Functional Study On Peptide 1 Encoded By Infectious Myonecrosis Virus And Galectin As Well As Akirin In Shrimp Litopenaeus Vannamei

Shrimp is an important aquatic species with high economic value,especially in China.However,diseases caused by bacteria or virus have led to great loss of shrimp production.Hence,it is vital to study the mechanism of pathogen-host interactions and shrimp immune system in both fundamental and applicable aspects for shrimp breeding.In this study,Peptide1 of Infectious myonecrosis virus,Galectin and Akirin in shrimp Litopenaeus vannamei were investigated focusing on their roles in virus-host interactions and shrimp anti-bactiria immunity.Infectious myonecrosis is caused by Infectious myonecrosis virus(IMNV)which can cause shrimp mortality to 70% during all growth period,especially in Litopenaeus vannamei with skeletal muscle becoming apoptosis.Previous reports have predicted a small peptide 1(P1)encoded by IMNV open reading frame 1 which is a dsRNA binding domain(ds RBD).However,there is no any experimental evidence of P1 in its characteristics and its role in helping IMNV inhibiting shrimp immune system.Here,by using protein expression,gel shift assay,gel filtration analysis,RNAi reporter assay and subcellular localization we found P1 is a suppressor of RNA interference(RNAi)and it is most possible to suppress shrimp anti-viral RNAi when IMNV infecting shrimp.Galectin is an important pattern recognition receptor while Akirin is a nuclear transcription factor downstream of NF-?B pathway.Thus,through cDNA cloning,real-time quantitative PCR,protein expression,RNAi,the characterization and initial immune functions of Litopenaeus vannamei galectin and Akirin were reported.Below is the detailed data.1.P1 binds dsRNA through the C-terminal “?????” type dsRBDAs the uncertainty of start codon in P1 open reading frame,there are two possible versions,namely LP1(long P1,1-196 aa)and SP1(short P1,56-196 aa).Both LP1 and SP1 were expressed in E.coli system and the Electrophoretic mobility shift assays(EMSAs)showed that both LP1 and SP1 bind 90 bp dsRNA but seemingly form large complex and can not exit gel wells.Combining our initial coserved domain prediction results,we doubted thatboth LP1 and SP1 might form homo-oligomers through its N-terminal protein binding domain and further form large complex when binding dsRNA.Then the only C-terminal of P1(SSP1)was tested to do EMSA and found that it shifts well and exit the gel wells.The mutants of SSP1 losing the ability to bind dsRNA makes us clear about the important residues(H128?K151?K154)involved in dsRNA binding.The further titration assays of SSP1 with 90 bp dsRNA and 622 bp dsRNA showed us that 1 unit of SSP1 binds 8-9 bp dsRNA which follow the rule that a unit of classical “?????”dsRBD binds 8-10 bp dsRNA.2.LP1 or SP1 forms homo-oligomers through its N-terminal protein binding domainThe EMSAs showed us the indirect evidence that LP1 or SP1 might form homo-oligomers with its N-terminal domain.In order to make it clear,LP1,SP1 or SSP1 were used to do chemical crosslinking assay,fast protein liquid chromatography and results clearly showed that P1 forms homo-oligomers through its N-terminal protein binding domain.A further mutants experiment showed the two motifs 64-ILKLL-68 and 79-ILHLL-83 are involved in protein binding.Multi-Angle Light Scattering showed SP1 exists at least in the form of dimmers.3.P1 is an RNAi suppressorThe RNAi reporter assay in Drosophila S2 R+ cells showed that P1 inhibits host RNAi through both its C-terminal dsRNA binding domain and N-terminal protein binding domain independently.A further RNAi reporter assay using the mutants of P1 found that dsRNA binding of P1 is one way in suppressing RNAi.Besides,SSP1 but not NLP1 inhibits siRNA-induced RNAi through binding 21 nt siRNA.In order to know how NLP1 inhibits RNAi,two-step pull down was adopted using S2 cell extracts expressing NLP1 on the prediction that NLP1 might bind some components of RNAi pathway and inhibits its role during processing dsRNA.Howerver,we did not find an interesting protein like this.Then the E.coli expressed NLP1 was used to do EMSA and found that NLP1 could not binds dsRNA as well.Thus,the mechanism of NLP1 inhibiting RNAi needs further study.4.P1 locates in the nucleus and cytoplasm of S2 cellsUsing the con-focal microscopy we found LP1 locates in both nucleus and cytoplasm.In order to know how LP1 enters into nucleus,LP1 sequence was analysed by using an online SUMO-prediction Server,and found three potential SUMO motifs located in N-terminal.Then SSP1-EGFP was constructed which did not contain the three potential SUMO motifs but SSP1-EGFP still locates in both nucleus and cytoplasm which means P1 entering nucleus might through an unknown way or in a random way.An in-vitro pull down assay and co-IP with LP1 and LvUBC9 or LvcSUMO did not show that LP1 can bind any of the two proteins.5.Cloning and immune functions of shrimp Litopenaeus vannamei galectinA galectin cDNA was cloned in shrimp Litopenaeus vannamei(LvGal).Its predicted protein contains 338 amino acides and a classical carbohydrates recognition domain at its N-terminal.Besides,we found partial residues in LvGal which resemble carbohydrates binding motif.Tissue expression of LvGal using quantative real time PCR showed that it expressed in all examined tissues with higher expression in haemocytes and gills.LvGal is expressed significantly higher post Vibrio anguillarium infection.Western blot showed that the E.coli expressed LvGal binds Vibrio anguillarium and Micrococcus lysodeikticus in vitro.Besides,Lv Gal was found to agglutinate Vibrio anguillarium.However,it did not show inhibition activity against Vibrio anguillarium or Micrococcus lysodeikticus.Lastly,LvGal promotes bacteria phagocytosis in shrimp haemocytes.6.Cloning and immune functions of shrimp Litopenaeus vannamei AkirinAkirin is a highly conserved nucleus protein and has been demonstrated its role in regulating NF-?B dependent anti-microbial peptides(AMPs)transcription.Here,partial Akirin cDNA sequence was cloned in shrimp Litopenaeus vannamei(LvAkirin).The predicted protein of LvAkirin has 212 amino acides with two classical nucleus location signals at its N-terminal 24–30: PHKRRC and 78–81: KRRK.BLAST analysis showed that LvAkirin is closest to M.japonicus shrimp Akirin.Tissue expression showed LvAkirin is constitutively expressed in all examined tissues with higest level in testis and relatively high in haemocytes.Post Vibrio parahaemolyticus infection,the mRNA expression of LvAkirin increased significantly at 2,6 and 24 hours.RNAi was used to examine whether LvAkirin has some affects on the transcription of AMPs.We found that three AMPs including anti-lipopolysaccharide factorAA-K,CrustinP and Penaeidin3 a were down-expressed at some points post LvAkirin was silenced.Hence,we predicted that LvAkirin might function in regulating AMPs transcription in shrimp but it needs more fundamental data to make it clear.In total,this thesis describes how P1 encoded by Infectious myonecrosis virus suppresses host RNAi,found the role of LvGal in helping bacteria-phagocytosis of hemocytes and explored the potential role of Lv Akirin in regulating AMPs transcription.These studies will provide functional data in understanding shrimp immune system and helping shrimp breeding work.