China Animal Husbandry and Veterinary Medicine ›› 2019, Vol. 46 ›› Issue (9): 2535-2542.doi: 10.16431/j.cnki.1671-7236.2019.09.005

• Biotechnology • Previous Articles     Next Articles

Screening and Analysis of Transcription Factors Regulating Tβ4 Gene in Min Pig

ZHANG Dongjie1,2, WANG Liang1,2, LIU Yang2,3, LIU Di1,2,3   

  1. 1. Institute of Animal Husbandry, Heilongjiang Academy of Agricultural Science, Harbin 150086, China;
    2. Key Laboratory of Combining Farming and Animal Husbandry, Ministry of Agriculture and Rural Affairs, Harbin 150086, China;
    3. College of Animal Science and Technology, Northeast Agricultural University, Harbin 150030, China
  • Revised:2019-03-26 Online:2019-09-20 Published:2019-09-21

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Abstract: 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.

Key words: Min pig; Tβ4 gene; promoter; luciferase activity; enhancer element

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