Cloning of RASSF gene in Litopenaeus vannamei and its expression analysis in response to stimulation of different pathogens

【Objective】To identify the molecular structure,tissue expression patterns and expression characteristics in response to infections of different pathogens of RASSF gene in Litopenaeus vannamei(LvRASSF),and to provide basic data for in-depth research on the role of RASSF gene in the innate immunity of L.vannamei. 【Method】The open reading frame(ORF) sequence of the LvRASSF gene was cloned by PCR technology,bioinformatics analysis was performed and phylogenetic tree was constructed. Real-time fluorescence quantitative PCR was used to analyze the expression of LvRASSF gene in 12 tissues and the expression changes in mRNA after the gene was infected by different pathogens. 【Result】The ORF sequence of LvRASSF gene was 669 bp,encoding 222 amino acid residues with protein molecular weight of 89.2 kD.The theoretical isoelectric point(pI) was 5.06. LvRASSF amino acid sequence included one RA domain and one SARAH domain. The phylogenetic tree constructed based on the similarity of RASSF amino acid sequences showed that L. vannamei was closer to Penaeus monodon and P. chinensis. LvRASSF gene was expressed in all tissues,with the highest relative expression in the hemocytes and the lowest in the eyestalk. After LvRASSF gene was infected by white spot syndrome virus(WSSV) and Decapod iridescent virus 1(DIV1),its relative expression in the gills tissues of L. vannamei was extremely significantly up-regulated(P<0.01, the same below),while the expression was extremely significantly downregulated in hemocytes after a short-term extremely significant up-regulation. In addition, the relative expression of LvRASSF gene after infection with Vibrio parahaemolyticus(VP)was integrally up-regulated in the gills and hemocytes of L. vannamei. 【Conclusion】The typical RA domain and SARAH domain of RASSF family exist in LvRASSF protein which is relatively conserved in evolution. LvRASSF gene is widely expressed in various tissues of L.vannamei,with the highest relative expression in hemocytes. The relative expression levels of LvRASSF gene infected by different pathogens show extremely significant changes in hemocyte and gill tissues,indicating that LvRASSF gene plays an important role in the disease resistance and immunity of L. vannamei.