植物sRNA对昆虫脂代谢基因的跨界调控机制

Many signaling molecules that transmitted among insects and plants regulate the interaction of insects and plants. Small RNA (sRNA) is one of these signaling molecules. Our preliminary dates showed that dcl1 mutation significantly reduces the growth of cotton bollworm feeds on plants. The expressions of lipid metabolism genes are also significantly changed in bollworms fed with dcl1 mutant plants compared with bollworms fed with CK plants. Moreover, the fat content and the lipid droplet content of bollworms feed with dcl1 mutant plants are much lower than those of bollworms feed with CK plants. We then removed the bollworm guts and found plant sRNAs accumulate in the rest organs of bollworm. These sRNAs potentially target bollworm genes and regulate their expressions. These results suggested that these plant sRNAs can regulate the lipid metabolism of insects.. Next, we will determine the regulation of lipid metabolic enzyme by plant sRNAs in insects with in vitro luciferase, RNA co-immunoprecipitation and co-localization assays. The function of these sRNAs in bollworms will be validated by directly injecting the agomir/antagomir into bollworms. The cross-kingdom regulations of these plant sRNAs in insects will also be determined in sRNA over-expression and knockout plants. This proposed study not only will reveal the biological functions of cross-kingdom sRNAs, but also will identify key genes involved in pest-plant interaction. The overall goal of this proposal is developing novel technology to control the pests and benefit crop production.

昆虫与植物相互作用过程中，许多信号分子在其间传递和作用。sRNA作为机体间传递的重要信号分子，参与调控植物和动物的防御反应。项目前期研究发现棉铃虫取食dcl1突变体拟南芥后体重显著降低，总脂肪含量降低，脂滴量减少，脂代谢相关基因表达明显变化；sRNA高通量测序和定量验证发现去除消化道棉铃虫中出现拟南芥sRNA，且与脂代谢酶基因有潜在结合。在此基础上项目拟开展如下研究：1）采用体外荧光素酶、RNA免疫共沉淀和共定位实验，确定拟南芥sRNA对棉铃虫脂代谢酶的调控作用；2）昆虫过表达或沉默植物sRNA，分析其对总脂肪含量及脂代谢基因和蛋白表达的影响；3）植物过表达/沉默sRNA以及昆虫RNAi/rescue实验，阐明植物sRNA对昆虫脂代谢的调控机制。研究结果不仅将发现昆虫与植物互作中防御机制的调控节点，还将揭示植物sRNA跨界调控昆虫脂代谢的作用机制，对指导抗虫育种和害虫有效治理具有重要意义。

昆虫与植物相互作用过程中，许多信号分子在其间传递和作用。sRNA作为机体间传递的重要信号分子，参与调控植物和动物的防御反应。项目研究了AGO1参与调控拟南芥抗棉铃虫的新机制，发现了miR5021作为一个重要抗虫信号通路的关键分子，与AGO1结合调控JA信号通路的受体基因COI1，增强植物抗性，这将对棉铃虫的生物防治提供重要的科学依据。研究结果不仅将发现昆虫与植物互作中防御机制的调控节点，还将揭示植物sRNA跨界调控昆虫脂代谢的作用机制，对指导抗虫育种和害虫有效治理具有重要意义。.为了揭示sRNA降解机制，我们首先利用Northern blot发现5’-3’方向的核酸外切酶xrn4突变体中部分miRNA*s积累量显著上升，但miRNA前体的积累水平并没有发生变化，此结果暗示AtXRN4可能通过对miRNA*降解通路的调控影响了miRNA*的积累。由于AtAGO2是结合miRNA*的核心蛋白，继而我们利用Co-IP和BiFC技术验证了AtXRN4与AtAGO2蛋白在降解和储存RNA的加工小体（processing body, P-body）中互作。研究结果发现了AtXRN4在加工小体内通过与AtAGO2的互作调控miRNA*的降解。. 此外，我们还发现，miR-8-5P负调控了谷氨酸至γ-氨基丁酸合成通路上的关键基因——GAD，导致脑内兴奋性神经递质和抑制性神经递质合成和积累发生变化，从而引起飞蝗发生型变。群居型飞蝗抑制miR-8-5P能够上调GAD的表达，并增加γ-氨基丁酸的合成，导致群居型飞蝗的行为向散居型显著趋近；研究结果揭示了miRNA介导的表型可塑性分子机理以及对飞蝗型变的调控机制。