绵羊皮肤组织VEGF-A基因可变剪切体存在的证实与鉴定
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摘要
为了克隆绵羊血管内皮生长因子A(VEGF-A)基因,探寻该基因与绵羊毛囊发育及毛用性状形成的潜在关系,试验采用RT-PCR方法从小尾寒羊与新吉细毛羊皮肤组织克隆出VEGF-A基因mRNA并对其进行生物信息学分析。结果表明:以皮肤组织cDNA为模板,RT-PCR扩增出3条特异性条带;克隆测序结果显示所有条带均为VEGF-A基因序列,片段长度依次为828,825,756,624 bp。828 bp和825 bp片段属于全长VEGF-A,其中825 bp片段在3′非编码区缺失3个碱基;756 bp和624 bp片段由可变剪切产生,分别为外显子6缺失和外显子6,7双缺失,编码蛋白与人类VEGF-A165和VEGF-A121类似;核苷酸序列比对发现,VEGF-A基因mRNA仅存在3个SNP位点;蛋白结构域分析发现,三种类型VEGF-A蛋白均含有完整的血小板衍生生长因子(PDGF)结构域。说明试验成功地从绵羊皮肤组织中克隆到VEGF-A基因并证实可变剪切突变体的存在。
In order to clone the vascular endothelial growth factor A(VEGF-A) gene and explore the relationship of VEGF-A gene and hair follicle development or wool traits in sheep. In this study, the mRNA of VEGF-A gene was cloned by RT-PCR in skin tissue from Xinji Fine wool sheep and Small-tail Han sheep. In RT-PCR study, three bands were amplified and identified as isoforms of VEGF-A gene by cloning and sequencing. The length of isoforms is 828,825,756 and 624 bp respectively. The amplicons with 828 bp and 825 bp belong to the full-length VEGF-A type, although the 825 isoform has 3 nucleotides deletion in 3′ UTR. Both of 756 and 624 bp isoforms were produced by alternative splicing(AS), and the exon 6 was deleted in 756 bp isoform and both exon 6 and 7 were deleted in 624 bp isoform. The 756 and 624 bp isoforms encode the similar proteins to VEGF-A165 and 121 which were identified in human. The protein domain annotation shown there was PDGF domain in all isoforms of VEGF-A. The identification of multiply AS isoforms mRNA in VEGF-A gene in sheep skin which provides the opportunity to explore the gene function of VEGF-A in sheep skin.
In order to clone the vascular endothelial growth factor A(VEGF-A) gene and explore the relationship of VEGF-A gene and hair follicle development or wool traits in sheep. In this study, the mRNA of VEGF-A gene was cloned by RT-PCR in skin tissue from Xinji Fine wool sheep and Small-tail Han sheep. In RT-PCR study, three bands were amplified and identified as isoforms of VEGF-A gene by cloning and sequencing. The length of isoforms is 828,825,756 and 624 bp respectively. The amplicons with 828 bp and 825 bp belong to the full-length VEGF-A type, although the 825 isoform has 3 nucleotides deletion in 3′ UTR. Both of 756 and 624 bp isoforms were produced by alternative splicing(AS), and the exon 6 was deleted in 756 bp isoform and both exon 6 and 7 were deleted in 624 bp isoform. The 756 and 624 bp isoforms encode the similar proteins to VEGF-A165 and 121 which were identified in human. The protein domain annotation shown there was PDGF domain in all isoforms of VEGF-A. The identification of multiply AS isoforms mRNA in VEGF-A gene in sheep skin which provides the opportunity to explore the gene function of VEGF-A in sheep skin.
引文
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[19] ARCONDéGUY T,LACAZETTE E,MILLEVOI S,et al.VEGF-A mRNA processing,stability and translation:a paradigm for intricate regulation of gene expression at the post-transcriptional level[J].Nucleic Acids Res,2013,41(17):7997-8010.
[20] QI M,HUANG X,ZHOU L,et al.Four polymorphisms of VEGF (+405C>G,-460T>C,-2578C>A,and -1154G>A) in susceptibility to psoriasis:a meta-analysis[J].DNA Cell Biol,2014,33(4):234-244.
[21] COHEN T,GITAY-GOREN H,SHARON R,et al.VEGF121,a vascular endothelial growth factor (VEGF) isoform lacking heparin binding ability,requires cell-surface heparan sulfates for efficient binding to the VEGF receptors of human melanoma cells[J].J Biol Chem,1995,270(19):11322-11326.
[2] DVORAK H F,BROWN L F,DETMAR M,et al.Vascular permeability factor/vascular endothelial growth factor,microvascular hyperpermeability,and angiogenesis[J].Am J Pathol,1995,146(5):1029-1039.
[3] MATSUMOTO K,EMA M.Roles of VEGF-A signalling in development,regeneration,and tumours[J].J Biochem,2014,156(1):1-10.
[4] TARALLO V,DE FALCO S.The vascular endothelial growth factors and receptors family:Up to now the only target for anti-angiogenesis therapy[J].Int J Biochem Cell Biol,2015,64:185-189.
[5] TISCHER E,MITCHELL R,HARTMAN T,et al.The human gene for vascular endothelial growth factor.Multiple protein forms are encoded through alternative exon splicing[J].J Biol Chem,1991,266(18):11947-11954.
[6] KAZEMI M,CARRER A,MOIMAS S,et al.VEGF121 and VEGF165 differentially promote vessel maturation and tumor growth in mice and humans[J].Cancer Gene Ther,2016,23(5):125-132.
[7] MEIRON M,ANUNU R,SCHEINMAN E J,et al.New isoforms of VEGF are translated from alternative initiation CUG codons located in its 5’UTR[J].Biochem Biophys Res Commun,2001,282(4):1053-1060.
[8] XU J,DOU T,LIU C,et al.The evolution of alternative splicing exons in vascular endothelial growth factor A[J].Gene,2011,487(2):143-150.
[9] GONG B,LIANG D,CHEW T G,et al.Characterization of the zebrafish vascular endothelial growth factor A gene:comparison with vegf-A genes in mammals and Fugu[J].Biochim Biophys Acta,2004,1676(1):33-40.
[10] ZHAO J,LIU N,LIU K,et al.Identification of genes and proteins associated with anagen wool growth[J].Anim Genet,2017,48(1):67-79.
[11] LIU N,LI H,LIU K,et al.Identification of skin-expressed genes possibly associated with wool growth regulation of Aohan fine wool sheep[J].BMC Genet,2014,15:144.
[12] 张立春,马惠海,曹阳,等.绵羊血管内皮生长因子D基因克隆与表达分析[J].黑龙江畜牧兽医,2016(06上):1-6,11.
[13] 张立春,李梦姝,曹阳,等.绵羊VEGF-C基因可变剪切体克隆与序列分析[J].中国畜牧杂志,2018,54(4):89-92.
[14] SHIBUYA M.Structure and function of VEGF/VEGF-receptor system involved in angiogenesis[J].Cell Struct Funct,2001,26(1):25-35.
[15] DOLGIN E.The most popular genes in the human genome[J].Nature,2017,551(7681):427-431.
[16] WU X J,ZHU J W,JING J,et al.VEGF165 modulates proliferation,adhesion,migration and differentiation of cultured human outer root sheath cells from central hair follicle epithelium through VEGFR-2 activation in vitro[J].J Dermatol Sci,2014,73(2):152-160.
[17] ZHANG Q L,LI J P,LI Y M,et al.Expression and localization of the vascular endothelial growth factor and changes of microvessel density during hair follicle development of Liaoning cashmere goats[J].Genet Mol Res,2013,12(4):6424-6432.
[18] CHEN H L,LIU K.Vascular endothelial growth factor polymorphisms and coronary artery disease:a systemic review and meta-analysis[J].Int J Cardiol,2014,172(1):e220-224.
[19] ARCONDéGUY T,LACAZETTE E,MILLEVOI S,et al.VEGF-A mRNA processing,stability and translation:a paradigm for intricate regulation of gene expression at the post-transcriptional level[J].Nucleic Acids Res,2013,41(17):7997-8010.
[20] QI M,HUANG X,ZHOU L,et al.Four polymorphisms of VEGF (+405C>G,-460T>C,-2578C>A,and -1154G>A) in susceptibility to psoriasis:a meta-analysis[J].DNA Cell Biol,2014,33(4):234-244.
[21] COHEN T,GITAY-GOREN H,SHARON R,et al.VEGF121,a vascular endothelial growth factor (VEGF) isoform lacking heparin binding ability,requires cell-surface heparan sulfates for efficient binding to the VEGF receptors of human melanoma cells[J].J Biol Chem,1995,270(19):11322-11326.