过表达和敲减ame-miR-13b对意大利蜜蜂幼虫肠道内基因表达的影响

【目的】微小RNA(microRNAs, miRNAs)在昆虫的繁殖、发育和免疫等重要生命活动的调控过程中扮演关键角色。本研究旨在探究在意大利蜜蜂Apis mellifera ligustica幼虫个体水平过表达与敲减ame-miR-13b对其靶基因表达的影响，为深入探究ame-miR-13b调控意大利蜜蜂幼虫肠道发育的分子机理提供理论和实验依据。【方法】根据ame-miR-13b的核苷酸序列设计合成相应的模拟物mimic-ame-miR-13b和抑制物inhibitor-ame-miR-13b及其对照mimic-NC和inhibitor-NC，混入饲料后饲喂意大利蜜蜂3日龄幼虫，每12 h更换一次饲料，连续饲喂6次，以分别过表达和敲减ame-miR-13b；通过RT-qPCR检测过表达和敲减ame-miR-13b后意大利蜜蜂幼虫肠道中ame-miR-13b及其靶基因Ecr, Egfr和P450 18a1的表达量。【结果】与mimic-NC饲喂组相比, mimic-ame-miR-13b饲喂组中ame-miR-13b在意大利蜜蜂4和6日龄幼虫肠道中均显著上调表达；与饲喂inhibitor-NC组相比，inhibitor-ame-miR-13b饲喂组中ame-miR-13b在4日龄幼虫肠道中下调表达，在5和6日龄幼虫肠道中显著下调表达。与mimic-NC饲喂组相比，过表达ame-miR-13b组中意大利蜜蜂6日龄幼虫肠道中Egfr的表达量显著降低，而Ecr和P450 18a1的表达量均显著升高。与inhibitor-NC饲喂组相比，敲减ame-miR-13b组中意大利蜜蜂6日龄幼虫肠道中Egfr上调表达但不显著，Ecr显著上调表达，而P450 18a1显著下调表达。【结论】通过饲喂法在意大利蜜蜂幼虫个体中成功实现了miRNA的过表达和敲减；ame-miR-13b与Egfr之间存在潜在的负调控关系。

Impact of overexpression and knockdown of ame-miR-13b on the expression of genes in larval gut ofApis mellifera ligustica

【Aim】MicroRNAs (miRNAs) play a pivotal role in important life processes such as reproduction, development and immunity in insects. The aim of this study is to explore the impact of overexpression and knockdown of ame-miR-13b on the expression of target genes in Apis mellifera ligustica larvae, so as to provide theoretical and experimental bases for further investigation of regulation mechanism of ame-miR-13b underlying the development of A. m. ligustica larval gut. 【Methods】 Based on the nucleotide sequence of ame-miR-13b, the sequences of the corresponding mimic-ame-miR-13b and inhibitor-ame-miR-13b and their counterpart controls mimic-NC and inhibitor-NC were designed and synthesized, and then mixed with the diet which was used to feed the 3-day-old larvae for 6 times with the diet changed every 12 h to perform overexpression and knockdown of ame-miR-13b, respectively. RT-qPCR was performed to detect the expression levels of ame-miR-13b and target genes Ecr, Egfr and P450 18a1 in the larval gut of A. m. ligustica after overexpression and knockdown of ame-miR-13b. 【Results】 The expression levels of ame-miR-13b in the guts of the 4- and 6-day-old larvae of A. m. ligustica fed with mimic-ame-miR-13b were significantly up-regulated as compared to that in the group fed with mimic-NC, and those in the guts of the 4-day-old larvae and in the guts of the 5- and 6-day old larvae fed with inhibitor-ame-miR-13b were down-regulated and significantly down-regulated, respectively, as compared to that in the group fed with inhibitor-NC. After overexpression of ame-miR-13b, the expression level of Egfr in the gut of the 6-day-old larvae was significantly decreased, while those of Ecr and P450 18a1 were significantly increased as compared to that in the group fed with mimic-NC. After knockdown of ame-miR-13b, the expression of Egfr in the gut of the 6 day-old larvae was up-regulated insignificantly, that of Ecr was significantly up-regulated, whereas that of P450 18a1 was significantly down-regulated as compared to that in the group fed with inhibitor-NC. 【Conclusion】 Overexpression and knockdown of miRNA in larval individuals of A. m. ligustica are successfully achieved by feeding method. There is a potential negative regulatory relationship between ame-miR-13b and Egfr.