尼罗罗非鱼TNF-R1和TNF-R2基因克隆及表达  

Cloning and expression analysis of Nile tilapia TNF-R1 and TNF-R2 gene

作　　者：叶航宇 葛岩岩 吴金英 詹绪良 YE Hangyu;GE Yanyan;WU Jinying;ZHAN Xuliang(School of Life Sciences,Sun Yat-sen University/Institute of Aquatic Economic Animals and Guangdong Provincial Key Laboratory for Aquatic Economic Animals/Guangdong Provincial Engineering Technology Research Center for Healthy Breeding of Important Economic Fish,Guangzhou 510275,China)

机构地区：[1]中山大学生命科学学院/水生经济动物研究所暨广东省水生经济动物良种繁育重点实验室/广东省重要经济鱼类健康养殖工程技术研究中心,广东广州510275

出　　处：《中山大学学报（自然科学版）》2020年第5期144-155,共12页Acta Scientiarum Naturalium Universitatis Sunyatseni

基　　金：现代农业产业技术体系专项资金(CARS-46);广州市科技计划重点项目(201904020043);现代农业人才支撑计划项目(2016-2020)。

摘　　要：为揭示TNF-α受体基因TNF-R1和TNF-R2在尼罗罗非鱼(Oreochromis niloticus)免疫过程的作用,本研究利用RACE技术获得了TNF-R1和TNF-R2的cDNA。结果表明,TNF-R1 cDNA全长2275 bp,ORF区1335bp,编码444个氨基酸;TNF-R2 cDNA全长1719 bp,ORF区1476 bp,编码491个氨基酸。TNF-R1含3个富含半胱氨酸结构域(cysteine-rich domain,CRD)和一个死亡结构域(death domain,DD),而TNF-R2有4个CRD,缺少DD。系统进化分析表明:其TNF-R1和TNF-R2分别与其他脊椎动物TNF-R1和TNF-R2基因同源性很高,在进化树中TNF-R1、TNF-R2各自聚类且两者之间分支明显。实时荧光定量PCR结果表明:TNF-R1和TNF-R2在该鱼体的不同组织中均有表达,其中在鳃、头肾和脾脏中表达水平最高,而在肌肉、垂体中表达量低。用脂多糖(LPS)和多聚肌苷酸-胞苷酸(PolyⅠ:C)分别刺激离体孵育的该鱼头肾和脾脏白细胞可不同程度上调TNF-α、TNF-R1和TNF-R2的表达。无乳链球菌(Streptococcus agalactiae)感染该鱼可显著上调头肾和脾脏中3种基因的表达。本研究对揭示TNF-R1和TNF-R2免疫调控机制,提高罗非鱼健康养殖水平具有一定参考价值。To reveal the role of TNF-αreceptor genes TNF-R1 and TNF-R2 in the Nile tilapis(Oreochromis niloticus)immune processes,we cloned the full-length cDNA sequences of TNF-R1 and TNFR2 from Nile tilapia by RACE.Sequence analysis showed that the full length of TNF-R1 cDNA was 2275 bp,containing an open reading frame of 1335 bp,and encoding a protein with 444 amino acids;the full length of TNF-R2 cDNA was 1719 bp,containing an open reading frame of 1476 bp,and encoding a protein with 491 amino acids.Protein structure prediction showed that TNF-R1 have three cysteinerich domains and an intracellular death domain,while TNF-R2 have four cysteine-rich domains and without death domain.Systematic evolutionary analysis showed that the tilapia TNF-R1 and TNF-R2 were highly homologous with those of other vertebrates.Phylogenetic tree showed that TNF-R1 and TNF-R2 were clustered separately.By qRT-PCR,the highest expression levels of TNF-R1 and TNFR2 mRNA were found in the gill,head kidney and spleen,and the lowest levels were in pituitary and muscle.The expression of TNF-α,TNF-R1 and TNF-R2 genes can be increased to varying degrees by stimulating the tilapia kidneys and spleen white cells incubated with LPS and PolyⅠ:C,respectively.The expressions of three genes in the head,kidney and spleen can be increased after intra-peritoneal injection of Streptococcus agalactiae.This study has some reference value to show the immune regulation mechanisms of TNF-R1 and TNF-R2 to improve the level of healthy breeding of tilapia.

关 键 词：TNF-Α TNF-R1 TNF-R2 罗非鱼 免疫 无乳链球菌 

分 类 号：Q17[生物学—水生生物学]