褶牡蛎金属硫蛋白基因的克隆及组织表达分析  

Cloning and Expression of Metallothionein Gene in Oyster Ostrea plicatula

作　　者：阎光宇[1,2,3] 孙继鹏[1,3] 易瑞灶 苏永全[2] 

机构地区：[1]国家海洋局第三海洋研究所,福建厦门361005 [2]厦门大学,福建厦门361005 [3]国家海洋局海洋生物资源综合利用工程技术研究中心,福建厦门361005

出　　处：《水产科学》2017年第5期634-641,共8页Fisheries Science

基　　金：厦门海洋研究开发院共建项目(K150104);国家海洋局第三海洋研究所基本科研业务费专项资金资助项目(海三科2013014)

摘　　要：金属硫蛋白是一类广泛存在于生物体中的低分子质量、富含半胱氨酸、高度诱导性的内源金属结合蛋白。采用RACE技术,首次获得了褶牡蛎金属硫蛋白基因的全长cDNA序列。该序列全长500bp,由长54bp的5′非翻译区,122bp的3′非翻译区和324bp的开放阅读框组成,共编码107个氨基酸。该蛋白序列中半胱氨酸含量丰富(28%),不含芳香族氨基酸,富含金属硫蛋白典型的Cys-X(1-3)-Cys结构,存在软体动物等无脊椎动物金属硫蛋白特征序列,是金属硫蛋白家族成员。荧光定量PCR法,测定褶牡蛎体内3种组织(内脏团、鳃、外套膜)中金属硫蛋白mRNA组织特异性表达以及重金属(Cd^(2+)、Zn^(2+))慢毒胁迫效应。结果表明,金属硫蛋白mRNA在褶牡蛎内脏团中相对表达量最高;褶牡蛎内脏团金属硫蛋白mRNA表达量与重金属胁迫时间呈现出一定时间效应关系,从胁迫开始至7d表现为正调,而7~10d开始为负调,Cd^(2+)胁迫和Zn^(2+)联合Cd^(2+)胁迫下金属硫蛋白mRNA表达量显著增加,最大表达量为对照组的42.5倍(Cd),Zn^(2+)联合Cd^(2+)胁迫表现为拮抗作用。本试验结果为生产实践上在多种重金属污染下应用金属硫蛋白作为生物标志物监测水域污染状况,保护水生生态环境提供了依据。A 500 bp full-length cDNA sequence of metallothionein（named as Op-MT）gene as a kind of low molecular weight,rich cysteine,and highly induced endogenous metal binding protein was obtained from oyster Ostrea plicatulausing rapid amplification of cDNA end（RACE）technique,which consists of a54 bp 5′untranslated region（UTR）,a 122 bp 3′UTR and a 324 bp open reading frame（ORF）.The OpMT-clone encodes 107 amino acids,and in the encoded protein,no any aromatic amino acid,was found and the cysteines accounted for 28% of the total amino acid,demonstrating the typical structure with Cys-X（1-3）-Cys type.The predicted protein sequence analysis showed that Op-MT as a member of the metallothionein family is characterized by the conserved features of invertebrate′s MTs.Moreover,real-time PCR showed the differential expression of MT mRNA in the oyster,with the maximum in hepatopancreas under the natural state.Additionally,MT mRNA levels demonstrated an up-regulation in a time-dependent manner due to Cd2＋and Zn2＋＋ Cd2＋exposure during 2—7 day and down-regulated during 7—10 day.MT mRNA had no obvious difference to the control when exposed to zinc and the effect of cadmium and the zinc combining cadmium were significant（P0.001）,and the maximum expression level of MT mRNA appeared when exposed to cadmium（42.5 times for reference）.Zinc exerted antagonistic effects on uptake of cadmium.The findings show MT gene is a potential pollution of cadmium in biological markers and provide a basis for the application of MT in the production practice to monitor the water pollution status and protection of the aquatic environment.

关 键 词：褶牡蛎 金属硫蛋白 基因 组织表达 

分 类 号：S917[农业科学—水产科学]