脊尾白虾抗细胞凋亡因子DAD1基因的克隆及其组织表达分析  

cDNA cloning and expression analysis of defender against cell death 1 (DAD1) gene in Exopalaemon carinicauda

作　　者：段亚飞[1,2] 刘萍[1] 李吉涛[1] 李健[1] 高保全[1] 陈萍[1] 

机构地区：[1]农业部海洋渔业可持续发展重点实验室,中国水产科学研究院黄海水产研究所,山东青岛266071 [2]中国水产科学研究院南海水产研究所,农业部渔业资源开发利用重点实验室,广东广州510300

出　　处：《中国水产科学》2014年第1期44-52,共9页Journal of Fishery Sciences of China

基　　金：国家863计划项目(2012AA10A409);国家虾产业技术体系项目(CARS-47);公益性行业(农业)科研专项(201103034);中央及公益性科研院所基本科研业务费项目(2013A0701);山东省自主创新专项(2013CXC80202)

摘　　要：取健康脊尾白虾（Exopalaemoncarinicauda）成体，体长（5．81±0．32）cm，体质量（1．18±0．35）g。根据本实验室已构建的脊尾白虾血细胞全长CDNA文库进行EST测序分析，获得脊尾白虾抗细胞凋亡因子DADl基因的cDNA全长并命名为EcDADl基因。该序列全长645bp，包括5非编码区184bp，开放阅读框345bp和3非编码区116bp，共编码114个氨基酸，预测分子量为12．85kD，理论等电点为8．75。同源性分析表明，脊尾白虾抗细胞凋亡因子EcDAD1氨基酸序列与其他物种DAD1有高度同源性，与斑节对虾（Penaeusmonodon）的相似性最高，为92％；与其他无脊椎动物的相似性为73％～80％。系统进化分析表明，脊尾白虾EcDAD1与斑节对虾的DAD1紧密聚为一支。荧光定量PCR分析结果表明，EcDAD1基因在脊尾白虾血细胞、鳃、肝胰腺、肌肉、卵巢、肠、胃和眼柄中均有表达，其中卵巢中的相对表达量最高。感染鳗弧菌（Vibrioanguillarum）和WSSV后6h和12h，脊尾白虾血细胞和肝胰腺中EcDAD1的表达量较对照组均显著增加（P〈0．01），且不同时间点间差异显著，表明EcDAD1基因可能在脊尾白虾抵抗病原体入侵机体的过程中具有重要作用。The full-length eDNA of defender against cell death 1 （DAD1） gene was first isolated and characterized from the hemocyte eDNA library of Exopalaemon carinicauda, which was named EcDAD1. The full length eDNA sequence of EcDAD1 is 645 bp, which contains a 184 bp 5-UTR （untranslated region）, 116 bp Y-UTR, and 345 bp open reading frame （ORF） that encodes 114 amnio-acid polypeptides, which has the molecular mass of 12.85 kD and isoeleetric point （pI） of 8.75. Homology analysis revealed that the amnio acid sequences of EeDAD1 had highly identifies with DAD1 of other species, for example, it had 92% identity with DAD1 ofPenaeus monodon, and had 73%-80% identifies with DAD1 of other invertebrates. The phylogenetic analysis showed that E. carinicauda EcDAD1 was in the same branch as that ofP monodon. The expression level ofEcDAD1 gene in different tissues were analyzed by quantitative real-time PCR. The results showed that EcDAD1 existed in all tested tissues of E. carinicauda, including hemoeytes, gill, hepa- topancreas, muscle, ovary, intestine, stomach and eye stalk, and the the highest expression level ofEcDAD1 gene was in ovary. Six hours and 12 h after challenge by Vibrio anguillarum and white spot syndrome virus （WSSV）, the relative expression level of EcDAD1 was up-regulated extremely significantly （P〈0.01） in the hemocytes and hepatopanereas of E. carinicauda. The results implied that EcDADI might play an important role in the prawn immune system.

关 键 词：脊尾白虾 抗细胞凋亡因子(DAD1) 基因克隆 基因表达 

分 类 号：Q959[生物学—动物学] S917[农业科学—水产科学]