摘要
【目的】明确凡纳滨对虾(Litopenaeus vannamei)RASSF基因(LvRASSF)的分子结构、组织表达模式及其应答不同病原感染的表达特征,为深入研究LvRASSF基因在凡纳滨对虾先天性免疫中的功能及作用机制打下基础。【方法】利用PCR克隆得到LvRASSF基因开放阅读框(ORF)序列,并对其进行生物信息学分析及构建系统发育进化树,运用实时荧光定量PCR分析LvRASSF基因在12个组织中的表达情况及不同病原感染后的表达变化。【结果】LvRASSF基因ORF序列长669 bp,共编码222个氨基酸残基,蛋白分子量为89.2 kD,理论等电点(pI)为5.06。LvRASSF氨基酸序列包含1个RA结构域和1个SARAH结构域。基于RASSF氨基酸序列相似性构建的系统发育进化树显示,凡纳滨对虾与斑节对虾(Penaeus monodon)和中国对虾(Penaeus chinensis)的亲缘关系较近。LvRASSF基因在各组织中均有表达,其中在血细胞中的相对表达量最高,在眼柄中的最低。感染白斑综合征病毒(White spot syndrome virus,WSSV)和十足目虹彩病毒(Decapod iridescent virus 1,DIV1)后,LvRASSF基因在凡纳滨对虾鳃组织中的相对表达量极显著上调(P<0.01,下同),而在血细胞中短期极显著上调后又极显著下调;感染副溶血弧菌(Vibrio parahaemolyticus,VP)后LvRASSF基因在凡纳滨对虾鳃和血细胞中的相对表达量整体呈上调趋势。【结论】LvRASSF蛋白存在RASSF家族典型的RA结构域和SARAH结构域,在进化上较保守。LvRASSF基因广泛表达于凡纳滨对虾各组织中,以血细胞中的相对表达量最高。不同病原感染后LvRASSF基因在血细胞和鳃组织中相对表达量发生极显著变化,表明LvRASSF基因在凡纳滨对虾的抗病免疫中发挥重要作用。
【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.vanna-mei 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 vi-rus(WSSV)and Decapod iridescent virus 1(DIV1),its relative expression in the gills tissues of L.vannamei was ex-tremely significantly up-regulated(P<0.01,the same below),while the expression was extremely significantly down-regulated 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.
作者
肖伟
戚建英
姚元茂
史黎黎
XIAO Wei;QI Jian-ying;YAO Yuan-mao;SHI Li-li(Fisheries College,Guangdong Ocean University,Zhanjiang,Guangdong 524088,China;Shenzhen Institute,Guangdong Ocean University,Shenzhen,Guangdong 518120,China)
出处
《南方农业学报》
CAS
CSCD
北大核心
2023年第9期2665-2673,共9页
Journal of Southern Agriculture
基金
广东省自然科学基金面上项目(2022A1515011943)
深圳市自然科学基金面上项目(JCYJ20220530162017040)
广东省普通高校特色创新类项目(2019KTSCX055)。
