Cloning Expression And Function Analysis Of PDK4 And FGF10 Gene In Congjiang Xiang Pig

The PDK4 gene is a member of the pyruvate dehydrogenase kinase family,which plays an important role in the physiological processes such as lipid metabolism,glycolysis,tricarboxylic acid cycle and ATP formation.Some studies have shown that the PDK4 gene is an important candidate gene that affects fowl fat deposition.The FGF10 gene is a member of the fibroblast growth factor family and participates in the development and maturation of various organs.The study shows that FGF10 plays an important role in the development and metabolism of white adipose tissue.However,it is unclear whether PDK4 and FGF10 gene play a regulatory role in the differentiation of intramuscular preadipocyte from Congjiang Xiang Pig.In this study,we took Congjiang Xiang pig as the experimental subject,molecular cloning,real-time PCR,cell culture,overexpression and interference methods were used to elucidate the expression characteristics of PDK4 and FGF10 and their effects on thedifferentiation of intramuscular preadipocyte in Congjiang Xiang pig.The main results of this study are as follows:1.The results showed that the coding region of PDK4 gene was 1224 bp,encoding 407 amino acids.The coding region of FGF10 gene was 636 bp and encoded 211 amino acids.Homologous comparison was conducted with BLAST software,the homologies of nucleotide sequences of PDK4 gene with sheep,horse and human were 93%,92% and 91%.The homologies of nucleotide sequences of FGF10 gene with sheep,cattle,human and mouse were 94%,93%,93% and 90%.2.The expression levels in different tissues of PDK4 and FGF10 genes in Congjiang Xiang pig and Large White pig had been researched.It was detected by q RT-PCR method to analyze expression levels in ten different tissues such as heart,liver,spleen,lung,kidney,stomach,large intestine,small intestine,dorsal longissimus dorsi and fat.Real-time PCR results showed that: in different tissues of Congjiang pig,PDK4 gene expression in kidney tissue was higher than other tissues,with a higher expression in the stomach and adipose as well,FGF10 gene expression in stomach tissue was higher than other tissues,with a higher expression in the kidney and adipose as well,but both of PDK4 and FGF10 gene expression were the lowest in the longissimus muscle.In different tissues of Large White pig,both of PDK4 and FGF10 gene were expressed highest than other tissues,PDK4 gene expression in the longissimus muscle was the lowest,while the FGF10 gene expression was the lowest in the heart.3.The intramuscular preadipocyte from Congjiang Xiang pig had been cultured and induced.The longissimus dorsi had been collected from the 5-day-old Guizhou Congjiang Xiang pig,type ? collagenase was adopted to digest the cells,and intramuscular preadipocyte cells were separated and cultured.Intramuscular preadipocytes were induced and cultured,and the expression of PDK4 and FGF10 gene was detected by Real-time PCR.The results showed that the intramuscular preadipocyte cells of congjiang Xiang pig were successfully cultured.The expression of PDK4 and FGF10 gene was increased at the early stage of induction,and the expression of PDK4 and FGF10 gene was the highest at 48 h after induction anddifferentiation.4.The expression vectors and interference vectors of PDK4 and FGF10 gene were transfected into intramuscular preadipocytes of Congjiang Xiang pig,the expressions of PPAR?,ACC,ATGL,ADIPOQ,FABP4,FAS and LPL after overexpression of PDK4 and FGF10 genes were detected by Real-time PCR.The results showed that the expressions of ACC,FABP4,FAS,LPLand PPAR? were increased,and the expressions of ATGL ? HSL were decreased after PDK4 and FGF10 gene were up-regulated,the expressions of ACC,ATGL,ADIPOQ,FABP4,FAS,LPL,PPAR? and HSL were decreased after down-regulation of PDK4 and FGF10 genes.