猪Nhlh2基因启动子活性及其表达调控的初步研究

doi:10.11843/j.issn.0366-6964.2018.04.002

摘要/Abstract

摘要：

旨在研究母猪Nhlh2基因启动子活性及其与转录因子之间的调控关系，为探索该基因的表达调控提供分子水平的依据，也为研究母猪繁殖机制提供一定的参考。本研究以猪卵巢组织DNA为模板，PCR扩增Nhlh2不同长度的启动子缺失片段，克隆到pGL3-basic载体构建启动子报告基因重组质粒，并将其转染到猪卵巢颗粒细胞，测定相对双荧光素酶活性值；通过染色质免疫共沉淀技术（ChIP），研究Nhlh2启动子与YY1、C/EBPβ蛋白之间的相互作用；随后构建转录因子超表达、突变载体和小干扰RNA（siRNA），利用双荧光素酶检测系统进行活性验证。经双荧光素酶报告系统检测发现，Nhlh2基因的P7（-238～＋129）区域启动子转录活性最高，-654～-238片段范围可能存在负调控元件，-238～-20区域可能存在正调控元件；通过生物信息学分析和ChIP验证，Nhlh2启动子区-101～-85和-153～-140区域分别是转录因子YY1和C/EBPβ的结合位点；突变YY1和C/EBPβ的结合位点后，P7的双荧光活性显著上调；超表达YY1和C/EBPβ后，P7的双荧光活性显著下调；干扰YY1和C/EBPβ的表达后，P7的双荧光活性显著上调。本研究确定了猪Nhlh2基因的核心启动子区域为-238～+129，YY1和C/EBPβ分别结合在Nhlh2基因启动子区的-101～-85和-153～-140区域，抑制猪Nhlh2基因的转录活性。

Abstract:

The present study aimed to explore the promoter activity of Nhlh2 gene and the relationship between the promoter activity and transcription factors in cells, and further provide a theoretical basis for the regulating mechanism of Nhlh2 gene in pigs. Based on the template of porcine ovarian genomic DNA, PCR was used to amplify different length deletion fragments of Nhlh2 gene promoter. These fragments were cloned into pGL3-basic to build the promoter recombinant plasmid, and then were transfected into the porcine ovarian granulosa cells. The relative luciferase activities of these plasmids were further measured by using the dual luciferase assay system. Chromatin immunoprecipitation assay (ChIP) was used to confirm the interaction between YY1, C/EBPβ and the promoter of Nhlh2. Finally, the overexpression and mutant vectors, small interfering RNA (siRNA) of YY1 and C/EBPβ were constructed, the luciferase activity was detected by dual luciferase reporter assay system. Results showed that transcription activity of P7 (—238-+129) region was the highest, —654-—238 was the potential region containing the negative regulatory element, and —238-—20 was the potential region containing the positive regulatory element; After the bioinformatic analysis and ChIP test, we found the binding sites of YY1 and C/EBPβ at Nhlh2 gene promoter were —101-—85 and —153-—140, respectively. After mutating the binding sites of YY1 and C/EBPβ, the transcription activity of P7 was significantly up-regulated; The overexpression of YY1 and C/EBPβ significantly reduced the transcription activity of P7, and the transcription activity was significantly up-regulated by YY1-siRNA and C/EBPβ-siRNA. These results indicate that the core promoter region of Nhlh2 is —238-+129. Furthermore, YY1 and C/EBPβ respectively bound at —101-—85 and —153-—140 of Nhlh2 gene promoter to inhibit the transcription of Nhlh2 gene in pigs.

中图分类号:

S828.2

温丽娟, 刘发伟, 林长光, 陈子韬, 张哲, 张豪, 李加琪, 袁晓龙. 猪Nhlh2基因启动子活性及其表达调控的初步研究[J]. 畜牧兽医学报, 2018, 49(4): 667-674.

WEN Li-juan, LIU Fa-wei, LIN Chang-guang, CHEN Zi-tao, ZHANG Zhe, ZHANG Hao, LI Jia-qi, YUAN Xiao-long. Analysis of the Promoter Activity and Expression Regulation of Porcine Nhlh2 Gene[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2018, 49(4): 667-674.

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https://www.xmsyxb.com/CN/Y2018/V49/I4/667

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