Functional Study Of Cathepsin D And Serine Protease Inhibitor 3 Of Eriocheir Sinensis In The Process Of Spiroplasma Infection

The Chinese mitten crab, Eriocheir sinensis, is an economically important species,which occupies a large proportion in Chinese freshwater aquaculture industry. However,in recent years, with the expansion of cultivation scale and the continuous improvement of intensification, various diseases caused by bacteria, viruses, parasites and other pathogens have increasing seriously. At the same time, the diseases caused great economic losses to the development of E. sinensis. Tremor disease (TD) is one of the most serious diseases of E. sinensis. Professor Wang identified and isolated the pathogen of TD, which was a novel bacteria - Spiroplasma by ultrastructure observation, bacteria cultivation in vitro, infection experiment, and officially named it as Spiroplasma eriocheiris. Early researches showed that S.eriocheiris entered into crab through the gills or surface. Firstly, S.eriocheiris invaded its target cells-hemocytes and propagated. Then the pathogens were carried to neural and muscle organization and other tissues by blood circulation. Finally, the pathogen caused the crab appendages tremor at the later stage of infection. Therefore, it is very important to study the infection mechanism of S. eriocheiris and the immune defense mechanism of the crab.On the basis of previous research, two proteins of the crab Eriocheir sinensis,Cathepsins D and serine protease inhibitor 3 (EscatD and Esserpin-3) were studied. In this present work, the functions in the immune response mechanism of E. sinensis when challenged with S.eriocheiris were studied by gene cloning, bioinformatics analysis and gene interference experiments. This paper will provide theoretical basis for further exploring of the immune defense mechanism of E. sinensis. The main results of this paper include the following three aspects:1. EscatD gene cloning, bioinformatics analysis, protein prokaryotic expression and related functions studiesThe Cathepsin D of E. sinensis (named EscatD) was successfully amplified by PCR from the hemocytes cDNA. The results showed that the full-length cDNA of the gene was 2748 bp. Among them, 5 ’untranslated region (UTR) was 140 bp, 3’UTR was 1450 bp, open reading frame was 1158 bp, encoding 385 amino acids. The molecular weight and isoelectric point of EscatD was about 41.66 kDa and 6.53. Sequence analysis showed that EscatD protein was composed of signal peptide, precursor domain and mature domain. Homology analysis showed that the amino acid sequence of EscatD was highly conserved with other species, and the consistency was between 70% and 85 %. The evolutionary analysis showed that EscatD had a close genetic relationship with shrimp, which was consistent with the traditional classification. The expression plasmid pet-28a-EscatD was constructed in vitro. The recombinant protein EscatD was found in the form of inclusion bodies and purified. Tissue expression showed that EscatD expressed in all tested tissues of crabs. The expression levels in the hemocytes,intestine and nerve of the crab were relatively high, and the expression level in the heart,muscle and gills was less. The above results will lay an important foundation for the further study of the function of EscatD.2. Immune function of EscatDEscatD belongs to the cathepsin D family, and recent studies have shown that EscatD plays an important role in the congenital immune response of crustaceans. qRT-PCR results showed that the expression level of EscatD mRNA was obviously higher in S. eriocheiris group than that of the control group. After RNAi of EscatD, the expression lever of the downstream genes JNK, ERK, P38 and Relish were all decreased. After RNAi of EscatD and infectioned with S. eriocheiris, the copy numbers of S. eriocheiris and the mortality rate of crabs were increased. This further demonstrates that EscatD plays an important immune role in the infection of the S.eriocheiris to the crabs.3. Immunological function of Esserpin-3Esserpin-3 belongs to the serine protease inhibitor family. Recent studies have shown that Esserpin-3 plays an important role in the congenital immune response of crustaceans. qRT-PCR results showed that the expression level of Esserpin-3 mRNA was obviously higher in S. eriocheiris group than that of the control group. After RNAi of Esserpin-3, the expression lever of the upstream gene LGBP was increased and the downstream gene proPO was increased. After RNAi of EscatD and infectioned with S.eriocheiris, the copy numbers of S. eriocheiris and the mortality rate of crabs were decreased. This further demonstrates that Esserpin-3 plays an important immune role in the infection of the S. eriocheiris to the crabs.