Cloning And Activity Analysis Of Pig Pyruvate Dehydrogenase Kinase 4 (Pdk4) Gene Promoter

Pyruvate dehydrogenase kinase 4 (PDK4), is one isoform of PDK isoenzymes, which belongs to the family of adenosine triphosphatase(ATPase). PDK4 gene leads to complete inactivation to enzymatic activity by phosphorylation to the El a subunit of Pyruvate dehydrogenase complex (PDC) and thus contributing to the regulation of glucolysis, TCA cycle and ATP genesis.Previously researches have showed that porcine PDK4 gene had significant difference expressing in Meishan and LargeWhite. The polymorphism in PDK4 had significant orhighly dominant significant effect on rate of lean to fat (RLF), drip loss rate, water holding capacity, color value of longissimus dorsal muscle (CVLD), muscle fatcontent and muscle water content (MWC).To discover it, we set up a sire experiment to analysis the gene’s promoter. It’s promoter region was firstly analyzed, and then it’s core sequences and major function region of the promoter was determined. The 5’flanking region and different fixed 3’terminal fragments of Pig PDK4 were cloned and recombined into pGL3-basic plasmids. After DNA sequencing confirmation, the recombined vectors were transfected into mouse embryonic fibroblasts cells 3T3 (MEF-3T3) and PK15 (pig kidney cells), respectively, and then the promoter activities of different fragments of 5’flanking region of PDK4 gene were determined by qualitative assays including quantitative RT-PCR and luciferase reporter gene analysis assay. The main results are as follows:1, In order to investigate the tissue-specific distribution of PDK4 gene, real-time PCR were performed to measure gene expression. Expression of PDK4 gene exists in every tissue of the newborn RongChang pig and the expression of PDK4 were significantly different in Each organization (P< 0.01). The expression level of newborn porcine PDK4 was significantly highest in small intestine than other tissues (P<0.01). The lowest expression level was detected in heart and brain.2, The 5’deletion fragments of Pig PDK4 were cloned and recombined into pGL3-basic plasmids. The recombined vectors were pGL3-122/+224 (346bp), pGL3-325/+224 (2092bp), pGL3-504/+224 (728bp), pGL3-985/+224 (1209bp), pGL3-1868/+224 (2092bp) and pGL3-2680/+224 (2904bp).3, By comparative deletion analysis of the Sus PDK4 promoter in PK15 and MEF-3T3 cells,we found that the progressive 5’deletion series and their luciferase activities in PK15 were significantly greater than in MEF-3T3. So, PK15 is the specific expressed cell for PDK4 gene.4, By the analysis of the transfection results and prediction of TESS software, the core sequences of PDK4 promoter was determined and the possible transcription factor binding sites was predicted.The studies showed that the long DNA fragment, sequence from +224 to -122, and sequence up to -325 of 5’flanking region, had the stronger, basal, or maximal promoter activities, respectively. This may be relative to the CCAAT/Enhancer-binding Proteinβ.Further studies showed that there were positive (-325/-122), (-2680/-1868) and negative (-1860/-325) regulatory domains, respectively. This may be to the stimulatory protein 1, myocyte enhancer factor-2 and myogenic determining factor respectively. The recombinant reporter plasmids containing different deduced fragments of PDK4 promoter were successfully constructed, and its core and major regulatory regions were found.