Molecular Cloning And Evolution Analysis Of MIF And SOCS Gene Family In Teleost Fish

In this study, we initially discovered and cloned the macrophage migration inhibitory factor (MIF) and SOCS gene family including 55 genes assigned to 12 members in teleostei aided by developed bioinformatic methods. Moreover, the molecular evolution and phylogenetics, tissue distribution, expression and preliminary function of them were further studied.In part one, we develop some bioinformatic methods of identifying immune-related genes in fish, which include the usage of databases, prediction of ORF and UTR in genomic sequences and correction of mistakes found in genomic DNAwith examples. Sofwwares including GeneMaper, GetBLAT and TransSeq were devised to facilitate the gene prediction process. All of these lay solid foundation for the subsequent molecular cloning, biological function and evolution analysis of genes.In part two, we performed databases searches and identified MIF gene from various organisms including Fundulus heteroclitus, Ictalurus punctatu, Haplochromis chilotes, Tetraodon nigroviridis, Salmo sala, Oncorhynchus mykiss, Danio rerio and Takifugu rubripes. Then, the molecular cloning, southern blotting and evolution analysis of Tetraodon MIF have been done. And recombinant TnMIF protein and rabbit anti-MIF serum were obtained. The expression and function of MIF was studied by RT-PCR, Q-PCR , western blotting and immuno-histochemical analysis.In the third part of this dissertation, we initially identified the 55 SOCS-like genes from Tetraodon nigroviridis, Gasterosteus aculeatus, Takifugu rubripes, Danio rerio and Oryzias latipes, among which SOCS3b, SOCS5b, SOCS8 and SOCS9 are novel fish-specific SOCS members. Based on newly-discovered genes, the molecular origin and evolution analysis of SOCS family in vertebrates were performed. Phylogenetic analyses indicated that the SOCS gene family can be divided into two subfamilies: (i) Type I SOCS, including vertebrate SOCS4-7,SOCS5b,SOCS9 and invertebrate SOCS members; (ii) Type II SOCS, including CISH, SOCS1-3, SOCS3b and SOCS9. We present a model for the evolution history of SOCS multi-gene family in vertebrates, considering that the family was developed through at least cubical gene duplication events during vertebrate evolution process.