广西巴马小型猪MyoD1基因克隆及其真核表达载体的构建

doi:10.16431/j.cnki.1671-7236.2019.04.003

Abstract

Abstract:

This study was aimed to clone and analyze myoblast-determining 1(MyoD1) gene in Guangxi Bama Mini-pig,and construct the eukaryotic expression vector of MyoD1 gene,which would lay the foundation on the regulation of skeletal muscle of MyoD1 gene in Guangxi Bama Mini-pig.The coding sequence of porcine MyoD1 gene was cloned by RT-PCR using the liver tissue,and the nucleotide and protein sequences were analyzed by bioinformatics.The eukaryotic expression vector was constructed and verified in C2C12 cells by PCR,double digestion and transfection.The results showed that the coding region of MyoD1 gene in Guangxi Bama Mini-pig was 960 bp of length and encoded 319 amino acids.The homology of Guangxi Bama Mini-pig with Sus scrofa,Bos taurus,Bubalus bubals,Ovis aries,Equus cabalus,Homo sapiens,Mus musculus,Rattus norvegicus and Canis lupus was 99.7%,92.8%,92.9%,92.6%,91.5%,82.9%,82.9%,82.0% and 90.9%,respectively.The phylogenetic tree analysis showed MyoD1 gene of Guangxi Bama Mini-pig was highly conservative in different species.The protein structure analysis indicated that MyoD1 protein was an extramembrane protein,and there was a MyoD family-named MyoD domain in the 4-116th amino acid.There was a very high similarity in the high structure of MyoD1 protein among the Guangxi Bama Mini-pig,Sus scrofa and Homo sapiens.The expression vector pEGFP-N1-MyoD1 with MyoD1 gene was successfully constructed in Guangxi Bama Mini-pig and expressed in C2C12 cells with a green fluorescent signal,which indicated that MyoD1 gene was successfully expressed on C2C12 cells.

Key words: Guangxi Bama Mini-pig; MyoD1 gene; muscle cells; eukaryotic expression vector

CLC Number:

FENG Lingli, ZHANG Ruimen, HUANG Ye, XIA Panjie, ZHANG Mingyuan, LIANG Jing, LAN Ganqiu. Cloning and Eukaryotic Expression Vector Construction of MyoD1 Gene in Guangxi Bama Mini-pig[J]. , 2019, 46(4): 977-985.

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Cloning and Eukaryotic Expression Vector Construction of MyoD1 Gene in Guangxi Bama Mini-pig

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