Characterization And Functional Analysis Of Stimulator Of Interferon Gene(PsSTING) In Pelodiscus Sinensis

Chinese soft-shelled turtle(Pelodiscus sinensis)is an important freshwater economic breeding species in China.Due to the increasement of the breeding density and the deterioration of the water environment,the artificial breeding of Chinese soft-shelled turtle has caused frequent diseases,which restricted the development of Chinese soft-shelled turtle breeding industry.Among these dieases,the Chinese soft-shelled turtle diseases caused by the virus have not been found effective prevention drugs.Many studies have shown that stimulator of interferon genes(STING)is an important mediator in the body’s antiviral innate immune,which stimulates the production of type I interferon to play an immunomodulatory effect.Therefore,our study used immunological and molecular biology methods to research the characterization and function of the PsSTING in the Chinese soft-shelled turtle in order to explore its mechanism of action in the innate immune response and provide a theoretical basis for the prevention and control of the Chinese soft-shelled turtle disease.The research results are as follows:(1)The PsSTING gene was cloned from the liver tissue of Chinese soft-shelled turtle using RACE technology.Its cDNA is 2145 bp in length,including a coding region of 1152 bp which encods a protein composed of 383 amino acids without a signal peptide.Its molecular weight(Mw)is 44.16 kDa and the isoelectric point(pI)is 6.75 and it contains 4 transmembrane regions(TMs),2 endoplasmic reticulum retention motifs(R20E21R22,R82H83R84),1 helicase domain(CTD)and multiple dimerization sites(DD).The homology comparison and the phylogenetic tree showed that the STING protein of the Chinese soft-shelled turtle had a higher homology(over 70%)with the order of the turtles,followed by the homology with the same reptiles,the lowest origin(below 35%)compaed with invertebrates.(2)The qRT-PCR results showed that the PsSTING gene was widely distributed in all detected Chinese soft-shelled turtle tissues.The tissues with higher expression levels were spleen,liver,and small intestine,with the lowest expression level in blood cells.After LPS,poly(I:C)0～96 h stimulation,PsSTING mRNA appeared to be up-regulated first and then down-regulated to varying degrees,indicating that PsSTING was involved in innate immunity.(3)The western blot analysis showed that the stimulation of A.hydrophila,LPS and poly(I:C)would induce PsSTING expression to increase and reach the highest value at 48 hpi in the liver and small intestine tissue,which was consistent with the mRNA expression level,indicating that the stimulation of A.hydrophila,LPS and poly(I:C)would affect the protein expression level.The immunofluorescence results showed that PsSTING protein was expressed in liver cells,around the medullary body of the spleen,in the Goblet cells and the brush border of small intestinal epithelial cells and,and was significantly up-regulated with the induction of stimuli.(4)The RNA interference results showed that PsSTING gene-specific siRNA interference could significantly reduce its mRNA expression level in the small intestine after 72 h(p<0.05).In addition,after PsSTING gene silencing,the genes TBK1,IRF3,IRF7,STAT6,IFN-β in the IFN signaling pathway were significantly down-regulated.(5)The dual luciferase test results showed that PsSTING protein could activate the promoters of pNF-κB and pIFN-β,indicating PsSTING was involved in the signaling pathway of NF-κB and IFN-β.