Study On The Function Of Vesicle-associated Membrane Protein And Calmodulin Kinase Ⅱ Of Eriocheir Sinensis In Spiroplasma Infection

Eriocheir sinensis(E.sinensis),an aquatic organism,is a delicacy loved by many people,which produces tremendous economic benefits every year.E.sinensis farming is the pillar industry of China’s freshwater aquaculture and has contributed to a huge economy value.However,following the advancement of aquaculture science and technology,intensive aquaculture has become a common method of river crab farming.With the rapid development,the occurrence of diseases caused by various bacteria,viruses,and parasites has restricted the river crab farming Progress,causing huge economic losses to this industry.Spiroplasma eriocheiris(S.eriocheiris)is an important pathogen discovered in recent years that can infect aquatic crustaceans such as E.sinensis,Procambarus clarkii,Penaeus vannamei,Macrobrachium rosenbergii and Macrobrachium nipponense.S.eriocheiris enters E.sinensis through the gills and surface of body,and then invades the hemocyte and multiplies in it.Then through hemocyte circulation to the whole body,S.eriocheiris infects muscles and nerves and other tissues to cause the crab appendages tremor and lead to the death.Because it caused the host to produce typical tremor symptoms,this disease was called the ‘tremor disease’ of E.sinensis.Due to the speciality and pathogenicity of spiroplasma,it is very important to study the infection mechanism of S.eriocheiris.The results of neurophosphorylation proteomics of E.sinensis after spiroplasma stimulation in Dr.Hou Libo’s graduation thesis showed that vesicle-associated membrane protein(EsVAMP)and calcium/calmodulin-dependent protein kinase II(EsCaMKⅡ)have significantly changed.It is suggested that these two proteins involved in the physiological process to resist spiroplasma infection in E.sinensis.This study mainly aims at EsVAMP and EsCaMKⅡ to carry out its function in the process of the spiroplasma infection.Bioinformatics analysis,double-stranded RNA interference technology,eukaryotic expression and other technologies were used in the research.This paper mainly includes the following three results:1.Cloning,bioinformatics analysis and organization distributionof EsVAMP and ESCAMKⅡRACE technology is used to clone EsVAMP and EsCaMKⅡ sequence.The results show that the length of VAMP is 1681 bp,which contains 395 bp open reading frame(ORF),90 bp 5’-non-coding region(UTR)and 1277 bp 3’-UTR.The similarity of EsVAMP protein sequences with those of L.vannamei,Nasonia vitripennis,Orussusabietinus and Bactrocera dorsalis were 96.67%,91.67%,89.69% and 88.00%,respectively.QPCR showed that EsVAMP was expressed highly in hemocyte and nerves,followed by gills,intestines and hepatopancreas,and lowly in heart and muscles.The total length of EsCaMK is 3314 bp,including 1605 bp ORF,90 bp 5’-UTR and 1619 bp 3’-UTR.The gene contains a 774 bp serine and threonine protein kinase catalytic domain.Between Es CAMKⅡ protein sequence and Periplaneta Americana,Cryptotermes secundus,Zootermopsis nevadensis and Photinus pyralis CAMK Ⅱ protein sequence similarity were 83.43%,83.58%,82.87% and 84.69%,respectively.q PCR showed that the expression of EsCaMKⅡ is higher in hemocyte,nerve,gill and muscle,and is very low in the hepatopancreas,gut and heart.2.The function of EsVAMP involved in S.eriocheiris infectionThe interference experiment was carried out by synthesizing EsVAMP dsRNA and injected into E.sinensis.Through the detection of interference efficiency,it was verified that ds RNA successfully suppressed the expression of EsVAMP.The S.eriocheiris copy number and the mortality of crab increased significantly after EsVAMP RNAi.Meanwhile,EsVAMP were connected for eukaryotic expression.The green fluorescence and western blot were used to determine whether the transfection was successful into macrophages.Afterwards,the cells were stimulated with S.eriocheiris,the cell morphology and cell viability of the transfection group were significantly better than the control group.In the transfection group,the number of S.eriocheiris copies was significantly lower than that in the control group.The above experimental results indicate that EsVAMP plays an important role in the process of S.eriocheiris infection in E.sinensis.3.The function of EsCaMKⅡ of E.sinensis involved with S.eriocheirisThe interference experiment was carried out by synthesizing EsCaMKⅡds RNA and injected it into E.sinensis.Through the detection of interference efficiency,it was verified that ds RNA successfully inhibited the expression of EsCaMKⅡ in E.sinensis.The S.eriocheiris copy number and the mortality rate of crab increased significantly.The serine and threonine protein kinase catalytic domain is the main structural domain of EsCaMKⅡ.This functional domain is connected to GFP vector for eukaryotic expression.The green fluorescence and western blot were used to determine whether the transfection was successful into macrophages.After spiroplasma infection,the cell morphology and cell viability of the transfection group were significantly better than the control group.In the transfection group,the number of spiroplasma copies was significantly lower than that in the control group.The above experimental results indicate that EsCaMKⅡ plays an important role in the process of S.eriocheiris infection in E.sinensis.