EST ANALYSIS AND IMMUNE-RELATED GENE IDENTIFICATION OF GRASS CARP THYMUS IN RESPONSE TO VIRUS INFECTION

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.