意大利蜜蜂工蜂幼虫肠道中ame-miR-bantam对靶基因USP和P300的表达调控

doi:10.16380/j.kcxb.2022.11.001

昆虫学报 ›› 2022, Vol. 65 ›› Issue (11): 1401-1410.doi: 10.16380/j.kcxb.2022.11.001

• 研究论文 • 下一篇

意大利蜜蜂工蜂幼虫肠道中ame-miR-bantam对靶基因USP和P300的表达调控

胡颖^1,#, 张文德^1,#, 王思懿¹, 张凯遥¹, 任中民¹, 吉挺³, 蔺哲广³, 赵红霞⁴, 陈大福^1,2,*, 郭睿^1,2,*

(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. 扬州大学动物科学与技术学院, 江苏扬州 225000; 4. 广东省科学院动物研究所, 广州 510260)

出版日期:2022-11-20 发布日期:2022-12-02

Regulation of the expression of target genes USP and P300 by ame-miR-bantam in the larval gut of Apis mellifera ligustica workers

HU Ying^1,#, ZHANG Wen-De^1,#, WANG Si-Yi¹, ZHANG Kai-Yao¹, REN Zhong-Min¹, JI Ting³, LIN Zhe-Guang³, ZHAO Hong-Xia⁴, CHEN Da-Fu^1,2,*, GUO Rui^1,2,*

(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. College of Animal Science and Technology, Yangzhou University, Yangzhou, Jiangsu 225000, China; 4. Institute of Zoology, Guangdong Academy of Sciences, Guangzhou 510260, China)

Online:2022-11-20 Published:2022-12-02

摘要/Abstract

摘要： 【目的】本研究旨在通过饲喂法对意大利蜜蜂Apis mellifera ligustica工蜂幼虫肠道中ame-miR-bantam进行过表达与敲减，明确ame-miR-bantam对靶基因USP和P300表达的调控作用。【方法】通过饲喂ame-miR-bantam模拟物(mimic-ame-miR-bantam)和抑制物(inhibitor-ame-miR-bantam)及相应的阴性对照mimic-NC-ame-miR-bantam和inhibitor-NC-ame-miR-bantam，对意大利蜜蜂工蜂4-6日龄幼虫肠道组织中的ame-miR-bantam分别进行过表达和敲减。利用生物信息学软件进行ame-miR-bantam的靶向预测及分析。通过RT-qPCR检测4-6日龄意大利蜜蜂工蜂幼虫肠道中ame-miR-bantam的过表达和敲减效果及靶基因USP和P300的相对表达量。【结果】相较于饲喂mimic-NC-ame-miR-bantam，饲喂mimic-ame-miR-bantam后，ame-miR-bantam在意大利蜜蜂4-6日龄工蜂幼虫肠道中均显著上调；相较于饲喂inhibitor-NC-ame-miR-bantam，饲喂inhibitor-ame-miR-bantam后，ame-miR-bantam在4日龄工蜂幼虫肠道中显著下调，在5日龄和6日龄工蜂幼虫肠道中下调表达。ame-miR-bantam共靶向222个基因，可注释到细胞进程等35个GO条目和Wnt信号通路等160条KEGG通路。相比于饲喂mimic-NC-ame-miR-bantam的阴性对照组，过表达ame-miR-bantam后，4日龄工蜂幼虫肠道中USP和P300的表达量均为上调；5日龄工蜂幼虫肠道中USP的表达量下调，P300显著下调表达；6日龄工蜂幼虫肠道中USP和P300均显著下调表达。相比于饲喂inhibitor-NC-ame-miR-bantam的阴性对照组，敲减ame-miR-bantam后，4和5日龄工蜂幼虫肠道中USP和P300的表达量均为上调；6日龄工蜂幼虫肠道中USP显著上调表达，P300的表达量上调。【结论】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中ame-miR-bantam的有效过表达和敲减；幼虫肠道中ame-miR-bantam可负调控USP和P300的表达。

关键词: 意大利蜜蜂, 微小RNA, ame-miR-bantam, 模拟物, 抑制物, 肠道, 发育

Abstract: 【Aim】 The aim of this study is to clarify the regulatory effects of ame-miR-bantam on the expression of target genes USP and P300 by performing overexpression and knockdown of ame-miR-bantam in the larval gut of Apis mellifera ligustica workers through feeding method. 【Methods】 Overexpression and knockdown of ame-miR-bantam in tissues of the larval gut of the 4-6-day-old workers of A. m. ligustica were conducted by feeding mimic-ame-miR-bantam and inhibitor-ame-miR-bantam, and their corresponding negative controls mimic-NC-ame-miR-bantam and inhibitor-NC-ame-miR-bantam of ame-miR-bantam, respectively. Target genes of ame-miR-bantam were predicted and analyzed using bioinformatic software. RT-qPCR was used to detect the overexpression and knockdown effects of ame-miR-bantam and the relative expression levels of target genes USP and P300 in the larval gut of the 4-6-day-old workers of A. m. ligustica. 【Results】 Compared with feeding with mimic-NC-ame-miR-bantam, feeding with mimic-ame-miR-bantam resulted in significant up-regulation of ame-miR-bantam in the larval gut of the 4-6-day-old workers of A. m. ligustica. Compared with feeding with inhibitor-NC-ame-miR-bantam, feeding with inhibitor-ame-miR-bantam resulted in significant down-regulation of ame-miR-bantam in the larval gut of the 4-day-old workers, and down-regulation of ame-miR-bantam in the larval gut of the 5- and 6-day-old workers. ame-miR-bantam can target a total of 222 genes, which can be annotated to 35 GO terms such as cellular process and 160 KEGG pathways such as Wnt signaling pathway. After overexpression of ame-miR-bantam, the expression levels of USP and P300 in the larval gut of the 4-day-old workers were up-regulated, that of USP in the larval gut of the 5-day-old workers was down-regulated, that of P300 in the larval gut of the 5-day-old workers was significantly down-regulated, those of USP and P300 in the 6-day-old larval gut were significantly down-regulated as compared to those in the negative control group fed with mimic-NC-ame-miR-bantam. After knockdown of ame-miR-bantam, the expression levels of USP and P300 in the larval gut of the 4- and 5-day-old workers were up-regulated, that of USP in the larval gut of the 6-day-old workers was significantly up-regulated whereas that of P300 in the larval gut of the 6-day-old workers was up-regulated as compared with those in the negative control group fed with inhibitor-NC-ame-miR-bantam. 【Conclusion】 Effective overexpression and knockdown of ame-miR-bantam in the larval gut of A. m. ligustica workers can be achieved by feeding with mimic and inhibitor, respectively, and ame-miR-bantam in the larval gut may negatively regulates the expression of USP and P300.

Key words: Apis melliferae ligustica, microRNA, ame-miR-bantam, mimic, inhibitor, gut, development

胡颖, 张文德, 王思懿, 张凯遥, 任中民, 吉挺, 蔺哲广, 赵红霞, 陈大福, 郭睿. 意大利蜜蜂工蜂幼虫肠道中ame-miR-bantam对靶基因USP和P300的表达调控[J]. 昆虫学报, 2022, 65(11): 1401-1410.

HU Ying, ZHANG Wen-De, WANG Si-Yi, ZHANG Kai-Yao, REN Zhong-Min, JI Ting, LIN Zhe-Guang, ZHAO Hong-Xia, CHEN Da-Fu, GUO Rui. Regulation of the expression of target genes USP and P300 by ame-miR-bantam in the larval gut of Apis mellifera ligustica workers[J]. Acta Entomologica Sinica, 2022, 65(11): 1401-1410.

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意大利蜜蜂工蜂幼虫肠道中ame-miR-bantam对靶基因USP和P300的表达调控

Regulation of the expression of target genes USP and P300 by ame-miR-bantam in the larval gut of Apis mellifera ligustica workers

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