The Investigation Of The Role Of Tyrosine Phosphorylated Protein And Cytoplasmic Dynein In White Spot Syndrome Virus Infection

In the present work, the dynamic of tyrosine phosphorylation level in hemocytes ofshrimp (Fenneropenaeus chinensis) was examined by phospho-specific flowcytometry (FCM) during WSSV infection, and tyrosine phosphorylated proteins inhemocytes were identified by western blotting and mass spectrometry(Matrix-assisted laser desorption/ionization tandem mass spectrometry:MALDI-TOF-MS/MS), and the gene expression levels of three identified proteinswere further detected by quantitative real-time RT-PCR (qRT-PCR).In order to investigate whether cytoplasmic dynein is involved in WSSV infection,rapid amplification of cDNA ends (RACE) was applied to clone the intermediatechain cDNA sequence of cytoplasmic dynein of Fenneropenaeus chinensis(FcDYNCI). The expression profile of FcDYNCI in hemocyte was investigatedduring WSSV infection by quantitative real-time RT-PCR (qRT-PCR).Immunofluorescence confocal microscopy was used to examine the localizations ofFcDYNCI and WSSV in hemocytes of the WSSV-infected shrimp. RNA interference(RNAi) was also applied to knock down the FcDYNCI gene and its effect on WSSVgenes expression in hemocyte was investigated. The resultant data would facilitatebetter understanding of WSSV transport in host cells. The dissertation included thefollowing six parts:(1) The change in the level of protein tyrosine phosphorylation in hemocytes ofshrimp (Fenneropenaeus chinensis) was detected by phospho-specific flow cytometryduring white spot syndrome virus (WSSV) infection. Post WSSV infection, thehemocytes could be classified into two subsets (R2and R3) with up-ordown-regulated tyrosine phosphorylation level, whereas the geometric mean level oftyrosine phosphorylation in total hemocytes was significantly increased and exhibited two peaks at1and12hour post infection.(2) In present work, the change of protein tyrosine phosphorylation in shrimphemocytes was investigated by FCM, western blotting and confocal microscopyduring WSSV infection. All the results showed that the level of protein tyrosinephosphorylation was significantly up-regulated with a similar pattern of fluctuation.Confocal microscopic observation revealed that the tyrosine phosphorylated proteinswere localized in nucleus as well as cytoplasm, and five tyrosine phosphorylatedproteins were detected in hemocytes by western blotting usingphosphotyrosine-specific antibody, which were identified to be histone H2A, histoneH3, histone H4, alpha-2-macroglobulin (A2M) and heat shock protein70(HSP70) bymass spectrometry. Moreover, the expression levels of h2a, h3and h4were examinedto be significantly up-regulated post WSSV infection by quantitative real-timeRT-PCR.(3) The full-length cDNA of cytoplasmic dynein intermediate chain (FcDYNCI) wasfirst cloned from the hemocyte of Fenneropenaeus chinensis, which consists of2582bp and encodes a polypeptide of660amino acids (Aa). The predicted proteinmolecular mass was73.40kDa, and the estimated isoelectric point was5.16. Multiplesequence alignment showed that FcDYNCI shared high identities with DYNC1I2from four species in Insecta. These suggested that FcDYNCI was a member ofcytoplasmic dynein1family and might be clustered into isoform2.(4) The FcDYNCI mRNA was most highly expressed in hemocytes, which wassignificantly up-regulated post WSSV infection. At12h post infection (hpi), confocalmicroscopic observation showed that WSSV virions could be co-localized with thecytoplasmic dynein in hemocytes.(5) After silencing by specific FcDYNCI dsRNA, the FcDYNCI mRNA level and theprotein amount of FcDYNCI in hemocytes both exhibited a significant reduction, andthe expression levels of three WSSV genes ie1, wsv477and vp28all exhibited thegreatest decreases at24hpi with approximate1/2700,1/3000and1/150of the controllevel respectively.(6) The prediction of STRING revealed that FcDYNCI had a probability of80%to interact with Rab7. In order to investigate the interaction between these two proteins,the rabbit anti-Rab7antibodies were used to precipitate Rab7and its reactingproteins through co-immunoprecipitation. The results of SDS-PAGE showed that aprotein with a molecular weight of74kDa existed in the sediments. However theresults of MS indicated that this protein can not match with FcDYNCI.