小菜蛾中肠Cry1Ac和Cry2Aa毒素结合蛋白Polycalin受体功能的鉴定

Although several receptor proteins of Cry toxins have been identified, our understanding of mechanism in toxins action and resistance to these toxins still remains unclear. In recent years, it has been reported that polycalin, a protein found in the midgut of lepidopteran insects such as silkworm and diamondback moth, binds to Cry1Ac toxin. Our previous results suggested another Lepidoptera-toxic toixn Cry2Aa binding to Polycalin in Plutella xylostella. Based on obtaining of full-length sequence of Polycalin from P.xylostella, the differences of temporal and spatial expression of gene will be analyzed by real-time quantitative PCR and the difference in Polycalin gene expression between before and after Cry1Ac/Cry2Aa treated will also be studied to confirm the function of gene. The prokaryotic expression and purification of Polycalin protein are helpful for further validation of interaction between Polycalin protein and toxins by molecular interaction device Forte Bio octet.To verify the role and function of Polycalin in toxins action, eukaryotic expression combined with cytotoxicity test will be carried out to clarify the possible participation manner of Polycalin and other indentified receptor protein (ABCC2 and CAD) from P. xylostella in toxin action. Crispr/Cas9 will be used to validate the physiological function of polycalin as a receptor protein in vivo. In a word, the function of toxin-binding protein polycalin from P. xylostella will be studied thoroughly as potential receptor protein of Cry1Ac and Cry2Aa by in vivo and vitro experiments, respectively. This project will provide a insight into the deeper understanding of mechanism of Cry toxins, and help to develop approaches for the cross resistance management and continuable application of Cry1Ac/Cry2A bivalent genetically modified organism.

多种Cry毒素受体蛋白已被鉴定，但我们对毒素作用机制及抗性机制的理解仍不完善。小菜蛾中肠Polycalin已被报道是Cry1Ac的结合蛋白，而我们的前期实验结果表明小菜蛾Polycalin也可与同样对鳞翅目昆虫具有毒性作用的Cry2Aa结合。本项目拟在获取小菜蛾Polycalin序列全长的基础上通过荧光定量PCR分析小菜蛾Polycalin基因时空表达差异及毒素作用下的表达差异；原核表达纯化和分子互作进一步验证此蛋白与毒素的结合特征；真核表达结合细胞毒性试验研究此蛋白在毒素作用过程中的参与方式；Crispr/Cas9技术明确此蛋白在毒素作用下的试虫体内的生理功能。从基因、蛋白和在体三方面对Polycalin作为Cry1Ac和Cry2Aa共有受体蛋白的功能展开研究，为进一步阐明和完善Cry毒素作用机制奠定理论基础，对治理Cry1Ac/Cry2A交互抗性具有指导意义。

转Bt基因作物防虫害的持续应用正受到来自靶标害虫抗性产生的威胁。对Cry毒素受体及其潜在增效机制的研究有助于靶标害虫的防控和抗性治理。本研究通过PCＲ技术从小菜蛾幼虫中肠克隆获得脂质运载蛋白Polycalin (PxPolycalin)蛋白基因的全长序列，采用实时定量PCR技术研究Polycalin蛋白在小菜蛾不同发育阶段、3龄幼虫不同组织及用Cry1Ac和Cry2Aa毒素饲喂3龄幼虫后的表达量。该蛋白在3龄幼虫体内表达量最高，而幼虫腹部中该蛋白的表达量则高于其他部位。同时，Cry1Ac毒素作用下，Polycalin基因表达量呈现浓度依赖性下降，而Cry2Aa毒素作用下表达量变化差异不显著。原核表达并纯化的Polycalin蛋白片段PxPolycalinf（G2209-A2942）拥有与Cry1Ac毒素结合的能力，并在生测试验中增效Cry1Ac毒素的杀虫活性，同时，PxPolycalinf多克隆抗体降低了Cry1Ac毒素的杀虫活性。ELISA试验结果显示了PxPolycalin与小菜蛾幼虫中肠BBMV的浓度依赖性结合。表达PxPolycalinf的昆虫细胞Sf9对Cry1Ac毒素不敏感，但PxPolycalinf增效了Cry1Ac毒素对表达了小菜蛾ABCC2蛋白的Sf9细胞毒性。免疫共定位和ELISA试验显示PxPolycalinf与Sf9细胞膜互作，并与细胞提取物成浓度依赖性结合。RNA干扰后小菜蛾3龄幼虫的PxPolycalin表达量以及Cry1Ac毒素对试虫的毒性均显著下降。以上研究内容提供了PxPolycalin作为Cry1Ac毒素潜在靶标受体的证据，以及其关键序列蛋白在增效Cry1Ac毒素活性中的重要作用，为进一步完善Cry毒素毒理机制并提出抗性治理策略提供了参考和借鉴。