中文摘要
靶标害虫对Bt蛋白的抗性风险是影响Bt水稻可持续应用和产业化发展的关键因素。本项目以Bt水稻靶标害虫二化螟为研究对象，采用F1/F2代家系和诊断剂量相结合的方法对我国转基因水稻环境释放地福州的二化螟种群进行检测，已初步明确田间二化螟种群对Bt毒蛋白的敏感性产生分化，同时也表明，该方法是二化螟田间早期抗性检测的一种有效方法；在室内采用Bt毒饲料饲喂的方法对二化螟种群进行抗性筛选，获得了对Cry1Ac和Cry1Ab毒蛋白耐受的筛选种群；通过PCR及RACE技术获得了二化螟Bt受体蛋白- - 氨肽酶-N基因的全长序列，利用实时荧光定量PCR对氨肽酶-N基因的表达谱进行分析，发现其均在二化螟幼虫3龄期和胸部表达量最高；构建氨肽酶-N基因表达载体，对受体蛋白进行体外表达，表达产物纯化后并进行Western印迹杂交，明确受体蛋白与Bt毒素的结合区片段，然后进行体内功能验证，进而明确其与Bt毒素抗性的关系。在此基础上，建立二化螟Bt早期抗性的分子检测技术。该研究对于阐明二化螟对Bt抗性产生的分子机制，建立和完善水稻害虫对Bt的抗性监测与预警体系，实施预防性的抗性治理策略具有重要意义。
英文摘要
Resistance risk of target pests is an importnant factor to influence the sustainable utilization and healthy development of Bt rice. Chilo suppressalis (Walker) is one of the major target pests of Bt rice. The initial Bt tolerance were determined in Fuzhou populations of C. suppressalis with the method of F1/F2 iso-female line and diagnostic dose bioassay. The results showed that susceptibility to Cry1Ac in C. suppressalis population was differentiated significantly and early genetic resistance detecting technique was established. Simultaneously, high tolerant populations to Bt toxins were screened with Bt toxin diet incorporation bioassay. Full length cDNA sequences of aminopeptidase-N (APN) of C. suppressalis BBMV were cloned by degenerative PCR combined with RACE techniques and the temporal and spatial expressions of APN genes were analyzed further by real-time quantitative PCR technique. High expression levels were exhibited in the 3rd instar and thorax tissue. APN genes were recombined into the expression vetor for overexpression in Escherichia coli. Western-blotting was conducted for the expressed fusion protein to indentify the binding segments of APN with Bt toxin.Function of the binding segments were examined in vivo to show the relationship between binding regions and Bt resistance. DNA-based early resistance detecting technique was established based on these results. The study is very meaningful in elucidating the molecular mechanism of C. suppressalis resistance to Bt toxin, establishing and improving the monitoring and forecasting system for resistance to Bt rice, and implementing proactive resistance management strategy.
结题摘要
靶标害虫对Bt蛋白的抗性风险是影响Bt水稻可持续应用和产业化发展的关键因素。本项目以Bt水稻靶标害虫二化螟为研究对象，采用F1/F2代家系和诊断剂量相结合的方法对我国转基因水稻环境释放地福州的二化螟种群进行检测，已初步明确田间二化螟种群对Bt毒蛋白的敏感性产生分化，同时也表明，该方法是二化螟田间早期抗性检测的一种有效方法；在室内采用Bt毒饲料饲喂的方法对二化螟种群进行抗性筛选，获得了对Cry1Ac和Cry1Ab毒蛋白耐受的筛选种群；通过PCR及RACE技术获得了二化螟Bt受体蛋白- - 氨肽酶-N基因的全长序列，利用实时荧光定量PCR对氨肽酶-N基因的表达谱进行分析，发现其均在二化螟幼虫3龄期和胸部表达量最高；构建氨肽酶-N基因表达载体，对受体蛋白进行体外表达，表达产物纯化后并进行Western印迹杂交，明确受体蛋白与Bt毒素的结合区片段，然后进行体内功能验证，进而明确其与Bt毒素抗性的关系。在此基础上，建立二化螟Bt早期抗性的分子检测技术。该研究对于阐明二化螟对Bt抗性产生的分子机制，建立和完善水稻害虫对Bt的抗性监测与预警体系，实施预防性的抗性治理策略具有重要意义。