ame-miR-79负调控意大利蜜蜂工蜂幼虫肠道中靶基因CYP450和FG的表达

【目的】本研究旨在通过饲喂ame-miR-79的模拟物(mimic)和抑制物(inhibitor)对意大利蜜蜂Apis mellifera ligustica工蜂幼虫肠道内的ame-miR-79分别进行过表达和敲减，探究ame-miR-79对幼虫肠道内靶基因表达的调控作用。【方法】通过分子克隆与Sanger测序验证意大利蜜蜂工蜂幼虫肠道内ame-miR-79的序列真实性。通过饲喂mimic-miR-79和inhibitor-miR-79对意大利蜜蜂工蜂4-6日龄幼虫肠道内的ame-miR-79分别进行过表达和敲减。采用相关生物信息学软件进行ame-miR-79的靶基因预测与分析。通过RT-qPCR检测ame-miR-79的过表达和敲减效果及过表达和敲减ame-miR-79后靶基因的相对表达量。【结果】ame-miR-79在意大利蜜蜂工蜂幼虫肠道内真实存在。与无义模拟物(nonsense mimic, mimic-NC)组相比，mimic-miR-79组的4-6日龄幼虫肠道内ame-miR-79的表达量皆极显著上调；与无义抑制物(nonsense inhibitor, inhibitor-NC)组相比，inhibitor-miR-79组的4和5日龄幼虫肠道内ame-miR-79的表达量显著下调，6日龄幼虫肠道内ame-miR-79的表达量极显著下调。ame-miR-79共靶向303个基因，涉及27个GO条目和179条KEGG通路。相较于mimic-NC组，靶基因细胞色素P450基因CYP450在mimic-miR-79组的4和5日龄幼虫肠道内均极显著下调表达，而在6日龄幼虫肠道内上调表达；靶基因fringe糖基化转移酶(fringe glycosyltransferase, FG)基因在4日龄幼虫肠道内下调表达，在5日龄幼虫肠道内显著下调表达，而在6日龄幼虫肠道内上调表达。相较于inhibitor-NC组，CYP450在inhibitor-miR-79组的4日龄幼虫肠道内极显著上调表达，在6日龄幼虫肠道内上调表达，而在5日龄幼虫肠道内下调表达；FG的表达水平在4日龄幼虫肠道内显著上调，在5和6日龄幼虫肠道内极显著上调。【结论】ame-miR-79在意大利蜜蜂工蜂幼虫肠道内真实存在；通过饲喂模拟物和抑制物能分别实现意大利蜜蜂工蜂幼虫肠道内ame-miR-79的有效过表达和敲减；ame-miR-79负调控意大利蜜蜂工蜂幼虫肠道内CYP450和FG的表达。

ame-miR-79 negatively regulates the expression of target genesCYP450 andFGin the larval guts ofApis mellifera ligusticaworkers

【Aim】 The objective of this study is to further investigate the role of ame-miR-79 in regulating the expression of target genes in the larval guts of Apis mellifera ligustica workers by performing overexpression and knockdown of ame-miR-79 in the larval guts by feeding with mimic and inhibitor of ame-miR-79, respectively. 【Methods】 Molecular cloning and Sanger sequencing were conducted to confirm the sequence authenticity of ame-miR-79 in the larval guts of A. m. ligustica workers. Overexpression and knockdown of ame-miR-79 in the 4-6-day-old larval guts of A. m. ligustica workers were performed through feeding with mimic-miR-79 and inhibitor-miR-79, respectively. Prediction and analysis of target genes of ame-miR-79 were carried out with related bioinformatic software. The overexpression and knockdown effects of ame-miR-79 and the relative expression levels of target genes after overexpression and knockdown of ame-miR-79 were examined by RT-qPCR. 【Results】 ame-miR-79 truly exists in the larval gut of A. m. ligustica workers. Compared with the nonsense mimic (mimic-NC) group, the mimic-miR-79 group showed extremely significantly up-regulated expression of ame-miR-79 in the 4-6-day-old larval guts. In comparison with the nonsense inhibitor (inhibitor-NC) group, the inhibitor-miR-79 group showed significantly down-regulated expression of ame-miR-79 in the 4- and 5-day-old larval guts and extremely significantly down-regulated expression of ame-miR-79 in the 6-day-old larval gut. ame-miR-79 can target 303 genes, which are associated with 27 GO terms and 179 KEGG pathways. In comparison with the mimic-NC group, the mimic-miR-79 group showed extremely significantly down-regulated expression of the cytochrome P450 gene CYP450 in the 4- and 5-day-old larval guts and up-regulated expression of CYP450 in the 6-day-old larval gut, while showed down regulated expression of fringe glycosyltransferase (FG) gene in the 4-day-old larval gut, significantly down-regulated expression of FG in the 5-day-old larval gut, and up-regulated expression of FG in the 6-day-old larval gut. Compared with the inhibitor-NC group, the inhibitor-miR-79 group exhibited extremely significantly up-regulated expression of CYP450 in the 4-day-old larval gut, up-regulated expression of CYP450 in the 6-day-old larval gut, and down regulated expression of CYP450 in the 5-day-old larval gut, while significantly up-regulated expression of FG in the 4-day-old larval gut and extremely significantly up-regulated expression of FG in the 5- and 6-day-old larval guts. 【Conclusion】 ame-miR-79 truly exists in the larval guts of A. m. ligustica workers. Effective overexpression and knockdown of ame-miR-79 in the larval guts of A. m. ligustica workers can be achieved via feeding with mimic and inhibitor, respectively. ame-miR-79 negatively regulates the expression of CYP450 and FG in the larval guts of A. m. ligustica workers.