鸡gga-miR-31-5p 启动子真核表达载体的构建及其转录因子结合位点预测

doi:10.3969/j.issn.1004-1524.2022.04.07

浙江农业学报 ›› 2022, Vol. 34 ›› Issue (4): 713-719.DOI: 10.3969/j.issn.1004-1524.2022.04.07

鸡gga-miR-31-5p 启动子真核表达载体的构建及其转录因子结合位点预测

邓哲宇(), 王乙婷, 王颖洁, 胡菜, 吴宇慧, 赵宗仪, 左其生, 张亚妮()

扬州大学动物科学技术学院,江苏省动物遗传繁育与分子设计重点实验室,江苏扬州 225009

收稿日期:2020-10-12 出版日期:2022-04-25 发布日期:2022-04-28
通讯作者: 张亚妮
作者简介:*张亚妮,E-mail: ynzhang@yzu.edu.cn
邓哲宇(1999—),男,江苏常州人,本科生,主要从事动物胚胎工程与遗传工程研究。E-mail: 363657260@qq.com
基金资助:
扬州大学大学生科技创新基金(X20190618);扬州大学大学生科技创新基金(X20200642);江苏高校优势学科建设工程

Construction of eukaryotic expression vector of chicken gga-miR-31-5p promoter and prediction of its transcription factor binding sites

DENG Zheyu(), WANG Yiting, WANG Yingjie, HU Cai, WU Yuhui, ZHAO Zongyi, ZUO Qisheng, ZHANG Yani()

Key Laboratory of Animal Genetics Breeding and Molecular Design of Jiangsu Province, College of Animal Science and Technology, Yangzhou University, Yangzhou 225009, Jiangsu,China

Received:2020-10-12 Online:2022-04-25 Published:2022-04-28
Contact: ZHANG Yani

摘要/Abstract

摘要：

为确定鸡gga-miR-31-5p启动子基本活性区域,并分析调控其转录的重要转录因子,初步探究影响鸡gga-miR-31-5p的表达调控机制,采用PCR法扩增鸡gga-miR-31-5p启动子,插入到pEGFP-N1载体,构建重组载体。利用菌液PCR及测序鉴定阳性重组质粒。将重组载体转染DF-1细胞系,观察其启动活性。利用在线预测网站预测gga-miR-31-5p启动子区域的转录因子结合位点。结果显示,成功克隆出gga-miR-31-5p启动子片段,并成功构建其pEGFP重组载体。体外转染结果表明,该区域具有启动活性,并发现鸡gga-miR-31-5p启动子区域存在RARα、ER、c-Jun、GR等重要转录因子结合位点。研究初步确定了鸡gga-miR-31-5p启动子区域,同时发现该区域存在重要转录因子结合位点,该结果为后续探讨进一步研究gga-miR-31-5p启动子的生物学功能奠定基础。

关键词: 鸡, gga-miR-31-5p, 启动子, 转录因子

Abstract:

The purpose of this study was to determine the basic active region of chicken gga-miR-31-5p promoter, analyze the important transcription factors regulating its transcription and explore the expression and regulation mechanism of chicken gga-miR-31-5p. The promoter sequence of chicken gga-miR-31-5p was amplified by PCR and inserted into the pEGFP-N1 vector to construct the recombinant vector. The positive recombinant plasmids were identified by PCR with bacterial solution and sequencing, and then transfected into DF-1 cell lines to observe their priming activity. The transcription factor binding sites in gga-miR-31-5p promoter region were predicted using online prediction software. The results showed that the gga-miR-31-5p promoter fragment was cloned and its pEGFP recombinant vector was constructed successfully. The transfection results in vitro showed that the region had promoter activity, and important transcription factor binding sites such as RARα, ER, c-Jun and GR were found in the promoter region of chicken gga-miR-31-5p. The above results indicated that the promoter region of chicken gga-miR-31-5p was preliminarily determined, and the important transcription factor binding sites were found in this region, which lays a foundation for further study on the biological function of gga-miR-31-5p promoter.

Key words: chicken, gga-miR-31-5p, promoter, transcription factors

中图分类号:

S831

邓哲宇, 王乙婷, 王颖洁, 胡菜, 吴宇慧, 赵宗仪, 左其生, 张亚妮. 鸡gga-miR-31-5p 启动子真核表达载体的构建及其转录因子结合位点预测[J]. 浙江农业学报, 2022, 34(4): 713-719.

DENG Zheyu, WANG Yiting, WANG Yingjie, HU Cai, WU Yuhui, ZHAO Zongyi, ZUO Qisheng, ZHANG Yani. Construction of eukaryotic expression vector of chicken gga-miR-31-5p promoter and prediction of its transcription factor binding sites[J]. Acta Agriculturae Zhejiangensis, 2022, 34(4): 713-719.

图/表 5

图1 gga-miR-31-5p启动子片段PCR扩增结果

Fig.1 PCR results of gga-miR-31-5p promoter fragment M,DL5000 Marker;1,pEGFP-miR31-2180.

图2 gga-miR-31-5p启动子系列载体菌液PCR结果

Fig.2 PCR results of gga-miR-31-5p promoter M, DL5000 Marker;N, pEGFP-N1;1 and 2, pEGFP-miR31-2180.

图3 pEGFP-miR-31-2180载体测序结果

Fig.3 Sequencing results of pEGFP-miR-31-2180 vectors

图4 鸡gga-miR-31-5p启动子-2 180~+1 bp片段的活性检测

Fig.4 Activity detection of gga-mir-31-5p promoter -2 180-+1 bp in chicken

表1 gga-miR-31-5p启动子区域转录因子预测

Table 1 Prediction of transcription factors in gga-miR-31-5p promoter region

启动子区域 Promoter region	转录因子 Transcription factors
-2 180~+1 bp	Antp,AP-1,ATF,ATF-a,C/EBP,C/EBPalpha,C/EBPbeta,C/EBPdelta,C/EBPepsilon,C/EBPgamma,CFF,c-Jun,CP1,CPE_binding_pro,CREB,CRE-BP1,Dl,ER,ETF,Ftz,GATA-1,GCN4,GCR1,GLO,GR,Hb,HNF-1,HNF-1C,HNF-3,HNF-4alpha1,HOXA4,HSF1_(long),HSTF,ICSBP,IRF-1,LyF-1,MEB-1,NF-1,NF-E2,Oct-1,Oct-11,Oct-2.1,Odd,p40x,Pit-1,Pit-1a,PR,PU.1,RAP1,RAR-alpha1,REB1,REV-ErbAalpha,Sp1,SRF,T3R-alpha,TBP,Zen-1

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鸡gga-miR-31-5p 启动子真核表达载体的构建及其转录因子结合位点预测

Construction of eukaryotic expression vector of chicken gga-miR-31-5p promoter and prediction of its transcription factor binding sites

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