水牛Oct4、Nanog基因的克隆及其在水牛胎儿成纤维细胞中的表达

doi:10.3864/j.issn.0578-1752.2014.02.019

摘要/Abstract

摘要： 【目的】转录因子Oct4、Nanog是调节干细胞多能性相关的转录因子，在干细胞的分化和胚胎发育中有重要作用。本研究旨在克隆水牛Oct4和Nanog基因、分析基因序列和蛋白结构、构建逆转录表达载体，并在水牛体细胞中表达，为进一步研究其在水牛早期胚胎发育中的作用，以及为建立水牛胚胎干细胞系和诱导多能干细胞系（iPSC）奠定基础。【方法】分别从水牛生殖嵴和体细胞中提取RNA和DNA，将RNA反转录成第1链cDNA，参照牛基因序列（Oct4：NM_174580，Nanog：NM_001025344）设计特异性引物，采用RT-PCR和PCR分别扩增Oct4和Nanog的编码区序列（the coding sequences，CDS）和DNA全长序列，其中Oct4全长DNA分3段扩增，Nanog全长DNA分2段扩增；利用生物在线分析软件对水牛Oct4和Nanog进行蛋白质结构预测和同源性比对；采用EcoRⅠ、XholⅠ和XholⅠ、NotⅠ双酶切，T4连接酶，将Oct4和Nanog的CDS连接到逆转录病毒载体pMX上，构建逆转录表达载体pMX-Oct4和pMX-Nanog；采用组织块法培养水牛胎儿成纤维细胞（buffalo fetal fibroblasts，BFFs），逆转录表达系统经过病毒包装后产生的病毒上清液感染BFFs，感染12—15h后，继续培养48 h；通过RT-PCR和免疫荧光技术检测转基因在BFFs中的表达情况。【结果】Oct4 CDS全长1 083 bp，编码361个氨基酸，DNA全长4 509 bp，包括5个外显子和4个内含子；Nanog CDS全长903 bp，编码301个氨基酸，DNA全长4 473 bp，包括4个外显子和3个内含子；将Oct4和Nanog基因序列提交到GenBank，分配的基因登录号分别为JN991003和JN991004；Oct4氨基酸序列与牛、猪、人和鼠相应氨基酸的同源性分别为98%、96%、91%和81%，Nanog氨基酸序列与牛、猪、人和鼠相应氨基酸的同源性分别为90%、81%、69%和47%；Oct4和Nanog蛋白结构与小鼠相应蛋白的结构相似，分别含有本家族特有的POU结构域和HOX同源结构域；成功构建表达Oct4和Nanog的逆转录病毒载体pMX-Oct4和pMX-Nanog；逆转录病毒系统pMX介导的Oct4和Nanog基因能转入BFFs中，在mRNA水平和蛋白水平都表达，阴性对照中不表达。【结论】分别克隆了水牛Oct4和Nanog的DNA序列全长和CDS，其基因序列和氨基酸序列在物种间高度保守；逆转录病毒转基因方法将Oct4和Nanog基因成功地转入BFFs并表达。逆转录病毒系统介导目的基因表达的转基因方法可以应用于水牛转基因研究和水牛iPSC生产。

关键词: 水牛 , Oct4基因 , Nanog基因 , 克隆 , 异位表达

Abstract: 【Objective】Oct4 and Nanog were the pluripotency-related transcription factors, and play important roles in the differentiation of stem cell and embryo development. In this study, the buffalo Oct4 and Nanog genes were cloned, the gene sequences and protein sequences were analysed, and eukaryotic expression vectors were constructed. This study will provide a good foundation for investigating the functions of Oct4 and Nanog, especially their roles in the embryo development, establishing buffalo embryonic stem cell lines and inducing pluripotent stem cell lines (iPSC). 【Method】The RNA and DNA were extracted from fetus germ ridge and somatic cells, and the RNA was reverse-transcribed into first-strand cDNA. The primers were designed specifically according to the bovine gene sequences (Oct4：NM_174580，Nanog：NM_001025344). The coding sequences (CDS) and DNA fragments of Oct4 and Nanog were cloned by RT-PCR and PCR, respectively, three fragments for Oct4 gene and two fragments for Nanog. The protein structure prediction and homology comparison were analysed by online software. After digestion with enzymes (EcoRⅠ, XholⅠfor Oct4 and XholⅠ, NotⅠfor Nanog), the CDS of Oct4 and Nanog were inserted into the pMX retrovirus vector using T4 ligase, respectively, named pMX-Oct4 and pMX-Nanog. Buffalo fetal fibroblasts (BFFs) were cultured by the method of tissue explants. The retrovirus supernatant was collected from the retroviral system after virus packaging, and infected the BFFs 12-15 h. After culturing for 48 h, the ectopic expression of Oct4 and Nanog in the BFFs were detected by RT-PCR and immunofluorescence assay.【Result】The results showed that the CDS and DNA length of Oct4 was 1083 bp and 4509 bp, respectively, encoding 361 amino acids, and containing 5 exons and 4 introns. The CDS and DNA length of Nanog was 903 bp and 4473 bp, respectively, encoding 361 amino acids, and containing 4 exons and 3 introns. The gene sequences were submitted to GenBank online, and the accession numbers were JN991003 and JN991004, respectively. The amino acids of buffalo Oct4 and Nanog exhibited high homology with bovine, pig, human and mouse, the percentage of conservatism displayed Oct4 (98%, 96%, 91% and 81%) and Nanog (90%, 81%, 69% and 47%). Buffalo Oct4 and Nanog protein structures were similar to that of mice, Oct4 containing POU-domain and Nanog containing HOX-domain. Using the retrovirus vectors pMX-Oct4 and pMX-Nanog, buffalo Oct4 and Nanog genes could be transfected into BFFs, and their mRNA and protein could be expressed in the BFFs. 【Conclusion】 The CDS and DNA fragments of buffalo Oct4 and Nanog were cloned, their base sequence and amino acid sequences were conservation during evolution. Oct4 and Nanog were transfected into BFFs and expressed in BFFs using the retrovirus transgene method. The retrovirus-based target genes transfer could be used in the buffalo genetically modified research and buffalo iPSC generation.

Key words: buffalo , Oct4 gene , Nanog gene , cloning , ectopic expression

邓彦飞, 刘庆友, 邓海莹, 罗婵, 杨素芳, 石德顺. 水牛Oct4、Nanog基因的克隆及其在水牛胎儿成纤维细胞中的表达[J]. 中国农业科学, 2014, 47(2): 394-402.

DENG Yan-Fei, LIU Qing-You, DENG Hai-Ying, LUO Chan, YANG Su-Fang, SHI De-Shun. Cloning of Buffalo (Bubalus bubalis) Oct4 and Nanog Genes and Ectopic Expression in Buffalo Fetal Fibroblasts[J]. Scientia Agricultura Sinica, 2014, 47(2): 394-402.

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