The Clone Of PK,PEPCK And Their Response Under Different Levels Of Carbohydrate In Cyprinus Carpio

Glucose metabolism is including the pathway of glycolysis and gluconeogenesis.Pyruvate kinase(PK)and Phosphoenolpyruvate carboxylase kinase(PEPCK)are the key enzymes of glycolysis and gluconeogenesis.The experiment use Ukraine scale carp as experimental subject,cDNA of PK and PEPCK were analysed with the technique of rapid-amplification of cDNA ends and touch down PCR,sequences analysis,homologous alignment,building of phylogenetic tree and protein prediction were investigated with the application of DNAStar,NCBI and other softwares or databases.The Ukraine scales carps were divided into three groups,the first two groups were fed with a low-carbohydrate diet,high-carbohydrate diet at 9:00,the liver,brain,kidney,heart,intestine and muscle were removed and frozen at 15:00.The third group was fasted for two days,the same treatment was did at 9:00 on the third day.Semi-quantitative PCR was used to detect the expression of PK and PEPCK in the six organizations of the three groups,and analysis the expression of these two genes of Ukraine scale carps in each organization under different nutritional status.1.Full length cDNA of PK was 1913 bp,including 154bp3' untranslated regions with polyadenylation tail and 143 bp 5' untranslated regions.Its open reading frame(ORF)was 1617 bp and encoded a 538 amio acid(Mr 58.72kDa),the isoelectric point(PI)was 6.57.It has active site and domain boundaries.The PK of Ukraine scales carp had high conservatism,its identities were as high as 84%~92% Ukraine scales carp and other fishes,and it was 68% between Ukraine scales carp and mammals.2.Full length cDNA of PEPCK was 2567 bp,including 526 bp 3' untranslated regions with polyadenylation tail and 154 bp 5' untranslated regions.Its open reading frame(ORF)was 1911 bp and encoded a 636 amio acid(Mr 69.76kDa),the isoelectric point(PI)was 7.89.Which had the active site,GTP binding site,the metal binding site and the substrate binding site.The PEPCK of Ukraine scales carp had high conservatism,its identities were as high as 87%~96% Ukraine scales carp and other fishes,and it was 68% between Ukraine scales carp and mammals.3.Semi-quantitative PCR results showed that the fishes of fasting group,PEPCK and PK were expressed in each organization,PEPCK were highly expressed in liver and kidney,PK were highly expressed in liver,kidney and intestine;the fishes of low-carbohydrate diet group,PEPCK were highly expressed in liver,PK were expressed in each organization,which were highly expressed in liver,brain and heart;the fishes of high-carbohydrate diet group,PEPCK were weakly expressed in liver,kidney and intestine,PK were highly expressed in liver and brain,but which were weakly expressed in heart and muscle.