棉铃虫CYP6B6基因的RNAi沉默及其转棉花抗虫机理研究

The cytochrome P450 monooxygenases have important roles in the metabolism of endogenous and exogenous compounds. CYP6B6 is a cytochrome P450 isolated from cotton bollworm, which not only participates in endogenous metabolism of some substances which have important physiological functions, such as hormones, fatty acid, but also metabolizes some xenobiotics such as allelochemicals and insecticides. Transcription of CYP6B6 can be enhanced by toxic compounds, leading to increased expression of the protein and thus increased detoxi?cation, to make the cotton bollworm possess high tolerance to insecticides. On the basis of the previous work we intend to investigate CYP6B6 by RNAi technique. To select the optimum RNAi fragment in the interfering fragments designed by the CYP6B6 sequence, we will clone the target gene fragment relating to systemic RNAi, and express dsRNA in bacteria HT115 (DE3), then analyze whether the target gene silencing can be induced by FQ-PCR and immunohistochemistry, and the growth and development of the cotton bollworm can be disturbed by feeding dsRNA. Based on the optimum RNAi fragment of the CYP6B6，the RNAi vector of the target gene which is drived by 35S promoter is constructed, and then transform it to the cotton plants to get the transgenic cotton plants by pollen-tube pathway. To evaluate pest-resistant effect of the dsRNA expressed by the transgenic cotton plants, we will investigate the inhibition of the CYP6B6 expression, tolerance to insecticides and disturbance to the growing development of the cotton bollworm after feeding the transgenic cotton plants. These results will not only provide a new technical method for the biological control of Helicoverpa armigera, but also introduce a new strategy for other pests management.

细胞色素P450 CYP6B6是棉铃虫抵御外源有毒物质的关键基因之一，不仅参与一些具有重要生理功能的内源性物质如激素、脂肪酸的代谢，而且通过CYP6B6基因过表达使棉铃虫免受植物次生物质等有毒物质的伤害，还增强对杀虫剂的耐受性。在前期工作的基础上，引入RNAi沉默技术策略，用大肠杆菌表达针对CYP6B6基因的dsRNA，以饲喂方式将表达的dsRNA引入棉铃虫体内，用荧光定量PCR和免疫组化的方法研究其对CYP6B6的抑制表达效果、对棉铃虫生长发育及其对常用杀虫药剂耐受性的变化，综合分析获得最佳的干扰片段。进而构建棉花表达干扰载体，用花粉管通道法转化棉花，获得表达与CYP6B6基因相匹配dsRNA的棉花，检测棉铃虫取食后对CYP6B6基因表达的抑制、杀虫剂的耐受性以及对生长发育的影响，评价棉花表达dsRNA的抗虫效果。这不仅为棉铃虫的高效防治提供新的技术手段，而且也为其它害虫治理提供新策略。

细胞色素P450 CYP6B6是棉铃虫抵御外源有毒物质的关键基因之一，不仅参与一些具有重要生理功能的内源性物质如激素、脂肪酸的代谢，而且通过CYP6B6基因过表达使棉铃虫免受植物次生物质等有毒物质的伤害，还增强对杀虫剂的耐受性。在前期工作的基础上，针对棉铃虫P450 CYP6B6，基于原核表达的dsRNA，以饲喂的方式将能表达dsRNA的工程菌引入棉铃虫体内。结果表明工程菌在室温条件下，pH值为 7.0的KH2PO4/K2HPO4缓冲溶液中，能稳定保持24h；dsC1的能很好地抑制靶基因的表达，明显提高棉铃虫的死亡率，延迟棉铃虫幼虫的生长发育，化蛹率也明显降低；同时还提高棉铃虫对毒死蜱和3-氟氯氰菊酯的敏感度，继而将其转导到棉花里，通过4年的田间筛选和室内检测，获得转基因阳性棉花株系3株，棉铃虫幼虫对这3个株系表现出强烈的拒食性，被迫取食后不仅使靶基因CYP6B6表达量减少，而且幼虫的死亡率显著提高、蜕皮受阻以及幼虫生长发育受到明显影响，上述结果这不仅为棉铃虫的高效防治提供新的技术手段，而且也为其它害虫治理提供新策略。