昆虫学报 ›› 2022, Vol. 65 ›› Issue (4): 460-468.doi: 10.16380/j.kcxb.2022.04.006

• 研究论文 • 上一篇    下一篇

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

祝智威1,#, 王杰1,#, 隆琦1, 许雅静1, 冯睿蓉1, 刘佳美1, 赵浩东1朱乐冉1, 侯海青1, 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. 福建农林大学蜂疗研究所, 福州 350002)
  • 出版日期:2022-04-20 发布日期:2022-03-24

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

ZHU Zhi-Wei1,#, WANG Jie1,#, LONG Qi1, XU Ya-Jing1, FENG Rui-Rong1, LIU Jia-Mei1, ZHAO Hao-Dong1, ZHU Le-Ran1, HOU Hai-Qing1, 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, Fujian Agriculture and Forestry University, Fuzhou 350002, China)
  • Online:2022-04-20 Published:2022-03-24

摘要: 【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, EgfrP450 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EcrP450 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。

关键词: 意大利蜜蜂, 幼虫, 肠道, 微小RNA, ame-miR-13b, 靶基因

Abstract: 【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.

Key words: Apis mellifera ligustica, larva, gut, microRNA, ame-miR-13b, target gene