The Mechanism Of JAK Gene Of Scylla Paramaosain In The Innate Immune Function And Hemocyte Proliferation

JAK(Janus kinase)belongs to the family of non-receptor tyrosine kinases.In mammals,the JAK-STAT signaling pathway has been proved to be involved in multiple biological processes such as hematopoietic function,embryonic development,tissue homeostasis,innate immunity and adaptive immunity.In this study,we mainly used RNAi technology to inhibit the expression of JAK gene in S.paramamosain,to explore the immune function of JAK in S.paramamosain.Quantitative Real-time PCR(q RT-PCR)was used to detect the expression level of JAK gene in each tissue.The results showed that the expression level of JAK gene was the highest in the intestine,while the expression level was low in hemocyte and heart.Through RNAi technology,JAK gene expression was successfully knocked down.After the JAK gene was knocked down,the expression of typical innate immune molecules and signal transduction factors in S.paramamosain was detected.The results showed that the expression levels of several important immune genes were significantly down-regulated.The expression levels of hematopoietic hormone Astakine were particularly significant.After the JAK gene was knocked down,the activity of phenoloxidase(PO),superoxide dismutase(SOD)and lysozyme(LZM)decreased significantly,and the content of reactive oxygen species(ROS)increased significantly.These results indicate that JAK gene can affect part gene expression of immune signaling pathway and enzyme activity.The expression of JAK gene in S.paramamosain was significantly up-regulated after infection with Vibrio alginolyticus within 72 h.Survival analysis showed that the death rate of S.paramamosain infected with V.alginolyticus was significantly increased after JAK gene was knocked down.The results of hemocyte phagocytosis showed that after JAK gene was knocked down,the ability of hemocyte to phagocytize V.alginolyticus was significantly up-regulated,and the apoptosis rate of hemocyte was also significantly up-regulated.These results suggest that JAK gene could participate in the immune response to V.alginolyticus by regulating hemocyte phagocytosis and apoptosis.The expression level of JAK gene in S.paramamosain significantly up-regulated at 48 h after WSSV infection,and decreased gradually with the passage of time,but the expression level at 72 h was still significantly higher than that at 0 h.After the JAK gene was knocked down by the JAK-ds RNA treatment,the ability of hemocyte to phagocytize WSSV was significantly up-regulated,and the apoptosis rate of hemocyte was also significantly up-regulated,while the total hemocyte counts was significantly decreased.Survival analysis and WSSV copy number showed that the mortality and WSSV copy number of S.paramamosain were significantly up-regulated after JAK gene was knocked down.The above results indicate that interference with JAK gene expression may weaken the immunity of S.paramamosain resistance to WSSV infection,and JAK gene could participate in immune response to WSSV.In the Astakine immunovalidation study,the expression level of the hematopoietic hormone Astakine was significantly down-regulated within 72 h after the JAK gene was interfered.Through detecting the hemocyte proliferation of S.paramamosain after Astakine-ds RNA treatment.The results showed that the total number of hemocyte and the hemocyte proliferation of S.paramamosain significantly decreased,but the apoptosis rate of hemocyte increased.The above results indicated that Astakine gene was involved in proliferation and apoptosis of hemocyte.Through detecting the hemocyte proliferation related indicators of S.paramamosain after JAK-ds RNA treatment,and the results were the same as Astakine-ds RNA treatment.The above results indicate that JAK gene also plays a positive regulatory role in hemocyte proliferation of S.paramamosain.Combined with the influence of JAK gene on hematopoietic hormone Astakine,JAK gene could affect hemocyte proliferation function by regulating hematopoietic hormone Astakine.In conclusion,this study revealed the mechanism of JAK gene in the innate immunity of S.paramamosain.It was proved that JAK gene is involved in the immune response of S.paramamosain to pathogens(WSSV or V.alginolyticus)and plays an important role.In addition,it was found that JAK can affect hemocyte proliferation function by regulating the expression of hematopoiesis hormone Astakine,which provided a theoretical basis for improving the innate immune function of S.paramomosain in the future.