猪紧密连接蛋白1基因克隆及其shRNA片段的筛选

doi:10.16431/j.cnki.1671-7236.2016.06.002

›› 2016, Vol. 43 ›› Issue (6): 1405-1412.doi: 10.16431/j.cnki.1671-7236.2016.06.002

猪紧密连接蛋白1基因克隆及其shRNA片段的筛选

曹丽华, 王萍, 郝文艳, 代小丽, 陈培芳, 石德顺, 李湘萍

广西大学, 亚热带农业生物资源保护与利用国家重点实验室, 南宁 530004

收稿日期:2015-12-21 出版日期:2016-06-20 发布日期:2016-07-11
通讯作者: 石德顺, 李湘萍 E-mail:ardsshi@gxu.edu.cn;xiangpingli@163.com
作者简介:曹丽华(1991-),女,湖南益阳人,硕士,研究方向:动物基因工程与胚胎技术,E-mail:420269676@qq.com
基金资助:
国家自然科学基金(31560632);广西自然科学基金项目(2012GXNSFCB053002、2014GXNSFAA118084);国家留学回国基金项目(BLH120499)

Cloning of Pig ZO-1 Gene and Screening on its Effective shRNA Fragments

CAO Li-hua, WANG Ping, HAO Wen-yan, DAI Xiao-li, CHEN Pei-fang, SHI De-shun, LI Xiang-ping

State Key Laboratory of Subtropical Agricultural Biological Resources Protection and Utilization, Guangxi University, Nanning 530004, China

Received:2015-12-21 Online:2016-06-20 Published:2016-07-11

摘要/Abstract

摘要： 试验旨在筛选有效抑制猪紧密连接蛋白1 (zonula occludens-1,ZO-1)基因表达的shRNA干扰片段,为后期利用RNA干扰技术深入了解ZO-1基因在猪卵母细胞成熟过程中的功能奠定基础。本研究首先克隆了猪ZO-1基因,并构建了其真核表达载体,而后筛选了有效抑制ZO-1基因表达的shRNA干扰片段。结果表明,猪ZO-1基因编码区长度为3 036 bp,多重序列比对结果显示,猪ZO-1基因核苷酸序列与牛、羊、马和人相应序列的同源性分别为88%、88%、87%和85%。构建的pDsRed-N1-ZO-1真核蛋白融合表达载体经脂质体法转染HEK293T细胞后,可观察到清晰的RFP红色荧光蛋白表达。将靶质粒与shRNA干扰质粒共转染HEK293T细胞48 h后,与对照组相比,可观察到红色荧光表达得到抑制。实时荧光定量PCR结果显示,设计合成的2条shRNA对猪ZO-1基因表达均有抑制效果,其中ZO-1 shRNA201的抑制效果显著高于ZO-1 shRNA1276 (77.8%和67.0%,P < 0.05)。

关键词: 猪; ZO-1基因; 克隆; shRNA

Abstract: The aim of this study was to screen effective shRNA fragments targeting pig zonula occludens-1 (ZO-1) gene,which would lay foundation to further explore its function in pig oocytes in vitro maturation (IVM) by RNAi technology.Pig ZO-1 gene was cloned and its eukaryotic expression vector was constructed,its effective shRNA fragments were screened.The results showed that the CDS length of cloned pig ZO-1 gene was 3 036 bp.The results of sequence multialigned showed that the sequence of pig ZO-1 gene shared 88%,88%,87% and 85% homology with Taurus,Ovis aries, Equns caballus and Homo sapiens,respectively.Clear RFP red fluorescent was observed in transfected cells with the pDsRed-N1-ZO-1 eukaryotic expression plasmid transfected into HEK293T cells by X-tremeGENE HP DNA transfection reagent.Two shRNA fragments targeting pig ZO-1 gene were designed and synthesized,weaker RFP red fluorescent was observed in co-transfected cells with the target and interfered shRNA plasmid groups.The Real-time PCR results showed that the two designed shRNAs could effectively inhibit the expression of pig ZO-1 mRNA,shRNA201 fragment had significantly higher inhibition effect than that of shRNA1276 fragment (77.8% and 67.0%,P < 0.05).

Key words: pig; ZO-1 gene; clone; shRNA

中图分类号:

Q785

曹丽华, 王萍, 郝文艳, 代小丽, 陈培芳, 石德顺, 李湘萍. 猪紧密连接蛋白1基因克隆及其shRNA片段的筛选[J]. , 2016, 43(6): 1405-1412.

CAO Li-hua, WANG Ping, HAO Wen-yan, DAI Xiao-li, CHEN Pei-fang, SHI De-shun, LI Xiang-ping. Cloning of Pig ZO-1 Gene and Screening on its Effective shRNA Fragments[J]. , 2016, 43(6): 1405-1412.

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猪紧密连接蛋白1基因克隆及其shRNA片段的筛选

Cloning of Pig ZO-1 Gene and Screening on its Effective shRNA Fragments

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