中肠干细胞增殖与分化介导的二化螟Cry1Ab抗性机制研究

Resistance risk of Chilo suppressalis (Walker), to Bacillus thuringiensis（Bt）insecticidal protein is the key factor to influence sustainable utilization of Bt rice. Molecular resistant mechanism of C. suppressalis to Bt protein should be identified, before effective insect resistance management strategies are implemented. Enhanced midgut regeneration has been previously suggested as a potential resistance mechanism to Cry protein. Our study is focused on alterations in the midgut response to Cry1Ab intoxication as a mechanism potentially resulting in C. suppressalis resistance to Cry1Ab protein. The primary goal of this study is to identify the factors related to the proliferation and differentiation of stem cell that activates the midgut epithelium regeneration and healing response and the characterization of alterations in this process that result in resistance to Cry1Ab. The main work includes that the characteristic of proliferation and differentiation of stem cell in midgut of resistant and susceptible strains of C. suppressalis. Transcriptome and proteome of mature cell and metabolome of mature cell secretion from resistant and susceptible strains of C. suppressalis will be obtained by RNA-seq, iTRAQ, electrophoresis/HPLC and mass spectrum techniques, respectively. Related differential expressed genes regulating the proliferation and differentiation of midgut stem cell will be recognised based on comprehensive analysis of systems biology data from transcriptome, proteome and metabolome. RNA interference technique will be used to test the functions of these differential expressed genes. Simultaneously, silenced efficiency will be examined further by qRT-PCR. Bioactivity responses of stem cell are detected between RNAi and control treatments. Susceptible variations of C. suppressalis larva to Cry1Ab protein and histopathological strucural changes in the midgut epthelium will be further detected between RNAi and control treatments, if bioactivity response of stem cell is increased in some RNAi treatements. Differential expressed proteins are heterologously expressed in E. coli. and their functions are validated by testing bioactivity responses of stem cell. Ultimately, some key factors regulating the proliferation and differentiation of stem cell involved ..in midgut regeneration and healing are identified based on these researches and its further regulatory effect in resistance of C. suppressalis to Cry1Ab is elucidated. The study is very important to elucidate the new molecular mechanism of C. suppressalis resistance to Bt insecticidal protein and implement insect resistance management. Besides, the broader impacts of this research is the identification of crucial healing pathways for the development of novel biocontrol methods and insecticides.

二化螟对Bt杀虫蛋白的抗性演化是影响Bt水稻可持续应用的关键因素之一，明确其抗性产生的机制是实施害虫抗性治理策略的重要基础。由干细胞增殖和分化介导的中肠再生和修复是昆虫对Bt蛋白抗性产生的重要机制之一。本项目在明确二化螟Cry1Ab抗、感品系中肠干细胞增殖和分化能力存在显著差异的基础上，通过抗、感品系成肠细胞转录组、蛋白质组和分泌物的代谢组学等系统生物学数据的综合比较分析，获得与Cry1Ab抗性相关的干细胞增殖和分化的差异表达候选基因；通过RNA干扰、蛋白的体外表达、干细胞生物活性检测和Bt蛋白的生物测定等方法进一步验证候选基因的功能，鉴定出与二化螟Cry1Ab抗性相关的中肠干细胞增殖和分化的相关因子，进而揭示干细胞增殖和分化介导的中肠组织再生和修复对二化螟Cry1Ab抗性的调控作用。该研究对于揭示害虫对Bt蛋白抗性产生的多元分子机制和实施害虫抗性治理均具有重要意义。

二化螟对Bt杀虫蛋白的抗性演化是影响Bt水稻可持续应用的关键因素之一，明确其抗性产生的机制是实施害虫抗性治理策略的重要基础。由干细胞增殖和分化介导的中肠再生和修复是昆虫对Bt蛋白抗性产生的重要机制之一。本项目在获得二化螟Cry1Ab抗、感品系的基础上，分离并纯化了二化螟中肠干细胞，建立了利用流式细胞仪监测干细胞增殖和分化能力的方法，明确了Cry1Ab抗、感品系中肠干细胞的增殖和分化能力存在显著差异，通过抗、感品系成肠细胞转录组、蛋白质组和分泌物的代谢组学等系统生物学数据的综合比较分析，获得一系列与Cry1Ab抗性相关的干细胞增殖和分化的差异表达候选基因；通过RNA干扰、蛋白的异源表达、干细胞生物活性检测和Bt蛋白的生物测定等方法进一步验证候选基因的功能，鉴定出成肠细胞顶端突出物分泌的芳基储存蛋白家族可能是诱导中肠干细胞增殖和分化的关键因子，进而调控二化螟Cry1Ab抗性。研究结果解析了干细胞增殖和分化介导的中肠组织再生和修复对二化螟Cry1Ab抗性的调控作用，对揭示害虫对Bt蛋白抗性产生的多元分子机制和实施害虫抗性治理均具有重要意义。在本项目的支持下，以第一/通讯作者发表了学术论文9篇，其中SCI论文6篇；以第一发明人获国家发明专利2项；培养研究生6名。