The Mechanism Research Of Marsupenaeus Japonicus Crustin-like Gene In Vibrio Alginolyticus And White Spot Syndrome Virus Infection

Crustin is one kind of antimicrobial peptides(AMPs)which play a key role in innate immunity of crustaceans.In this study,we aimed to determine the function of crustin-like in kuruma shrimp,Marsupenaeus japonicus white spot syndrome virus(WSSV)or Vibrio alginolyticus infection.The entire 660 bp crustin-like gene sequence with a 507 bp open reading frame(ORF)encoding a168-aa-long protein was cloned from M.japonicus.The 5? and 3? untranslated regions(UTRs)comprised 72 bp and 81 bp,respectively.The clone also included a poly(A)tail.The crustin-like peptide had an estimated molecular mass of 16.22 kDa and a theoretical pI of 8.37.Sequence homology and phylogenetic analysis showed that this gene is mainly exists in shrimps and some of crabs.Among the known species,M.japonicus crustin-like peptide showed the closest relationship with that of L.vannamei.The real-time quantitative PCR(RT-qPCR)results showed that crustin-like gene was primarily expressed in gills,and low expression in hemocytes,hepatopancreas,muscles and digestive gland.By the double-strand RNA(dsRNA)interference technology,we constructed the dsRNA prokaryotic expression vector in vitro and the expression of crustin-like gene was successfully inhibited by the addition of dsRNA.The results of important shrimp immune genes detection found that a number of known immune-related genes in shrimp innate immune system were affected when crustin-like gene was silenced,and partial immune pathway signal transduction related genes such as immune deficiency homolog(IMD),Rab7,L-lectin,mitogen-activated protein kinase(MAPK),P53,prophenoloxidase(pro PO)and Rho were down-regulated significantly while nitric oxide synthase(NOS),myosin and tumor necrosis factor-?(TNF-?)were up-regulated significantly in the hemocytes at 24 h after CRU-dsRNA treatment.Shrimp immunocompetence experiments showed that the total hemocytes counts(THC)decreased,and phenoloxidase(PO)and superoxide dismutase(SOD)activity reduced after crustin-like gene knocked down.These results suggested that crustin-like may play an important role in phagocytosis,apoptosis and some major immune factors.V.alginolyticus challenged experiment showed that crustin-like was over-expressed in shrimp hemocytes after challenged with V.alginolyticus.The results of shrimp mortality experiment showed that cumulative mortality of shrimp was significantly increased after crustin-like gene silenced.It suggested that shrimp crustin-like has an obviously antibacterial effect.The phagocytosis experimentshowed there was a higher phagocytosis rate of hemocytes after CRU-dsRNA treatment.The result indicated that V.alginolyticus may be able to use crustin-like to avoid phagocytosis of shrimp hemocytes.The RT-qPCR results showed that crustin-like was over-expressed in shrimp haemocytes after challenged with WSSV.WSSV copies expriment and Kaplan-Meier survival analysis showed that after crustin-like gene knocked out,WSSV copies were significantly reduced,and shrimp survival rate was significantly increased.When silenced the crustin-like,the results of phagocytosis showed that the phagocytic rate of shrimp hemocytes on WSSV decreased significantly,and the apoptotic result showed that the apoptosis of shrimp hemocytes increased obviously.These findings confirmed that crustin-like can suppress shrimp hemocytes apoptosis,while WSSV can positively take advantage of crustin-like to benefit its own replication in shrimps.This is the first time that reveals the key role of crustin-like in shrimp virus infection.Moreover,the research provides an important theoretical basis for studying the mechanism of innate immunity of shrimp and WSSV infection.