Molecular Cloning, Characterization And Immune Stimii Of Three Immune-related Genes In Sinocyclocheilus Grahami

Sinocyclocheilus grahami is a precious species of fish in Dianchi Lake. Due to water pollution, habitat degradation, overfishing and invasion of exotic fish species, S. grahami has experienced a sharp population contraction. In this study, we investigated three S. grahami innate immune-related genes, providing a theoretical basis for exporing the S. grahami innate immune system and its defense.The detailed achievements are listed as follow:First of all, we cloned myeloid differentiation protein88gene, an important adapter protein in toll like receptor signaling pathways. It encodes a284amino acid residues, containing a DEATH domain and a TIR domain. S. grahami MyD88have three conserved regions and high degree of similarity with these of other fish species and mammals. Phylogenetic analysis showed that S. grahami MyD88had a close genetic relationship with Cyprinus carpio, Carassius carassius, and Danio rerio.Secondly, Hepcidin gene was cloned from cDNA library of S. grahami. The gene contains an open reading frame of261bp nucleotides, encoding86amino acid residues. The mature hepcidin contains25amino acids including8cysteine. Moreover, LEAP-2gene was obtained by transcriptome sequencing. LEAP-2contains an open reading frame of279bp, encoding92amino acids, wherein the mature LEAP-2is consist of44amino acid residues including four cysteine. Phylogenetic analysis showed hepcidin and LEAP-2belong to two antimicrobial peptide families.Moreover, the levels of MyD88, hepcidin, and LEAP-2were investigated in liver of individuals challenged with Staphyloccocus aureus and LPS and poly â… :C by quantitative real-time RT-PCR. After stimulation, MyD88and hepcidin expression levels appeared to increase regularly. Upregulation of Myd88was earlier than upregulation of hepcidin, while the upregulation of hepcidin last longer than Myd88. However, LEAP-2expression levels changed obviously and irregularly. These results suggest that the three genes are involved and played important role in the innate immune of S. grahami.