大黄鱼肿瘤坏死因子受体基因(TNFR)的克隆及其在各组织和副溶血弧菌感染下的表达  

Cloning of Tumor Necrosis Factor Receptor Gene(TNFR) and Its Expression in Different Tissues and Response to Vibrio parahaemolyticus Challenge in Large Yellow Croaker(Larimichthys crocea)

作　　者：孙从正 韩坤煌 黄贻涛[1] 刘家富 张子平[1] 谢芳靖[1] 王艺磊[1] 

机构地区：[1]农业部东海海水健康养殖重点实验室,集美大学水产学院,厦门361021 [2]福建省国家级大黄鱼原种场,宁德市富发水产有限公司,宁德352103

出　　处：《农业生物技术学报》2014年第3期333-342,共10页Journal of Agricultural Biotechnology

基　　金：福建省农业科技重点项目(No.2013N0024);福建省教育厅项目(No.JA09156);集美大学创新团队基金项目(No.2010A001)

摘　　要：肿瘤坏死因子(tumor necrosis factor,TNF)与肿瘤坏死因子受体(TNF receptor,TNFR)超家族在机体的先天免疫和获得性免疫方面都发挥重要作用。本研究从本实验室构建的大黄鱼(Larimichthys crocea)EST(expressed sequence tag)数据库中筛选出TNFR(LcTNFR)基因片段,利用SMART-RACE技术克隆出大黄鱼LcTNFR基因cDNA全序列,全长2 016 bp,其中5'非翻译区(untranslated region,UTR)64 bp,3'非翻译区602 bp,开放阅读框(open reading frame,ORF)1 350 bp。ORF编码449个氨基酸(GenBank登录号:KF432416)。预测的蛋白质等电点为5.47,分子量为49.7 kD。对LcTNFR蛋白三维结构进行分析发现,其含有6个α螺旋,不含β折叠。Blast比对分析显示,与斑马宫丽鱼(May landia zebra)的TNFR 10B-like氨基酸序列一致性最高,达74%;与斑马鱼(Danio rerio)卵巢型TNFR一致性较高,达51%;与鲫鱼(Carassius auratus)TNFRⅠ的一致性为47%。鱼类TNFR进化树分析进一步表明,LcTNFR与TNFR 10B-like、TNFRⅠ及卵巢型TNFR聚为一大支。荧光定量PCR结果显示,TNFR基因在大黄鱼各组织中均有表达,在卵巢的表达水平最高,显著高于精巢及其它组织;其次在免疫相关组织脾脏也有较高的表达。副溶血弧菌(Vibrio parahaemolyticus)感染后的第2天和第4天,LcTNFR基因在大黄鱼脾脏组织中的表达量达到最高,显著高于对照组(P<0.05),到第8天逐渐回落到对照组水平。本结果可为大黄鱼免疫学研究及抗病相关分子标记的开发提供参考依据。The tumor necrosis factor （TNF） and tumor necrosis factor receptor （TNFR） superfamilies play crucial roles in both innate and adaptive immunity. In this study, the fragment of TNFR （LcTNFR） was identified from the EST（expressed sequence tag）database of large yellow croaker （Larimichthys crocea）, and the full length cDNA of LcTNFR of 2 016 bp was obtained using SMART-RACE method, consisting of a 5＇ UTR of 64 bp, an ORF of 1 350 bp and a 3＇ UTR of 602 bp（GenBank accession No KF432416）. The deduced protein was composed of 449 amino acids, with a predicted pI of 5.47 and an estimated molecular mass of 49.7 kD. Protein structure prediction by SWISS-MODEL found that LcTNFR contained 6 ~t-helix and 0 13- fold. Blast analysis results revealed that LcTNFR had the highest identity with Maylandia zebra 10B-like （74%）, followed with Danio rerio ovarian TNFR （51%） and with Carassi~ auratus TNFR I （47%）. Phylogenetic tree of the TNFR amino acid sequences analysis furtherly indicated that LcTNFR together with TNFR 10 B - like, TNFR type I and ovarian TNFR formed a branch. The results of quantitative Real-time PCR （qRT-PCR） showed that LcTNFR was expressed ubiquitously in examined tissues of male and female fishes. The expression level of LcTNFR was significantly higher in ovary than that in other tissues including testis, and the expression level of LcTNFR in spleen, which is an organ involved in immune function, was also higher than that in other examined tissues. In the spleen, the expression level of LcTNFR was up-regulated significantly （P 〈 0.05） at the 2 day and 4 day after Vibrio parahaemolyticus challenge, and then it restored to the control level at 8 day, The results may provide insight into the immunology research and the development of disease-resistance genetic markers for large yellow croaker.

关 键 词：大黄鱼 肿瘤坏死因子受体(TNFR) 副溶血弧菌 组织表达 定量PCR 

分 类 号：S941.42[农业科学—水产养殖]