东北狍PDGFA基因cDNA克隆及表达分析

Cloning and expression analysis of PDGFA gene cDNA from northeastern Roe deer

为了研究血小板衍生生长因子A（PDGFA）基因在狍茸生长发育中的调控作用,试验采用分子克隆技术得到PDGFA基因全部编码区的cDNA序列并结合生物信息学方法进行分析。结果表明：获得的东北狍PDGFA基因cDNA长度为653 bp,其中包括594 bp的开放阅读框（ORF）,编码197个氨基酸。PDGFA基因编码蛋白为不稳定蛋白,相对分子质量为22 259.65,等电点为8.53,具备信号肽,属于分泌性蛋白。蛋白质二级结构由α-螺旋、扩展链及无规则卷曲结构组成。通过构建系统进化树发现,狍与白尾鹿亲缘关系最近,与野牦牛、水牛、野猪、绵羊的亲缘关系较近,与原鸡、人、褐家鼠、斑马鱼的亲缘关系较远。实时荧光定量PCR分析显示,PDGFA基因在狍茸顶端组织不同组织层表达存在差异,其中前软骨层和软骨层的相对表达量显著高于皮肤层和间充质层（P〈0.05）。

In order to study the regulation of PDGFA gene on the growth and development of northeastern Roe deer antler, molecular cloning technique was used to obtain the cDNA sequence including complete coding region of PDGFA gene and bioinformatics methods were used to analyze the cDNA sequence. The results showed that the cDNA length of PDGFA gene was 653 bp, including an open reading frame（ORF） of 594 bp, encoding 197 amino acids. The protein encoded by PDGFA gene was an unstable protein. The relative molecular weight of the protein was 22 259.65 and theoretical isoelectric point was 8.53. PDGFA protein had signal peptide and belonged to secretory protein. The secondary structure of PDGFA protein consisted of alpha helix, random coil and extended chain. The phylogenetic tree showed that Roe deer had the closest genetic relationship with Odocoileus virginianus texanus. Roe deer was closely related to Bos grunniens, Bubalus bubalis, Sus scrofa and Ovis aries musimon. It was far genetic relationship with Gallus gallus, Homo sapiens, Rattus norvegicus and Danio rerio. The qPCR analysis showed that the expression of PDGFA gene was different in different tissue of Roe deer antler, and the relative expression levels of PDGFA gene in pre-cartilage and cartilage were significantly higher than that in skin and mesenchymal（P0.05）.