Cloning And Tissue Distribution Of Carbohydrate Metabolic Key Enzymes In Silurus Meridionalis

The most important pathways are glycolysis and gluconeogenesis of hepaticglucose metabolism in fish. Glucokinases,6-phosphofructo-1-kinase and pyruvatekinase are key enzymes in the glycolysis. Phosphoenolpyruvate carboxykinase,fructose-1,6-bisphosphatase and glucose-6-phosphatase are key enzymes in thegluconeogenesis. Glucokinase is the first key enzyme in the glycolysis.Phosphoenolpyruvate carboxykinase is the first key enzyme of the gluconeogenesis.This paper selects Silurus meridionalis as experimental subject, and clone GK cDNAfragment and PEPCK cDNA fragment by regular PCR. In order to get informationabout GK and PEPCK expression in Silurus meiridionalis, tissues distribution wasdone by real-time fluorescent quantification PCR. This paper has two sections:1. GK cDNA fragment and PEPCK cDNA fragment clone of Silurusmeridionalis and sequence analysis.The cDNA of Silurus meridionalis GK was amplified by RT-PCR using primersdesigned based on conserved region of multiple alignment of the published sequencesof GK genes in Genbank. The amplified fragment was sequenced after purifying anT-A clone. The same to the cDNA of Silurus meridionalis PEPCK. The glucokinasegene fragment was successfully cloned whose length was688bp from thehepatopancreas of Silurus meridionalis. The result of BLAST showed the targetfragment had83%nucleotide identity with the corresponding segment of Cyprinuscarpio. The deduce amino acids sequence encoded216aa. The phosphoenolpyruvatecarboxykinas gene fragment was successfully cloned whose length was705bp fromthe hepatopancreas of Silurus meridionalis. The result of BLAST showed the targetfragment had84%nucleotide identity with the corresponding segment of Cyprinus carpio. The deduce amino acids sequence encoded230aa.2. Tissue distribution of GK mRNA and PEPCK mRNA in Silurus meridionalisGK mRNA tissue distribution was done in6tissues of Silurus meridionalis byreal-time fluorescent quantification PCR(SYBR Green I fluorescent dye), usingβ-actin primer amplification of Nile tilapia that determine it linear growth for actin.Experimental result has showed GK mRNA was mainly expressed in the liverand in the brain,although it was also found in the pancreas,the muscle. Expression ofGK mRNA in the brain was0.464times as much as in the liver, whereas it was0.177times and0.064times in the pancreas and in the muscle than in the liver. It showedthat GK occurred in minute amounts in the pancreas and in the muscle, but it was notexpression in the heart and in the kidney. It was the situation with the results ofprevious of research, and it was further evidence of the result that GK was mainly inthe liver and in the brain. In a word, the liver is the main place of GK of fish.The research measured the expression of PECPK mRNA from6tissues(liver,pancreas, kidney, heart, muscle, brain). The experimental result showing theexpression of PEPCK mRNA of Silurus meridionalis was mainly in the liver.Expression of PEPCK mRNA in the kidney was0.332times than in the liver. A smallnumber of expression was found in the pancreas, however little expression was foundin the heart, the muscle, the brain. Like the previous studies of mammal and the otherfishes, this study showed the expression of PEPCK of Silurus meridionalis is mainlyin the liver.