杂交鳢CmaZAP-70基因的克隆、表达及其功能分析  

Cloning, Expression and Functional Analysis of CmaZAP-70 from Hybrid Snakehead(Channa maculata♀×Channa argus♂)

作　　者：刘付翠 赵飞[1] 谭爱萍[1] 孔璐璐 何山 张瑞泉[1] 邓玉婷 姜兰[1] LIU Fu-Cui;ZHAO Fei;TAN Ai-Ping;KONG Lu-Lu;HE Shan;ZHANG Rui-Quan;DENG Yu-Ting;JIANG Lan(Key Laboratory of Fishery Drug Development,Ministry of Agriculture and Rural Affairs/Key Laboratory of Aquatic Animal Immune Technology of Guangdong Province/Pearl River Fisheries Research Institute,Chinese Academy of Fishery Sciences,Guangzhou 510380,China;College of Fishery and Life Science,Shanghai Ocean University,Shanghai 201306,China)

机构地区：[1]中国水产科学研究院珠江水产研究所/农业农村部渔用药物创制重点实验室/广东省水产动物免疫技术重点实验室,广州510380 [2]上海海洋大学水产与生命学院,上海201306

出　　处：《农业生物技术学报》2019年第11期2052-2062,共11页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金(No.31402338);现代农业产业技术体系专项(No.CARS-46);广东省渔港建设和渔业产业发展专项(No.A201601B07);广东省级鱼病防治专项(粤财农[2014]585号);广东省自然科学基金(No.2015A030310137)

摘　　要：酪氨酸蛋白激酶ZAP-70(70 kD zeta-associated-protein,ZAP-70)是启动T细胞受体(T cell receptor,TCR)信号转导的关键因子,在T细胞增殖、活化过程中发挥重要作用。为探索杂交鳢(Channa maculata♀×Channa argus♂)ZAP-70(CmaZAP-70)在抗菌感染过程中的作用及其在信号转导中的功能,本研究克隆获得CmaZAP-70基因(GenBank No.MK134563)的ORF,分析其对舒伯特气单胞菌(Aeromonas schubertii)和鰤鱼诺卡氏菌(Nocardia seriolae)感染的应答特性,构建真核表达质粒pEGFP-N1-ZAP70,检测其过表达对核因子κB(nuclear factor kappa-B,NF-κB)和活化蛋白1(activating protein 1,AP-1)活性的影响。序列分析显示,CmaZAP-70基因的完整ORF为1818 bp,编码605个氨基酸;CmaZAP-70含两个串联的SH2结构域和1个SH1激酶结构域;与哺乳动物ZAP-70某些磷酸化位点及催化基序具有极高的保守性;进化树中CmaZAP-70与其他鱼类ZAP-70聚为一支,并与欧洲鲈(Dicentrarchus labrax)亲缘性最近。qRT-PCR检测发现,CmaZAP-70在健康杂交鳢所检的10个组织中广泛表达,于脾脏中表达最高,皮肤中表达最低(P<0.05)。在杂交鳢分别感染两种病原菌后,CmaZAP-70在肝脏、脾脏和头肾中的表达主要呈上调趋势;其中感染舒伯特气单胞菌后,CmaZAP-70在肝脏和脾脏于第1天,头肾在第2天达到表达峰值(P<0.05),而感染鰤鱼诺卡氏菌后,均在第3天出现表达上调峰值(P<0.05)。双荧光素酶报告基因结果显示pEGFP-N1-ZAP70的过表达显著增强转录因子NF-κB和AP-1的活性。上述结果表明,CmaZAP-70在防御病原菌感染应答过程中发挥重要作用。本研究为进一步揭示鱼类TCR信号通路提供了理论基础。As a key factor in the initiation of T cell receptor(TCR)signal transduction,tyrosine protein kinase ZAP-70(70 kD zeta-associated-protein)is critical to the proliferation and activation of T cells,contributing to the fight against bacterial infection.However,current studies on ZAP-70 in fish are quite limited.In this study,a series of experiments were conducted to uncover the potential role of ZAP-70 in hybrid snakehead(Channa maculata♀×Channa argus♂)(CmaZAP-70),a feature economic species in China.The complete ORF of CmaZAP-70 gene(GenBank No.MK134563)was cloned and its response to Aeromonas schubertii and Nocardia seriolae infection was analyzed.The structure,homology and phylogenetic tree of CmaZAP-70 sequence were also analyzed.Moreover,the plasmid pEGFP-N1-ZAP70 was constructed to detect the effect of overexpression on the activity of nuclear factor kappa-B(NF-κB)and activating protein 1(AP-1).Sequence analysis showed that the complete ORF of CmaZAP-70 gene was 1818 bp,which encoded 605 aa with relative molecular weight and theoretical point of 68.2 kD and 8.06,respectively.SignalP 4.1 Server analysis showed that CmaZAP-70 was a non-secretory protein without signal peptide,which contained 2 tandem SH2 domains and 1 SH1 kinase domain.In addition,CmaZAP-70 was highly conserved with some phosphorylation sites and catalytic motifs of mammal ZAP-70.Phylogenetic analysis showed that CmaZAP-70 was clustered into 1 group with other teleost and it was clustered closely with Dicentrarchus labrax,which indicated that 2 species had high homology.Moreover,the qRT-PCR results showed that CmaZAP-70 was widely expressed in 10 tissues of healthy hybrid snakehead,with the highest expression in spleen(P<0.05)and relatively highly in the thymus,head kidney and blood,whereas it was expressed at lowest levels in the skin(P<0.05).The expression changes of CmaZAP-70 were similar and mainly up-regulated in the liver,spleen,and head kidney at different time points after infection with the pathogens A.schubertii and N.seriolae,re

关 键 词：杂交鳢 ZAP-70 基因表达 转染 T细胞受体(TCR)信号转导 

分 类 号：S917.4[农业科学—水产科学]