中国畜牧兽医2016，Vol.43Issue(8)：1951-1959,9.DOI:10.16431/j.cnki.1671-7236.2016.08.004
水牛 MBD3基因克隆分析及其真核表达载体的构建
Cloning,Analysis and Construction of Eukaryotic Expression Vector of Buffalo MBD3 Gene
摘要
Abstract
In this study ,through cloning buffalo MBD3 gene and analyzing the biological informa‐tion of MBD3 gene sequence ,and constructing the expression vector of buffalo MBD3 ,to provide a basis for the function research of buffalo MBD3 gene on embryo development and iPCSs .The total RNA was extracted from buffalo fresh ovary ,and MBD3 gene was amplified and sequenced , the sequence was systemically analysed with bioinformatics techniques .And the MBD3 CDS was cloned into the pEGFP‐C1 vector .Then the recombinant plasmid pEGFP‐C1‐MBD3 was trans‐ferred into the HEK293T cells and buffalo fetal fibroblasts (BFF) ,the expression was analyzed by RT‐PCR ,Western blotting and fluorescence microscope .The results showed that 898 bp of MBD3 gene fragment including whole 774 bp CDS was cloned and sequenced ,and encoded 257 amino acids .The multiple sequence alignment and analysis of phylogeny tree showed that MBD3 gene was highly conserved in the process of evolution .Especially the MBD domain ,the MBD do‐main of buffalo MBD3 gene shared 100% of similar nucleotide sequence with Bos taurus ,and shared 97% of similar nucleotide sequence with Homo sapiens ,Sus scrof a and Pan troglodytes . The recombinant plasmids pEGFP‐C1‐MBD3 were transferred into HEK293T cells and BFF ,fluo‐rescence observation ,RT‐PCR and Western blotting were used to analyze the expression of buffa‐lo MBD3 .The results suggested that the expression vector of buffalo MBD3 gene was successfully constructed .T he study laid the foundation for the function research of MBD 3 on embryo develop‐ment and inducing of iPCSs .关键词
水牛/MBD3基因/DNA甲基化/表观遗传/干细胞Key words
buffalo/MBD3 gene/DNA methylation/epigenetic/stem cell分类
生物科学引用本文复制引用
粟小平,肖宁,罗西尔,雷薇,崔奎青,石德顺,刘庆友..水牛 MBD3基因克隆分析及其真核表达载体的构建[J].中国畜牧兽医,2016,43(8):1951-1959,9.基金项目
国家自然基金（31260552）；国家高技术研究发展计划（863计划） （）
