| Shrimp aquaculture is threatened by many diseases,among which white spot disease(WSD)caused by white spot syndrome virus(WSSV)is one of the major diseases.With the prawn farming areas and scales expanding constantly,WSSV had emerged the variation between different strains accordingly.The differences between different isolates may lead to WSSV virulence variation.This paper mainly analyzes the WSSV variation through the shrimp culture zone in 2015-2016.Furthermore,RNAi technology was used to silence wsv001 and wsv025 and wsv045 genes to study the effect of these genes on the WSSV proliferation.The mainly research contents and results show as follows:Chapter Ⅰ: To understand the variations of WSSV wsv001,wsv025 and wsv045 in different regions of C hina,we investigated 104 WSSV-positive samples collected in disease outbreak areas during 2015 and 2016.The tested samples were from Shandong,Hebei,Zhejiang,Tianjin,Hainan and Guangdong Province respectively.Using specific primers,we amplified WSSV-positive samples with PCR.The samples for amplification of the wsv001 fragment reached 25%,the amplification of wsv025 fragment reached 23.08%,the amplification of wsv045 fragment reached 45.19%.No mutation in wsv025 and wsv045 genes was found.However,the mutation of wsv001 was confirmed in this experiment compared with the CN01.Sequence alignment showed that the samples from Jimo,C hangyi,Weihai and Tianjin had 18 amino acids(aa)deletion;The samples from Changyi,Shouguang and Zhoushan appeared 121 amino acids deletion;The samples from Weifang and C hangyi showed 3 and 20 amino acids insertion,respectively.All of the mutation leaded to structure change of collegen surperfamily.The samples from Changyi,Shouguang,Jimo,Zhoushan,Weihai and Weifang had amino acid mutation on 8 sites.Mutation position of amino acid focus on 202 aa,280 aa,307 aa,310 aa,311 aa,314 aa,364 aa and 366 aa.In conclusion,wsv001 genes and their encoded amino acids had obvious deletion and variation in different areas of C hina.These results suggested that there were certain degrees of prevalence and variation in WSSV,which caused white spot disease in most parts of China,with clear variations between the different strains in wsv001.Chapter Ⅱ: In order to understand the WSSV wsv001,wsv025 and wsv045 protein structure,the specific primers were designed,and the recombinant vector pBAD/gⅢA-001、pBAD/gⅢA-025、p BAD/gⅢA-045 were constructed.The bacterial liquid was expanded cultured and induced to express protein.The expression of the proteins was detected by SDS-PAGE and Western-blot analysis.The results showed that wsv025 and wsv045 were successfully expressed with low expression quantity,but the wsv001 was no expressed.Chapter Ⅲ: To reveal the function of wsv001,wsv025 and wsv045,RNA interference was employed.In this study,the samples of litopenaeus vannamei,were intramuscularly injected with double-strand RNA(ds RNA)corresponding to wsv001、wsv025,wsv045 and an unrelated dsRNA of a green fluorescence protein(GFP)gene.Quantitative RT-PCR analysis showed that gene-specific ds RNA could lead to higher inhibition of WSSV replication comparing with the GFP group.The result showed that the expression of WSSV was decreased after wsv001,wsv025 and wsv045 silencing,which was consistent with the results of infection experiments that the death rate of shrimp was reduced after the genes were disturbed,and wsv045 can inhibited the death of shrimp to certain extent.In conclusion,the results will benefit to understand the maturation and roles of wsv001、wsv025,wsv045. |
