不同种属H1启动子驱动miR-1在PK15细胞中的表达情况

【目的】筛选出能高效表达外源性成熟miR-1的真核表达载体,为揭示miR-1参与猪肌肉发育及肉质性状形成的分子机制奠定基础。【方法】分别克隆巴马小型猪miR-1前体序列、H1启动子序列及人类H1启动子序列,并构建不同种属H1启动子驱动其表达的真核表达载体,利用脂质体法转染PK15细胞后,采用荧光定量RT-PCR（qRT-PCR）检测不同种属H1启动子表达载体驱动miR-1表达效率。【结果】经酶切鉴定和测序分析证实,成功构建了3个miR-1过表达载体（pEGFP-miR-1、pEGFP-hH1-miR-1和pEGFP-pH1-miR-1）,以其转染PK15细胞24和48 h后,荧光显微镜观察发现...

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Published in南方农业学报 Vol. 46; no. 11; pp. 2020 - 2025
Main Author 郭晓萍 陈时锦 蒋钦杨 杨秀荣 郭亚芬 黄建芳 兰干球
Format Journal Article
LanguageChinese
Published 广西大学动物科学技术学院,南宁,530005 2015
Subjects
H1启动子
miR-1
PK15细胞
巴马小型猪
真核表达载体
eukaryotic expression vector
Bama mini pig
巴马小型猪
miR-1
PK15细胞
H1启动子
真核表达载体
PK15 cell
H1 promoter
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ISSN2095-1191
DOI10.3969/j:issn.2095-1191.2015.11.2020

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Summary:【目的】筛选出能高效表达外源性成熟miR-1的真核表达载体,为揭示miR-1参与猪肌肉发育及肉质性状形成的分子机制奠定基础。【方法】分别克隆巴马小型猪miR-1前体序列、H1启动子序列及人类H1启动子序列,并构建不同种属H1启动子驱动其表达的真核表达载体,利用脂质体法转染PK15细胞后,采用荧光定量RT-PCR（qRT-PCR）检测不同种属H1启动子表达载体驱动miR-1表达效率。【结果】经酶切鉴定和测序分析证实,成功构建了3个miR-1过表达载体（pEGFP-miR-1、pEGFP-hH1-miR-1和pEGFP-pH1-miR-1）,以其转染PK15细胞24和48 h后,荧光显微镜观察发现绝大部分细胞都发出绿色荧光,且含有H1启动子细胞的荧光强于无H1启动子细胞。转染pEGFP-pH1-miR-1细胞、转染pEGFP-hH1-miR-1细胞、转染pEGFP-miR-1细胞的miR-1相对表达量分别为105.02、99.00和79.65,其miR-1表达水平均极显著高于转染pEGFP-C 1细胞和空白对照组细胞（P〈0.01）。【结论】重组质粒pEGFP-miR-1、pEGFP-hH1-miR-1和pEGFP-pH1-miR-1均能在PK15细胞中高效表达miR-1,即猪源与人源的H1启动子均能提高外源性miR-1表达,可用于后续的miR-1功能研究。
Bibliography:45-1381/S
Bama mini pig; H1 promoter; miR-1; PK15 cell; eukaryotic expression vector
GUO Xiao-ping, CHEN Shi-jin, JIANG Qin-yang, YANG Xiu-rong, GUO Ya-fen, HUANG Jian-fang, LAN Gan-qiu （College of Animal Science and Technology, Guangxi University, Nanning 530005, China）
[Objective]In order to lay the foundation for further research on molecular mechanisms of miR-1 involved in porcine muscle development and meat quality traits,the eukaryotic expression vectors that could express efficiently exogenous mature miR-1 were screened out.[Method]The sequences of porcine pre-miR-1,H1 promoter and human H1 promoter were cloned,respectively.And H1 promoter-controlled miR-1 expression vectors of different species were constructed.Then the recombinant expression vectors were transfected into PK15 cells by liposome-mediated transfection method,and the expression efficiency of miR-1 was analyzed by fluorescent quantitative qRT-PCR.[Result]The enzyme digestion and sequencing results demonstrated that three recombinant vectors（
ISSN:2095-1191
DOI:10.3969/j:issn.2095-1191.2015.11.2020