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

Abstract

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

CLC Number:

Q785

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.

References

[1] Zicarelli L,Esposito L,Campanile G,et al.Effects of using vasectomized bulls in artificial insemination practice on the reproductive efficiency of Italian buffalo cows[J].Anim Reprod Sci,1997,47(3):171-180.
[2] Hai T,Teng F,Guo R,et al.One-step generation of knockout pigs by zygote injection of CRISPR/Cas system[J].Cell Research,2014,24(3):372-375.
[3] Ni W,Qiao J,Hu S,et al.Efficient gene knockout in goats using CRISPR/Cas9 system[J].PLoS One,2014,9(9):e106718.
[4] Mali P,Yang L,Esvelt K M,et al.RNA-guided human genome engineering via Cas9[J].Science,2013,339(6121):823-826.
[5] Takahashi K,Yamanaka S.Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors[J].Cell,2006,126(4):663-676.
[6] Hou P,Li Y,Zhang X,et al.Pluripotent stem cells induced from mouse somatic cells by small-molecule compounds[J].Science,2013,341(6146):651-654.
[7] 廖荣荣,颉孝贤,马育芳,等.诱导多能干细胞技术在猪转基因研究中的应用前景[J].中国畜牧兽医,2013,40(6):90-95.
[8] Deng Y,Liu Q,Luo C,et al.Generation of induced pluripotent stem cells from buffalo (Bubalus bubalis) fetal fibroblasts with buffalo defined factors[J].Stem Cells Dev,2012,21(13):2485-2494.
[9] Tyler J K,Kadonaga J T.The "dark side" of chromatin remodeling:Repressive effects on transcription[J].Cell,1999,99(5):443-446.
[10] Zhang Y,Ng H H,Erdjument-Bromage H,et al.Analysis of the NuRD subunits reveals a histone deacetylase core complex and a connection with DNA methylation[J].Genes Dev,1999,13(15):1924-1935.
[11] Hendrich B,Abbott C,Mcqueen H,et al.Genomic structure and chromosomal mapping of the murine and human MBD1,MBD2,MBD3,and MBD4 genes[J].Mamm Genome,1999,10(9):906-912.
[12] Le Guezennec X,Vermeulen M,Brinkman A B,et al.MBD2/NuRD and MBD3/NuRD,two distinct complexes with different biochemical and functional properties[J].Mol Cell Biol,2006,26(3):843-851.
[13] Hendrich B,Bird A.Identification and characterization of a family of mammalian methyl-CpG binding proteins[J].Mol Cell Biol,1998,18(11):6538-6547.
[14] Yildirim O,Li R,Hung J,et al.Mbd3/NURD complex regulates expression of 5-hydroxymethylcytosine marked genes in embryonic stem cells[J].Cell,2011,147(7):1498-1510.
[15] Hainer S J,Fazzio T G.Regulation of nucleosome architecture and factor binding revealed by nuclease footprinting of the ESC genome[J].Cell Reports,2015,13(1):61-69.
[16] Chen Y,Damayanti N P,Irudayaraj J,et al.Diversity of two forms of DNA methylation in the brain[J].Front Genet,2014,5:1-13.
[17] Hendrich B,Guy J,Ramsahoye B,et al.Closely related proteins MBD2 and MBD3 play distinctive but interacting roles in mouse development[J].Genes Dev,2001,15(6):710-723.
[18] Zviran A,Rais Y,Mor N,et al.MBD3/NuRD is a key inhibitory module during the induction and maintenance of naïve pluripotency[J].Jacob Hanna,2015,Epub of publish.
[19] Wade P A.Methyl CpG binding proteins:Coupling chromatin architecture to gene regulation[J].Oncogene,2001,20(24):3166-3173.
[20] Ohki I,Shimotake N,Fujita N,et al.Solution structure of the methyl-CpG binding domain of human MBD1 in complex with methylated DNA[J].Cell,2001,105(4):487-497.
[21] Allen H F,Wade P A,Kutateladze T G.The NuRD architecture[J].Cellular and Molecular Life Sciences:CMLS,2013,70(19):3513-3524.
[22] Rais Y,Zviran A,Geula S,et al.Deterministic direct reprogramming of somatic cells to pluripotency[J].Nature,2013,502(7469):65-70.
[23] Shimbo T,Du Y,Grimm S A,et al.MBD3 localizes at promoters,gene bodies and enhancers of active genes[J].PLoS Genetics,2013,9(12):e1004028.
[24] Luo M,Ling T,Xie W,et al.NuRD blocks reprogramming of mouse somatic cells into pluripotent stem cells[J].Stem Cells,2013,31(7):1278-1286.
[25] Dos Santos R L,Tosti L,Radzisheuskaya A,et al.MBD3/NuRD facilitates induction of pluripotency in a context-dependent manner[J].Cell Stem Cell,2014,5(2):102-110.
[26] Günther K,Rust M,Leers J,et al.Differential roles for MBD2 and MBD3 at methylated CpG islands,active promoters and binding to exon sequences[J].Nucleic Acids Research,2013,41(5):3010-3021.
[27] Cui Y,Irudayaraj J.Dissecting the behavior and function of MBD3 in DNA methylation homeostasis by single-molecule spectroscopy and microscopy[J].Nucleic Acids Res,2015,43(6):3046-3055.

Cloning,Analysis and Construction of Eukaryotic Expression Vector of Buffalo MBD3 Gene

PDF (PC)

Knowledge

Abstract

Cite this article

share this article

References

Related Articles 15

Recommended Articles

Metrics

Comments

[1]	XU Yuanyuan, ZHENG Haiying, YANG Chunyan, DENG Tingxian, HUANG Chenqian, FENG Chao, LU Xingrong, DUAN Anqin, MO Xia, MA Xiaoya, SHANG Jianghua. Effects of Inhibition of BMP/Smad Signaling Pathway on the Growth and Steroid Hormone Synthesis of Buffalo Ovarian Granulosa Cells [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(6): 2354-2362.
[2]	XIAO Peng, SHANG Jianghua, YANG Chunyan, LI Mengqi, DUAN Anqin, MA Xiaoya, FENG Chao, HUANG Chenqian, ZHANG Bo, ZHOU Jinchen, WEI Kelong, ZHENG Wei, ZHENG Haiying. Effects of Alpha-linolenic Acid on Buffalo Ovarian Granulosa Cells Cultured in vitro [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(6): 2380-2387.
[3]	YAO Jiawei, HUANG Yujie, CHEN Zhisheng, WANG Bingyun, ZHANG Hui. Immunomodulatory Effects of Dimethyl Alpha-ketoglutarate Pretreatment on Canine Adipose-derived Mesenchymal Stem Cells [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(4): 1642-1652.
[4]	LIU Bo, LI Meilin, LIU Xu, LIANG Shuangying, MU Baolong, ZHAO Wenting, GE Yansong. Effects of Adipose-derived Stem Cell Conditioned Medium Alleviates Oxidative Stress Injury Induced by Sodium Taurocholate and Trypsin [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(2): 798-806.
[5]	WANG Zhilong, LIU Li, ZHANG Lu, GAO Shuai. Research Progress of Porcine Pluripotent Stem Cell Lines [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(1): 67-75.
[6]	GAO Xiaomin, ZHOU Shujian, CHEN Chen, JIN Jing, HU Cai, ZHANG Chen, ZUO Qisheng, ZHANG Yani, CHEN Guohong, LI Bichun. Regulation of STAT1 and Histone Acetylation Modification on lncRNA-BMP4 Transcription in Chickens [J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(9): 3321-3332.
[7]	GAO Yefan, SONG Hanan, WANG Yunan, WU Yue, NIU Ruili, ZONG Xianchun, GUAN Weijun. Study on the Isolation, Culture and Differentiation Potential of Limbal Stem Cells from Beijing You Chickens [J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(9): 3372-3381.
[8]	LU Wengeng, SI Linqing, TIAN Chunyu, YUAN Siqi, XIE Hexin, ZHOU Jiwei, JIN Jidong, HAN Yinghao. Establishment of Oxidative Stress Model of Bovine Endometrial Epithelial Cells and the Effects of Bovine Adipose-derived Mesenchymal Stem Cells on Cell Migration [J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(7): 2506-2514.
[9]	ZAN Gengxiu, QIN Yingchao, ZHU Chao, WANG Xiuqi. Research Progress of JAK/STAT Signal Pathway Regulating Intestinal Mucosal Regeneration and Helper T Cell Response [J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(6): 2079-2087.
[10]	YAN Yelian, ZHANG Mengya, LIU Qiuchen, WANG Xin, XU Changzhi, ZONG Yanfeng, ZHU Zhihua, WU Sucheng, SONG Yu, LI Yunsheng, ZHANG Yunhai, CAO Zubing. Research Progress of Epigenetic Reprogramming in Mammalian Somatic Cell Nuclear Transfer Embryo Development [J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(6): 2259-2269.
[11]	TANG Lin, ZHANG Junfang, WANG Ying, SUN Bin, WANG Enze, SHIN Jongsuh, GUO Panpan, JIN Xin, YAN Changguo, LI Xiangzi, LI Qiang. Anti-apoptotic Effect of Canine Adipose-derived Mesenchymal Stem Cells on Endoplasmic Reticulum Stress Model of Severe Acute Pancreatitis in vitro [J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(5): 1840-1851.
[12]	LIU Xinyong, TAN Zhengzhun, LI Hui, HUANG Jian, LUO Hua, ZENG Linghu, CHENG Juanru, ZHONG Huapei, YANG Chunyan, WANG Xiaoli, QIN Guangsheng. Effects of Heat Stress on Blood Biochemical Indexes of Murrah and Nili-Ravi Buffaloes [J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(2): 521-528.
[13]	TIAN Yangqing, WANG Yayuan, ANG Yanfen, YAN Han, LIU Xin, YAN Yulin. Research Progress on Therapeutic Mechanism and Application of Canine Hematopoietic Stem Cell Transplantation [J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(2): 791-799.
[14]	MA Xingshu. Advances on Trained Immunity of Poultry [J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(12): 4756-4775.
[15]	DONG Bowen, CUI Xiaozhen, BAI Rui, ZHENG Mingxue, ZHANG Li, LYU Xiaoling, REN Yuhong. Effects of EGF and FGF-2 on Proliferation and Differentiation of Porcine Subcutaneous Adipose Neural Crest Stem Cells [J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(12): 4878-4886.