绵羊胎盘生长因子基因可变剪切异构体克隆与表达分析

The Cloningand Expression Pattern Analysis of Alternative SplicingIsoforms of Placental Growth Factor Gene from Sheep

为克隆绵羊胎盘生长因子(Placental growth factor,PGF)基因,并分析该基因在绵羊组织器官中的表达模式,本研究首先利用RT-PCR(Reverse Transcript Polymerase Chain Reaction,RT-PCR)方法从小尾寒羊与新吉细毛羊皮肤组织克隆出PGF基因mRNA片段,随后利用可变剪切(alternative splicing,AS)鉴别引物和qRT-PCR(Quantity reverse transcript polymerase chain reaction,qRT-PCR)方法对不同组织器官PGF基因AS及其表达模式进行分析.以两个品种绵羊皮肤组织cDNA为模板,RT-PCR扩增显示存在2条特异性扩增片段,克隆测序结果发现片段长度依次为1339bp和1276bp,分析发现1276bp片段是由于主转录本第7外显子缺失产生,为编码羧基末端缺失21个氨基酸的PGF蛋白突变体.该突变体并未引起血小板源生长因子(Platelet derived growth factor,PDGF)结构域改变,可能通过改变PDGF结构域下游单位酶切位点来影响PGF分子的成熟.PGF基因AS检测及qRT-PCR结果发现小尾寒羊与新吉细毛羊不同组织器官PGF基因可变剪切突变体组织分布及其表达模式存在明显差异.本研究证明绵羊皮肤组织中表达PGF基因且存在AS现象,为进一步研究PGF基因在绵羊毛囊发育及毛用性状形成中的作用奠定了基础.

In order to clone the placental growth factor (PGF) gene and explore the expression pattern in different organs of sheep.In the present study,the PGF mRNAs were cloned by RT-PCR from skin cDNAs in both Small-tail Han sheep (STH) and Xin-ji Fine Wool sheep (XJ). The alternative splicing (AS) isoforms were verified by specific primers,and total PGF mRNA expression level was tested by qRT-PCR in different organs in both STH and XJ respectively by conserved region primers.The RT-PCR showed there were two specific amplicons when the skin cDNAs were templet, and they were identified as PGF mRNA with a length of 1339 bp and 1276 bp respectively.The 1276 bp isoform was produced by AS proceed by deleting the Exon 7 and coding a novel PGF protein with 21 amino acids deletion in C end.However,the deletion mutant didn’t destroy the PDGF(Platelet derived growth factor,PDGF) domain and might to affect the mature of PGF protein by changing the protein enzyme site close to the PDGF domain.The AS isoforms and total mRNA expression measurement results showed that there were remarkable distinct both on AS isoforms and mRNA expression between STH and XJ sheep.In conclusion,this study approved the existence of PGF and its AS isoform in sheep skin and it had different expression pattern among organs between STH and XJ sheep.These data provide an opportunity to explore the PGF function on hair follicle development and wool traits.