陆川猪PLIN5基因的克隆和序列分析

Cloning and sequence analysis of PLIN5 gene in Luchuan pig

为了研究陆川猪背最长肌围脂滴蛋白5(PLIN5,Perilipin5)基因的结构和功能,试验对陆川猪PLIN5基因进行RT-PCR扩增,所得产物经转化获得含目的基因的重组质粒pMD19-T-PLIN5,经菌落PCR扩增和测序鉴定正确后利用生物信息学方法对陆川猪PLIN5基因的结构、理化性质及其编码蛋白信号肽、跨膜结构、亚细胞定位等进行分析,并探讨了陆川猪与其他物种PLIN5氨基酸序列的进化关系。结果表明:PLIN5基因编码区(CDS)序列长度为1 377 bp,编码458个氨基酸,与NCBI上公布的野猪PLIN5基因序列中的编码区存在4个碱基差异,均为错义突变;陆川猪PLIN5蛋白不存在信号肽,没有跨膜结构域,不存在N糖基化位点;二级结构中α-螺旋占47.16%,无规则卷曲占43.67%,延伸链占9.17%;陆川猪PLIN5基因在不同物种及进化过程中具有较高的保守性,该基因编码的蛋白符合一般Perilipin超家族的结构特征。

In order to study Perilipin 5 ( PLIN5) in longissimus dorsi muscle of Luchuan pig and further investigate its structure and function of PLIN5 gene in Luchuan pig,the PLIN5 gene of Luchuan pig was amplified by RT-PCR and the obtained product was transformed. And then the recombinant plasmid pMD19-T-PLIN5 containing the target gene PLIN5 was acquired and confirmed by colony PCR and sequencing. The structure,physicochemical properties,and the encoded protein signal peptide,transmembrane structure,and subcellular localization of the PLIN5 gene in Luchuan pig were detected by bioinformatics methods. Meanwhile the evolutionary relationship of the amino acid sequence of PLIN5 between Luchuan pig and other species were analyzed. The results showed that the length of PLIN5 gene CDS region is 1 377 bp and it encoded 458 amino acids. It differed from the CDS region in the Sus scorfa PLIN5 gene sequence published on NCBI by 4 bases, and all of them were missense mutations. There was no signal peptide,transmembrane domain,and N glycosylation site in PLIN5 protein of Luchuan pig.The ratios of α-helix,random coil and extending strand in the secondary structure were 47. 16%,43. 67% and 9. 17%, respectively.PLIN5 gene was highly conservative in different species and evolution process. It demonstrated that the protein encoded by the PLIN5 gene in Luchuan pig was conformed to the structural features of the general Perilipin superfamily.