Study On The Mechanism Of Astakine Gene In Innate Immune Function And Hematocyte Proliferation Of Scylla Paramamosain

Astakine is the only known molecule as hematopoietic growth factor in crustaceans,which is most abundant in half granulocytes and mainly secreted from half granulocytes.Astakine,with molecular mass between 8-14 k Da,contains a conserved domain containing 10 spacing conserved cysteine,which is homologous to the structure PROKs vertebrates.In this study,we use RNAi techniques to suppress Scylla paramamosain Astakine gene expression,and then explore the role of Astakine genes in S.paramamosain congenital immune function.With quantitative real-time PCR,the expression of Astakine genes in tissues showed the highest expression in hemocytes,but low expression in muscle,gonad,hepatopancreas and heart.After Astakine gene was suppressed,the expression of S.paramamosain typical innate immune molecules and signal transduction factors was detected.The expression of several important immune genes in S.paramamosain was significantly down-regulated,especially the JAK,Propo,and hemocyanin gene.After inhibiting Astakine gene expression,the activity of phenoloxidase(PO)decreased significantly,but the activity of superoxide dismutase(SOD)hardly changes.All results indicate that Astakine gene can affect some immune signaling pathways and enzyme activities.The expression of Astakine genes in the first 12 h of Vibrio alginolyticus infection was significantly up-regulated and then decreased over time;the number of hemocytes increased significantly,while the number of hemocytes decreased significantly when the Astakine gene was knocked down.The apoptosis rate of hemocytes was up-regulated and the phagocytosis of hemocytes decreased significantly,indicating that S.paramamosain Astakine genes may participate in S.paramamosain immune response to V.alginolyticus by regulating phagocytosis and apoptosis.Survival analysis showed that the cumulative mortality of V.alginolyticus infected S.paramamosain increased significantly after Astakine was suppressed.After WSSV infection,S.paramamosain Astakine gene expression was significantly up-regulated within 24 h,declined at 48 h,and rising again at 72 h.After Astakine-ds RNA treatment,the total number of hemocytes decreased significantly,the apoptosis rate of hemocytes was significantly upregulated.After Astakine gene was inhibited,the copy numbers of WSSV were decreased significantly.Survival analysis showed that the cumulative mortality rate of WSSV-infected S.paramamosain increased significantly after Astakine gene was suppressed.These results suggest that knockdown of Astakine gene expression may weaken the resistance of S.paramamosain to WSSV infection,and Astakine genes are involved in S.paramamosain immune response to WSSV.After the Astakine was knocked down,the total number and proliferation ability of hemocytes decreased significantly and the apoptosis rate of hemocytes increased in S.paramamosain,indicating Astakine genes are involved in S.paramamosain hemocytes proliferation function.To sum up,this study reveals the molecular mechanism of Astakine genes in S.paramamosain innate immunity,proves that Astakine genes play an important role in the immune response to pathogens(WSSV or Vibrio alginolyticus),and provides a theoretical basis for further exploration the molecular mechanism of Scylla paramamosain innate immunity.