摘要
本研究旨在对广西沼泽型水牛MYF5基因进行克隆和分析,并构建其真核表达载体。应用RT-PCR方法从水牛肌肉组织中扩增MYF5基因,测序鉴定后对其核苷酸序列和蛋白质序列进行生物信息学分析,同时构建该基因真核表达载体,并在细胞水平验证所构建载体的正确性。结果表明:水牛MYF5基因编码区序列长度为768 bp,编码255个氨基酸,其核苷酸序列与牛、山羊、猪、人、猩猩和狼相应序列的相似性分别为99%、98%、94%、91%、90%和89%;水牛MYF5基因在不同物种及进化过程中具有高度保守性;MYF5蛋白为膜外蛋白,具有MYOD家族标志性MYOD结构域;构建水牛MYF5基因真核表达载体pMXs-MYF5,转染HEK293T、水牛颗粒细胞后产生绿色荧光信号,表明细胞表达MYF5基因。
The purpose of this study was to clone and analyze MYF5 gene of Guangxi buffalo,and construct its eukaryotic expression vector.The MYF5 gene sequence was amplified from buffalo muscle tissue by RT-PCR.After identification by sequencing,its nucleotide and protein sequence were analyzed by bioinformatics method.At the same time,the eukaryotic expression vector was constructed,and the correctness of constructed vector was certificated by RT-PCR.The results showed that the length of encoding region of the buffalo MYF5 gene was 768 bp,encoding for a protein of 255 amino acids.The nucleotide sequences of the buffalo MYF5 shared 99%,98%,94%,91%,90%and 89%similarities with that of Bos Taurus,goats,pigs,human,gorillas and wolves,respectively.It was speculated that the MYF5 gene was highly conserved in the different species and the process of evolution based on the results of system evolution analysis.The MYF5 protein is extramembranous protein with MYOD domain of the MYOD family.The eukaryotic expression vector pMXs-MYF5 of the buffalo MYF5 gene was constructed successfully,and transferred into HEK293T and buffalo granulosa cells.Fluorescence observation and RT-PCR confirmed that both cells expressed the buffalo MYF5 gene.
作者
张瑞门
邓彦飞
奉玲丽
崔佳瑜
潘雨
石德顺
韦英明
杨素芳
ZHANG Rui-men;DENG Yan-fei;FENG Ling-li;CUI Jia-yu;PAN Yu;SHI De-shun;WEI Ying-ming;YANG Su-fang(State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources,Guangxi University,Guangxi Nanning 530004,China;College of Animal Science and Technology,Guangxi University,Guangxi Nanning 530004,China)
出处
《中国畜牧杂志》
CAS
北大核心
2018年第10期100-105,共6页
Chinese Journal of Animal Science
基金
国家自然科学基金(31760334)
国家现代农业产业技术体系广西创新团队建设项目(nycytxgxcxtd-09-01)
广西自然科学基金(桂科AA17204051
2015GXNSFAA139080)
关键词
水牛
MYF5基因
克隆
载体构建
骨骼肌发育
Buffalo
MYF5 gene
Cloning
Vector construction
Skeletal muscle development
