| The Chinese mitten crab,Eriocheir sinensis,is an economically important freshwater species for aquaculture in China.The aquaculture of E.sinensis is a rapidly developing industry in Jiangsu province of China,currently.However,the diseases caused by bacteria,viruses and parasites have flourished within booming E.sinensis cultures,and threatened the sustainability of the aquaculture of E.sinensis.Tremor disease(TD)is one of the most devastating diseases of E.sinensis.A spiroplasma was previously identified as a novel causative pathogen of the disease and named Spiroplasma eriocheiris.The preliminary researches showed that S.eriocheiris entered in crab body through the gills or surface of body,invaded its target cells-hemocytes and propagated in it.Then the bacteria were carried to the connective tissues of neural,muscle and other tissues though blood circulation.The pathogen caused the crab tremor and dead at later stage of infection.As the target cells of S.eriocheiris,the hemocytes of crab paly an important role in the process of the crab against the infection of the bacteria.Therefore,to explore the mechanism of S.eriocheiris infection the crab and the host immune defense mechanism could help us to find ways to reduce the economic losses caused by S.eriocheiris.Our team has identified different hemocytes proteins of E.sinensis with iTRAQ during spiroplasma infection and selected two important proteins,Rab7 and 14-3-3?,to conduct preliminary studies to confirm their participation in the process of immune of the crab against to the infection of S.eriocheiris.Based on this,the functions of these two proteins were further studied.The related interacting microRNA of each gene was screened.RNA interference and recombinant protein overexpression experiments were performed in vitro to verify their immune function.The specific research contents are as follows: 1.Preliminary study on the identification and function of EsRab7 and Es14-3-3? related microRNAsIn this study,it was further confirmed in vitro that the transcriptions of EsRab7 and Es14-3-3? proteins were up-regulated after S.eriocheiris stimulation,confirming that these two proteins are involved in the process of immune of crab.Two microRNAs,miR-131-3p and miR-2309,which were able to target the EsRab7 and Es14-3-3? genes,were screened,respectively.Tissue distributions of these two miRNAs in crab were examined.The results showed that both of them were highest in hepatopancreas,followed by nerves,and also in hemolymph.After stimulating crabs with S.eriocheiris,the expression profiles of miR-131-3p and miR-2309 in hemocytes and nerve were detected.The results showed that both of them were significantly down-regulated in hemocytes and nerve.These results indicate that the regulatory between miR-131-3p and EsRab7 gene as well as miR-2309 and Es14-3-3Z gene play an important role in the process of immune of crab against S.eriocheiris.2.Immune function analysis of EsRab7 protein during infection of S.eriocheirisIn this part,RNA interference technology was used to successfully interfere with the expression of EsRab7 gene in the hemocytes of crabs before stimulated by S.eriocheiris.The morphology,viability,antibacterial peptides expressions and spiroplasma copy number were detected.The results showed that the cell death,viability and antimicrobial peptides expressions in the EsRab7-silencing group were significantly lower than those in both the control group and the inhibitor group.The S.eriocheiris copy number was significantly higher than that of the control group and the inhibitor group.After overexpression of EsRab7-GFP protein in Drosophila S2 cells and stimulation with S.eriocheiris,the cell morphology,viability and S.eriocheiris copy number were detected.The results showed that the cells in the overexpressing group survived the most.The cell viability was significantly higher than that in the control group.And the S.eriocheiris copy number was significantly lower than that in the control group.The results above suggest EsRab7 palys an important role in the process of the crab respond to S.eriocheiris.3.Immune function analysis of Es14-3-3Z protein during infection of S.eriocheirisIn this part,RNA interference technology was used to successfully interfere with the expression of Es14-3-3? gene in the hemocytes of crabs before stimulated by S.eriocheiris.The morphology,viability,immune related genes expression and spiroplasma copy number were detected.The results showed that the cell death,viability,antimicrobial peptides,immunocytokine EsRelish and EsERK expressions in the Es14-3-3? silencing group were significantly lower than those in the control group and the inhibitor group while the S.eriocheiris copy number was significantly higher than those of the control group and the inhibitor group.EsRab7 transcript levels also positively correlated with the change in Es14-3-3?.After overexpression of Es14-3-3?-GFP protein in Drosophila S2 cells and stimulation with S.eriocheiris,the cell morphology,viability and S.eriocheiris copy number were detected.The results showed that the cells in the overexpressing group survived the most.The cell viability was significantly higher than that in the control group.And the S.eriocheiris copy number was significantly lower than that in the control group.The above results suggest Es14-3-3? palys an important role in the process of the crab respond to S.eriocheiris. |
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