陆川猪PDK4基因序列分析、真核表达载体构建及组织表达分析

试验旨在了解陆川猪丙酮酸脱氢激酶4（pyruvate dehydrogenase kinase 4，PDK4）基因CDS区序列信息及其所编码蛋白的结构和功能，构建PDK4基因的真核表达载体，分析PDK4基因在陆川猪不同组织中的表达情况，以期为阐明PDK4基因在陆川猪生长发育过程中的分子机制奠定基础。采用RT-PCR技术扩增陆川猪皮下脂肪PDK4基因CDS区，利用生物信息学软件预测分析其结构与功能，并利用常规分子克隆技术将其插入真核表达载体中获得pEGFP-N1-PDK4，用脂质体法将重组质粒转染3T3-L1细胞并观察荧光，用实时荧光定量PCR检测PDK4基因mRNA在陆川猪心脏、肝脏、脾脏、肺脏、肾脏、背最长肌、皮下脂肪中的表达情况。结果显示，陆川猪PDK4基因CDS区全长1 224 bp，编码407个氨基酸，与NCBI上公布的野猪PDK4基因CDS区同源性达99.8%。对陆川猪PDK4基因所编码的蛋白进行生物信息学分析发现，其分子质量约为46.144 ku，原子总数为6 509个，理论等电点（pI）为7.21，带正电荷和负电荷的氨基酸数均为42个。PDK4蛋白可能有2个N-糖基化位点、33个磷酸化位点。亚细胞定位结果发现，PDK4蛋白有34.8%存在于线粒体，30.4%存在于细胞质，26.1%存在于细胞核，质膜和液泡膜各占4.3%。细胞试验发现，对照组和试验组均发出荧光，相较于对照组，试验组中PDK4表达量极显著升高（P<0.01），PDK4基因在皮下脂肪中表达丰度最高，随之为肝脏、肺脏、心脏、脾脏和肾脏，在背最长肌中表达量最低，而且在皮下脂肪中的表达量极显著高于背最长肌（P<0.01）。本试验成功扩增出PDK4基因CDS区并构建了真核表达载体，成功对其结构和功能进行预测分析，为研究陆川猪皮下脂肪沉积的遗传改良提供了参考依据。

Sequence Analysis,Eukaryotic Expression Vector Construction and Tissue Expression Analysis of PDK4 Gene in Luchuan Pigs

This study was aimed to understand the sequence information of pyruvate dehydrogenase kinase 4 (PDK4) gene CDS region and the structure and function of the encoded protein in Luchuan pigs,construct a eukaryotic expression vector of PDK4 gene,and analyze the expression of PDK4 gene in different tissues,which laid a foundation for elucidating the molecular mechanism of PDK4 gene in the growth and development of Luchuan pigs.The CDS region of PDK4 gene in subcutaneous fat of Luchuan pigs was amplified by RT-PCR technology,the structure and function of PDK4 gene were predicted and analyzed using bioinformatics software.It was inserted into a eukaryotic expression vector to obtain pEGFP-N1-PDK4,the recombinant plasmid was transfected into 3T3-L1 cells by the liposome method,and then the fluorescence was observed.The expression of PDK4 gene mRNA in heart,liver,spleen,lung,kidney,longissimus dorsi muscle and subcutaneous fat in Luchuan pigs were detected by Real-time quantitative PCR.The results showed that the CDS region of PDK4 gene in Luchuan pigs was 1 224 bp in length,encoding 407 amino acids,and had 99.8% homology with the CDS region of PDK4 gene in Sus scrafa published on NCBI.Bioinformatics analysis of the protein encoded by PDK4 gene in Luchuan pigs revealed that its molecular mass was 46.144 ku,the total number of atoms was 6 509,the theoretical isoelectric point (pI) was 7.21,and the number of positively and negatively charged amino acids was 42,respectively.There were 2 N-glycosylation sites and 33 phosphorylation sites of PDK4 protein.Subcellular localization results showed that 34.8% of PDK4 protein was present in mitochondria,30.4% was present in cytoplasm,26.1% was present in nucleus,and plasma membrane and vacuole membrane each accounted for 4.3%.Compared with control group,the expression of PDK4 gene in experimental group extremely significantly increased (P<0.01).The expression of PDK4 gene was most abundantly expressed in subcutaneous fat,followed by liver,lung,heart,spleen and kidney,it had the lowest expression in longissimus dorsi muscle,and the expression of subcutaneous fat was extremely significantly higher than that of longissimus dorsi muscle (P<0.01).This experiment successfully amplified the CDS region of PDK4 gene and constructed a eukaryotic expression vector containing it,and successfully predicted its structure and function,providing a theoretical basis for studying the genetic improvement of subcutaneous fat deposition in Luchuan pigs.