Studies On The Functions Of VEGF Signaling Pathway In Shrimp During WSSV Infection

VEGF signaling pathway plays important roles in inducing endothelial cell proliferation,migration,angiogenesis,vascular permeability and inhibition of apoptosis in mammals.In the previous study,we found that the transcription levels of the key genes in VEGF signaling pathway increased significantly after WSSV infection to shrimp.However,the role of VEGF signaling in shrimp during WSSV infection was not clear.In the present study,we systematically studied the roles of VEGF signaling pathway in shrimp during WSSV infection.The data will not only enrich the theoretical knowledge of shrimp innate immunitye,but also provid theoretical guidance on the prevention strategy to WSSV disease.The major progresses achived in the thesis are as follows:1.The full length cDNAs of five VEGF genes,named LvVEGF1-LvVEGF5 were isolated and identified from L.vannamei.Sequences analysis showed that their deduced amino acid sequences contained a typical PDGF/VEGF domain and a cysteine knot motif(CXCXC).They showed different tissue expression profiles.LvVEGF1 was predominantly expressed in lymphoid organ(Oka).LvVEGF2 was mainly detected in gill and hemocytes.LvVEGF3 was mainly expressed in hemocytes.LvVEGF4 was mainly detected in hemocytes and gill while LvVEGF5 was mainly expressed in hemocytes and intestines.The transcriptional levels of different VEGF genes showed apparent up-regulation during WSSV infection.Shrimp with VEGF genes silenced by double-stranded RNA injection showed a lower cumulative mortality rate after WSSV infection compared with control group without RNA interference.These results indicated that VEGFs genes played an important role during WSSV infection to shrimp.2.Two VEGFRs genes,LvVEGFR1 and LvVEGFR2,were isolated from L.vannamei.Their deduced amino acid sequences contained a typical IG-like domains and PK domains for VEGFR family.Differential expression profile in shrimp tissues were found for them.LvVEGFR1 was mainly expressed in hemocytes and intestine while LvVEGFR2 is mainly expressed in hemocytes and Oka.In addition,the transcriptional levels of LvVEGFR1 and LvVEGFR2 could be obviously up-regulated after WSSV infection.Silencing of LvVEGFR1 and LvVEGFR2 by dsRNA interference could reduce the mortality of shrimp during WSSV infection.These data showed that VEGFRs played important roles during WSSV infection to shrimp.3.In order to understand the interaction between different LvVEGFs and LvVEGFRs in shrimp,we detected the interactions between VEGFs and VEGFRs at RNA and protein level.The transcriptional expression of LvVEGFR1 was apparently down-regulated when LvVEGF1,LvVEGF3 or LvVEGF5 was silenced and the expression of LvVEGFR2 was apparently down-regulated after LvVEGF2,LvVEGF3 or LvVEGF5 was silenced.The expression of LvVEGFR1 was apparently up-regulated after injection of recombinant LvVEGF3 protein.And the expression of LvVEGFR2 was apparently up-regulated after injection of recombinant LvVEGF1,LvVEGF2 or LvVEGF3 protein.Yeast two-hybrid experiments further confirmed tha tall five LvVEGFs could interact with LvVEGFR1,while only LvVEGF2 and LvVEGF3 could interact with LvVEGFR2.These results suggested that VEGFs participated in WSSV infection via interacting with different VEGFRs in shrimp.4.In order to know the roles of VEGFs or VEGFRs in regulating the expressions of the genes in VEGF signaling pathway,the expressions of the downstream genes in VEGF signaling pathway were analyzed after VEGFs or VEGFRs were silenced.The results showed that silencing of VEGFs and VEGFRs genes in L.vannamei resulted in the decrease of the expression of LvFAK and LvPI3 K.Based on above data,we suggested the outlines of VEGF signaling pathway functions in WSSV infection to shrimp.