：1）完成其雌成蜂毒液小肽组学分析，及小肽组分的筛选与鉴定；2）利用全基因组扫描，完成蝶蛹金小蜂抗菌肽基因的鉴定、注释与进化分析；3）成功克隆蝶蛹金小蜂毒液源几丁质结合蛋白PpCBP、Pacifastin丝氨酸蛋白酶抑制因子PpPLPI，以及Kazal型蛋白酶抑制因子PpKazal编码基因克隆与序列分析；4）完成上述3个基因的重组表达，明确3个基因及其所编码蛋白的表达模式的组织特异性，及其蜂发育动态的变化；5）明确PpCBP能与几丁质特异性结合，PpPLPI及PpKazal能显著抑制寄主体内原酚氧化酶的激活过程，削弱寄主体液免疫反应；6）明确PpPLPI具有1个β-折叠结构，PpKazal则具有2个β-折叠和1个α-螺旋结构，PpPLPI及PpKazal上均各自含有6个半胱氨酸残基，分别能够形成3对二硫键，其上还分别含有1个活性位点；7）发现半胱氨酸残基及其所形成的二硫键，以及活性位点均对PpPLPI及PpKazal生物学功能起关键作用；8）PpPLPI及PpKazal分别与雪花莲凝集素GNA融合重组后，其产物经饲喂后可显著削弱寄主害虫的天然免疫反应，并能显著延长其发育历期及降低其生存率；9）相关研究成果已发表SCI期刊论文8篇，授权和申报国内发明专利各1项。上述研究结果，一方面为深入揭示寄生蜂毒液组成与功能，及功能组分调控寄主先天免疫反应的机理研究提供新结果与新方法，另一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。