水牛MBD3基因克隆分析及其真核表达载体的构建

doi:10.16431/j.cnki.1671-7236.2016.08.004

摘要/Abstract

摘要：

本研究旨在克隆水牛MBD3基因，进行生物信息学分析，并构建MBD3基因的真核表达载体，为研究MBD3基因在水牛胚胎发育及诱导多能干细胞（iPSCs）中的作用奠定基础。试验从卵巢组织中提取总RNA，反转录得到cDNA，并以此为模板，应用RT-PCR克隆得到MBD3基因，测序并应用相关的生物学软件进行分析；将MBD3基因连接至真核表达载体pEGFP-C1，再将携带目的基因的重组质粒转染HEK293T细胞和水牛胎儿成纤维细胞（BFF），利用RT-PCR及Western blotting方法分析目的基因的表达。结果表明，克隆获得了898 bp的水牛MBD3基因序列，其中编码区全长774 bp，编码257个氨基酸。通过对MBD3基因核苷酸序列的多重比对及进化树分析，MBD3基因在进化中高度保守，特别是MBD结构域，水牛与牛的同源性为100%，与人、猪、猩猩的同源性均为97%。将水牛MBD3基因真核表达载体转染HEK293T细胞和BFF，通过荧光观察、RT-PCR及Western blotting方法鉴定表明，成功构建了水牛MBD3基因的真核表达载体。本研究克隆得到了水牛的MBD3基因，并成功构建了MBD3基因的真核表达载体，为进一步研究MBD3基因在水牛胚胎发育及iPSCs诱导上的作用奠定了基础。

关键词: 水牛; MBD3基因; DNA甲基化; 表观遗传; 干细胞

Abstract:

In this study,through cloning buffalo MBD3 gene and analyzing the biological information 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 transferred 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 domain of buffalo MBD3 gene shared 100% of similar nucleotide sequence with Bos taurus,and shared 97% of similar nucleotide sequence with Homo sapiens,Sus scrofa and Pan troglodytes.The recombinant plasmids pEGFP-C1-MBD3 were transferred into HEK293T cells and BFF,fluorescence observation,RT-PCR and Western blotting were used to analyze the expression of buffalo MBD3.The results suggested that the expression vector of buffalo MBD3 gene was successfully constructed.The study laid the foundation for the function research of MBD3 on embryo development and inducing of iPCSs.

Key words: buffalo; MBD3 gene; DNA methylation; epigenetic; stem cell

中图分类号:

Q785

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.

SU Xiao-ping, XIAO Ning, LUO Xi-er, LEI Wei, CUI Kui-qing, SHI De-shun, LIU Qing-you. Cloning,Analysis and Construction of Eukaryotic Expression Vector of Buffalo MBD3 Gene[J]. , 2016, 43(8): 1951-1959.

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