害虫基因干扰中的dsRNA序列相关脱靶效应

RNA interference with dsRNA is high efficient for insect pests, and also easy to be achieved in field by engineering plants and microorganisms. Thus, new pest control technology could be developed. However, bio-safety concerning with off-target effects seriously depressed the development of this technique. Now, some work has been done on siRNA off-target effects. But little is known for dsRNA off-target effects, especial for the factors influencing its occurrence. For this reason we present this application. The research will employ Helicoverpa armigera and Tribolium castaneum as test insects, and homologous genes with high similarity from P450 super family will be cloned. Among these genes a target gene will be selected and its conserved and special sequence fragments will be cut for synthesis of dsRNA mosaic molecules. These dsRNA molecules are designed with different similarity, continuous matching fragments or modified sites. Then, series of experiments with these dsRNA will be carried out and RNA interference effects on target and non-target genes will be checked. Finally, the molecular characters of dsRNA influencing off-target effects, the rule for off-target occurrence, and the molecule related mechanism for off-target occurrence will be analyzed. This study will provide good knowledge for dsRNA off-target effects, reveal the possibility for control of off-target effects, and promote the development of a new pest control technology with RNA interference.

用dsRNA进行害虫基因干扰不仅高效，而且便于利用工程生物野外实施，进而开创害虫防控的新途径。但脱靶效应带来的潜在安全隐患严重制约了这一新技术的发展。目前国内外对siRNA的脱靶效应进行了不少研究，但对dsRNA的脱靶效应，尤其是影响其发生的相关因素，知之甚少。因此，本研究立项拟利用基因干扰难易程度不同的棉铃虫和赤拟谷盗为试虫，克隆其P450超家族基因，获取相似性较高的系列同源基因，从中选取靶标基因，利用其保守序列和专一性序列片段，拼接合成与已知P450基因相似性不同、连续匹配序列长短不一、以及经过不同修饰的系列dsRNA嵌合分子，通过基因干扰组合试验，检测不同分子对靶标和非靶标基因的干扰效果，进而分析影响脱靶效应的dsRNA分子特征、脱靶效应的发生规律，以及与序列匹配相关的脱靶机制，揭示控制脱靶效应的可能性，为全面阐释dsRNA的脱靶效应，推动农业害虫RNAi控制技术的发展提供理论依据。

用dsRNA进行害虫基因干扰不仅高效，而且便于利用工程生物野外实施，进而开创害虫防控的新途径。但脱靶效应带来的潜在安全隐患严重制约了这一新技术的发展。目前国内外对siRNA的脱靶效应进行了不少研究，但对dsRNA的脱靶效应，尤其是影响其发生的相关因素，知之甚少。因此，本研究立项拟利用基因干扰难易程度不同的棉铃虫和赤拟谷盗为试虫，克隆其P450超家族基因，获取相似性较高的系列同源基因，从中选取靶标基因，利用其保守序列和专一性序列片段，拼接合成与已知P450基因相似性不同、连续匹配序列长短不一、以及经过不同修饰的系列dsRNA嵌合分子，通过基因干扰组合试验，检测不同分子对靶标和非靶标基因的干扰效果，进而分析影响脱靶效应的dsRNA分子特征、脱靶效应的发生规律，以及与序列匹配相关的脱靶机制，揭示控制脱靶效应的可能性，为全面阐释dsRNA的脱靶效应，推动农业害虫RNAi控制技术的发展提供理论依据。