| Litopenaeus vannamei occupies very important position in Marine aquaculture of China, and is the most important variety of the crustaceans breeding industry in China. In recent years, the total area and output of breeding rise steadily, driving the local economic development. But there are still a lot of problems,especially the effect of the shrimp virus diseases. It caused a huge economic losses to the farmers and a serious threat to the sustainable development of shrimp industry,but it was still no effective prevention drugs and means so far. As we known, shrimp lacks specific immune system, and it depends on a variety of immune factors to resist diseases. This thesis aim is to clone immune related genes of litopenaeus vannamei and to carry on the analysis initially in order to help the prevention of litopenaeus vannamei disease and provide the new thought of the shrimp own disease-resistant ability.This research uses SMART technology constructs the full-length cDNA library of the hepatopancreas of litopenaeus vannamei, with drops degree of1.2x106pfu/mL, and recombination rate of97%. The size of LD-PCR amplification fraction is in0.2KB-3.5KB, and it exists a lighter belt in1.0KB place, the quality of the constitutive library is eligible. Then randomly pick the positive cloning and take sequenced, and take it in homology comparation with the cDNA sequence in GenBank database sequence, finding that the length of the library have Rab gene family member, Rab5B.This research gets part of the cDNA sequence of Rab5B and Rab6A gene of litopenaeus vannamei by cloning, and the length of open reading frames of them are respectively378bp and442bp, coding126and147amino acids. By comparison of BLAST protein, we found that both have structure domain of NTP enzyme super family, and showing similar structure with other members of the family of Rab. No tissue specificity was got by pathogens to further (SPF) attack in external poisoning experiment of SPF litopenaeus vannamei, combined with RT-PCR, but showed little higher expression just in the immune tissue of shrimp. In contrast, both of them showed higher expression quantity in tissues of IHHNV virus infection shrimp than that in normal ones.We got the full-length sequence of cDNA of Rab7in Litopenaeus vannamei through3’RACE and5’RAC, with the length of767bp, the open reading box (ORF) of618bp, which coding205amino acids, and the molecular weight is about23.33kDa, and the theory isoelectric point is5.85. The5’end and the3’ end respectively exist non-translation sections of88bp and61bp.Online analysis showed that the main structure of Rab7precursor protein domain is consistent with the known Rab protein family, presenting strong conservatism in the main function domain between species and just difference of amino acid sequence at the end of precursor protein. Half quantitative PCR displays normal expression quantity and no obvious difference of the Rab7gene in litopenaeus vannamei organization, but higher in IHHNV virus infection prawn, and highest in the antiviral prawn. |
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