Cloning And Expression Analysis Of SIMP And TRAFs In The Grass Carp Ctenopharyngoden Idellus

The grass carp (Ctenopharyngoden idellus) is an important species in aquaculture industry in China. The culture of grass carp has been threatened seriously by many kinds of diseases. It is thus important and necessary to develop some effective methods for the control of diseases. The present study aimed to clone some immune genes for the grass carp, with their expression analysis. The three immune genes obtained are concerned with glycosylation modification, tumor immune and cell apoptosis.The SIMP cDNA full sequence of grass carp has been cloned using RACE-PCR and PCR. The genomic structure including the promoter region has been obtained by PCR and Genome Walking method. In addition, two apoptosis related genes were cloned for the first time in the grass carp, i.e. TNFR-associated factor 1 (gcTRAF1) and TNFR-associated factor 2 (gcTRAF2).The gcSIMP full length cDNA is 4384 bp, including a 1117 bp 5′UTR (untranslated region), a 2418 bp open reading frame, and a 849 bp 3′UTR. The putative protein of gcSIMP is 805 aa. The gcSIMP spans over more than 24,212 bp in length, containing 16 exons and 15 introns. The gcSIMP promoter contains many putative transcription factor binding sites, such as Oct-1, GCN4, YY1, Sp1, Palpha, TBP, GATA-1, C / EBPbeta, and five C/ EBPalp binding sites. The gcTRAF1 full length cDNA is 2235 bp, including a 250 bp 5′UTR (untranslated region), a 1659 bp open reading frame, and a 326 bp 3′UTR. The putative protein of gcTRAF1 is 552 aa. The gcTRAF2 full length cDNA is 3162 bp, including a 60 bp 5′UTR (untranslated region), a 1611 bp open reading frame, and a 1491 bp 3′UTR. The putative protein of gcTRAF2 is 536 aa.The organ distribution of the three genes was analyzed both in the transcription and protein levels. By real time PCR analysis, the transcription products of the cloned genes were detected in different tissues of healthy grass carp, and the three genes had different patterns of tissue distribution. Western blotting analyses revealed that both gcTRAF1 and gcTRAF2 protein existed broadly in examined tissues with the highest expression level in heart and lowest in trunk kidney.