Cloning,Identification And The Role Of Innate Immunity Of Grass Carp (Ctenopharyngodon Idella) STAT4

Signal transduction and Activator of Transcription(STAT)protein plays an important role in cell development and differentiation as well as Signal transduction and transcriptional regulation.STAT4,as a member of the STAT family,is also activated by JAK kinase,phosphorylated and dimerized into the nucleus to activate(ISG)interferon-induced gene expression,thus up-regulating the expression of type Ⅱinterferon,which plays an important regulatory role in inflammatory immunity.As one of the most important farmed fish in China(20% of the freshwater farmed fish),the prevention and control of fish diseases in grass carp is the top priority.As the most threatening disease to the cultivation of grass carp,hemorragic disease of grass carp is definitely one of the focuses of our research.In order to study the role of STAT4 in its inflammatory immunity,in this study,we first cloned the open reading frame(ORF)of STAT4 of grass carp,which was 2202 bp in length and encoded a 734 amino acid polypeptide chain.Then the phylogenetic tree comparison of STAT4 with other cyprinid fishes and mammals showed that it has the highest homology with faecnow minnow.Through multiple sequence alignment,we found that the STAT4 of grass carp had the highest similarity with that of zebrafish,and interestingly,the STAT1 of grass carp showed higher similarity with STAT4 of other other species.The domain of STAT4 protein was analyzed using the online tool SMART,which contains two domains,STAT-INT(N-terminal domain)and SH2,and shows high conserved.After RT-PCR detection,we found that under normal state,the expression level was the highest in the intestine and gill,and the expression level was very low in the brain.In CIK cells,Poly I:C stimulation was added according to the time gradient.RT-PCR results showed that STAT4 expression was significantly up-regulated after Poly I:C stimulation,and reached the peak at 24 hours,indicating that STAT4 had an obvious response to stimulation,suggesting that STAT4 may play a role in innate immunity.Through overexpression and interference,it was proved that STAT4 in grass carp can positively regulate IFN-Ⅰ and IFN-γ2 in grass carp,further demonstrating its role in innate immunity.The dimerization of STAT4 was proved by immunoprecipitation experiment,and in the following subcellular localization experiment,we verified that STAT4 in grass carp had nucleation phenomenon under Poly I:C stimulation,and preliminaryexplored the mechanism of its regulation of interferon in grass carp.In conclusion,the role of STAT4 in innate immunity of grass carp was preliminary explored in this study,and STAT4 was cloned and identified,which proved that STAT4 played a role in innate immunity of grass carp by upregulation of Ⅰ/ alpha-interferon,laying a foundation for further research in the future.