版纳微型猪近交系BARD1基因克隆、mRNA表达及蛋白质功能生物信息学分析
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摘要
BARD1基因在精子形成过程中起重要作用。以Gen Bank下载的牛、鼠BARD1基因序列以及猪EST序列克隆猪BARD1基因,设计特异引物克隆版纳微型猪近交系(BMI)BARD1基因。应用q PCR分析15个重要组织的mRNA表达谱,并对蛋白质序列进行功能生物信息学分析,构建多物种系统进化树。研究获得了BMI BARD1 2 334 bp的编码区序列(Gen Bank登录号KU705640,对应的氨基酸登录号AOC89058),编码777个氨基酸,蛋白质分子量(Mw)为86.15 ku,等电点(p I)为8.84。多组织荧光定量表达分析表明:BARD1基因在睾丸和尿道球腺中高表达,在肝脏、脾脏、肺、十二指肠、结肠中中度表达,在心脏、肾脏、胃脏、脑、肌肉、精囊腺、前列腺、附睾中低表达。功能生物信息学分析表明,BARD1蛋白质存在3个保守结构域,无跨膜结构,无信号肽序列;N末端疏水,C末端亲水;有6类功能活性位点,位于细胞核的概率是94.1%。系统进化分析表明,BMI与牛、羊的亲缘关系最近。
BARD1 gene plays an important role in the process of spermatogenesis. Referring to cattle BARD1 gene sequence and EST sequences of pig from Gen Bank,using silicon cloning strategy,we obtained pig BARD1 gene sequence. Subsequently we designed specific primers and amplified Banna mini-pig inbred line( BMI) BARD1 gene. q PCR was applied to analyze mRNA expression profiles of 15 important tissues. Protein sequence was used to carry out functional bioinformatics analysis and construct phylogenetic tree. A coding sequence of 2 334 bp( Gen Bank accession number:KU705640,the corresponding amino acid sequence accession number: AOC89058) of BMI BARD1was obtained,which encodes a protein of 777 amino acids,molecular weight( Mw) 86. 15 ku,and isoelectric point( p I) 8. 84. Fluorescence quantitative expression analysis showed that BARD1 gene expressed highly in testis and antiprostate,moderately in liver,spleen,lung,duodenum and colon,weakly in heart,kidney,stomach,brain,muscle,seminal vesicle gland,prostate gland and epididymis. Functional bioinformatics analysis indicated that BARD1 protein contained three conserved domains,no transmembrane region,no signal peptide sequences,its N-terminal was hydrophobic while C-terminal was hydrophilic,it had six kinds of functionally active sites,with a percentage of94. 1 to be located in cell nucleus. Phylogenetic analysis demonstrated that BMI had the closest relationship with cattle and goat.
BARD1 gene plays an important role in the process of spermatogenesis. Referring to cattle BARD1 gene sequence and EST sequences of pig from Gen Bank,using silicon cloning strategy,we obtained pig BARD1 gene sequence. Subsequently we designed specific primers and amplified Banna mini-pig inbred line( BMI) BARD1 gene. q PCR was applied to analyze mRNA expression profiles of 15 important tissues. Protein sequence was used to carry out functional bioinformatics analysis and construct phylogenetic tree. A coding sequence of 2 334 bp( Gen Bank accession number:KU705640,the corresponding amino acid sequence accession number: AOC89058) of BMI BARD1was obtained,which encodes a protein of 777 amino acids,molecular weight( Mw) 86. 15 ku,and isoelectric point( p I) 8. 84. Fluorescence quantitative expression analysis showed that BARD1 gene expressed highly in testis and antiprostate,moderately in liver,spleen,lung,duodenum and colon,weakly in heart,kidney,stomach,brain,muscle,seminal vesicle gland,prostate gland and epididymis. Functional bioinformatics analysis indicated that BARD1 protein contained three conserved domains,no transmembrane region,no signal peptide sequences,its N-terminal was hydrophobic while C-terminal was hydrophilic,it had six kinds of functionally active sites,with a percentage of94. 1 to be located in cell nucleus. Phylogenetic analysis demonstrated that BMI had the closest relationship with cattle and goat.
引文
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[16]Irminger-Finger I,Soriano J V,Vaudan G,et al.In vitro repression of Brca1-associated RING domain gene,Bard1,induces phenotypic changes in mammary epithelial cells[J].The Journal of Cell Biology,1998,143(5):1329-1339.
[2]Andre P A,Prele C M,Vierkotten S,et al.BARD1 mediates TGF-βsignaling in pulmonary fibrosis[J].Respiratory Research,2015,16:118.
[3]Irminger-Finger I,Leung W C,Li J,et al.Identification of BARD1 as mediator between proapoptotic stress and p53-dependent apoptosis[J].Molecular Cell,2001,8(6):1255-1266.
[4]Irminger-Finger I,Ratajska M,Pilyugin M.New concepts on BARD1:Regulator of BRCA pathways and beyond[J].The International Journal of Biochemistry&Cell Biology,2016,72:1-17.
[5]Morris J R,Keep N H,Solomon E.Identification of residues required for the interaction of BARD1 with BRCA1[J].The Journal of Biological Chemistry,2002,277(11):9382-9386.
[6]Li M,Yu X.Function of BRCA1 in the DNA damage response is mediated by ADP-ribosylation[J].Cancer Cell,2013,23(5):693-704.
[7]Weiss M,Vigier M,Hue D,et al.Pre-and postmeiotic expression of male germ cell-specific genes throughout 2-week cocultures of rat germinal and sertoli cells[J].Biology of Reproduction,1997,57(1):68-76.
[8]Sinha Hikim A P,Lue Y,Diaz-Romero M,et al.Deciphering the pathways of germ cell apoptosis in the testis[J].The Journal of Steroid Biochemistry and Molecular Biology,2003,85(2-5):175-182.
[9]Feki A,Jefford C E,Durand P,et al.BARD1 expression during spermatogenesis is associated with apoptosis and hormonally regulated[J].Biology of Reproduction,2004,71(5):1614-1624.
[10]Yu P,Zhang L,Li S,et al.Screening and analysis of porcine endogenous retrovirus in Chinese Banna minipig inbred line[J].Transplant Proceedings,2004,36(8):2485-2487.
[11]Zeng R,Zeng Y Z.Molecular cloning and characterization of SLA-DR genes in the 133-family of the Banna mini-pig inbred line[J].Animal Genetics,2005,36(3):267-269.
[12]Huo J L,Wang P,Zhao Y,et al.Molecular cloning,mRNA expression and characterization of a novel FAIM1 gene from Chinese Banna mini-pig inbred line(BMI)[J].Journal of Animal and Veterinary Advances,2012,11(8):1080-1086.
[13]Wang P,Huo H L,Wang S Y,et al.Cloning,sequence characterization,and expression patterns of members of the porcine TSSK family[J].Genetics and Molecular Research,2015,14(4):14908-14919.
[14]Joukov V,Chen J,Fox E A,et al.Functional communication between endogenous BRCA1 and its partner BARD1 during xenopus laevis development[J].Proceedings of the National Academy of Sciences of the United States of America,2001,98(21):12078-12083.
[15]Brzovic P S,Meza J E,King M C,et al.BRCA1 RING domain cancer-predisposing mutations[J].The Journal of Biological Chemistry,2001,276(44):41399-41406.
[16]Irminger-Finger I,Soriano J V,Vaudan G,et al.In vitro repression of Brca1-associated RING domain gene,Bard1,induces phenotypic changes in mammary epithelial cells[J].The Journal of Cell Biology,1998,143(5):1329-1339.