亚洲玉米螟防卫腰带长体茧蜂寄生的免疫识别受体βGRP2鉴定及其作用机制研究

On the process of immune recognition of host insects on parasitoids, the combination of parasitoid associated molecular structures (PAMS) and immune recognition receptors plays a crucial role in innate immunity system of insects. The parasitic system of Macrocentrus cingulum and the larvae of host insect, Ostrinia furnacalis, will be investigated in this research project. To characterize the PAMS of Macrocentrus cingulum, the RNA abstracted from ovary and larvae of M. cingulum will be deeply sequenced for the transcriptomic analysis. The elution material of eggs and calyx fluid of M. cingulum will also be detected with Nano-LC MS/MS for proteomics analysis.The βGRP2, a kind of candidate immune recognition receptors, will be purified from the hemolymph of O. furnacalis larvae, and the βGRP2 protein will be prokaryotic expressed. The polyclone antibody of βGRP2 will be prepared. The binding abilities of βGRP2 with eggs of parasitic wasps will be analyzed with FACS for the idendification of the PRRs. Real time PCR and In situ hybridization will be applied to detect the dynamic variation of βGRP2 gene on the mRNA level, and the function of βGRP2 will be investigated by RNA interference techanique. The real time PCR, immunomicroscopy, IIFA and enzymic analysis will be used to study the effects of βGRP2 gene on the expressions of prophenoloxidase(PPO) gene, prophenoloxidase activating proteinase (PAP) gene and serine proteinase inhibitor 1 (Serpin 1) gene in this parasitic system. This study will not only reveal the immune recognition molecular mechanism that host insect used to overcome the parasitism from parasitic wasps, but also provide a method for creation of novel insecticide and a new vision for control pest insect using this kind of parasitic wasps in future.

寄主昆虫对寄生蜂进行分子免疫识别过程中，寄生蜂相关分子结构（PAMS）和寄主体内免疫识别受体的结合起到至关重要的作用。本项目以腰带长体茧蜂及其寄主昆虫亚洲玉米螟为研究体系，对腰带长体茧蜂卵巢和幼虫采用RNA-seq深度测序技术进行转录组分析，结合对寄生蜂卵洗脱物和萼液成分Nano-LC MS/MS蛋白质组数据，分析PAMS；纯化亚洲玉米螟幼虫血淋巴中候选免疫识别受体βGRP2，并原核表达βGRP2；制备抗体；通过FACS分析寄生蜂卵与βGRP2结合能力，鉴定βGRP2受体。通过定量PCR和原位杂交检测βGRP2在mRNA水平上动态变化；通过RNAi验证βGRP2功能。通过定量PCR、免疫电镜、间接免疫荧光和酶学实验检测βGRP2基因与PPO、PAP和serpin1基因表达相关性。该研究不但可揭示寄主昆虫免疫识别寄生蜂寄生的分子机制，而且可为新农药创制，充分利用寄生蜂防控害虫提供崭新的视角。

因项目执行中，在βGRP2基因的系统进化分析中采纳SCI审稿专家建议，将βGRP2基因改名为βGRP3基因。因此，本结题报告中全部以βGRP3基因表示。寄主昆虫对寄生蜂进行分子免疫识别过程中，寄生蜂相关分子结构（PAMS）和寄主体内免疫识别受体的结合起到至关重要的作用。本项目以腰带长体茧蜂及其寄主昆虫亚洲玉米螟为研究体系，对腰带长体茧蜂卵巢和幼虫采用RNA-seq深度测序技术进行转录组分析，结合对寄生蜂卵洗脱物和萼液成分Nano-LC MS/MS 蛋白质组数据，分析PAMS；克隆了寄主亚洲玉米螟幼虫体内的候选模式识别受体βGRP3基因，并原核和真核表达和纯化了β GRP3蛋白，制备了抗体；进行了功能分析；研究了βGRP3在寄主昆虫免疫识别寄生蜂过程中和对细菌侵染的应答过程调控的分子作用机制。通过定量PCR检测β GRP3在mRNA水平上动态变化；通过RNAi验证βGRP3功能。通过定量PCR、免疫电镜、间接免疫荧光和酶学实验检测βGRP3基因与PPO、PAP和Serpin1基因表达相关性。该研究不但可揭示寄主昆虫免疫识别寄生蜂寄生的分子机制，而且可为新农药创制，充分利用寄生蜂防控害虫提供崭新的视角。