| Diseases caused by white spot syndrome virus (WSSV) are the greatest challenge to the worldwide shrimp aquaculture. Thus the main problem for us is to prevent and inhibition the spread of the virus. And one of the critical solvents is to study the innate immunity of shrimp. By improving the antiviral immunity of shrimp, we can control shrimp diseases much more efficiently. Since Marsupenaeus japonicus is one of the most important kinds of aquaculture products, it is taken as the investigated object in our study. We constructed and characterized a Yeast two hybrid cDNA library and identified and characterized several immune-relevant genes, which would be helpful, to some extend, for shrimp diseases control.Taking the hemocyte and pancreas of Marsupenaeus japonicus as material, the mixed yeast two- hybrid cDNA library was constructed by the technologies of CLONTECH SMART and homologous recombination. The results showed that 1.4×106cfu/ml recombinants were obtained from the library and the recombination efficiency researches more than 90%. The insert fragments of recombinants varied in size. The yeast two- hybrid cDNA library of Marsupenaeus japonicus was proved to be successfully constructed and reliable by a primary screening using Marsupenaeus japonicus TCTP (Translationally Controlled Tumor Protein) as bait.With nested PCR and RACE, we identified two kinds of MAPK, ERK1/2 and p38, in Marsupenaeus japonicus. According to lots of researches in other species, both of ERK1/2 and p38 are key factors functioning in many activities in cells, including the antiviral immunity. And that is why we want to investigate their roles in shrimp innate immunity. By analyzing their sequences, we found that both ERK1/2 and p38 of Marsupenaeus japonicus have similar conserved domains and motifs with their homologies. Furthermore, based on the results of western blot, the diphosphorylated level ERK1/2 of Marsupenaeus japonicus was found to be specifically induced by the entry of WSSV into host cells. However, there are also some phenomena uncovered in Marsupenaeus japonicus ERK1/2 and p38 different from other species. First, it was showed by western blot, there is only one kind of ERK1/2 in Marsupenaeus japonicus, but not ERK1 and ERK2. Secondly, the protein deduced by Marsupenaeus japonicus p38 gene is smaller than p38s in other species because of the lack of nearly 100 amino acids in its C-terminal. To investigate the reasons, we need some more researches which might also contribute to the study of these genes'functions in shrimp innate immunity against virus.Centaurin-α1 is reported by our study as a new immune-relevant gene in Marsupenaeus japonicus antiviral immunity. With the Real time PCR, the elementary relationship between centaurin-α1 and WSSV infection was discovered. During the early stage of WSSV infection, the gene transcription expression of centaurin-α1 could be induced to a high level, while in the virus-resistant shrimps, it was inhibited. Such interesting phenomena might suggest the bivalent role of centaurin-α1 during WSSV infection and host cell defense. In order to further study the functions of centaurin-α1, on one hand, we got the full-length sequence of it by semi-nested PCR. And the analysis showed that centaurin-α1 of Marsupenaeus japonicus had one ArfGAP and two PH conserved domains which represented its possible functions. On the other hand, the RT-PCR and immunofluorescence indicated that centaurin-α1 distributed in every tissue of Marsupenaeus japonicus and assembled mainly in cytoplasm.
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