亚洲玉米螟载脂蛋白apolipophorinⅢ识别和免疫防卫腰带长体茧蜂寄生的分子机理

The molecular mechanism of recognition and immune defence of apolipophorin Ⅲ (apoLpⅢ) in innate immunity will be studied in the parasitic system of Macrocentrus cingulum and host insect Ostrinia furnacalis in this project. The apoLpⅢ protein will be purified with column chmatography from the hemolymph of O. furnacalis larvae, and the polyclonal antibody of apoLpⅢ will be prepared. The mutants of apoLpⅢ gene will be constructed by site-directed mutagenesis, and the mutant protein will be expressed and purified from Escherichia coli strain BL21(DE3). The secondy structures of mutant protein will be detected with circular dichroism(CD) and nuclear magnetic resonance (NMR) to reveal the relationship between conformational change and Leucines in the α-helics of apoLpⅢ protein. The content of apoLpⅢ protein in the hemolymph of parasitized O. furnacalis larvae will be detected by ELISA. The wild type and mutant type of apoLpⅢ protein will be incubated with M. cingulum eggs in vitro to observe the recognition and adhension of apoLpⅢ protein on eggs. The ratio of parasitism, phenoloxidase (PO) acticity and ratio of encapsulation on eggs of parasitic wasp or Sephadex beads in O. furnacalis larvae which apoLpⅢ gene expression is inhibited by RNA interference will be analyzed. Furthermore, the rescue method will be applied on O. furnacalis larvae by injecting wild type and mutant type of apoLpⅢ protein, and then the ratio of parasitism, PO acticity and ratio of encapsulation on eggs of parasitic wasp or Sephadex beads in O. furnacalis larvae will be detected. qPCR, indirect immunofluorescence assay (IIFA) and colloidal gold labelled immuno-electron microscope will be used to verify the spatial and temporal consistency on transcriptional and translational level in vivo. The GST Pull down and CoIP methods will be applied to confirm the presence of apoLpⅢ/PPO complex in vitro and in vivo. This research will be helpful to illuminate the molecular mechanism of immune defence in parasitic wasp and host insect, and will also provide the fundation for novel insectcide development based on the functional and structural researches on apoLpⅢ.

本项目以腰带长体茧蜂与亚洲玉米螟为寄生体系，研究亚洲玉米螟apolipophorinⅢ识别和免疫防卫寄生蜂的分子机理。拟纯化亚洲玉米螟血淋巴中apoLpⅢ制备抗体；制备apoLpⅢ突变体蛋白，进行构象转换验证。检测被寄生幼虫体内apoLpⅢ含量，将apoLpⅢ蛋白体外孵育寄生蜂卵，观察apoLpⅢ与卵结合情况；测定RNAi后幼虫被寄生率、PO活性及对寄生蜂卵和Sephadex珠子包囊率；采用Rescue实验对RNAi后幼虫注射突变体apoLpⅢ蛋白，检测被寄生率、PO活性及对卵和Sephadex珠子包囊率，分析apoLpⅢ对寄生蜂卵的识别及PO功能的影响。采用qPCR、IIFA和胶体金免疫电镜验证被寄生幼虫体内apoLpⅢ与PPO时空表达一致性；通过Pull down和免疫共沉淀鉴定apoLpⅢ/PPO复合体。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。

本项目以腰带长体茧蜂与亚洲玉米螟为寄生体系，研究亚洲玉米螟apolipophorinⅢ（apoLpⅢ）识别和免疫防卫寄生蜂的分子机理。进行了被腰带长体茧蜂寄生的亚洲玉米螟转录组分析，亚洲玉米螟不同发育阶段不同组织中apoLpⅢ基因的表达分析，被寄生后亚洲玉米螟各组织的apoLpⅢ和PPO2相对表达水平分析，被寄生亚洲玉米螟幼虫体内免疫和营养代谢相关基因的转录水平分析，被寄生后亚洲玉米螟幼虫血淋巴中营养状况变化分析。构建了apoLpⅢ原核表达载体，表达和纯化apoLpⅢ蛋白，并制备apoLpⅢ蛋白多克隆抗体，进行了原核表达apoLpⅢ蛋白的质谱鉴定，apoLpⅢ野生型与突变体蛋白二级结构的圆二色谱分析，制备了apoLpⅢ突变体蛋白，进行了构象转换后活性验证。测定了apoLpⅢ基因RNAi对亚洲玉米螟幼虫apoLpⅢ、PPO2、抗菌肽Gloverin、Moricin和一氧化氮合成酶NOS1基因表达的影响，腰带长体茧蜂寄生对亚洲玉米螟幼虫apoLpⅢ和PPO2基因表达的影响，腰带长体茧蜂寄生对亚洲玉米螟幼虫不同组织中PO活性的影响，ds-apoLpⅢ注射后亚洲玉米螟血淋巴对Sephadex珠子的包囊程度的影响，ds-apoLpⅢ注射结合腰带长体茧蜂寄生对幼虫不同组织中PO活性的影响，分析了apoLpⅢ对寄生蜂卵的识别及PO功能的影响。采用IIFA和胶体金免疫电镜验证了被寄生幼虫体内apoLpⅢ与PPO时空表达一致性，通过Pull down鉴定了apoLpⅢ/PPO复合体。采用CRISPR/Cas9基因编辑技术，进行apoLpⅢ基因敲除突变制备，为后续apoLpⅢ功能验证实验提供基础实验材料。进行了部分亚洲玉米螟部分免疫相关基因的分子克隆，获得了15个基因的cDNA全长序列，为后续功能研究打下坚实的基础。本项目揭示了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。