小菜蛾中肠Cry非受体结合蛋白的鉴定与功能研究

Cry-receptor binding in the midgut BBMV of insect is the key step for insecticidal process of Cry proteins produced by Bacillus thuringiensis (Bt). Researches on those nonreceptor Cry-binding proteins in the midgut juice and their effect on Cry-receptor binding, will provide important issue to reveal the entire picture of the interaction between Cry and insect, will further our knowledges about the mechanism and specificity of Cry, and will provide new ideas on how to solve the resistance and increase the activity of current Bt products. In our previous work, we have identified putative Cry1Ab1-binding nonreceptor proteins from the midgut juice of Plutella xylostella. In this project, four Cry proteins, which exhibited high, moderate, low and none activities against P. xylostella, were subjected to isolate and identifiy their nonreceptor binding proteins from the midgut juice of P. xylostella, and then the function and mechanism of Cry-binding nonreceptor proteins were further elucidated. Firstly, all putative Cry-binding nonreceptor proteins were isolated from the midgut juice of P. xylostella by using Sepharose coupled with Cry, and were identified by LC-MS/MS, then were cloned, expressed and purified. Second, real Cry-binding nonreceptor proteins were screened by Co-IP and protein pull-down. Third, the function of Cry-binding nonreceptor proteins were identified by cofeeding Cry protein with its nonreceptor binding protein against P. xylostella, and those with obvious either enhancing or disturbing effect were further confirmed by silencing the nonreceptor protein via RNAi. Finally, the disturbing mechanism of selected nonreceptor Cry-binding proteins were clarified by mutation on the interaction face, which was identified by molecular docking, and assay against P. xylostella. The project here will establish a system for studying Cry-binding nonreceptor proteins from the insect midgut, will find out their variety and function, and will clarify how the selected Cry-binding nonreceptor proteins disturb the Cry-receptor binding.

Cry杀虫过程远比现有认识来得复杂，与受体结合是其中关键环节。研究害虫中肠中结合Cry的非受体蛋白及其对Cry-受体的影响，将为揭示Cry杀虫过程的全貌提供重要线索，有助于深入认识其杀虫机理和杀虫特异性，亦可为不改变工厂现有产品基础上解决害虫抗性和提高杀虫活性提供新的技术思路。拟以四种具不同杀小菜蛾活性的Cry及小菜蛾中肠液为材料，以揭示害虫中肠Cry非受体结合蛋白的种类、功能及机理为主线，通过非受体结合蛋白的分离鉴定及验证、Cry与非受体结合蛋白一起喂食的杀虫活性考察（RNAi进一步验证）、分子对接基础上关键结合界面的突变、验证及活性考察等研究，建立起害虫中肠Cry非受体结合蛋白研究体系，探明小菜蛾中肠液中Cry非受体结合蛋白的种类，明确其对Cry杀虫的影响，阐明抑制效应明显的非受体结合蛋白如何影响Cry与受体的结合。

抗性发展及一些重要害虫缺乏相应高效 Cry 是 Bt 产业发展的两大难题，对 Cry 杀虫过程的深入研究有助于难题的解决。长期以来，研究者们着重分离鉴定了害虫中肠 BBMV 上的 Cry 结合蛋白（受体和候选受体），对深入研究 Cry 杀虫机理和指导 Cry 的改造发挥了重要作用。项目组从害虫BBMV之外的Cry结合蛋白（非受体）这一新视角，开展非受体的鉴定与功能研究。. 与福建农林大学合作发现galectin-14是Cry11Aa的非受体，喂食galectin-14降低了Cry11Aa的杀虫活性，Cry11Aa与其受体ALP1的结合受到galectin-14的干扰。基于分子对接揭示了其干扰机制：galectin-14与ALP1的互作界面，与Cry11Aa-ALP1的互作界面发生了部分重叠，其中重叠区段涉及Cry11Aa-ALP1互作的关键区域及关键残基。这一结果揭示了非受体干扰Cry-受体互作影响杀虫活性的机理，深化了对Cry机理的认识，为Cry的改造提供了新思路。. 发现碱性保幼激素抑制蛋白（BJSP-1）是Cry7Ab4与Cry2Ab12的非受体。喂食Cry7Ab4使小菜蛾末龄幼虫血淋巴和脂肪体中的BJSP-1表达水平显著升高，同时显著降低了末龄幼虫游离JH的水平。这一结果揭示了Cry蛋白通过其非受体调控保幼激素水平进而抑制害虫生长发育的分子机理，为抑虫Cry资源的开发利用提供了理论基础和技术思路。. 发现害虫Cathepsin B和其共生菌来源的PFKA为Cry41的非受体。Cry41的结合，改变了Cathepsin B的活性口袋，显著提高了其酶活，可能因此导致细胞凋亡加速进而致死害虫。基于互作关键位点构建的Cry41突变体对Cathepsin B酶活的影响效应发生了相应改变。Cry41的结合，显著抑制了其非受体PFKA的酶活，降低了PFKA产生菌的数量，可能因此影响了害虫的生长发育。这一结果揭示了两种非受体途径的Cry作用机理，构建了基于Cry-非受体互作的Cry改造技术。. 综上，项目组建立了害虫Cry非受体结合蛋白研究技术体系，鉴定了若干Cry非受体，阐明了部分Cry非受体的功能，完成了既定的研究任务。