Characterization Of Two Signal Transduction Factors Of Litopenaeus Vannamei And Chitin Binding Proteins Of Tubeworm (Ridgeia Piscesae) From Hydrothermal Vent

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 inhibit the spread of the virus. And one of the critical solutions is to study the innate immunity of shrimp. By improving the antiviral immunity of shrimps, we can control shrimp diseases much more efficiently. Since Litopenaeus vannamei is one of the most important aquaculture species, we take it as the investigated object in our study. We have made a primary research on the characterization of ERK (extracellular signal-regulated kinase) and Src tyrosine kinase which may contribute to the signaling mechanism of ERK and Src in the WSSV infection.In this investigation, based on the results of western blot, the phosphorylated ERK of Litopenaeus vannamei was found to be specifically induced during the infection of WSSV, which occurred in the early stage of WSSV proliferation. Meanwhile, viral DNA production could be significantly decreased in specific ERK inhibitor-treated primary cultured crayfish hemocyte. Furthermore, a specific 500 bp dsRNA (dsRNA-ERK) was used to induce gene silencing in Litopenaeus vannamei shrimp. It was found that the transcription and expression of erk gene were silenced by the sequence-specific dsRNA. As revealed by quantitative PCR, the dsRNA caused a significant reduction in viral DNA production of WSSV-infected shrimps.Src tyrosine kinase is reported by our study as a new immune-relevant gene in Litopenaeus vannamei. With the RT PCR, the elementary relationship between Src and WSSV infection was discovered. During the late stage of WSSV infection, the gene transcription expression of Src could be induced to a high level. In order to further study the functions of Src, we got the full-length sequence of it by 3'RACE. And the analysis showed that Src of Litopenaeus vannamei had one tyrosine catalytic domain and two SH conserved domains which represented its possible functions. On the other hand, the RT PCR indicated that Src distributed in every tissue of Litopenaeus vannamei.In previous study, we constructed cDNA library of tube worm (Ridgeia piscesae) and sequencing result showed that it contains twenty three chitin-binding proteins (CBPs). With high transcription level and diversity, each protein contains 1-3 different types of chitin binding domain(s). In view of the important role in chitin degradation and the special habitats of the tube worms, we further studied the functional characterization of four typical chitin-binding proteins. After analyzed, the nucleotide sequences of signal peptides were moved off and the fragments encoding chitin-binding proteins were amplified by PCR, inserted into pIZ-FLAG Vector and successfully expressed in Hi5 cells. In the chitin affinity assay, the results demonstrated that the four FLAG-CBPs can bind toα-chitin respectively. Meanwhile, the supernatant of cell lysate including the expressed product FLAG-CBPs was able to enhance the activity of chitinase in the process of degradingα-chitin andβ-chitin respectively. In-depth study will be taken to verify other biological functions of chitin binding proteins and optimize their ability of catalyzation.