苏云金芽孢杆菌Cry1Ab毒素对亚洲玉米螟ABC转运蛋白的分子互作研究

Transgenic plants producing Bacillus thuringiensis (Bt) toxins are threatened by insect resistance after massive planting. Mutation of ATP binding cassette transporter (ABC transporter protein) and its varied transcript are reported to be involved in Cry1A toxin resistance against various Lepidopteran strains. This study uses immunohistochemistry to reveal the localization of ABC transporter in Asian corn borer larvae; on the basis of clarifying ABC transporter binds Cry1Ab toxin with high affinity, we will analyze the functional role of ABC transporter as Cry1Ab binding receptor through RNAi and Crispr/Cas9 technologies, aiming to demonstrate that ABC transporter is the functional target of Cry1Ab; we will also testify the alteration of Cry1Ab toxicity on S2 cell with the expression of ABC transporter and mutation, and further analyze the functional region that ABC transporter interacts with Cry1Ab. This study will provide fundamental knowledge for different strategies to cope with Asian corn borer resistance, and also for delaying Cry resistance having ABC transporter as resistance target.

植物转苏云金芽孢杆菌（Bacillus thuringiensis 简称Bt）的大量种植使靶标害虫产生了抗性。ABC转运蛋白基因的突变和表达量的改变参与了多种鳞翅目害虫对Bt Cry1A毒素的抗性调节。本项目采用免疫组化的方法对其在玉米螟体内的表达进行组织定位，在证明ABC转运蛋白与Cry1Ab具有亲和力的基础上，运用RNAi和Crispr/Cas9技术验证其为Cry1Ab毒素的功能性受体蛋白, 明确ABC转运蛋白为Cry1Ab的的作用靶标；构建蛋白突变体后，测定Cry1Ab对表达ABC转运蛋白和突变体的S2细胞的活力变化，确定ABC转运蛋白与Cry1Ab的作用功能域。本研究将为亚洲玉米螟抗性机理奠定基础，也为延迟以ABC转运蛋白为抗性靶标的Cry毒素的抗性形成提供理论依据。

协同分子生物学和反向遗传学的主流研究手段，通过RACE克隆了ABC转运蛋白的3K全长片段，并截取了部分基因作为抗原，用于制备抗体。本研究检测了ABC转运蛋白的时空表达模式，发现ABC转运蛋白在幼虫期二龄到五龄时期表达量比其他时期表达量显著升高，其中在五龄期表达量最高，主要表达组织为前肠和中肠。本研究构建了亚洲玉米螟RNAi基因沉默系统，首次通过涂抹法实现了对ABC转运蛋白在亚洲玉米螟体内的基因沉默，检测了沉默后该基因在五天内的表达量变化，确认了体表涂抹法具有极高的沉默效率。构建了亚洲玉米螟CRISPR/Cas9 基因编辑系统，使用具有明显表型的Abd-A基因测试了基因编辑系统在亚洲玉米螟体内的成功构建，并对ABC转运蛋白进行靶向敲除，成功获得了ABC转运蛋白G0代和G1代突变体，但由于致死率较高，无法利用足够的杂合体交配以筛选出纯合体，进而做进一步的Cry1Ab毒素的生测试验。研究结果可谓其他鳞翅目害虫Bt作用机制和受体蛋白互作机制研究提供理论基础和新思路。