广西巴马小型猪MyoD1基因克隆及其真核表达载体的构建

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

本研究旨在对广西巴马小型猪成肌细胞决定基因1(myoblast-determining 1,MyoD1)进行克隆分析,并构建MyoD1基因真核表达载体pEGFP-N1-MyoD1,为进一步研究该基因在广西巴马小型猪骨骼肌发育中的调控作用奠定基础。以广西巴马小型猪的肝脏组织为材料,应用RT-PCR技术克隆得到广西巴马小型猪MyoD1基因编码序列,对其核苷酸序列和蛋白质序列进行生物信息学分析,并构建该基因的真核表达载体,经过PCR和双酶切验证,通过脂质体转染,将重组质粒转染C2C12细胞,在细胞水平验证了该载体的正确性。结果显示,广西巴马小型猪MyoD1基因编码区序列长960 bp,编码319个氨基酸,核苷酸序列与猪、牛、水牛、绵羊、马、人、小家鼠、褐家鼠、家犬的同源性依次为99.7%、92.8%、92.9%、92.6%、91.5%、82.9%、82.9%、82.0%和90.9%;系统进化树分析表明,广西巴马小型猪MyoD1基因在不同物种及进化过程中具有高度保守性;蛋白质结构分析表明,MyoD1蛋白为膜外蛋白,在第4-116位氨基酸处存在1个MyoD家族标志性的MyoD结构域,对广西巴马小型猪、猪和人的MyoD1蛋白高级结构比较发现其具有极高的相似性。本研究构建的广西巴马小型猪MyoD1基因表达载体pEGFP-N1-MyoD1,转染C2C12细胞后产生绿色荧光信号,表明MyoD1基因在C2C12细胞中成功表达。

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 MyoD 1 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.