绵羊皮肤组织VEGF-A基因可变剪切体存在的证实与鉴定

The discovery and identification of alternative splicing isoforms of VEGF-A gene from Sheep skin tissue

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828,825,756,624bp。828bp和825bp片段属于全长VEGF-A,其中825bp片段在3′非编码区缺失3个碱基;756bp和624bp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 fulllength 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-A 165 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.