MOLECULAR CLONING AND EXPRESSION ANALYSIS OF FRUCTOSE-1,6-BISPHOSPHATASE IN GIBEL CARP

Abstract

Fructose-1,6-bisphosphatase is one of the key rate-limiting enzymes in gluconeogenesis,which plays important roles in carbohydrate metabolism.Mammals have two isoforms,liver and muscle fructose-1,6-bisphosphatase encoded by Fbp1 and Fbp2 respectively.Gibel carp is widely cultured as an economic fish in China.However,the fructose-1,6-bisphosphatase gene has not been elucidated in teleosts,especially its tissue distribution in adult fish and spatiotemporal expression in embryogenesis.In this study,we cloned the full-length cDNA of gibel carp Fbp1 by RACE polymerase chain reaction from the gastrula embryo SMART cDNA library,and we also examined its expression pattern in the tissues of adult fish and the developmental process of embryos in this fish by gene specific primers.The full length sequence of CagFbp1 consists of 1170 base pairs which encodes 337 amino acids.Multiple alignment and phylogenetic analysis showed that the cloned gene was liver Fbp of gibel carp.The tissue expression pattern analysis by RT-PCR with specific primers showed that CagFbp1 was expressed in the liver,brain,heart,spleen,kidney,intestine,muscle and ovary,and the expression in the liver was obviously higher than others.At the same times,there were two protein bands in liver by western blot analysis,one was common in the detected tissue except muscle,and the other was specific for liver.However,only one band emerged in the muscle,which was specific for muscle tissues.Mature eggs and ontogenic analysis by RT-PCR and western blotting with specific primers showed that both the transcripts and proteins of CagFbp1 were maternal.The transcripts were increased from gastrula and reached a higher level in neurula till hatching.Interestingly,there was a new band with smaller molecular weight other than the maternal proteins after tail bud stage,which was similar to the liver specific band.These results indicated that the fructose-1,6-bisphosphatase gene cloned in gibel carp was liver isoform,and there might be at least two isoenzymes,the liver fructose-1,6-bisphosphatase and muscle one in teleost.