LncRNA作为ceRNA靶向miRNA调控螺虫乙酯抗性棉蚜ACC过表达机制

Further analysis of resistance mechanism is an important basis for resistant cotton aphid control. LncRNA function as a competitive non coding RNA which sequesters miRNAs to regulate expression level of other transcripts sharing common miRNA response elements. Preliminary results showed that overexpression of the mutated ACC is the main reason account for the high levels spirotetramat resistance in cotton aphid, while ACC mRNA expression level was post transcriptional regulated by miR-3016 and miR-276 which expression was significantly decreased in resistant aphid; In addition, high-throughput sequencing and bio-information analysis revealed that the miR-276/miR-3016 response elements occurred in the resistant specific overexpressed lncRNAs. Therefore, we suggest that lncRNA may be involved in the inhibition of miR-276 and miR-3016, thus affecting the function of miRNA silencing ACC in resistant aphid. This project aims to obtain the complete sequence of the lncRNAs, identify response elements of miR-276/miR-3016 and testify the function of lncRNA to the expression of miR-276/miR-3016 and ACC mRNA. Finally elucidate the role of lncRNA-miR-276/miR-3016-mRNA (ACC) pathway in the spirotetramat resistance of cotton aphid. This will make up for the gap of the lncRNA-miRNA-mRNA regulatory pathway in the mechanism of insect resistance. At the same time, this study also provides a new perspective for study of resistance mechanism.

深入解析抗药性机制是抗性棉蚜治理的重要基础。前期结果表明，靶标基因ACC突变及其过量表达导致棉蚜对螺虫乙酯产生高水平抗性，其表达受 miR-276 和 miR-3016调控，且抗性棉蚜miR-276和 miR-3016 表达量显著降低；另外，高通量测序筛选出抗性棉蚜特异性高表达的 lncRNA含有 miR-276/miR-3016 的应答元件。因此，推测lncRNA可能靶向抑制miR-276和miR-3016，从而减弱miRNA对 ACC转录后沉默功能。本项目拟获得这些lncRNA全序列，鉴定出miR-276/miR-3016的应答元件并验证其功能，阐明lncRNA-miRNA-mRNA 调控途径在棉蚜螺虫乙酯抗药性形成中的作用。这将弥补lncRNA-miRNA-mRNA调控途径参与昆虫对外源化合物耐受机制的空白。同时，本项研究也为昆虫对外源化合物耐受性形成机制提供了新的研究视角。

深入解析抗药性机制是抗性棉蚜治理的重要基础。长链非编码 RNA (lncRNA) 是指长度超过200 bp，未翻译成蛋白质非编码RNA。其主要功能是直接或者间接的调控信使RNA的表达，最终调控细胞内的各种生化生理进程以及有机体的发育。为了进一步研究棉蚜这种世界性害虫的抗药性机制，我们探究了是否lncRNA能够调控棉蚜关键的解毒代谢酶乙酰辅酶A羧化酶（ACC）和细胞色素P450酶（P450）来介导棉蚜对螺虫乙酯抗性。在棉蚜中首次鉴定出6059条lncRNA；螺虫乙酯抗性和敏感品系中有874个差异表达的lncRNA，其中440个lncRNA在SR品系中高表达，434个lncRNA在SS品系中低表达。差异表达的lncRNA大部分的归于调控代谢途径，结合活性以及细胞膜功能。LncRNA: MSTRG.28822能够调控ACC基因的表达从而介导棉蚜螺虫乙酯抗性，但不是作为分子海绵方式抑制miR-276和miR-3016而调控ACC表达；同时证明了转录因子基因C/EBP 和 C/EBPzeta可以调控 MSTRG.28822的表达。转基因果蝇实验验证了CYP380C6和 CYP6CY21 介导了棉蚜螺虫乙酯抗性；并证明了MSTRG.36649.2/5 和 MSTRG.71880.1 能够通过除分子海绵模式的其他方式来调控CYP6CY21和CYP380C6 的表达。本研究研究证明了LncRNA参与昆虫对外源化合物耐受性形成，表明的害虫抗性进化及其调控的复杂和多样性，为理解和深入研究抗性适应性进化奠定一定基础。