大熊猫RPL35A基因的克隆及超表达研究
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摘要
探究大熊猫核糖体蛋白基因RPL35A及其编码蛋白的特点,以进一步研究其编码蛋白的功能,为深入开展大熊猫分子生物学研究及大熊猫基因保护等积累科学资料;本研究以大熊猫骨骼肌为材料,采用RT-PCR和降落-PCR方法克隆RPL35A基因的cDNA基因组序列,IPTG法诱导基因在BL21细胞系中的表达;大熊猫RPL35A基因的cDNA序列为337 bp,编码110个氨基酸;该基因的基因组序列长1 275 bp,包含2个外显子和1个内含子;RPL35A基因在BL21细胞系中的表达产物与组氨酸标签蛋白融合形成18 k Da的融合蛋白;该基因的cDNA序列、基因组序列中的外显子片段及其编码的蛋白序列与已报道的人、牛、小家鼠、褐家鼠等物种的对应序列同源性很高,蛋白质理化性质和功能位点基本相同,这说明该基因及其编码蛋白在进化过程中非常保守,可用于分子进化方面的研究,在物种进化关系研究中具有一定的参考意义和研究价值;同时,该基因可在原核生物中表达出具有活性的产物,可进一步开展纯化和蛋白质功能的研究。
To futher investigate the function of encoded protein,gather scientific data for molecular biology study of giant panda and protect gene resources of this endangered species,this paper explores the characteristics of ribosomal protein L35A gene(RPL35A) and its encoded protein.RT-PCR and touch-down PCR technology are used to clone the cDNA and the genomic sequence of RPL35A from giant panda and overexpressed in BL21 by IPTG.The results show that the length of cDNA is 333 bp encoding 110 amino acids and the genomic sequence is 1 275 bp in length possessing two exons and one introns.The expression protein of RPL35A in BL21 and N-terminally Histagged protein give rise to the accumulation of an expected 18 kDa polypeptide fusion protein.The cDNA and its deduced amino acid sequence share a high homology with that of mammals reported,including Homo sapiens,Bos Taurus,Mus musculus and Rattus norvegicus.And their molecular weight as well as isoelectric point are basically the same.All of these illustrate that gene RPL35 A and its encoded protein are very conservative in the process of evolution.These findings can be used in the study of molecular evolution.It is of certain reference significance and research value in phylogenic relationship.Meanwhile,its active overexpression in prokaryotic organism shows that further study on protein purification and its function can be conducted
To futher investigate the function of encoded protein,gather scientific data for molecular biology study of giant panda and protect gene resources of this endangered species,this paper explores the characteristics of ribosomal protein L35A gene(RPL35A) and its encoded protein.RT-PCR and touch-down PCR technology are used to clone the cDNA and the genomic sequence of RPL35A from giant panda and overexpressed in BL21 by IPTG.The results show that the length of cDNA is 333 bp encoding 110 amino acids and the genomic sequence is 1 275 bp in length possessing two exons and one introns.The expression protein of RPL35A in BL21 and N-terminally Histagged protein give rise to the accumulation of an expected 18 kDa polypeptide fusion protein.The cDNA and its deduced amino acid sequence share a high homology with that of mammals reported,including Homo sapiens,Bos Taurus,Mus musculus and Rattus norvegicus.And their molecular weight as well as isoelectric point are basically the same.All of these illustrate that gene RPL35 A and its encoded protein are very conservative in the process of evolution.These findings can be used in the study of molecular evolution.It is of certain reference significance and research value in phylogenic relationship.Meanwhile,its active overexpression in prokaryotic organism shows that further study on protein purification and its function can be conducted
引文
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[14]杜玉杰,侯怡铃,郝彦哲,等.大熊猫(Ailuropoda melanoleuca)RPS24基因c DNA克隆及序列分析[J].西华师范大学学报(自然科学版).2009,30(2):109-114.
[15]王祖秀,郝彦哲,侯万儒.大熊猫NADH-辅酶Q氧化还原酶铁硫蛋白亚基6基因(NDUFS6)的序列分析[J].兽类学报.2008,28(3):321-324.
[16]SONG Y,HOU YL,HOU WR,et al.c DNA,genomic sequence cloning and overexpression of the ribosomal protein S13 gene in the giant panda(Ailuropoda melanoleuca)[J].Genetics and molecular research,2011,10(1):121-132.
[17]DU YJ,HOU YL,HOU WR.Nucleotide sequences of an important functional gene hnRNPA2/B1 from Ailuropoda melanoleuca and Ursus thibetanus mupinensis and its potential value in phylogenetic study[J].Nucleosides,nucleotides&nucleic acids,2014,33(1):18-30.
[18]DU YJ,HOU YL,HOU WR.Cloning and overexpression of an important functional gene ATP6V1F encoding a component of vacuolar ATPase from the Giant Panda(Ailuropoda melanoleuca)[J].Mol Biol Rep,2012,39(5):5761-6.
[19]李永明,赵玉琪.实用分子生物学方法手册[M].北京:科学出版社,1998,327-332.
[2]DRAPER DE,REYNALDO LP.RNA binding strategies of ribosomal proteins[J].Nucl Acids Res,1999,27:381-388.
[3]FEO S,DAVIES B,FRIED M,et al.The mapping of seven intron-containing ribosomal protein genes shows they are unlinked in human genome[J].Genomics,1992,13(1):201-207.
[4]COLOMBO P,READ M,FRIED M,et al.The human L35a ribosomal protein(RPL35A)gene is located at chromosome band3q29-qter.Genomics,1996,32(1),148-150.
[5]王平,曹焯,刘维新,等.初生大熊猫的组织学观察[J].科学通报,1980,(13):623.
[6]胡锦矗.大熊猫繁殖的效率[J].野生动物,1988,(5):32.
[7]侯万儒.大熊猫生命表及相关数据指标的修订[J].动物学研究,2000,21(5):361-366.
[8]DU Y J,HOU Y L,HOU W R,et al.Molecular characterization of a gene POLR2H encoded an essential subunit for RNA polymerase II from the Giant Panda(Ailuropoda Melanoleuca)[J].Molecular Biology Reports,2013,40(2):1495-1498.
[9]PENG R,ZENG B,MENG X X,et al.The complete mitochondrial genome and phylogenetic analysis of the giant panda(Ailuropoda melanoleuca)[J].Gene,2007,397(1/2):76-83.
[10]QIAO Q,MA Q Y,ZHU H,et al.Study on the cloning,in vitro expression and bioactivity of Ailuropoda melanoleuca Qinling subspecies interleukin-2[J].Zhi,2009,25(6),479-482.
[11]ZHANG T,HOU W R,HOU Y L,et al.c DNA,genomic sequence cloning and overexpression of ribosomal protein s20 gene(RPS20)from the Giant Panda(Ailuropoda melanoleuca)[J].African Journal of Biotechnology.2009,8(21),5627-5632.
[12]杜玉杰,侯万儒,彭正松,等.大熊猫酸性核糖体磷酸蛋白P1基因(RPLP1)c DNA克隆及序列分析[J].兽类学报.2008,28(1):75-80.
[13]侯万儒,陈瑜,彭正松,等.大熊猫辅酶Q-细胞色素C氧化还原酶的辅酶Q连接蛋白基因(QP-C)c DNA的克隆及序列比较[J].兽类学报.2007,27(2):190-194.
[14]杜玉杰,侯怡铃,郝彦哲,等.大熊猫(Ailuropoda melanoleuca)RPS24基因c DNA克隆及序列分析[J].西华师范大学学报(自然科学版).2009,30(2):109-114.
[15]王祖秀,郝彦哲,侯万儒.大熊猫NADH-辅酶Q氧化还原酶铁硫蛋白亚基6基因(NDUFS6)的序列分析[J].兽类学报.2008,28(3):321-324.
[16]SONG Y,HOU YL,HOU WR,et al.c DNA,genomic sequence cloning and overexpression of the ribosomal protein S13 gene in the giant panda(Ailuropoda melanoleuca)[J].Genetics and molecular research,2011,10(1):121-132.
[17]DU YJ,HOU YL,HOU WR.Nucleotide sequences of an important functional gene hnRNPA2/B1 from Ailuropoda melanoleuca and Ursus thibetanus mupinensis and its potential value in phylogenetic study[J].Nucleosides,nucleotides&nucleic acids,2014,33(1):18-30.
[18]DU YJ,HOU YL,HOU WR.Cloning and overexpression of an important functional gene ATP6V1F encoding a component of vacuolar ATPase from the Giant Panda(Ailuropoda melanoleuca)[J].Mol Biol Rep,2012,39(5):5761-6.
[19]李永明,赵玉琪.实用分子生物学方法手册[M].北京:科学出版社,1998,327-332.
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