ame-miR-14在意大利蜜蜂工蜂幼虫肠道发育过程的调控作用

Regulatory role of ame-miR-14 in the developmental process of the larval guts of Apis mellifera ligustica(Hymenoptera:Apidae)workers

【目的】本研究旨在揭示ame-miR-14在意大利蜜蜂Apis mellifera ligustica工蜂幼虫肠道发育过程的调控作用。【方法】通过Stem-loop RT-PCR和Sanger测序分别验证ame-miR-14在意大利蜜蜂工蜂6日龄幼虫肠道中的表达和序列真实性。饲喂ame-miR-14的模拟物(mimic-ame-miR-14)和抑制物(inhibitor-ame-miR-14)及其相应的阴性对照mimic-NC和inhibitor-NC对ame-miR-14分别进行过表达和敲降,利用RT-qPCR检测意大利蜜蜂工蜂4-6日龄幼虫肠道中ame-miR-14的表达量。利用生物信息学软件预测ame-miR-14的靶基因并进行相关分析。利用RT-qPCR检测ame-miR-14的过表达和敲降后其靶基因FoxO和Hedgehog在4-6日龄幼虫肠道中的相对表达量。【结果】ame-miR-14在意大利蜜蜂工蜂6日龄幼虫肠道中真实存在和表达。相较于饲喂mimic-NC,饲喂mimic-ame-miR-14后ame-miR-14表达量在意大利蜜蜂工蜂4-6日龄幼虫肠道中均为显著上调;相较于饲喂inhibitor-NC,饲喂inhibitor-ame-miR-14后ame-miR-14表达量在意大利蜜蜂工蜂4-6日龄幼虫肠道中皆为显著下调。ame-miR-14共靶向309个基因,可注释到45条KEGG通路和36个GO条目;进一步分析发现ame-miR-14可靶向14个生长发育相关基因,并与FoxO和Hedgehog之间存在潜在的靶向关系。过表达ame-miR-14后,相较于mimic-NC组,mimic-ame-miR-14组的4日龄幼虫肠道内FoxO表达量下调,5和6日龄幼虫肠道内FoxO表达量均为显著下调;敲降ame-miR-14后,相较于inhibitor-NC组,inhibitor-ame-miR-14组4日龄幼虫肠道内FoxO表达量下调,5日龄幼虫肠道内FoxO表达量显著上调,6日龄幼虫肠道内FoxO表达量上调。与mimic-NC组相比,过表达ame-miR-14后mimic-ame-miR-14组的Hedgehog表达量在4-6日龄幼虫肠道内皆显著下调;与inhibitor-NC组相比,敲降ame-miR-14后inhibitor-ame-miR-14组的Hedgehog表达量在4-6日龄幼虫肠道内均为上调。【结论】ame-miR-14在意大利蜜蜂工蜂幼虫肠道内真实存在和表达;通过饲喂模拟物和抑制物能够分别实现意大利蜜蜂工蜂幼虫肠道内ame-miR-14的有效过表达和敲降;ame-miR-14通过负调控FoxO和Hedgehog的表达潜在参与调节幼虫肠道发育。

【Aim】The objective of this study is to unravel the regulatory role of ame-miR-14 in the developmental process of the larval guts of Apis mellifera ligustica workers.【Methods】The expression and sequence authenticity of ame-miR-14 in the 6-day-old larval guts of A.m.ligustica workers were proved by Stem-loop RT-PCR and Sanger sequencing,respectively.Overexpression and knockdown of ame-miR-14 were conducted by feeding its corresponding mimic(mimic-ame-miR-14)and inhibitor(inhibitor-ame-miR-14),and their corresponding negative controls mimic-NC and inhibitor-NC,respectively,and then RT-qPCR was used to detect the expression levels of ame-miR-14 in the 4-6-day-old larval guts of A.m.ligustica workers.Bioinformatic software was used to predict and analyze the target genes of ame-miR-14.RT-qPCR was employed to detect the relative expression levels of the target genes FoxO and Hedgehog in the 4-6-day-old larval guts after overexpression and knockdown of ame-miR-14.【Results】ame-miR-14 truly exists and is expressed in the 6-day-old larval guts of A.m.ligustica workers.The expression levels of ame-miR-14 in the 4-6-day-old larval guts of A.m.ligustica workers fed with mimic-ame-miR-14 were significantly up-regulated as compared to those fed with mimic-NC.The expression levels of ame-miR-14 in the 4-6-day-old larval guts of A.m.ligustica workers fed with inhibitor-ame-miR-14 were significantly down-regulated as compared to those fed with inhibitor-NC.In total,ame-miR-14 can target 309 genes,which could be annotated to 45 KEGG pathways and 36 GO terms.Further analysis showed that ame-miR-14 can target 14 genes associated with growth and development and have potential targeting relationship with target genes FoxO and Hedgehog.After overexpression of ame-miR-14,the expression level of FoxO in the 4-day-old larval gut of the mimic-ame-miR-14 group was down-regulated and those in the 5-and 6-day-old larval guts significantly down-regulated as compared to those in the mimic-NC group.After knockdown of ame-miR-14,the expression level of FoxO in the 4-day-old larval gut of the inhibitor-ame-miR-14 group was down-regulated,while that in the 5-day-old larval gut was significantly up-regulated and that in the 6-day-old larval gut was up-regulated as compared to those in the inhibitor-NC group.After overexpression of ame-miR-14,the expression levels of Hedgehog in the 4-6-day-old larval guts of the mimic-ame-miR-14 group were significantly down-regulated in comparison with those in the mimic-NC group,whereas those in the 4-6-day-old larval guts of the inhibitor-ame-miR-14 group were up-regulated in comparison with those in the inhibitor-NC group.【Conclusion】ame-miR-14 truly exists and is expressed in the larval guts of A.m.ligustica workers.Effective overexpression and knockdown of ame-miR-14 in the larval guts of A.m.ligustica workers can be achieved by feeding mimic and inhibitor,respectively.ame-miR-14 potentially participates in regulation of the larval gut development by negatively regulating the expression of FoxO and Hedgehog.