miR-145-4调控蛋鸭卵泡发育机制的研究

doi:10.16431/j.cnki.1671-7236.2022.03.002

中国畜牧兽医 ›› 2022, Vol. 49 ›› Issue (3): 809-816.doi: 10.16431/j.cnki.1671-7236.2022.03.002

• 专题：水禽遗传繁育与营养 • 上一篇下一篇

miR-145-4调控蛋鸭卵泡发育机制的研究

吴艳^1,2, 皮劲松¹, 张昊¹, 梁振华¹, 杜金平¹, 潘爱銮¹, 申杰¹, 蒲跃进¹

1. 湖北省农业科学院畜牧兽医研究所, 武汉 430064;
2. 动物胚胎工程及分子育种湖北省重点实验室, 武汉 430064

收稿日期:2021-11-01 出版日期:2022-03-05 发布日期:2022-03-03
通讯作者: 皮劲松 E-mail:pijinsong@sina.com
作者简介:吴艳,E-mail:wuyanwh@163.com。
基金资助:
国家自然科学基金（32072709）；湖北省自然科学基金（2020CFB655）；湖北省重点研发计划（2020BBA034）；湖北省技术创新专项（2019ABA084）；财政部和农业农村部：国家现代农业产业技术体系资助（CARS-42）；湖北省农业科学院领军人才项目（L2018017）；湖北省农业科技创新中心项目（2016-620-000-001-023）

Study on the Mechanism of miR-145-4 Regulating Follicle Development in Egg Ducks

WU Yan^1,2, PI Jinsong¹, ZHANG Hao¹, LIANG Zhenhua¹, DU Jinping¹, PAN Ailuan¹, SHEN Jie¹, PU Yuejin¹

1. Institute of Animal Husbandry and Veterinary, Hubei Academy of Agricultural Science, Wuhan 430064, China;
2. Key Laboratory of Animal Embryo Engineering and Molecular Breeding of Hubei Province, Wuhan 430064, China

Received:2021-11-01 Online:2022-03-05 Published:2022-03-03

摘要/Abstract

摘要： 【目的】本研究旨在揭示miR-145-4在蛋鸭卵泡发育中的作用及调控机制。【方法】分离培养蛋鸭卵泡颗粒细胞，转染miR-145-4相似物（mimic）、抑制物（inhibitor）后，利用实时荧光定量PCR法检测细胞增殖凋亡相关基因的表达情况，采用RNAhybrid和Targetscan程序预测miR-145-4的靶基因及其与靶基因磷脂酰肌醇-3-激酶催化亚单位α（phosphatidylinositol-3-kinase catalytic subunit α，PIK3CA）3'-UTR的结合位点，分别构建PIK3CA基因3'-UTR的野生型和突变型双荧光素酶载体，与miR-145-4 mimic和mimic-NC共转染蛋鸭卵泡颗粒细胞后，采用双荧光素酶报告系统验证miR-145-4与靶基因PIK3CA的结合关系，最后采用实时荧光定量PCR法检测miR-145-4对蛋鸭卵泡颗粒细胞中PIK3CA基因表达的影响。【结果】实时荧光定量PCR结果显示，与对照组相比，过表达miR-145-4后CyclinB2基因的表达量极显著降低（P<0.01），BCL2基因的表达量极显著增加（P<0.01）；抑制miR-145-4后，CyclinB2基因表达量极显著升高（P<0.01），BCL2基因表达量极显著降低（P<0.01）。预测结果显示，miR-145-4与PIK3CA基因3'-UTR存在结合位点。PCR扩增和测序结果显示，PIK3CA基因3'-UTR的野生型和突变型载体构建成功。双荧光素酶检测结果显示，与突变型及空载体共转染组相比，野生型组双荧光素酶活性显著降低（P<0.05），说明miR-145-4能够与PIK3CA基因3'-UTR结合。实时荧光定量PCR结果表明，在蛋鸭卵泡颗粒细胞中过表达miR-145-4后，PIK3CA基因表达量显著降低（P<0.05），而抑制miR-145-4后，PIK3CA基因表达量极显著升高（P<0.01）。【结论】 miR-145-4通过正向调控靶基因PIK3CA促进蛋鸭卵泡颗粒细胞的凋亡，进而调控蛋鸭的卵泡发育。

关键词: 蛋鸭; miR-145-4; PIK3CA; 颗粒细胞凋亡

Abstract: 【Objective】 The purpose of this study was to reveal the role and regulatory mechanism of miR-145-4 in follicular development for egg duck.【Method】 The egg duck follicular granulosa cells were isolated and cultured.After miR-145-4 mimic and inhibitor were transfected, the expression of cell proliferation and apoptosis related genes were detected by Real-time quantitative PCR.RNAhybrid and Targetscan programs were used to predict the target gene and predict the potential binding sites of miR-145-4 to the 3'-UTR of phosphatidylinositol-3-kinase catalytic subunit α (PIK3CA) gene.The wild type and mutant type dual-luciferase reporter vector of PIK3CA gene 3'-UTR were constructed and co-transfeacted with miR-145-4 mimic and mimic-NC into duck granulose cells.The binding relationship between miR-145-4 and target gene PIK3CA was verified by double luciferase reporting system.And the effect of miR-145-4 on the expression of PIK3CA in granulosa cells of egg ducks was detected by Real-time quantitative PCR.【Result】 The results of Real-time quantitative PCR showed that after over expression of miR-145-4, compared with control group, the expression of CyclinB2 gene was extremely significantly decreased (P<0.01), and the expression of BCL2 gene was extremely significantly increased (P<0.01).After inhibiting miR-145-4, compared with control group, the expression of CyclinB2 gene was extremely significantly increased (P<0.01), and the expression of BCL2 was extremely significantly decreased (P<0.01).The predict results showed that miR-145-4 could be binding to PIK3CA gene 3'-UTR.PCR amplification and sequencing results indicated that the wild type and mutant type vectors of PIK3CA gene 3'-UTR were successfully constructed.The results of double luciferase assay showed that the double luciferase activity of wild-type group was significantly lower than that of mutant and empty vector co-transfection groups (P<0.05), indicating that miR-145-4 could bind to PIK3CA gene 3'-UTR.The results of Real-time quantitative PCR showed that the expression of PIK3CA gene decreased significantly after overexpression of miR-145-4 in egg duck follicular granulosa cells (P<0.05), while the expression of PIK3CA gene increased extremely significantly after inhibition of miR-145-4 (P<0.01).【Conclusion】 miR-145-4 promoted the apoptosis of follicle granulosa cells by positively regulating the target gene PIK3CA, and then regulated the follicular development of egg ducks.

Key words: egg duck; miR-145-4; PIK3CA; granulose cells apoptosis

中图分类号:

S834

吴艳, 皮劲松, 张昊, 梁振华, 杜金平, 潘爱銮, 申杰, 蒲跃进. miR-145-4调控蛋鸭卵泡发育机制的研究[J]. 中国畜牧兽医, 2022, 49(3): 809-816.

WU Yan, PI Jinsong, ZHANG Hao, LIANG Zhenhua, DU Jinping, PAN Ailuan, SHEN Jie, PU Yuejin. Study on the Mechanism of miR-145-4 Regulating Follicle Development in Egg Ducks[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(3): 809-816.

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miR-145-4调控蛋鸭卵泡发育机制的研究

Study on the Mechanism of miR-145-4 Regulating Follicle Development in Egg Ducks

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