调控小菜蛾对Cry1Ac毒素产生抗性的转录因子筛选及其分子机制研究

Bt is a kind of environment friendly microbial pesticides, widely applied throughout the world, however, the rising of pesticide resistance to Bt is seriously threatening the service life of Bt. Plutella xylostella is one of the most serious Bt resistant pests, in order to expound the mechanism of insect resistance to Bt toxins, solve the problem of pesticide resistance fundamentally, this project plans to fish transcription factors of resistance related genes ALP、ABCC2 and ABCC3 in diamondback moth, and then carry out gene cloning, structure analysis, in vitro expression and purification; determine the binding activity of transcription factor to its target gene, and the influence of transcription factors to RNA polymerase binding to DNA promoter, validate target gene expression regulated by transcription factors in living Sf9 cells; analyze the influence of transcription factor gene silencing to target gene expression and resistance in diamondback moth; identify the category of MAPK, and further analyze how MAPK signal pathway regulate resistance to Cry1Ac by transcription factors. From plutella xylostella receiving outside signal to activating MAPK signal pathway, then to transcription factors regulating the expression of ALP, ABCC2 and ABCC3, molecular mechanism of resistance to Bt Cry1Ac toxin in diamondback moth will be systematically studied.

Bt是一种环境友好型微生物杀虫剂，在世界范围内得到广泛应用，但害虫对Bt抗药性上升正严重威胁着Bt的使用寿命。小菜蛾是抗药性最为严重的害虫之一，为阐明其对Bt产生抗性的机制，从根本上解决抗药性问题，本项目拟应用Biacore技术垂钓小菜蛾抗性相关基因ALP、ABCC2和ABCC3的转录因子，对其进行基因克隆，结构分析，体外表达与纯化；测定转录因子与靶基因之间的亲和活性以及转录因子对RNA聚合酶与启动子结合活性的影响；在活体细胞Sf9中验证转录因子对靶基因的表达调控；分析转录因子基因沉默对靶基因表达量和小菜蛾抗性的影响；明确MAPK的类别，并进一步分析MAPK信号通路是如何通过转录因子调控小菜蛾抗性的。从小菜蛾接受外界信号激活MAPK信号通路，到转录因子调节ALP、ABCC2和ABCC3的表达量，进而使小菜蛾产生抗性，系统研究小菜蛾对Cry1Ac抗性的分子机制。

苏云金芽孢杆菌（Bacillus thuringiensis, 简称Bt）是一种对害虫高效、对人畜安全、对环境友好的微生物杀虫剂，被广泛应用于控制多种鳞翅目、双翅目和鞘翅目的害虫。Bt在减少害虫危害、增加作物产量、减少化学农药对环境的污染、改善作物品质等方面发挥了重要作用。但目前害虫对Bt产生了抗药性，小菜蛾是最早在田间发现的对Bt毒素产生抗性的昆虫，也是抗药性最为严重的害虫之一，其交互抗性广、抗性水平高、发展速度快，对十字花科蔬菜生产造成极大威胁。根据前期的研究结果，我们提出了一种全新的负调控机制来解释小菜蛾的抗性机理，即Bt毒素作用于小菜蛾后，小菜蛾中肠受体与Cry1Ac的结合激活了MAPK信号通路，MAP4K4增强表达。MAP4K4的增强表达进一步调节了PxmALP的表达量，使小菜蛾对Cry1Ac毒素产生抗性。本项目应用Biacore技术垂钓获得了小菜蛾抗性相关基因PxmALP的转录因子GAGA，其DNA结合位点为TTGCTCTCTC；应用Biacore分析了PxmALP启动子DNA与转录因子GAGA之间的亲和活性，从证明验证了GAGA为PxmALP的转录因子；应用RNAi技术沉默GAGA基因，测定了转录因子GAGA基因沉默对PxmALP表达量的影响和对小菜蛾抗性的影响，从反面验证了GAGA为PxmALP的转录因子；通过向小菜蛾体内注射信号通路抑制剂，明确了MAPK信号通路类别，并进一步阐明了GAGA转录因子通过p-JNK信号通路调控小菜蛾Cry1Ac受体基因PxmALP基因的表达量，进而对小菜蛾Cry1Ac抗性产生了影响。本项目获得的研究结果对转Bt基因植物／Bt制剂的抗药性治理、新型Bt杀虫蛋白的筛选以及延缓，甚至克服小菜蛾对Bt毒素的抗药性、提高作物产量和品质具有重要的理论和实践意义。