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调控民猪Tβ4基因转录因子的筛选与分析 预览

Screening and Analysis of Transcription Factors Regulating Tβ4 Gene in Min Pig
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摘要 为了筛选调控民猪胸腺β4(Tβ4)基因转录的增强子,探究该基因的表达调控机制,本研究以民猪基因组DNA为模板,通过PCR扩增Tβ4基因启动子区系列截短片段,与pMD18-T载体连接构建克隆质粒;通过双酶切和连接反应将系列截短片段定向连入pGL3-basic载体构建双荧光素酶重组质粒;将重组质粒转染PK15细胞系,利用双荧光素酶检测系统测定重组质粒的相对荧光素酶活性;根据相对荧光素酶活性的高低进一步筛选Tβ4基因的启动子核心区域;利用3个在线软件预测核心区域内的转录因子结合位点,根据预测结果,使用重叠PCR定点缺失转录因子结合位点构建突变载体,在PK15细胞中以野生型载体为对照检测突变载体的相对荧光素酶活性。结果表明,试验成功构建了6个Tβ4基因系列截短的启动子片段,其中5个片段具有明显的活性。经过两轮的双荧光素酶活性检测发现,-155~-105 bp区域为民猪Tβ4基因的启动子核心区域,经生物信息学分析发现,该区域存在E2F-1、MYBAS1和ELK-1转录因子的结合位点。利用定点缺失构建了3个转录因子缺失的突变载体,经双荧光素酶检测发现仅有ELK-1结合位点的缺失,会造成启动子活性的显著下降(P<0.05)。据此推测ELK-1是民猪Tβ4基因转录的正调控元件。 To screen the enhancer element of regulation Tβ4 gene transcription in Min pig,and explore the expression regulation mechanism of it,in this study,a series of truncated fragments of the Tβ4 gene promoter region were amplified by PCR using the genomic DNA in Min pig.PCR products were ligated into pMD18-T vector,the cloning plasmid was constructed.The cloning plasmid was ligated into pGL3-basic vector by double digestion and ligation reaction,the relative luciferase activity of this recombinant plasmid was determined in PK15 cell by dual luciferase detection system.The core region of Tβ4 promoter was further screened according to the number of relative luciferase activity.At the same time,three online softwares were used to forecast the binding site of transcription factor in promoter core region.Based on the forecast results,an overlapping PCR was used to site-directed delete in transcription factor binding site,the mutant vectors were constructed using these PCR products and transfected into PK15 cell using wild type plasmid as control.The results showed that six different lengths of Tβ4 gene promoter fragments were successfully constructed,five of them had significant activity.After two rounds of double luciferase activity assay,-155 to-105 bp region was determined as the core region of Tβ4 promoter in Min pig,through bioinformatics analysis,there were three transcription factor binding sites in this region,they were E2F-1,MYBAS1 and ELK-1.Three mutant vectors with deletion of transcription factors were constructed by site-directed deletion,only the deletion of ELK-1 binding site was found by double luciferase assay,which would result in a significant decrease in promoter activity(P<0.05).It was speculated that ELK-1 was a positive regulatory element of transcription of Tβ4 gene in Min pig.
作者 张冬杰 汪亮 刘洋 刘娣 ZHANG Dongjie;WANG Liang;LIU Yang;LIU Di(Institute of Animal Husbandry,Heilongjiang Academy of Agricultural Science,Harbin 150086,China;Key Laboratory of Combining Farming and Animal Husbandry,Ministry of Agriculture and Rural Affairs,Harbin 150086,China;College of Animal Science and Technology,Northeast Agricultural University,Harbin 150030,China)
出处 《中国畜牧兽医》 CAS 北大核心 2019年第9期2535-2542,共8页 China Animal Husbandry & Veterinary Medicine
基金 黑龙江省科技攻关项目(GC12B311) 黑龙江省科研机构创新能力提升专项(YC2016D001) 国家生猪产业技术体系(CARS-37)。
关键词 民猪 Tβ4基因 启动子 荧光素酶活性 增强子 Min pig Tβ4 gene promoter luciferase activity enhancer element
作者简介 张冬杰(1980-),女,黑龙江哈尔滨人,博士,副研究员,研究方向:地方猪遗传资源,E-mail:djzhang8109@163.com;通信作者:刘娣(1963-),女,吉林长春人,博士,教授,研究方向:动物遗传育种与繁殖,E-mail:liudi1963@163.com。
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