The thymus is the primary tissues for production of functional T lymphocytes in vertebrate. Histological studies showed that grass carp (Ctenopharyngodon idellus) display thymus tissue. In order to obtain much more molecular evidence for the physiological function of grass carp thymus, a SMART cDNA library was constructed with mRNAs derived from thymus of grass carp infected with grass carp hemorrhage virus (GCHV), and then EST (expressed sequence tags) analysis was performed. A total of 1933 ESTs longer than 220bp were yielded and sequenced. BLASTX analysis showed that 583 ESTs represent grass carp genes which are homologous to known genes in mammals (E-value≤1.00E 10-3，Identities≥30%), while the other 1350 ESTs share 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. 100 ESTs representing 59 different immune-related genes have 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 the immune related genes have various immune functions. This study reveals from molecular level that thymus is the important immune tissue grass carp and might play an important role in the immune response to virus infection. In addition, this result also showed that grass carp thymus express many novel genes that are functionally unknown so far.
Chemokines are a superfamily of chemotactic cytokines that coordinate the migration of leukocytes and play crucial role in host defenses and the development of immune system. Two full-length cDNA encoding a CC and a CXC chemokine were identified from the thymus cDNA library. The CC chemokine cDNA consists of 883 bp in full length and encodes a putative 101-amino-acid protein with a predicted signal peptide of 29 aa. The deduced protein has four cysteines that are also found in mammalian and fish CC chemokines in the same positions. The grass carp CC chemokine genocmic gene is composed of four exons and three introns. Homology and phylogenetic analysis revealed that the grass carp CC chemokine is most similar to human CCL25. Constitutive expression was observed in the 11 tissues of the healthy fish. When induced by poly I:C, the mRNA transcripts were upregulated in liver, gill and head kidney. The CXC chemokine cDNA is 618 bp encoding a putative 117-amino-acid protein. The deduced protein has a predicted 25 aa signal peptide at the N terminus and owns all the four cysteines in conserved positions as found in other CXC chemokines. The grass carp CXC chemokine genomic gene is composed of four exons and three introns. Homology and phylogenetic analysis revealed that the grass carp CXC chemokine is different from the known fish CXC chemokines. Constitutive expression was not detected in heart, brain and musle of healthy fish. Upon poly I:C induction, the chemokine transcripts were upregulated in skin, liver, spleen and head kidney. These results collectively suggested that both chemokines might be in relation to homeostatic functions and play an important role in response to dsRNA stimulation.