管氏肿腿蜂毒液抑制寄主血淋巴黑化功能蛋白及其作用靶标研究

Today, insect pests pose continuing rampant damage to plants. Chemical control is the heavily relied approach to insect pest management, which has caused many negative effects. There is an urgent need to accelerate the development and implementation of cost-effective, environmentally safe alternatives to synthetic chemical pesticides for pest control. As parasitoid venom has special physiological functions, venom proteins of parasitoid and their targets are potentially valuable resources for developing transgenic plant that is considered as a major breakthrough for designing novel biological control strategies. However, components of parasitoid venom and their acting targets are largely unexplored. As a consequence, on the basis of detailed physiological functions of venom from Scleroderma guani been well investigated in our laboratory, this project will conduct the following research contents: (1) isolation and identification of the venom proteins involved in inhibiting hosts hemolymph melanization from S. guani; (2) cloning and expression of the venom genes that are able to inhibit melanization; (3) identification of the hosts targets of the venom proteins that are able to inhibit melanization; (4) cloning and expression of the hosts target genes; and (5) investigation of the interaction between the identified venom proteins and their host targets. The goal of this proposal is to identify the venom proteins with ability to inhibit host hemolymph melanization from S. guani, and discover their host's targets. The results can offer to provide great insight into the venom components of parasitoid venom and the mechanism of parasitoid venom to manipulation the hosts. They are helpful for discovering novel insecticidal genes and RNAi target genes used to develop pest resistant genetically modified plants that target the insect immune system. In addition, they will facilitate the development of new innovative pest control tactics.

当前害虫持续猖獗危害，以化学农药防治为主的害虫控制途径带来诸多负面问题，急需寻找新途径以实现对害虫持续有效和经济安全的控制。因寄生蜂毒液具有独特的生理功能，可以寄生蜂毒液蛋白及其作用靶标为资源来研发新型转基因抗虫植物，以开辟新的害虫生物防治途径。然而，迄今寄生蜂毒液功能组分及其作用靶标尚不清晰。为此，本项目在前期系统研究了管氏肿腿蜂毒液生理功能的基础上，开展该蜂毒液抑制寄主血淋巴黑化功能蛋白分离鉴定及其基因克隆表达、该蜂毒液抑制寄主血淋巴黑化作用靶标鉴定及其基因克隆表达、以及该蜂毒液抑制寄主血淋巴黑化功能蛋白与其作用靶标互作验证等研究，最终揭示该蜂毒液抑制寄主血淋巴黑化功能蛋白及其作用靶标。研究结果，能为深入揭示寄生蜂毒液蛋白组分及其调控寄主的作用机理提供科学依据，对挖掘新杀虫基因及RNAi靶基因来研发针对害虫免疫系统的新型转基因抗虫植物具极大促进作用，有助于推动开发害虫控制新途径的发展。

本项目在前期研究发现管氏肿腿蜂粗毒液具有抑制寄主血淋巴黑化功能的基础上，旨在揭示其内在机制，围绕该蜂毒液抑制寄主血淋巴黑化功能蛋白及其寄主作用靶标的鉴定开展了系列研究，取得了以下研究结果：在明确了管氏肿腿蜂毒液器官超微结构及其基因背景的基础上，揭示了该蜂的毒液蛋白组分，并在该蜂毒液中首次鉴定获得了高分子量丝氨酸蛋白酶、M13家族金属蛋白酶、Vn5等新寄生蜂毒液蛋白；探明了管氏肿腿蜂的主要毒液蛋白及其基因在雌、雄成虫和毒液器官中的组织表达特征，发现其明显有别于隶属于茧蜂科、姬蜂科、金小蜂科等寄生蜂的主要毒液蛋白成分，更接近于社会性蜂类的主要毒液蛋白成分；克隆鉴定获得了丝氨酸蛋白酶1-3和超氧化物歧化酶3等4个具有抑制寄主血淋巴黑化功能的毒液蛋白基因，并明确了这些基因在不同发育阶段和组织的表达特征；明确了管氏肿腿蜂寄生对寄主黄粉甲蛹基因的转录和表达调控，揭示了受该蜂寄生调控的关键寄主免疫基因；明确了管氏肿腿蜂毒液丝氨酸蛋白酶2通过与寄主丝氨酸蛋白酶互作而抑制寄主血淋巴的黑化。研究结果，一方面提供了对寄生蜂毒液蛋白组分和进化及其抑制寄主体液免疫反应机制的新认识，另方面为后续发掘利用寄生蜂毒液功能蛋白及其作用靶标基因用于害虫防治提供了潜在的资源。