Study On The Toll Like Receptor And RIR-I Like Receptor Signal Pathways And Their Immune Functions In Pacific Oyster(Crassostrea Gigas)

Oyster is an important aquacultural species in China and even in the world. Oyster Herpesvirus-1(OsHV-1) has been found in many species of molluscans all over the world since herpes-like virus was first detected in Crassostrea virginica in 1972. OsHV-1 has been proved to be relative to the mortality of oyster larvae, and this mortality brought huge economic damage to the aquaculture. Up to date, there is still little research on the innate immunity of oyster to OsHV-1, and no effective solutions have been found to resolve the problems of the mortality and diseases brought by OsHV-1, blocking the the sustainable development of aquaculture. So research on the innate immunity of oysters to OsHV-1 enriches the theories of disease brought by the OsHV-1 and can reduce the economic damage of aquaculture.In the present study, the innate immunity of Pacific oyster(Crassostrea gigas) larvae and spat to OsHV-1 was analyzed with technology of cell biology, bioinformatics, molecular biology and immunology. The main sesults are as follows:TLRs and RLRs are important pattern recognition receptors(PRRs) in innate immune defense. The full-length cDNA of key molecules in TLR and RLR pathway were identified with rapid amplification of cDNA ends(RACE) PCR. Four TLR genes(named Cg05421 TLR, Cg17269 TLR, Cg21740 TLR and Cg27513 TLR) and two MyD88 genes(named CgMyD88-1 and CgMyD88-2) belonged to TLR pathway and eleven RIG-I genes(named from RIG-I-1 to RIG-I-11) and a MAVS gene(named CgMAVS) belonged to RLR pathway. Besides these gene, some ORF sequeces of TNF receptor associated factor(TRAF)(named CgTRAF2 and CgTRAF6) and Interferon regutory facror(IRF)(named Cg07268 IRF, Cg07269 IRFand Cg16646 IRF) were also identified. These genes have similar domains with those of vertebrate through SMART analysis, and the phylogenetic tree analysis of these genes was also completed.After cluster analysis of the C. gigas developmental RNA-seq data, 9 genes from the stably expressing clusters which were also orthologs of human housekeeping genes were selected as the candidate internal controls along with the usually used housekeeping genes(18S rRNA, 28 rRNA, Elongation factor-1, β-actin and glyceraldehyde-3 phosphate-dehydrogenase). The stabilities of these genes in virus-infected and uninfected samples were analyzed by quantitative real time PCR(qRT-PCR) and GeNORM software, the results suggested that RL7 and RS18 could be used as internal controls for studying gene expression in normal growing oyster larvae and in Os HV-1 infected larvae. The expressing models of four TLR genes and eleven RIG-I genes in OsHV-1 infected developmental RNA-seq data were validated using qRT-PCR normalized by the selected internal controls and the expressing models of these genes with qRT-PCR validation were the same to those of RNA-seq. There was a peak of mRNA expression in three TLR genes before the OsHV-1 replicating rapidly and a peak of mRNA expression in one TLR gene and eleven RIG-I genes after C. gigas larvae mortality happened. A hypothesis was proposed base on the results: these three TLRs(Cg05421 TLR, Cg17269 TLR and Cg21740 TLR) may be involved in the recognision of OsHV-1 on the cell membrance while Cg27513 TLR and eleven RIG-I genes may be involved in the recognition of DNA or RNA of OsHV-1 in the cytoplasm. The subcellular location experiments of the four TLR genes were completed to validate the hypothesis, and the results were not complied with the hypothesis: all of the four TLR proteins were located in the endosome and cytoplasm. This was the first report that TLR genes of C. gigas were located in endosome. Besides this, two MyD88 genes were located in cytoplasm which complied with their function in the TLR pathway.To confirm the important roles of the key genes in TLR and RLR signal pathway to the OsHV-1 immunity, the supernatant containing OsHV-1 were injected to C. gigas. The mRNA expression of TLR genes and MyD88 genes were all up-regulated and had significant differences at some time points with the control(p < 0.05). The TIR domain of Cg27513 TLR could interact with CgMyD88-2 through the yeast two-hybrid experiments. All of the results indicated that MyD88 dependent TLR signal pathway in C. gigas was invovled in OsHV-1 immunity. The mRNA expression of RIG-I genes and MAVS genes in RLR pathwaym along with TRAF genes and IRF genes were also up-regulated by OsHV-1 and had significant differences at some time points with the control(p < 0.05). A RNAi technology was applied to inhibit the expression of MAVS using siRNA injection, the mRNA expression of CgTRAF2, CgTRAF6, Cg07268 IRF, Cg07269 IRF and Cg16646 IRF were also down-regulated with the same expression model with MAVS. These results indicated that CgTRAF2, CgTRAF6, Cg07268 IRF, Cg07269 IRF and Cg16646 IRF may belong to RLR pathway and the RLR pathway may also be involved in OsHV-1 immunity.In conclusion, there existed MyD88 dependent TLR signal pathway and MAVS dependent RLR signal pathway in C. gigas, and these two pathways may play important roles in the innate immune defense system of C. gigas to OsHV-1.