载脂蛋白apolipophorinⅢ介导NO调控寄主昆虫亚洲玉米螟免疫防卫的分子机理

本项目以腰带长体茧蜂与亚洲玉米螟为寄生体系，研究载脂蛋白apolipophorinⅢ介导NO调控寄主昆虫亚洲玉米螟免疫防卫的分子机理。拟表达纯化apoLpⅢ蛋白，制备多克隆抗体；利用qPCR、RNAi、Rescue分析apoLpⅢ对NO和抗菌肽（AMPs）的调控及对寄生蜂幼虫的影响；采用原位杂交、免疫电镜、Western blotting、EILSA、IIFA、FACS和qPCR分析apoLpⅢ转运NO机理；进行转录组测序和qPCR，分析apoLpⅢ、NOS基因、与AMPs表达相关基因调控关系；在个体和细胞水平上通过寄生、NO供体和NOS抑制剂处理、NOS RNAi、过表达NOS，检测NO浓度及相关AMPs的转录水平和蛋白水平分析，研究NO对AMPs表达信号通路中转录因子Dorsal和Relish的调控作用，揭示寄主昆虫免疫防卫寄生蜂的分子机理，为利用apoLpⅢ进行新农药创制提供基础。

The molecular mechanism of immune defence that apolipophorin Ⅲ (apoLpⅢ) mediates nitro oxide (NO) to regulate the innate immunity in the parasitic system of Macrocentrus cingulum and host insect Ostrinia furnacalis will be studied in this project. The apoLpⅢ protein will be expressed and purified with affinity chromatography, and the polyclonal antibody of apoLpⅢ will be prepared. Quantitative real time-PCR (qPCR), RNA interference (RNAi) and Rescue experiments will be performed to analyze the regulation of apoLpⅢ on NO concentration, expressions of antimicrobial peptides (AMPs), and the development of M. cingulum larvae in the body of O. furnacalis larvae. The situ hybridization, colloidal gold labelled immuno-electron microscope, Western blotting, EILSA, indirect immunofluorescence assay (IIFA), FACS and qPCR will be used to analyze the transit mechanism of NO by apoLpⅢ. The transcriptome sequencing of different tissues of O. furnacalis larvae paratisized by M. cingulum and qPCR will be conducted to research the regulation relationship among apoLpⅢ, NOS and other genes related to AMPs expressions. On the individual level and the cell level, some experiments will also be performed by the parasitism of M. cingulum, NO donor treatment, NOS inhibitor treatment, RNAi of NOS expression, overexpression of NOS, detection of NO concentration and the analyses of relevant AMPs on transcription level and protein level. This research is helpful to reveal the molecular mechanism of immune defense between host insect and parasitic wasps by illustrating how NO regulates the expression of transcription factors, such as Dorsal and Relish in AMPs signaling pathways, and will also provide the basis for novel insectcide development based on the functional and structural studies on apoLpⅢ.