转录因子ATF3对大鼠Gadd45β/γ基因启动活性的影响及其可能的结合部位
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国家自然科学基金资助(81273333,81471626, 31470853);江苏省高校自然科学研究面上项目(14KJB310006)


Construction of rat Gadd45β/γ promoter and identification of its binding sequence with ATF3
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    摘要:

    目的:构建大鼠生长阻滞及DNA损伤诱导基因45(growth arrest and DNA-damage-inducible gene 45,Gadd45)β/γ 启动子(全长和截短)荧光素酶报告质粒,并观察在大鼠肾小球系膜细胞(glomerular messangial cells,GMCs)中过表达激活转录因子3(activating transcription factor 3,ATF3)后分别对大鼠Gadd45β/γ基因启动活性的影响-同时筛选其可能的ATF3结合位点-方法:采用 PCR 技术,将扩增出的大鼠Gadd45β基因启动子全长(-1 105 ~ +236 nt)和Gadd45γ基因启动子全长(-913 ~ +72 nt)分别插入到荧光素酶报告基因载体pGL3-basic中,获得Gadd45β/γ基因启动子全长荧光素酶报告质粒(pGL3-Gadd45β/γ-FL),再将pGL3-Gadd45β/γ-FL分别与课题组前期构建的大鼠野生型ATF3过表达质粒(pIRES2/ATF3)共转染GMCs,检测其荧光素酶活性,以确定ATF3对Gadd45β/γ基因的启动作用-另用生物信息学软件预测Gadd45β/γ基因启动子上ATF3潜在的结合位点,并据此构建4个Gadd45β和3个Gadd45γ基因启动子截短片段的荧光素酶报告质粒-将Gadd45β/γ基因启动子全长和各截短片段的荧光素酶报告质粒与pIRES2/ATF3共转染GMCs,再行荧光素酶活性测定,以筛选ATF3的结合位点-结果:菌液PCR及核酸测序证实,大鼠Gadd45β/γ基因启动子 (全长和截短)的荧光素酶报告质粒均构建成功-将pGL3-Gadd45β/γ-FL分别和pIRES2/ATF3共转染GMCs发现,Gadd45β/γ基因启动子活性均显著增加-而将Gadd45β启动子全长及4个截短质粒分别与pIRES2/ATF3共转染GMCs后显示,pGL3-Gadd45β-4的启动活性显著低于pGL3-Gadd45β-FL-pGL3-Gadd45β-1-pGL3-Gadd45β-2和pGL3-Gadd45β-3-提示ATF3可能结合在Gadd45β基因启动子的(-146 ~ +23 nt)区域-同样,将Gadd45γ启动子全长及3个截短质粒分别与pIRES2/ATF3共转染GMCs后显示,pGL3-Gadd45γ-2-pGL3-Gadd45γ-3的启动活性显著低于pGL3-Gadd45γ-FL和pGL3-Gadd45γ-1-提示ATF3可能结合在Gadd45γ基因启动子的(-456 ~ -61 nt)区域,且这段区域可能包含1个以上ATF3结合位点-结论:成功构建了大鼠Gadd45β/γ基因启动子全长及各截短片段荧光素酶报告质粒,并初步确定了ATF3在Gadd45β/γ基因启动子上的结合区域-

    Abstract:

    Objective:To construct luciferase reporter plasmids of full-length and truncated promoters of growth arrest- and DNA-damage-inducible gene 45 (Gadd45) β/γ and detect their activity in rat GMCs in response to activating transcription factor 3 (ATF3) overexpression respectively, screening the possible binding sites for ATF3. Methods: Rat Gadd45β promoter (-110 5 ~ +236 nt) and Gadd45γ promoter (-913 ~ +72 nt) were amplified by PCR, and then cloned into the luciferase reporter plasmids (pGL3-basic) separately. The recombinant plasmids (pGL3-Gadd45β/γ-FL) were transfected into GMCs accompanied with rat ATF3 expression plasmid (pIRES2/ATF3) and the luciferase activity was detected to determine the role of ATF3 in Gadd45β/γ gene transcription. Meanwhile, the potential ATF3 binding sites within Gadd45β/γ promoter were predicted by bioinformatics software. Based on the predicted results, four truncated Gadd45β promoter luciferase reporters and three truncated Gadd45γ promoter luciferase reporters were constructed. Subsequently, the full-length as well as different truncated promoter luciferase reporters of Gadd45β/γ were transfected into GMCs accompanied with pIRES2/ATF3, and the luciferase activity was then detected to identify the ATF3 binding sites. Results: It was verified that different kinds of plasmids were all constructed correctly by PCR analysis and nucleotide sequencing. GMCs co-transfected with pGL3-Gadd45β/γ-FL and pIRES2/ATF3 displayed increased luciferase activity of Gadd45β and Gadd45γ promoter, respectively. In addition, pGL3-Gadd45β-4 co-transfected with pIRES2/ATF3 in GMCs showed notably reduced Gadd45β promoter activity than that of pGL3-Gadd45γ-FL, pGL3-Gadd45β-1, pGL3-Gadd45β-2, pGL3-Gadd45β-3 co-transfected with pIRES2/ATF3, indicating that the region of rat Gadd45β promoter (-146 ~ +23 nt) might contain ATF3 binding element. Likewise, pGL3-Gadd45γ-2 and pGL3-Gadd45γ-3 co-transfected with pIRES2/ATF3 in GMCs exhibited acute reduction of Gadd45γ promoter activity than that of pGL3-Gadd45γ-FL and pGL3-Gadd45γ-1, indicating that the region of rat Gadd45γ promoter (-456 ~ -61 nt) might contain more than one ATF3 binding element. Conclusion: The full-length and truncated Gadd45β/γ promoter luciferase reporter plasmids were constructed successfully, and the ATF3 binding regions within Gadd45β/γ promoters were identified.

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周梦雅,何风霞,张 婧,刘 玉,虞天一,卢燕来,王璐璐,赵 聃,邱 文,王迎伟.转录因子ATF3对大鼠Gadd45β/γ基因启动活性的影响及其可能的结合部位[J].南京医科大学学报(自然科学版),2016,(1):26-32

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  • 收稿日期:2015-07-09
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  • 在线发布日期: 2016-01-30
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