高耐镉膨胀肾形虫种群金属硫蛋白基因的鉴定与诱导表达

为研究金属硫蛋白(Metallothionein, MT)对生物体抵抗重金属毒性的效应,获得一种具有镉高耐受性的膨胀肾形虫(Colpoda inflata)东北种群,该种群96h最高镉耐受浓度10 mg/L,其金属硫蛋白含量表现出与镉浓度、肾形虫种群增长率存在正相关关系。克隆获得金属硫蛋白Col-MT1基因,对基因序列和氨基酸序列特征分析表明,其为金属硫蛋白基因家族7a亚型的新成员。qRT-PCR实验证实, Col-MT1基因在60h、84h和108h三个时间点对5种浓度镉胁迫均上调表达,与镉浓度之间呈现出一定的剂量-效应关系。其分子调控机制还有待进一步研究。上述结果补充了原生动物MT基因数据库,为进一步揭示C. inflata MT基因的功能,以及应用于镉污染监测和环境修复奠定了基础。

IDENTIFICATION AND INDUCED EXPRESSION OF THE METALLOTHIONEIN GENE OF A HIGH CD-TOLERANCE COLPODA INFLATA POPULATION

Cadmium ranks first among the heavy metal pollutants under limited controll in water. Metallothionein is one of the key proteins that can bind to cadmium and regulate the oxidative stress response of organisms. The protozoan MT has been reported in two ciliates, Tetrahymena and Paramecium. In this study, we obtained a northeast population of Colpoda inflata with high cadmium tolerance. Metallothionein content of C. inflata showed a positive correlation with cadmium concentration and growth rate of population in five gradient concentration of cadmium stress experimental groups from 24—96h. The highest Cd-tolerance concentration was 10 mg/L in 96h. Metallothionein gene of Colpoda inflata was cloned and named Col-MT1. The gene sequence and amino-acid sequence were analyzed. The results showed that Col-MT1 had high homology with the amino acid sequences of other ciliates, and contains two conserved sites XXCXX and XCCX. It was a new member of subtype 7a of metallothionein gene family. TASSER protein model predicted that the secondary structure of Col-MT1 protein was composed of α-helix, β-folding and random crimp, accounting for 67.90%, 11.11% and 20.98%, respectively. SWISS-MODEL predicted that the 3D structure of Col-MT1 protein was the most similar to that of cyanobacteria metallothionein. qRT-PCR experiment confirmed that at 60h, 84h and 108h, the expression of Col-MT1 gene was up-regulated to different concentration cadmium and showed a dose-response relationship with cadmium concentration. The molecular mechanism of gene expression still needs further study. Above results supplemented the MT gene database of protozoa and lay a foundation for revealing the mechanism of C. inflata MT gene. As well as provide reference for monitoring and remediation of cadmium pollution.