双链RNA在转化质体中表达稳定性的探讨及其杀虫功能性研究

Colorado potato beetle (Leptinotarsa decemlineata; CPB) has been branded as an “international super pest.” which can cause the huge yield loss of potato plants. In previous study, transplastomic plants can accumulate high amount of dsRNAs that targeted against insect essential genes, and efficiently killed the CPB through RNA interference (RNAi). However, although the same cis-element (promoter) was used in the all constructs, we found that the expression levels of dsRNAs (ACT, SHR and ACT+SHR) in plastids are different and positively correlate with the mortality of CPB larvae. To elucidate the factors that influence the expression levels (stability) of dsRNAs expressed in plastids. Here we propose a strategy address this question. We will design dsRNA molecules that have variant GC content and different length and let them to be expressed in plastids. By testing expression levels of those dsRNAs in plastid and their lethality to potato beetle, we can evaluate the factors that affect dsRNAs stability in plastids which could associate with the effect of RNAi of CPB. Moreover, we will also design highly structured dsRNAs and have them to be expressed plastids to test if these modified dsRNAs are more stable and cause higher mortality rate of CPB. Taken together, this study will facilitate the future RNAi experiment design to control insect pest by precisely choose the suitable genes and their targeted sequences, and increase the dsRNAs expression levels in plastids. It will pave a way to generate transplastomic potato plants that are highly resistant to CPB and also apply to other crops against other insect pests.

马铃薯甲虫是一种世界性害虫，其危害严重影响马铃薯的产量。在前期研究中，我们采用质体转基因技术在马铃薯中表达针对甲虫特定基因的dsRNA，结果表明，相比核转化体系，质体转化体系极大地提高了dsRNA的表达水平，并起到了显著的植物保护和杀虫作用。但是，尽管使用相同的顺式调控原件(Prrn启动子)来控制表达不同靶基因对应的dsRNA，在转化质体中不同dsRNA的表达水平有所不同，且表达量高低和杀虫效果呈正相关性。为探索决定 dsRNA表达水平的可能因素和进一步提高杀虫效果，本研究将系统地设计不同长度、GC含量和高级结构的dsRNA，并在质体中表达，检测它们的表达水平(稳定性)和杀虫效果。本研究结果将有助于准确选择靶基因及靶基因对应序列，提高它们在质体中表达水平，为培育高抗虫性马铃薯质体转基因新品种，及未来用于其它抗虫性农作物的品种创新奠定基础。

马铃薯甲虫是一种世界性害虫，严重威胁着马铃薯的产量。质体介导RNAi抗马铃薯甲虫被认为是一种行之有效的新的抗虫技术，通过在质体中表达针对害虫必需基因的dsRNA，被害虫取食后诱导害虫本身的RNAi反应，沉默相应基因的达到抗虫的目的。但影响dsRNA在质体中的积累水平的因素还不是很清楚。本项目我们选取了马铃薯甲虫β-Actin基因（ACT），比较了不同长度的dsRNA诱导的RNAi效率。喂食体外合成dsRNA结果表明，虽然60 bp的dsRNA在马铃薯甲虫中仅诱导相对较低的RNAi反应，但200 bp及更长的dsRNA能引起相似的马铃薯甲虫幼虫的生长延缓和高致死率。当dsRNA从马铃薯质体基因表达时，我们发现dsRNA的积累水平与长度呈负相关，dsRNA积累水平与观察到的死亡率、抑制幼虫生长和抑制靶基因表达呈正相关。重要的是，表达200 bp dsRNA的转基因马铃薯植物比表达297 bp dsRNA的植物更好地保护马铃薯甲虫。因此，dsRNA的长度是影响其在成片中的积累水平的重要因素，因此决定了杀虫RNAi效应的强度。我们的发现将有助于设计优化的dsRNA表达载体以增强质体介导RNAi的抗虫效果。