高效氯氰菊酯亚致死剂量刺激大豆蚜生殖的分子机理

Application of insecticide spraying is one of important strategies to control pests. After insecticide applied in the field, the pests would be inevitably exposed to sublethal dosage of insecticide for a long period. It was an important reason and also a common phenomenon that sublethal dosage of insecticide can stimulate pest's reproduction, which lead to pest resurgence. However, there is limited knowledge about the molecular mechanism of this stimulating reproduction. Our preliminary research showed that sublethal dosage of beta-cypermethrin could significantly enhance the fecundity of F2 generation aphid. Aiding by the transcriptome sequencing and other molecular biology techniques, this project was designed to reveal following achievements in soybean aphid （Aphis glycines Matsumura）: (1) The quantity of significantly expression up-regulated genes in soybean aphid, comparing to the control after exposed the beta-cypermethrin sublethal dosage through transcriptome sequencing; (2) 3 to 5 genes were selected, which have close relation with reproduction and their mRNA expression levels were revealed during the reproductive periods in soybean aphid after exposed to beta-cypermethrin sublethal dosage; 3) Verify the function of above 3 to 5 genes through RNAi technology and enzyme-linked immunosorbent assay. Those three research facet could reveal the molecular mechanism of sublethal dosage of beta-cypermethrin enhancing soybean aphid reproduction. Completion of this project will elucidate that sublethal dosage of insecticide stimulating pest reproduction from the genetic level. Meanwhile, it has important theoretical and practical significance for guiding rationally and effectively to apply insecticides to control pests.

杀虫药剂的使用是控制害虫的重要措施之一。杀虫药剂在田间施用后，随着时间的推移害虫不可避免地暴露于亚致死剂量药剂的胁迫之下。亚致死剂量的药剂刺激害虫生殖力增强是导致害虫再猖獗的一个重要原因，但对其分子机理缺乏了解。本研究的前期结果表明高效氯氰菊酯亚致死浓度能够显著提高F2代大豆蚜的产蚜数。本项目拟以大豆蚜为对象，采用转录组测序等现代分子生物学技术，通过（1）转录组测序明确高效氯氰菊酯亚致死浓度刺激大豆蚜后其体内表达量上调的基因数量；（2）筛选3～5个与生殖相关的表达差异显著基因在不同产蚜期内其mRNA表达量的变化规律及（3）利用RNAi技术及酶联免疫测定验证上述3～5个基因的功能等三方面的研究，揭示高效氯氰菊酯亚致死剂量刺激大豆蚜生殖增强的分子机理。本项目的完成将初步从基因水平明确杀虫药剂亚致死剂量刺激害虫生殖的机理，对于指导生产实际中合理用药、有效控制害虫再猖獗具有重要的理论和实践意义。

杀虫剂施用田间后，会对目标害虫变成亚致死剂量，产生亚致死效应，使害虫种群数量在短时间内爆发。该项目的研究对揭示其机理和害虫抗性治理，保障农产品安全生产具有较重要的意义。.本项目研究了高效氯氰菊酯，马拉硫磷和吡虫啉亚致死浓度对大豆蚜虫发育历期，寿命和繁殖力的影响。0.01 mg L-1-0.02 mg L-1高效氯氰菊酯，0.1 mg L-1马拉硫磷，0.05mg L-1的吡虫啉都可以显著刺激大豆蚜的产蚜量。.转录组差异表达分析揭示了大豆蚜饲喂亚致死浓度的高效氯氰菊酯9h后，共有591个Unigene差异表达，其中314个Unigene表达上调，277个Unigene表达下调（p<0.05）。其中，差异表达极显著的上调Unigene有68个，下调Unigene 77个。.18S可作为处理大豆蚜虫的内参基因。10 mg L-1 的高效氯氰菊酯处理后，hem基因的表达量从第6小时开始持续增加，9小时与对照组的表达量大致相等，在12小时表达量达到最大值；POLA1基因的表达量从第3小时开始持续增加，8小时与对照组的表达量大致相等，在12小时表达量达到最大值；JHAMT基因的表达量从第6小时开始呈现下降趋势，在15小时表达量下降到最低水平。.RNA干扰结果表明：注射了体外合成 dsHem 片段的大豆蚜，12h和48h 后体内的 Hem 基因的表达量降低，与对照差异显著(P<0.01)。注射dsHem的大豆蚜7天内单头产蚜量为36.71头，而注射dsGFP的大豆蚜的产蚜量为46.63头，差异极显著p<0.01。说明注射dsHem能够影响大豆蚜成虫的平均产蚜量。大豆蚜内导入dsHem 后能直接影响到高效氯氰菊酯对大豆蚜的产蚜量，较对照组导入 dsGFP的产蚜量降低了 0.81倍，差异显著（P<0.05）。研究结果表明Hem基因参与调控了高效氯氰菊酯刺激产蚜过程。.该项目成功发明了一种高分子多糖人工膜，利用该膜能在24小时内检测出高效氯氰菊酯等多种农药的亚致死浓度对大豆蚜生殖的刺激效果，具有较好的应用前景。