Cry1Ac抗性棉铃虫中肠胰蛋白酶基因沉默的启动子调控

Cotton bollworm (Helicoverpa armigera) is one of the most serious insect pests of cotton. Transgenic cotton expressing Cry toxins derived from a soil bacterium, Bacillus thuringiensis (Bt), has been produced to target this pest. Bt cotton has been widely planted around the world, and this has resulted in efficient control of bollworm populations with reduced use of synthetic insecticides. However, evolution of resistance by this pest threatens the continued success of Bt cotton. To date, no field populations of bollworm have evolved significant levels of resistance; however, several laboratory-selected Cry-resistant strains of H. armigera have been obtained, which suggests that bollworm has the capacity to evolve resistance to Bt. In this study, we will analyze the mutations of trypsin promoter that maybe responsible for the silence of trypsin in the resistant H. armigera larvae. Activities of promoters of trypsin from susceptible and resistant strains will be determined in Sf9 cells by using of Dual-Luciferase reporter assay. In order to gain insight into silence of correlated with laboratory-selected resistance to Cry1Ac toxin in cotton bollworm, knockdown of trypsin in susceptible larvae and addition of trypsin expressed in Sf9 cells to the resistant larvae will be studied in bioassay. The purpose of this research project is to provide scientific evidence for molecular design of insecticidal gene and development of molecular monitoring techniques as well as delaying of evolution of Bt resistance.

棉铃虫是危害棉花最严重的害虫之一。Bt（Bacillus thuringiensis）棉花产生的Cry毒素对棉铃虫有高效毒杀作用。Bt棉花已在世界范围内商业化种植，有效控制棉铃虫种群数量，减少了化学农药使用。尽管没有发现对 Bt 棉花产生高水平抗性的棉铃虫田间种群，但室内持续筛选已培育出多个高水平抗性品系，表明存在棉铃虫对 Bt 棉花产生抗性的风险。本项目拟以实验室筛选的中肠胰蛋白酶基因（trypsin）沉默的棉铃虫抗性品系为研究对象，以启动子功能分析为主要研究线索，对抗感品系启动子序列差异分析、转录活性等方面进行深入分析，运用RNAi干扰、体内补偿及遗传连锁等手段来证明trypsin的沉默与抗性的关系，以期全面深入的阐明trypsin沉默的Cry1Ac抗性棉铃虫的启动子调控的分子机制，为Bt棉花的杀虫基因的分子设计、田间抗性的分子水平监测及延缓棉铃虫对Bt棉花的抗性演化提供科学依据。

Bt毒素须经过连续的水解、结合等反应，才能对昆虫具有毒性。其中任何一个环节的改变都会导致昆虫对Bt毒素产生抗性。产生抗性机制的主要因素也是Bt毒素作用机理中的关键环节。目前所发现的昆虫对Bt毒素抗性机制也不尽相同。本项目通过对实验室筛选的中肠胰蛋白酶基因（trypsin）沉默的棉铃虫抗性品系深入研究，以启动子功能分析为主要研究线索，对抗感品系启动子序列差异分析、转录活性等方面进行了深入分析，运用RNAi干扰、体内补偿及遗传连锁等手段证明了trypsin的沉默与抗性的关系，全面深入的阐明trypsin沉默的Cry1Ac抗性棉铃虫的启动子调控的分子机制，为Bt棉花的杀虫基因的分子设计、田间抗性的分子水平监测及延缓棉铃虫对Bt棉花的抗性演化提供科学依据。