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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.

The thymus is the primary tissues for production of functional T lymphocytes in vertebrate and is directly involved in defense mechanism. The grass carp ( Ctenopharyngodon idellus) is an integral part of fish culture and forms an important source of protein for human consumption in China. Histological studies showed that grass carp display thymus tissue, which is important for cellular immunity and humoral immunity and similar to mammals, becoming degenerated with fish ages. While the research from molecular level on grass carp thymus is rare. The underlying molecular mechanisms involved in the immune responses of fish thymus are poorly understood and its value as an indicator of immune competence is unknown. In order to obtain more molecular evidence for the physiological function of grass carp thymus, a cDNA library was constructed by using the SMART eDNA synthesis method with mRNAs derived from thymus of grass carp infected with grass carp hemorrhage virus (GCHV), and then EST ( expressed sequence tags) analysis was performed. SMART-prepared cDNA was full-length-enriched and well suitable for generation of cDNA libraries for various applications. Expressed sequence tags ( EST) were an efficient approach to characterize the transcripts of genes expressed in tissues exposed to a given set of environmental conditions. In this study, a total of 1933 ESTs longer than 220bp were yielded and sequenced. BLASTX analysis showed that 583 ESTs represent grass carp genes which were homologous to known genes in mammals (E-value = 30%), while the other 1350 ESTs shared low or no similarity by search of public protein databases in NCBI. Further analyses revealed that these genes, based on the functions of the homologous mammalian genes, could be classified into 6 categories, including immune-related, transcription and translation, metabolism, cell structure, signal transduction, cell proliferation, differentiation and apoptosis. Most of them encode proteins involved in biological processes, cell structure maintaining and immune response. Relatively few teleost genes involved in immune functions have been sequenced, compared to those from higher vertebrates. This study obtained a large number of immune-related genes that had not been previously reported for C. idellus, and which might represent key molecules for the understanding of the immune response to infection in fish thymus. 100 ESTs representing 59 different immune-related genes had been identified from the cDNA library. These genes encoding proteins involved in antigen processing and presentation, cell recognition, adhesion and activation, innate immune and so on. Many of these immune related genes had various immune functions. This study revealed from molecular level that grass carp thymus was the important immune tissue and might play an important role in the immune response to virus infection. In addition, this result also showed that grass carp thymus expressed many novel genes that were functionally unknown so far. Further investigations are needed to determine the function of these genes, as well as their role in the immune response. The generation of a collection of ESTs clones from grass carp thymus will provide the basis for functional genomics studies in this important organ.