敖汉细毛羊<i>BMP4</i>基因质粒的构建及在成纤维细胞中表达量的研究

doi:10.7668/hbnxb.201750810

摘要/Abstract

摘要： 旨在构建敖汉细毛羊BMP4基因的质粒及转染成纤维细胞后研究基因表达量的变化。以30日龄的敖汉细毛羊胚胎为研究对象。首先，通过RNA的提取反转录成cDNA参照GenBank中BMP4基因序列信息设计1对引物，通过PCR反应扩增获得BMP4基因片段、将得到的BMP4基因连接到pEASYTM-T1载体，构建pEASYTM-T1-BMP4重组质粒并转化大肠杆菌（E.coli）DH5α感受态细胞提取质粒进行酶切鉴定。鉴定正确后构建pcDNA3.1-BMP4重组质粒，转化大肠杆菌（E.coli）DH5α感受态细胞；其次对敖汉细毛羊的成纤维细胞进行分离培养，并将重组pcDNA3.1-BMP4表达载体转染成纤维细胞，后检测BMP4基因在mRNA和蛋白水平上表达量的变化。结果显示：pcDNA3.1-BMP4构建成功后转染成纤维细胞BMP4基因的mRNA和蛋白表达量均显著上升，且转染组的表达量极显著地高于对照组（P < 0.01）。成功构建了敖汉细毛羊BMP4基因的质粒，并且成功转染成纤维细胞，基因在细胞中过表达，结果可为进一步研究其功能奠定基础。

关键词: 敖汉细毛羊, 成纤维细胞, 质粒构建, BMP4基因转染, RT-PCR, Western Blot

Abstract: This study aims to construct the expression plasmid of BMP4 gene in AoHan sheep and explore the change of gene expression after transfection of fibroblasts. 30-day old AoHan sheep embryos were selected as the research subject. Firstly, a pair of primers was designed by reverse transcribing RNA into cDNA based on the sequence of BMP4 in GenBank. The BMP4 gene fragment was amplified by PCR reaction, and was ligated into pEASYTM-T1 vector to construct pEASYTM-T1-BMP4 recombinant plasmid, which was transformed into E.coli DH5α competent cells to extract the plasmid for restriction enzyme digestion. Then the fibroblasts of AoHan sheep were isolated and cultured, and the constructed plasmid pcDNA3.1-BMP4 was transfected into fibroblasts. The expression of BMP4 gene in fibroblasts was detected by quantitative PCR and Western Blot. The results showed that plasmid pcDNA3.1-BMP4 was successfully constructed by restriction enzyme digestion and sequencing, and BMP4 gene expression was significantly increased after transfection of fibroblasts. The expression level of transfection group was significantly higher than that of the control group. The plasmid was constructed and transfected into fibroblasts successfully. The result can be a basis for further study of its function.

Key words: AoHan fine wool sheep, Fibroblats, Plasmid construction, BMP4 transfection, RT-PCR, Western Blot

中图分类号:

Q78
S826.03

张梦瑶, 杨峰, 刘开东, 刘积凤, 柳楠, 贺建宁, 薛明. 敖汉细毛羊BMP4基因质粒的构建及在成纤维细胞中表达量的研究[J]. 华北农学报, 2019, 34(6): 227-234. doi: 10.7668/hbnxb.201750810.

ZHANG Mengyao, YANG Feng, LIU Kaidong, LIU Jifeng, LIU Nan, HE Jianning, XUE Ming. BMP4 Gene Plasmid Construction and Its Expression Amount in Fibroblasts of AoHan Fine Wool Sheep[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2019, 34(6): 227-234. doi: 10.7668/hbnxb.201750810.

http://www.hbnxb.net/CN/Y2019/V34/I6/227

参考文献

[1] 柳楠,王春亮,贺建宁,程明,刘开东,刘积凤,赵金山.敖汉细毛羊不同部位皮肤毛囊发育及形态结构研究[J].中国畜牧杂志,2015,51(17):1-5.doi:10.3969/j.issn.0258-7033.2015.17.001.
Liu N, Wang C L, He J N, Cheng M, Liu K D, Liu J F, Zhao J S. Study on skin hair follicle development and morphological of different boby parts of AoHan fine wool sheep[J].Chin J Anim Sci,2015,51(17):1-5.
[2] Hardy M H, Lyne A G. The pre-natal development of wool follicles in Merino sheep[J]. Division Anim Health Prod, 1956, 9(3):423-441.doi:10.1071/BI9560423.
[3] 吴瑜瑜,岳耀敬,郭婷婷,王天翔,郭健,李桂英,韩吉龙,杨敏,刘建斌,孙晓萍,李范文,何玉琴,杨博辉.中国超细毛羊(甘肃型)胎儿皮肤毛囊发育及其形态结构[J].中国农业科学,2013, 46(9):1923-1931.doi:10.3864/j.issn.0578-1752.2013.09.020.
Wu Y Y, Yue Y J, Guo T T, Wang T X, Guo J, Li G Y, Han J L, Yang M, Liu J B, Sun X P, Li F W, He Y Q, Yang B H.Study on fetal skin hair follicle development and morphological of China super-fine merino (Gansu type)[J].Scientia Agricultura Sinica,2013, 46(9):1923-1931.
[4] Stenn K S, Paus R. Controls of hair follicle cycling[J]. Physiol Rev, 2001, 81(1):449-494.doi:10.1152/physrev.2001.81.1.449.
[5] 柳楠,卜然,贺建宁,程明,刘开东,刘积凤,赵金山. Hox基因家族对细毛羊羊毛性状影响的研究[J].中国畜牧杂志,2014, 50(23):6-10.doi:10.3969/j.issn.0258-7033.2014.23.002.
Liu N, Pu R, He J N, Cheng M, Liu K D, Liu J F, Zhao J S.Effect of Hox gene family on wool traits of Fine-wool sheep[J].Chin J Anim Sci,2014, 50(23):6-10.
[6] Stenn K S. Molecular in sights into the hair follicle and its pathology:A review of recent developments[J].International Journal of Dermatology,2013,42(3):40-43.doi:10.1046/j.1365-4362.2003.01726.x.
[7] 李荣.内蒙古绒山羊BMP4、BMPR1B基因cDNA的克隆及BMPR1B在皮肤毛囊中的表达[D].呼和浩特:内蒙古农业大学,2007.
Li R.Cloning of BMP4 and BMPR1B gene cDNA from Inner Mongolia cashmere goat and expression of BMPR1B in skin hair follicle[D]. Hohhot:Inner Mongolia Agricultural University, 2007.
[8] Halloy J, Bernard B A, Loussouarn G, Goldbeter A. Modeling the dynamics of human hair cycles by a follicular automaton[J]. Biophysics, 2000, 97(15):8328-8333.doi:10.1073/pnas.97.15.8328.
[9] Van Exan R J, Hardy M H. A spatial relationship between innervation and the early differentiation of vibrissa folicles in the embryonic mouse[J]. Journal of Anatomy, 1981, 131(4):643-656.
[10] 吴江鸿,张文广,李金泉,闫祖威.内蒙古绒山羊毛囊发育不同时期皮肤中BMP4基因的表达[J].黑龙江畜牧兽医, 2009, 23(12):35-36. doi:10.3969/j.issn.1004-7034.2009.12.015.
Wu J H, Zhang W G, Li J Q,Yan Z W.Expression of BMP4 gene in skin of different stages of fluffy hair follicle development in Inner Mongolia[J].Heilongjiang Animal Science and Veterinary Medicin, 2009, 23(12):35-36.
[11] 毛青青,代蓉,沈思军,郭洪,万鹏程,周平,石国庆.BMP4对小鼠毛囊数量的影响[J].西北农业学报, 2012, 21(5):13-17.doi:10.3969/j.issn.1004-1389.2012.05.003.
Mao Q Q, Dai R,Shen S J, Guo H, Wan P C, Zhou P, Shi G Q. Influence of BMP4 on of hair follicles quantiy of mice[J].Acta Agriculturae Boreali-occidentalis Sinica, 2012, 21(5):13-17.
[12] 陈晓军,钟发刚,罗淑萍,王新华.多浪羊BMPR-1B基因多态性的初步研究[J].新疆农业科学,2004, 41(1):6-9.doi:10.3969/j.issn.1001-4330.2004.01.002.
Chen X J, Zhong F G, Luo S P, Wang X H. The primary studies on polymorphism of BMPR-1B Gene of Duolang sheep[J].Xinjiang Agricultural Sciences,2004, 41(1):6-9.
[13] 张梦瑶,杨峰,刘开东,刘积凤,柳楠,贺建宁.敖汉细毛羊Hoxa5基因重组质粒的构建及其在成纤维细胞中表达量的研究[J].中国兽医杂志,2018,54(10):25-29,35.
Zhang M Y, Yang F, Liu K D, Liu J F, Liu N, He J N.Construction of Hoxa5 gene recombinant plasmid and its expression in fibroblasts[J].Chinese Journal of Veterinary Medicine,2018,54(10):25-29,35.
[14] 张梦瑶,杨峰,刘开东,程明,王国义,刘积凤,柳楠,贺建宁.敖汉细毛羊DKK1基因重组质粒的构建及其在成纤维细胞中表达量的研究[J].中国畜牧兽医,2018,45(1):131-139.
Zhang M Y, Yang F, Liu K D, Cheng M, Wang G Y, Liu J F, Liu N, He J N.Construction of recombinant plasmid of DKK1 gene from shuhan fine wool sheep and its expression in fibroblasts[J].China Animal Husbandry and Veterinary Medicine,2018,45(1):131-139.
[15] 周建大.原代成体皮肤角质形成细胞、成纤维细胞的培养和转基因研究[D].长沙:中南大学,2004.
Zhou J D.Culture and transgenic study of primary adult keratinocytes and fibroblasts[D].Changsha:Central South University,2004.
[16] 张梦瑶,杨峰,刘开东,刘积凤,贺建宁,柳楠.利用同源重组技术构建DKK1/BMP4真核表达载体及共转染成纤维细胞表达的研究[J].华北农学报,2018,33(5):117-124.doi:10.7668/hbnxb.2018.05.017.
Zhang M Y, Yang F, Liu K D, Liu J F, He J N, Liu N.Construction of eukaryotic expression vector of DKK1/BMP4 gene by homologous recombination and its expression fibroblasts[J].Acta Agriculturae Boreali-Sinica, 2018,33(5):117-124.
[17] 王小佳,贺建宁,程明,刘开东,柳楠.BMP4基因在细毛羊肩部皮肤的表达及其与毛囊密度的关联分析[J].黑龙江畜牧兽医,2016(10):95-98,293.
Wang X J, He J N, Cheng M, Liu K D, Liu N. Association analysis between BMP4 gene expression in the shoulder skin of fine wool sheep and the hair follicle density[J].Heilongjiang Animal Science and Veterinary Medicin, 2016(10):95-98,293.
[18] Millar S E, Willert K, Salinas P C, Roelink H, Nuss R, Sussman D J, Barsh G S. WNT signaling in the control of hair growth and structure[J]. Developmental Biology,1999,207(1):133-149. doi:10.1006/dbio.1998.9140.
[19] Bazzi H, Fantauzzo K A, Richardson G D, Jahoda C A B, Christiano A M. The Wnt inhibitor, Dickkopf 4, is induced by canonical Wnt signaling during ectodermal appendage morphogenesis[J]. Developmental Biology,2007,305(2):498-507.doi:10.1016/j.ydbio.2007.02.035.
[20] Klopcic B, Maass T, Meyer E, Lehr H A, Metzger D, Chambon P, Mann A, Blessing M. TGF-β superfamily signaling is essential for tooth and hair morphogenesis and differentiation[J]. European Journal of Cell Biology,2007,86(11-12):781-799. doi:10.1016/j.ejcb.2007.03.005.
[21] Li A G, Koster M I, Wang X J. Roles of TGFβ signaling in epidermal/appendage development[J]. Cytokine and Growth Factor Reviews,2003,14(2):99-111.doi:10.1016/s1359-6101(03)00005-4.
[22] Richardson G D,Bazzi H,Fantauzzo K A,Waters J M,Crawford H,Hynd P,Christiano A M,Jahoda C A B. KGF and EGF signalling block hair follicle induction and promote interfollicular epidermal fate in developing mouse skin[J]. Development (Cambridge),2009,136(13):2153-2164. doi:10.1242/dev.031427.
[23] Fliniaux I, Mikkola M J, Lefebvre S, Thesleff I. Identification of dkk4 as a target of Eda-A1/Edar pathway reveals an unexpected role of ectodysplasin as inhibitor of Wnt signalling in ectodermal placodes[J]. Developmental Biology,2008,320(1):60-71.doi:10.1016/j.ydbio.2008.04.023.
[24] Botchkarev V A,Botchkareva N V,Sharov A A,Gilchrest B A,Funa K,Huber O. Modulation of BMP signaling by noggin is required for induction of the secondary (nontylotrich) hair follicles[J]. Journal of Investigative Dermatology,2002,118(1):3-10.doi:10.1046/j.1523-1747.2002.01645.x.
[25] Andl T,Ahn K,Kairo A,Chu E Y,Wine-Lee L,Reddy S T,Croft Nirvana J,Cebra-Thomas J A,Metzger D,Chambon P,Lyons K M,Mishina Y,Seykora J T,Crenshaw E B,Millar S E. Epithelial Bmpr1a regulates differentiation and proliferation in postnatal hair follicles and is essential for tooth development[J]. Development,2004,131(10):2257-2268. doi:10.1242/dev.01125.
[26] Van E R J,Hardy M H. A spatial relationship between innervation and the early differentiation of vibrissa follicles in the embryonic mouse[J]. Journal of Anatomy,1980,131(Pt4):643-656.doi:10.1007/BF00301823.
[27] 苏蕊,李金泉,张文广,尹俊,赵珺,常子丽.骨形态发生蛋白2(BMP2)基因在内蒙古绒山羊不同时期皮肤毛囊中的表达[J].中国农业科学,2008,41(2):559-563.doi:10.3864/j.issn.0578-1752.2008.02.033.
Su R, Li J Q, Zhang W G, Yi J, Zhao J, Chang Z L. The expression of BMP2 in the skin and hair follicles from different stage in Inner Mongolian cashmere goats[J].Scientia Agricultura Sinica,2008,41(2):559-563.

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BMP4 Gene Plasmid Construction and Its Expression Amount in Fibroblasts of AoHan Fine Wool Sheep

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