Study On The Function Of Eriocheir Sinensis Mannose Binding Protein (EsMBP) In The Process Of Spiroplasma Infection

Eriocheir sinensis tremor disease(TD)is a harmful aquatic disease that appeared in the 1990 s,which is named after the appearance of appendage tremor in the later stages of TD.The pathogen of TD is Spiroplasma eriocheiris,which threatens E.sinensis,Macrobrachium rosenbergii,Procambarus clarkii,Penacus vannamei and Macrobrachium nipponensis in recent years.Firstly,S.eriocheiris infects hemocytes of E.sinensis,and then flows through hemolymph to muscles and nerves.Finally,the appendages of E.sinensis shake until death.Due to the lack of adaptive immunity,E.sinensis resists the invasion of pathogens through innate immunity.It is particularly important to study the innate immune defense mechanism of E.sinensis.Based on the previous i TRAQ results,the mannose binding protein(EsMBP)of E.sinensis was identified as different expressed protein.On the basis of confirming the important immune function of EsMBP,this study main used bioinformatics analysis,recombination protein expression,double-stranded RNA interference,and screening for targeted interaction microRNAs to conduct experiments in vivo and in vitro.The research on the function of S.eriocheiris infection provides a theoretical basis for the effective control of TD.This paper mainly includes the following three results:1.The characteristics analysis and expression pattern of EsMBPIn this section,EsMBP was amplified by RACE.Sequence analysis revealed that EsMBP was 1153 bp in length and its ORF region contained 304 amino acids encoded by 915 bp.The encoded amino acid contains a signal peptide and CLECT region.The q RT-PCR results showed that EsMBP was the highest level of transcription in hemocytes.The transcription of EsMBP in hemocytes was significantly up-regulated after S.eriocheiris stimulation,indicating that EsMBP plays an important role in the infection of S.eriocheiris.Using in vitro prokaryotic expression technology,EsMBPCLECT domain were successfully expressed,purified and its polyclonal antibodies were prepared.It was found that the r EsMBP protein could be agglutinated and combined with S.eriocheiris in vitro.2.Functional study of EsMBP-CLECT recombinant protein and EsMBP ds RNA interference in the infection of S.eriocheirisAfter r EsMBP-CLECT protein was injected into E.sinensis,the PO activity in hemocytes was increased.After E.sinensis was infected with S.eriocheiris followed by r EsMBP-CLECT injection,the copy number of S.eriocheiris in hemocytes and the mortality of E.sinensis was significantly reduced.After the EsMBP gene was successfully silenced using ds RNA-EsMBP,PO activity involved in the innate immunity of E.sinensis was significantly reduced.After the EsMBP gene interference and the S.eriocheiris infection,the S.eriocheiris copy number in hemocytes and the mortality of E.sinensis was significantly increased compared with the control group.This further demonstrates that EsMBP silencing disrupts the innate immune system and reduces the host's ability to resist infection of S.eriocheiris.3.Functional and regulation between EsMBP and microRNAsTwo target RNAs(mi R-381-5p and mi R-2-3p)of the EsMBP gene were obtained by bioinformation analysis.The microRNA mi R-381-5p could successfully interacted with EsMBP gene by dual luciferase reporter gene and cell fluorescence detection.By mutating the EsMBP-3'UTR or mi R-381-5p,it was demonstrated that mi R-381-5p could target with EsMBP-3'UTR.The PO activity in hemocytes decreased significantly after EsMBP silencing with mi R-381-5p mimic.When treated with mi R-381-5p mimic and stimulated with S.eriocheiris,the hemocytes morphology was extremely poor,the hemocytes activity was decreased and the copy number of S.eriocheiris was significantly increased.On the contrary,when mi R-381-5p inhibitor was used to suppress the mi R-381-5p,the cell morphology was normal,cell activity was significantly increased,and the copy number of S.eriocheiris decreased compared to mi R-381-5p mimic group.It was found that mi R-381-5p interacts with EsMBP-3'UTR to silence EsMBP,reduced host PO activity and innate immunity,and aggravates host infection of S.eriocheiris.