牦牛Smad 4基因3'UTR区双荧光素酶载体构建及与bta-miR-146a的靶向验证

doi:10.11843/j.issn.0366-6964.2018.07.005

畜牧兽医学报 ›› 2018, Vol. 49 ›› Issue (7): 1366-1376.doi: 10.11843/j.issn.0366-6964.2018.07.005

牦牛Smad 4基因3'UTR区双荧光素酶载体构建及与bta-miR-146a的靶向验证

牛家强^1,2, 王玉恒^1,2, 索朗斯珠^1,2, 强巴央宗¹, 徐业芬^1,2*, 郭敏³, 程玲华³, 杨士承^1,2

1. 西藏农牧学院动物科学学院, 林芝 860000;
2. 西藏农牧学院西藏高原动物疫病研究自治区高校重点实验室, 林芝 860000;
3. 中国农业大学动物科技学院, 北京 100193

收稿日期:2017-11-27 出版日期:2018-07-23 发布日期:2018-07-23
通讯作者: 徐业芬,博士,教授,主要从事西藏高原动物繁殖生理学研究,E-mail:xzlzxyf@163.com
作者简介:牛家强(1975-),男,甘肃秦安人,副教授,主要从事西藏高原动物疾病和生理学研究,E-mail:463036297@qq.com;王玉恒(1994-),男,河北衡水人,硕士生,主要从事高原动物繁殖生理学研究,E-mail:450014811@qq.com。
基金资助:
国家自然科学基金项目（31460604）；西藏自治区科技厅厅校联合基金项目（XZ2017ZRG-25）；中西部高校综合实力提升计划：西藏农牧学院动物遗传育种与繁育学科建设（502000105）；国家肉牛牦牛产业技术体系项目（CARS-37）

Construction of Yak Smad 4 Gene 3'UTR Dual-luciferase Reporter Vector and Its Targeting Validation to bta-miR-146a

NIU Jia-qiang^1,2, WANG Yu-heng^1,2, SUOLANG Si-zhu^1,2, CHANGBA Yang-zong¹, XU Ye-fen^1,2*, GUO Min³, CHENG Ling-hua³, YANG Shi-cheng^1,2

1. Department of Animal Science, Tibet Agricultural and Animal Husbandry College, Linzhi 860000, China;
2. Provincial Key Laboratory of Tibet Plateau Animal Epidemic Disease Research, Tibet Agricultural and Animal Husbandry College, Linzhi 860000, China;
3. College of Animal Science and Technology, China Agricultural University, Beijing 100193, China

Received:2017-11-27 Online:2018-07-23 Published:2018-07-23

摘要/Abstract

摘要：

旨在鉴定牦牛Smad 4基因与bta-miR-146a的靶向关系，为探究它们在牦牛卵巢卵泡发育过程中的可能分子调控机制提供理论基础。利用miRBase数据库和MEGA 6.0软件分析miR-146a在不同物种中的保守性，同时利用TargetScan软件预测其潜在靶基因，通过构建psiCHECK2双荧光素酶报告基因载体进行靶向验证。生物信息学分析结果显示，miR-146a成熟序列在脊椎动物中具有高度的保守性，并预测到178个与卵巢卵泡发育相关的潜在靶基因，从中选取Smad 4基因分析发现其3'非编码区域（3'UTR）与bta-miR-146a存在结合位点；进一步成功构建牦牛Smad 4基因3'UTR野生型及突变型psiCHECK2双荧光素酶报告质粒，荧光素酶活性检测结果显示，bta-miR-146a mimic对牦牛Smad 4基因3'UTR野生型报告质粒荧光活性有明显的下调作用，分别与突变型及NC对照组差异显著（P<0.05）。本研究初步证实，牦牛Smad 4基因是bta-miR-146a的靶基因，从而提示bta-miR-146a可能通过调控Smad 4基因在牦牛卵巢卵泡发育过程中的表达发挥作用。

Abstract:

The aim of this study was to identify the targeting relationship between Smad 4 gene and bta-miR-146a, which would provide a theoretical basis for their regulating mechanism in follicular development of yak ovary. The conservation of miR-146a in different species was analyzed by miRBase database and MEGA 6.0 software. At the same time, TargetScan software was used to predict the potential target genes of miR-146a, and the targeting validation was performed by construction of psiCHECK2 dual-luciferase reporter vector. The results of bioinformatics analysis showed that the miR-146a mature sequence was highly conserved in vertebrates, and a total of 178 potential target genes related to ovarian follicular development were predicted, then, among these genes, Smad 4 gene was selected to be further analyzed, showing that its 3'UTR had the binding site to bta-miR-146a. The wild and mutant type psiCHECK2 dual-luciferase reporter vectors of 3'UTR of yak Smad 4 gene were successfully constructed, and the dual-luciferase assay result showed that the luciferase activity of wild vector of 3'UTR of yak Smad 4 gene was distinctly down-regulated by the bta-miR-146a mimics, and was significantly lower than that of mutant vector and NC group(P<0.05), respectively. In conclusion, this research preliminarily confirmed that yak Smad 4 gene was the target gene of bta-miR-146a, suggesting that bta-miR-146a might play roles in the development of yak follicles by regulating Smad 4 gene.

中图分类号:

S823.85

牛家强, 王玉恒, 索朗斯珠, 强巴央宗, 徐业芬, 郭敏, 程玲华, 杨士承. 牦牛Smad 4基因3'UTR区双荧光素酶载体构建及与bta-miR-146a的靶向验证[J]. 畜牧兽医学报, 2018, 49(7): 1366-1376.

NIU Jia-qiang, WANG Yu-heng, SUOLANG Si-zhu, CHANGBA Yang-zong, XU Ye-fen, GUO Min, CHENG Ling-hua, YANG Shi-cheng. Construction of Yak Smad 4 Gene 3'UTR Dual-luciferase Reporter Vector and Its Targeting Validation to bta-miR-146a[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2018, 49(7): 1366-1376.

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牦牛Smad 4基因3'UTR区双荧光素酶载体构建及与bta-miR-146a的靶向验证

Construction of Yak Smad 4 Gene 3'UTR Dual-luciferase Reporter Vector and Its Targeting Validation to bta-miR-146a

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