水牛HSD17B1基因启动子荧光素酶报告基因载体构建与分析

doi:10.16431/j.cnki.1671-7236.2021.10.002

摘要/Abstract

摘要： 试验旨在筛选水牛HSD17B1基因启动子活性区域及影响因素，并预测其转录结合因子，为探究该基因在水牛繁殖性能中的调控机理提供理论依据。以水牛血液基因组DNA为模板，PCR扩增得到3个HSD17B1基因启动子活性区域序列，并定向克隆至pGL3-promoter载体；将重组质粒转染到水牛卵泡颗粒细胞，通过双荧光素酶检测系统测定相对荧光素酶活性，并探究其与促黄体素（luteinizing hormone，LH）和促卵泡素（follicle stimulating hormone，FSH）的关系；利用生物信息学方法对HSD17B1基因启动子区进行转录结合因子预测。结果显示，本试验成功克隆了3个不同长度的HSD17B1基因启动子片段，并成功构建了双荧光素酶报告载体。经不同长度启动子片段的活性检测发现，pGL-pro-HSD17B1-1500活性最强，证实－866/－1 500 bp为HSD17B1基因核心启动子区域，表明该区域对HSD17B1基因转录调控有重要作用。荧光素酶活性检测结果显示，添加LH可增强HSD17B1基因启动子活性。生物信息学分析发现，HSD17B1基因启动子区存在6个转录因子结合位点：Sp1（－2 327/－2 317 bp）、HOXA4（－2 162/－2 146 bp）、Sp1（－1 409/－1 395 bp）、Sp1（－1 391/－1 380 bp）、Sp1（－1 345/－1 319 bp）和GATA1（－812/－801 bp），但无CpG岛，有1个TATA-box和2个CAAT-box。本研究成功构建了HSD17B1基因启动子荧光素酶报告载体，确定了HSD17B1基因启动子核心区域，并证明LH可增强启动子活性。

关键词: 水牛; HSD17B1基因; 启动子

Abstract: The research was designed to screen the active region and influence factors of buffalo HSD17B1 gene promoter, predict its transcription binding factor, and provide a theoretical basis for exploring the regulatory mechanism of HSD17B1 gene in buffalo reproductive performance. Using buffalo blood genomic DNA as template, PCR was used to amplify three active region sequences of HSD17B1 gene promoter, and then was directed to the pGL3-promoter vector. Recombinant plasmids were transfected into buffalo follicle granulosa cells, and the activity of relative luciferin enzymes was measured through a dual-luciferase detection system and their relationship to luteinizing hormone (LH) and follicle stimulating hormone (FSH) were explored. Transcription binding factor prediction was made in the HSD17B1 gene promoter region by bio-informational method. The results showed that three different length of HSD17B1 gene promoter fragments were successfully cloned and their dual-luciferase reporting vector were successfully constructed. The active detection of different length promoter fragments found that pGL-pro-HSD17B1-1500 was the most active, and -866/-1 500 bp was the core promoter region of HSD17B1 gene, indicating that this region played an important role in the transcription regulation of HSD17B1 gene. The luciferase activity test results showed that the addition of LH could enhance the activity of HSD17B1 gene promoter. Bio-information analysis of the HSD17B1 gene promoter region sequence found that there were 6 transcription factor binding site (Sp1(-2 327/-2 317 bp), HOXA4(-2 162/-2 146 bp), Sp1(-1 409/-1 395 bp), Sp1(-1 391/-1 380 bp), Sp1(-1 345/-1 319 bp) and GATA1(-812/-801 bp)), but no CpG island, and there were one TATA-box and two CAAT-box. The results indicated that the luciferase reporter gene vector of buffalo HSD17B1 gene promoter was successfully constructed, the core region of HSD17B1 gene promoter was determined, and the activity of the promoter was enhanced by LH adding.

Key words: buffalo; HSD17B1 gene; promoter

中图分类号:

陆杏蓉, 段安琴, 马小娅, 梁莎莎, 邓廷贤. 水牛HSD17B1基因启动子荧光素酶报告基因载体构建与分析[J]. 中国畜牧兽医, 2021, 48(10): 3512-3521.

LU Xingrong, DUAN Anqin, MA Xiaoya, LIANG Shasha, DENG Tingxian. Construction and Analysis of the Luciferase Reporter Gene Vector of HSD17B1 Gene Promoter in Buffalo[J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(10): 3512-3521.

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