广西巴马小型猪MyoD1基因克隆及其真核表达载体的构建
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摘要
本研究旨在对广西巴马小型猪成肌细胞决定基因1(myoblast-determining 1,MyoD1)进行克隆分析,并构建MyoD1基因真核表达载体pEGFP-N1-MyoD1,为进一步研究该基因在广西巴马小型猪骨骼肌发育中的调控作用奠定基础。以广西巴马小型猪的肝脏组织为材料,应用RT-PCR技术克隆得到广西巴马小型猪MyoD1基因编码序列,对其核苷酸序列和蛋白质序列进行生物信息学分析,并构建该基因的真核表达载体,经过PCR和双酶切验证,通过脂质体转染,将重组质粒转染C2C12细胞,在细胞水平验证了该载体的正确性。结果显示,广西巴马小型猪MyoD1基因编码区序列长960 bp,编码319个氨基酸,核苷酸序列与猪、牛、水牛、绵羊、马、人、小家鼠、褐家鼠、家犬的同源性依次为99.7%、92.8%、92.9%、92.6%、91.5%、82.9%、82.9%、82.0%和90.9%;系统进化树分析表明,广西巴马小型猪MyoD1基因在不同物种及进化过程中具有高度保守性;蛋白质结构分析表明,MyoD1蛋白为膜外蛋白,在第4-116位氨基酸处存在1个MyoD家族标志性的MyoD结构域,对广西巴马小型猪、猪和人的MyoD1蛋白高级结构比较发现其具有极高的相似性。本研究构建的广西巴马小型猪MyoD1基因表达载体pEGFP-N1-MyoD1,转染C2C12细胞后产生绿色荧光信号,表明MyoD1基因在C2C12细胞中成功表达。
This study was aimed to clone and analyze myoblast-determining 1(MyoD1) gene in Guangxi Bama Mini-pig,and construct the eukaryotic expression vector of MyoD1 gene,which would lay the foundation on the regulation of skeletal muscle of MyoD1 gene in Guangxi Bama Mini-pig.The coding sequence of porcine MyoD1 gene was cloned by RT-PCR using the liver tissue,and the nucleotide and protein sequences were analyzed by bioinformatics.The eukaryotic expression vector was constructed and verified in C2 C12 cells by PCR,double digestion and transfection.The results showed that the coding region of MyoD1 gene in Guangxi Bama Mini-pig was 960 bp of length and encoded 319 amino acids.The homology of Guangxi Bama Mini-pig with Sus scrofa,Bos taurus,Bubalus bubals,Ovis aries,Equus cabalus,Homo sapiens,Mus musculus,Rattus norvegicus and Canis lupus was 99.7%,92.8%,92.9%,92.6%,91.5%,82.9%,82.9%,82.0% and 90.9%,respectively.The phylogenetic tree analysis showed MyoD1 gene of Guangxi Bama Mini-pig was highly conservative in different species.The protein structure analysis indicated that MyoD1 protein was an extramembrane protein,and there was a MyoD family-named MyoD domain in the 4-116 th amino acid.There was a very high similarity in the high structure of MyoD1 protein among the Guangxi Bama Mini-pig,Sus scrofa and Homo sapiens.The expression vector pEGFP-N1-MyoD1 with MyoD1 gene was successfully constructed in Guangxi Bama Mini-pig and expressed in C2 C12 cells with a green fluorescent signal,which indicated that MyoD1 gene was successfully expressed on C2 C12 cells.
This study was aimed to clone and analyze myoblast-determining 1(MyoD1) gene in Guangxi Bama Mini-pig,and construct the eukaryotic expression vector of MyoD1 gene,which would lay the foundation on the regulation of skeletal muscle of MyoD1 gene in Guangxi Bama Mini-pig.The coding sequence of porcine MyoD1 gene was cloned by RT-PCR using the liver tissue,and the nucleotide and protein sequences were analyzed by bioinformatics.The eukaryotic expression vector was constructed and verified in C2 C12 cells by PCR,double digestion and transfection.The results showed that the coding region of MyoD1 gene in Guangxi Bama Mini-pig was 960 bp of length and encoded 319 amino acids.The homology of Guangxi Bama Mini-pig with Sus scrofa,Bos taurus,Bubalus bubals,Ovis aries,Equus cabalus,Homo sapiens,Mus musculus,Rattus norvegicus and Canis lupus was 99.7%,92.8%,92.9%,92.6%,91.5%,82.9%,82.9%,82.0% and 90.9%,respectively.The phylogenetic tree analysis showed MyoD1 gene of Guangxi Bama Mini-pig was highly conservative in different species.The protein structure analysis indicated that MyoD1 protein was an extramembrane protein,and there was a MyoD family-named MyoD domain in the 4-116 th amino acid.There was a very high similarity in the high structure of MyoD1 protein among the Guangxi Bama Mini-pig,Sus scrofa and Homo sapiens.The expression vector pEGFP-N1-MyoD1 with MyoD1 gene was successfully constructed in Guangxi Bama Mini-pig and expressed in C2 C12 cells with a green fluorescent signal,which indicated that MyoD1 gene was successfully expressed on C2 C12 cells.
引文
[1] 邹迪莎,于健.巴马小型猪动物模型在医学领域的研究进展[J].中国畜牧兽医,2017,44(4):1128-1134. ZOU D S,YU J.Research progress on animal model of Bama miniature pig in the field of medicine[J].China Animal Husbandry & Veterinary Medicine,2017,44(4):1128-1134.(in Chinese)
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[3] 祁钰钰.利用可控表达转基因小鼠模型研究MSTN对肌肉生长发育的影响[D].扬州:扬州大学,2016. QI Y Y.Study MSTN effects on muscle growth and development by controllable expressed transgenic mouse model[D].Yangzhou:Yangzhou University,2016.(in Chinese)
[4] 王辰.长链非编码RNA-MEG3对小鼠肌肉发育调控机制的研究[D].北京:中国农业科学院,2014. WANG C.Study on the regulation mechanism of long-chain non-coding RNA-MEG3 on muscle development in mice[D].Beijing:Chinese Academy of Agricultural Sciences,2014.
[5] LEGNINI I,DI TIMOTEO G,ROSSI F,et al.circ-znf609 is a circular rna that can be translated and functions in myogenesis[J].Molecular Cell,2017,66(1):22-37.
[6] GUO Y W,WANG J N,ZHU M F,et al.Identification of myod-responsive transcripts reveals a novel long non-coding RNA (lncRNA-AK143003) that negatively regulates myoblast differentiation[J].Scientific Reports,2017,7(1):2828.
[7] BORENSZTEIN M,MONNIER P,COURT F,et al.Mod and H19-Igf2 locus interactions are required for diaphragm formation in the mouse[J].Development,2013,140(6):1231-1239.
[8] 王红娜,孙洪新,张英杰,等.干扰MSTN对绵羊成肌细胞增殖分化及相关基因表达的影响[J].畜牧兽医学报,2018,49(1):46-54. WANG H N,SUN H X,ZHANG Y J,et al.Effects of interfering MSTN on proliferation and differentiation of sheep myoblasts and expression of related genes[J].Chinese Journal of Animal and Veterinary Sciences,2018,49(1):46-54.(in Chinese)
[9] 高宏飞,梁炳生,双卫兵.MyoD1和MyoG真核共表达载体构建及鉴定[J].中国组织工程研究,2013,17(37):6645-6651. GAO H F,LIANG B S,SHUANG W B.Construction and identification of eukaryotic co-expression vector carrying MyoD1 and MyoG[J].Journal of Clinical Rehabilitative Tissue Engineering Research,2013,17(37):6645-6651.(in Chinese)
[10] 项露颉,姜怀志,马龙,等.MyoD基因在不同品种肉羊肌肉中表达比较[J].中国兽医学报,2018,38(1):210-216. XIANG L J,JIANG H Z,MA L,et al.The expression of MyoD gene in the muscle of different mutton sheep[J].Chinese Journal of Veterinary Science,2018,38(1):210-216.(in Chinese)
[11] 刘馨怡.成纤维生长因子FGF21基因对成肌分化及肌纤维类型转化的调控[D].武汉:华中农业大学,2017. LIU X Y.Regulation of fibroblast growth factor FGF21 gene on myogenic differentiation and myofiber type transformation[D].Wuhan:Huazhong Agricultural University,2017.(in Chinese)
[12] 魏著英,白春玲,李光鹏.牛肌肉生长抑制素基因突变的遗传效应与育种应用[J].生物技术进展,2018,8(3):197-205. WEI Z Y,BAI C L,LI G P.Genetic effects and breeding application of myostatin gene mutation in beef cattle[J].Current Biotechnology,2018,8(3):197-205.(in Chinese)
[13] 赵金红,黄勇富,彭祥伟,等.肌分化因子MyoD的研究进展[J].上海畜牧兽医通讯,2007,6:12-13. ZHAO J H,HUANG Y F,PENG X W,et al.Research progress on muscle differentiation factor MyoD[J].Shanghai Journal of Animal Husbandry and Veterinary Medicine,2007,6:12-13.(in Chinese)
[14] 孙文娟.猪骨骼肌来源成肌和成脂细胞分化差异的分子机制[D].北京:中国农业大学,2017. SUN W J.Potential regulatory mechanism underlying distinct fate commitment of myogenic and adipogenic cells in skeletal muscle[D].Beijing:China Agricultural University,2017.(in Chinese)
[15] 张勇.骨髓间充质干细胞成肌分化及其对肌损伤修复的实验研究[D].重庆:第三军医大学,2002. ZHANG Y.Myogenic differentiation of mesenchymal stem cells in vitro and its graft in repair of muscle injury in mice[D].Chongqing:Third Military Medical University,2002.(in Chinese)
[16] RUDNICKI M A,LE GRAND F,MCKINNELL I,et al.The molecular regulation of muscle stem cell function[J].Cold Spring Harbor Symposia on Quantitative Biology,2008,73:323-331.
[17] MESHORER E,MISTELI T.Chromatin in pluripotent embryonic stem cells and differentiation[J].Nature Reviews Molecular Cell Biology,2006,7(7):540-546.
[18] 张雄,张勇,陈祥,等.猪肌分化因子1基因启动子区多态性及生物信息学研究[J].中国畜牧兽医,2016,43(5):1308-1315. ZHANG X,ZHANG Y,CHEN X,et al.Study on polymorphisms and bioinformatic of MyoD1 gene promoter region in pig[J].China Animal Husbandry & Veterinary Medicine,2016,43(5):1308-1315.(in Chinese)
[19] LATTANZI L,SALVATORI G,COLETTA M,et al.High efficiency myogenic conversion of human fibroblasts by adenoviral vector-mediated MyoD gene transfer.An alternative strategy for ex vivo gene therapy of primary myopathies[J].Journal of Clinical Investigation,1998,101(10):2119-2128.
[20] 付石军,郭时金,阚德英,等.牛肉质性状相关基因的研究进展[J].中国畜牧兽医,2013,40(9):158-165. FU S J,GUO S J,KAN D Y,et al.Research progress on meat quality candidate genes in cattle[J].China Animal Husbandry & Veterinary Medicine,2013,40(9):158-165.(in Chinese)
[21] KOOK S H,SON Y O,LEE K Y,et al.Hypoxia affects positively the proliferation of bovine satellite cells and their myogenic differentiation through up-regulation of MyoD[J].Cell Biology International,2008,32(8):871-878.
[22] NODA T,FUJINO T,MIE M,et al.Transduction of MyoD protein into myoblasts induces myogenic differentiation without addition of protein transduction domain[J].Biochemical and Biophysical Research Communications,2009,382(2):473-477.
[23] SZUHAI K,DE JONG D,LEUNG A Y,等.成人型梭行细胞横纹肌肉瘤高频发生MYOD1基因反式激活突变[J].临床与实验病理学杂志,2014,30(5):573. SZUHAI K,DE JONG D,LEUNG A Y,et al.Transactivation mutation of the MYOD1 gene is a frequent event in adult spindle cell rhabdomyosarcoma[J].Chinese Journal of Clinical and Experimental Pathology,2014,30(5):573.(in Chinese)
[24] 范红英,陈圆,曾凤群,等.不同日龄惠阳胡须鸡肌肉中IGF1、MyoD基因表达研究[J].惠州学院学报,2017,37(3):61-64. FAN H Y,CHEN Y,ZENG F Q,et al.Study on expression of IGF1 and MyoD genes in Huiyang bearded chicken at different ages[J].Journal of Huizhou University,2017,37(3):61-64.(in Chinese)
[25] 赵广珍,贾青,张翠翠,等.猪MyoD1基因多态性与肉质性状的相关分析[J].河北农业大学学报,2012,35(4):90-94. ZHAO G Z,JIA Q,ZHANG C C,et al.Relation between polymorphism of MyoD1 and meat quality traits in pig[J].Journal of Agricultural University of Hebei,2012,35(4):90-94.(in Chinese)
[26] 郭益文.MYOD下游长链非编码RNA的筛选及两个lncRNAs在成肌细胞分化中的功能研究[D].武汉:华中农业大学,2017. GUO Y W.Screening and identification of MYOD downstream long non-coding RNA and the function analysis of two lncRNAs during myoblast differentiation[D].Wuhan:Huazhong Agricultural University,2017.(in Chinese)
[27] 刘佳凡.长链非编码RNA lnc-mg促进成肌分化的效应及分子机制研究[D].广州:暨南大学,2017. LIU J F.The effect and mechanism of long non-coding RNA lnc-mg to promote myogenesis[D].Guangzhou:Jinan University,2017.(in Chinese)
[28] MUELLER A C,CICHEWICZ M A,DEY B K,et al.MUNC,a long noncoding RNA that facilitates the function of MyoD in skeletal myogenesis[J].Molecular and Cellular Biology,2015,35(3):498-513.
[29] 周迪,陈伟,赵佳福,等.地方黄牛MyoD1基因启动子多态性分析及其相关转录因子SF1验证[J].农业生物技术学报,2018,26(5):743-755. ZHOU D,CHEN W,ZHAO J F,et al.Polymorphism analysis of MyoD1 gene promoter and related SF1 transcription factor validation in local cattle (Bos taurus)[J].Journal of Agricultural Biotechnology,2018,26(5):743-755.
[2] 梅步俊,王志华.家畜育种中的基因组特征分析[J].基因组学与应用生物学,2016,35(4):788-796. MEI B J,WANG Z H.Analysis of genomic features in animal breeding[J].Genomics and Applied Biology,2016,35(4):788-796.(in Chinese)
[3] 祁钰钰.利用可控表达转基因小鼠模型研究MSTN对肌肉生长发育的影响[D].扬州:扬州大学,2016. QI Y Y.Study MSTN effects on muscle growth and development by controllable expressed transgenic mouse model[D].Yangzhou:Yangzhou University,2016.(in Chinese)
[4] 王辰.长链非编码RNA-MEG3对小鼠肌肉发育调控机制的研究[D].北京:中国农业科学院,2014. WANG C.Study on the regulation mechanism of long-chain non-coding RNA-MEG3 on muscle development in mice[D].Beijing:Chinese Academy of Agricultural Sciences,2014.
[5] LEGNINI I,DI TIMOTEO G,ROSSI F,et al.circ-znf609 is a circular rna that can be translated and functions in myogenesis[J].Molecular Cell,2017,66(1):22-37.
[6] GUO Y W,WANG J N,ZHU M F,et al.Identification of myod-responsive transcripts reveals a novel long non-coding RNA (lncRNA-AK143003) that negatively regulates myoblast differentiation[J].Scientific Reports,2017,7(1):2828.
[7] BORENSZTEIN M,MONNIER P,COURT F,et al.Mod and H19-Igf2 locus interactions are required for diaphragm formation in the mouse[J].Development,2013,140(6):1231-1239.
[8] 王红娜,孙洪新,张英杰,等.干扰MSTN对绵羊成肌细胞增殖分化及相关基因表达的影响[J].畜牧兽医学报,2018,49(1):46-54. WANG H N,SUN H X,ZHANG Y J,et al.Effects of interfering MSTN on proliferation and differentiation of sheep myoblasts and expression of related genes[J].Chinese Journal of Animal and Veterinary Sciences,2018,49(1):46-54.(in Chinese)
[9] 高宏飞,梁炳生,双卫兵.MyoD1和MyoG真核共表达载体构建及鉴定[J].中国组织工程研究,2013,17(37):6645-6651. GAO H F,LIANG B S,SHUANG W B.Construction and identification of eukaryotic co-expression vector carrying MyoD1 and MyoG[J].Journal of Clinical Rehabilitative Tissue Engineering Research,2013,17(37):6645-6651.(in Chinese)
[10] 项露颉,姜怀志,马龙,等.MyoD基因在不同品种肉羊肌肉中表达比较[J].中国兽医学报,2018,38(1):210-216. XIANG L J,JIANG H Z,MA L,et al.The expression of MyoD gene in the muscle of different mutton sheep[J].Chinese Journal of Veterinary Science,2018,38(1):210-216.(in Chinese)
[11] 刘馨怡.成纤维生长因子FGF21基因对成肌分化及肌纤维类型转化的调控[D].武汉:华中农业大学,2017. LIU X Y.Regulation of fibroblast growth factor FGF21 gene on myogenic differentiation and myofiber type transformation[D].Wuhan:Huazhong Agricultural University,2017.(in Chinese)
[12] 魏著英,白春玲,李光鹏.牛肌肉生长抑制素基因突变的遗传效应与育种应用[J].生物技术进展,2018,8(3):197-205. WEI Z Y,BAI C L,LI G P.Genetic effects and breeding application of myostatin gene mutation in beef cattle[J].Current Biotechnology,2018,8(3):197-205.(in Chinese)
[13] 赵金红,黄勇富,彭祥伟,等.肌分化因子MyoD的研究进展[J].上海畜牧兽医通讯,2007,6:12-13. ZHAO J H,HUANG Y F,PENG X W,et al.Research progress on muscle differentiation factor MyoD[J].Shanghai Journal of Animal Husbandry and Veterinary Medicine,2007,6:12-13.(in Chinese)
[14] 孙文娟.猪骨骼肌来源成肌和成脂细胞分化差异的分子机制[D].北京:中国农业大学,2017. SUN W J.Potential regulatory mechanism underlying distinct fate commitment of myogenic and adipogenic cells in skeletal muscle[D].Beijing:China Agricultural University,2017.(in Chinese)
[15] 张勇.骨髓间充质干细胞成肌分化及其对肌损伤修复的实验研究[D].重庆:第三军医大学,2002. ZHANG Y.Myogenic differentiation of mesenchymal stem cells in vitro and its graft in repair of muscle injury in mice[D].Chongqing:Third Military Medical University,2002.(in Chinese)
[16] RUDNICKI M A,LE GRAND F,MCKINNELL I,et al.The molecular regulation of muscle stem cell function[J].Cold Spring Harbor Symposia on Quantitative Biology,2008,73:323-331.
[17] MESHORER E,MISTELI T.Chromatin in pluripotent embryonic stem cells and differentiation[J].Nature Reviews Molecular Cell Biology,2006,7(7):540-546.
[18] 张雄,张勇,陈祥,等.猪肌分化因子1基因启动子区多态性及生物信息学研究[J].中国畜牧兽医,2016,43(5):1308-1315. ZHANG X,ZHANG Y,CHEN X,et al.Study on polymorphisms and bioinformatic of MyoD1 gene promoter region in pig[J].China Animal Husbandry & Veterinary Medicine,2016,43(5):1308-1315.(in Chinese)
[19] LATTANZI L,SALVATORI G,COLETTA M,et al.High efficiency myogenic conversion of human fibroblasts by adenoviral vector-mediated MyoD gene transfer.An alternative strategy for ex vivo gene therapy of primary myopathies[J].Journal of Clinical Investigation,1998,101(10):2119-2128.
[20] 付石军,郭时金,阚德英,等.牛肉质性状相关基因的研究进展[J].中国畜牧兽医,2013,40(9):158-165. FU S J,GUO S J,KAN D Y,et al.Research progress on meat quality candidate genes in cattle[J].China Animal Husbandry & Veterinary Medicine,2013,40(9):158-165.(in Chinese)
[21] KOOK S H,SON Y O,LEE K Y,et al.Hypoxia affects positively the proliferation of bovine satellite cells and their myogenic differentiation through up-regulation of MyoD[J].Cell Biology International,2008,32(8):871-878.
[22] NODA T,FUJINO T,MIE M,et al.Transduction of MyoD protein into myoblasts induces myogenic differentiation without addition of protein transduction domain[J].Biochemical and Biophysical Research Communications,2009,382(2):473-477.
[23] SZUHAI K,DE JONG D,LEUNG A Y,等.成人型梭行细胞横纹肌肉瘤高频发生MYOD1基因反式激活突变[J].临床与实验病理学杂志,2014,30(5):573. SZUHAI K,DE JONG D,LEUNG A Y,et al.Transactivation mutation of the MYOD1 gene is a frequent event in adult spindle cell rhabdomyosarcoma[J].Chinese Journal of Clinical and Experimental Pathology,2014,30(5):573.(in Chinese)
[24] 范红英,陈圆,曾凤群,等.不同日龄惠阳胡须鸡肌肉中IGF1、MyoD基因表达研究[J].惠州学院学报,2017,37(3):61-64. FAN H Y,CHEN Y,ZENG F Q,et al.Study on expression of IGF1 and MyoD genes in Huiyang bearded chicken at different ages[J].Journal of Huizhou University,2017,37(3):61-64.(in Chinese)
[25] 赵广珍,贾青,张翠翠,等.猪MyoD1基因多态性与肉质性状的相关分析[J].河北农业大学学报,2012,35(4):90-94. ZHAO G Z,JIA Q,ZHANG C C,et al.Relation between polymorphism of MyoD1 and meat quality traits in pig[J].Journal of Agricultural University of Hebei,2012,35(4):90-94.(in Chinese)
[26] 郭益文.MYOD下游长链非编码RNA的筛选及两个lncRNAs在成肌细胞分化中的功能研究[D].武汉:华中农业大学,2017. GUO Y W.Screening and identification of MYOD downstream long non-coding RNA and the function analysis of two lncRNAs during myoblast differentiation[D].Wuhan:Huazhong Agricultural University,2017.(in Chinese)
[27] 刘佳凡.长链非编码RNA lnc-mg促进成肌分化的效应及分子机制研究[D].广州:暨南大学,2017. LIU J F.The effect and mechanism of long non-coding RNA lnc-mg to promote myogenesis[D].Guangzhou:Jinan University,2017.(in Chinese)
[28] MUELLER A C,CICHEWICZ M A,DEY B K,et al.MUNC,a long noncoding RNA that facilitates the function of MyoD in skeletal myogenesis[J].Molecular and Cellular Biology,2015,35(3):498-513.
[29] 周迪,陈伟,赵佳福,等.地方黄牛MyoD1基因启动子多态性分析及其相关转录因子SF1验证[J].农业生物技术学报,2018,26(5):743-755. ZHOU D,CHEN W,ZHAO J F,et al.Polymorphism analysis of MyoD1 gene promoter and related SF1 transcription factor validation in local cattle (Bos taurus)[J].Journal of Agricultural Biotechnology,2018,26(5):743-755.