虾夷马粪海胆X染色体连锁凋亡抑制蛋白XIAP基因克隆及不同病原微生物刺激后的表达响应  

Cloning of X-linked inhibitor of apoptosis protein XIAP gene and its response to stimulation in sea urchin Strongylocentrotus intermedius challenged with pathogenic microorganisms

作　　者：刘圣美 尚凤芹 陈亚东[1] 常亚青[1] 王秀利[1] 仇雪梅[1] 刘洋[1] LIU Shengmei;SHANG Fengqin;CHEN Yadong;CHANG Yaqing;WANG Xiuli;QIU Xuemei;LIU Yang(Key Laboratory of Mariculture&Stock Enhancement in North China's Sea,Ministry of Agriculture and Rural Affairs,Dalian Ocean University,Dalian 116023,China)

机构地区：[1]大连海洋大学农业农村部北方海水增养殖重点实验室,辽宁大连116023

出　　处：《大连海洋大学学报》2022年第4期627-635,共9页Journal of Dalian Ocean University

基　　金：国家自然科学基金(31101923);辽宁省自然科学基金(2015020761);农业农村部北方海水增养殖重点实验室开放课题(2014-MSENC-KF-08)。

摘　　要：为研究X染色体连锁凋亡抑制蛋白(X-linked inhibitor of apoptosis protein,XIAP)在虾夷马粪海胆Strongylocentrotus intermedius(体质量为40~80 g)天然免疫系统中的作用,采用cDNA末端快速扩增(RACE)技术克隆获得虾夷马粪海胆XIAP基因的cDNA序列,并通过实时定量PCR分析了其在各组织中的分布,以及经脂多糖(lipopolysaccharide,LPS)、肽聚糖(peptidoglycan,PGN)、聚肌胞苷酸(polyinosinic-polycytidylic acid,Poly I:C)、强壮弧菌Vibrio fortis和葡聚糖(whole glucan particles,WGP)刺激后,XIAP基因在体腔细胞中的表达情况。结果表明:虾夷马粪海胆XIAP基因的cDNA长为4894 bp,其中,5′UTR为459 bp,开放阅读框为2331 bp,3′UTR为2104 bp,共编码776个氨基酸,具有3个BIR结构域和1个RING指环结构域,预测该蛋白相对分子质量为86960,等电点为5.96,为酸性蛋白;虾夷马粪海胆XIAP基因在检测的各组织中均有表达,其中,在卵巢中的表达量显著高于管足、体腔细胞、围口膜、肠和精巢(P<0.05);经PGN、LPS、Poly I:C、WGP和V.fortis刺激后,体腔细胞中的XIAP基因表达量显著上升(P<0.05),并在PGN、LPS和Poly I:C刺激后12 h,WGP刺激后24 h,V.fortis刺激后6 h时,XIAP基因表达量均达到最高值。研究表明,克隆获得的XIAP基因参与了虾夷马粪海胆的免疫应答,并在海胆的天然免疫中发挥重要作用。To investigate the role of X-linked inhibitor of apoptosis protein(XIAP)gene in the natural immune system of sea urchin Strongylocentrotus intermedius,the cDNA sequence of XIAP gene was cloned using rapid amplification of cDNA ends(RACE)technique,and distribution in various tissues including tube foot,peristomial membrane,intestine,and gonad and the expression of XIAP gene were analyzed in coelomocyte in the sea urchin exposed to stimulation including lipopolysaccharide(LPS),peptidoglycan(PGN),polyinosinic-polycytidylic acid(Poly I:C),Vibrio fortis and whole glucan particles(WGP)by real-time quantitative PCR.The results showed that the length of XIAP cDNA was 4894 bp,with 5′UTR of 459 bp,the open reading frame of 2331 bp and the 3′UTR of 2104 bp,encoding a total of 776 amino acids with three BIR structural domains and one RING finger structural domain,with a predicted relative molecular mass of 86960 and an isoelectric point of 5.96,as acidic protein.XIAP was expressed in all tissues examined,significantly higher expression in the ovary than that in the tube foot,coelomocyte,peristomial membrane,intestine and testis(P<0.05),after stimulation with PGN,LPS,Poly I:C,WGP and V.fortis.XIAP gene expression was up-regulated in coelomocyte significantly(P<0.05),with the maximal values 12 h after PGN,LPS and Poly I:C stimulation,and with the maximal values 24 h after WGP stimulation and 6 h after V.fortis stimulation.The findings indicated that the clonally obtained XIAP gene was involved in the immune response of the sea urchin and played an important role in its natural immunity.

关 键 词：虾夷马粪海胆 X染色体连锁凋亡抑制蛋白基因 实时荧光定量PCR 脂多糖 肽聚糖 葡聚糖 聚肌胞苷酸 强壮弧菌 

分 类 号：S917.4[农业科学—水产科学]