昆虫学报 ›› 2023, Vol. 66 ›› Issue (1): 1-10.doi: 10.16380/j.kcxb.2023.01.001

• 研究论文 •    下一篇

ame-miR-14在意大利蜜蜂工蜂幼虫肠道发育过程的调控作用

王紫馨1,#, 许雅静1,#, 张文德1, 张凯遥1, 吴鹰1, 刘佳美1, 朱乐冉1牛庆生3, 赵红霞4, 陈大福1,2,*, 郭睿1,2,*   

  1. (1. 福建农林大学动物科学学院(蜂学学院), 福州 350002; 2. 福建省蜂疗研究所, 福州 350002; 3. 吉林省养蜂科学研究所, 吉林 132000; 4. 广东省科学院动物研究所, 广州 510260)
  • 出版日期:2023-01-20 发布日期:2023-03-04

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

WANG Zi-Xin1,#, XU Ya-Jing1,#, ZHANG Wen-De1, ZHANG Kai-Yao1, WU Ying1, LIU Jia-Mei1, ZHU Le-Ran1, NIU Qing-Sheng3, ZHAO Hong-Xia4, CHEN Da-Fu1, 2,*, GUO Rui1, 2,*   

  1.  (1. College of Animal Sciences (College of Bee Science), Fujian Agriculture and Forestry University, Fuzhou 350002, China; 2. Apitherapy Research Institute of Fujian Province, Fuzhou 350002, China; 3. Apiculture Science Institute of Jilin Province, Jilin 132000, China; 4. Institute of Zoology, Guangdong Academy of Sciences, Guangzhou 510260, China)
  • Online:2023-01-20 Published:2023-03-04

摘要:  【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FoxOHedgehog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FoxOHedgehog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FoxOHedgehog的表达潜在参与调节幼虫肠道发育。

关键词:  , 意大利蜜蜂, 幼虫, 肠道, ame-miR-14, 靶基因, 调控, 发育

Abstract: 【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.

Key words: Apis melliferae ligustica, larva, gut, ame-miR-14, target gene, regulation, development