巴马香猪和杜长大猪的PDK4基因的克隆及组织表达分析

为了获得广西巴马香猪和杜长大猪丙酮酸脱氢酶激酶4 (PDK4)基因编码区序列(CDS),并研究PDK4基因在广西巴马香猪和杜长大猪不同组织中的mRNA表达差异,本研究利用基因克隆技术分别从巴马香猪和杜长大猪的背最长肌组织中提取总RNA,PCR扩增出PDK4基因CDS,并进行生物信息学分析。利用实时荧光定量PCR技术检测PDK4基因在这两品种猪不同组织中m RNA的相对表达量。结果表明:本研究分别成功克隆获得巴马香猪和杜长大猪PDK4基因CDS,长度为1 224 bp,这两品种猪的PDK4基因CDS同源性为100%,与GenBank报道的普通猪、人、鼠、马、羊的同源性分别为100%、91.0%、85.5%、92.3%、93.0%。基于PDK4基因碱基同源性构建的物种系统进化树可知,巴马猪和杜长大猪遗传距离是最近的,最远的是小鼠。两品种猪PDK4氨基酸组成中亮氨酸含量较高,占总氨基酸数的10.8%,同时发现PDK4蛋白具有较强的亲水性。巴马香猪和杜长大猪PDK4蛋白的高级结构中均包含有α螺旋、无规卷曲和延伸链。实时荧光定量PCR结果显示,巴马香猪和杜长大猪的不同组织PDK4基因表达量最高的为腹脂,最低的是脾。巴马香猪背最长肌中PDK4基因表达水平极显著高于杜长大猪,而杜长大猪的皮脂、腹脂、肝、脾以及肾中PDK4基因表达水平极显著高于巴马香猪。本试验成功克隆了巴马香猪和杜长猪的PDK4基因,并且检测了该基因在两品种猪不同组织中的基因表达水平的差异,为今后深入探讨PDK4基因在地方猪种脂质代谢和脂肪沉积方面发挥的作用奠定工作基础。

Cloning and Tissue Expression Analysis of PDK4 Genes in Bama Xiang Pig and DLY Pig

The objective of this study was to obtain the coding sequence(CDS) of the pyruvate dehydrogenase kinase 4(PDK4) gene and to study the mRNA expression difference of PDK4 gene in Guangxi Bama Xiang pig and DLY pig, the total RNA was extract from the longissimus dorsi muscle of Bama Xiang pig and DLY pig by gene cloning technology. The relative expression of PDK4 gene mRNA in different tissues of these two breeds was detected by Real-time quantitative PCR. The results showed that the CDS of PDK4 gene was 1 224 bp and the homology of PDK4 gene CDS was 100% in these two breeds. The homologies of nucleotide sequences of PDK4 gene with pig, human, mice, horse and sheep were 100%, 91.0%, 85.5%, 92.3% and 93.0% respectively. The evolution of the species in the PDK4 gene phylogenetic tree showed that the genetic distance between Bama Xiang pig and DLY pig was the closest, and the furthest was mice. The amino acid composition of PDK4 in the two breeds was higher, accounting for 10.8% of the total amino acids. It was found that PDK4 protein has strong hydrophilicity. The prediction of PDK4 secondary structure was that Bama Xiang pig and DLY pig contained αhelix, random coil and extended chain. The real-time PCR results that, in different tissues of Bama Xiang pig and DLY pig, abdominal subcutaneous fat was the highest and spleen was lowest. The longissimus dorsi tissue m RNA expression of PDK4 gene in Bama Xiang pig was significantly higher than DLY pig, but the mRNA expression of PDK4 gene in subcutaneous lipid, abdominal fat, liver, spleen and kidney of DLY pig was significantly higher than Bama Xiang pig. This study successfully cloned the PDK4 gene of Bama Xiang pig and DLY pig, and detected the relative expression of PDK4 gene in different tissues of Bama Xiang pig and DLY pig. The purpose of study provided scientific basis for further study on regulation of PDK4 gene on lipid metabolism and deposition.