嗜热四膜虫两类不同金属硫蛋白的功能补偿分析

ANALYSIS OF FUNCTIONAL COMPENSATION OF TWO DIFFERENT METALLOTHIONEIN SUBFAMILIES FROM TETRAHYMENA THERMOPHILA

为了分析嗜热四膜虫两类金属硫蛋白之间的关系,研究分别构建了MTT1-MTT3和MTT2-MTT4的基因敲除载体,通过同源重组获得敲除大核MTT1-MTT3和MTT2-MTT4的两种嗜热四膜虫突变体细胞株△MTT1-MTT3和△MTT2-MTT4。两种突变体细胞株暴露在Cd2+、Cu2+和H2O2的生长表现出显著不同,△MTT1-MTT3突变体细胞对Cd2+的耐受性显著下降,而△MTT2-MTT4突变体细胞对Cu2+和H2O2的耐受性均显著下降。实时荧光定量PCR分析不同突变体中其他MTT基因的表达变化,在△MTT2-MTT4突变体细胞株中,MTT5的表达水平下调,在500μmol/L Cu2+处理后,△MTT2-MTT4突变体细胞中MTT1、MTT3和MTT5表达相对野生型分别上调6.1、9.5和8.5倍。在△MTT1-MTT3突变体细胞中,MTT2、MTT4和MTT5的表达水平下调,当5μmol/L Cd2+处理后,△MTT1-MTT3突变体细胞株MTT5表达水平相对野生型上调2.9倍,而MTT2和MTT4表达水平相对野生型分别下降了4.9倍和2.5倍。结果表明嗜热四膜虫中的金属硫蛋白MTT1、MTT3和MTT5主要参与细胞的重金属解毒功能;而MTT2和MTT4主要参与细胞内正常的新陈代谢功能,不同的金属硫蛋白基因之间的表达存在相互调控和功能补偿。

Metallothioneins in Tetrahymena thermophila were divided into two subfamilies：7a including MTT1, MTT3 and MTT5 and 7b including MTT2 and MTT4. To explore whether these two subfamilies have functional compensation, the MTT1-MTT3 and MTT2-MTT4 gene knockout vectors were constructed. MTT1-MTT3 and MTT2-MTT4 somatic knockout strains were obtained by homologous recombination. We discovered that△MTT1-MTT3 mutant strains were more sensitive to Cd2＋ compared with the wild-type strain, and △MTT2-MTT4 mutant strains were more sensitive to Cu2＋and H2O2 compared with the wild-type strain. The diminished expression of MTT5 was observed in△MTT2-MTT4 strains, and 500μmol/L Cu2＋treatment upregulated the expression of MTT1, MTT3 and MTT5 in△MTT2-MTT4 strains. The expression level of MTT2, MTT4 and MTT5 decreased in △MTT1-MTT3 mutant strains, and the treatment of 5μmol/L Cd2＋ further diminished the expression level of MTT2 and MTT4 by 4.9 and 2.5 times, respectively, but the treatment of 5μmol/L Cd2＋induced the expression of MTT5. These results demonstrated that MTT1, MTT3 and MTT5 were mainly involved in detoxification, MTT2 and MTT4 were mainly participated in normal cellular metabolism. These two different MTT subfamily proteins are interactional and have functional compensation in Tetrahymena.