嗜酸氧化亚铁硫杆菌中金属转运基因的克隆与差异表达

吴学玲，张云静，刘代刚，段 红，范宏伟，刘学端

(中南大学 资源加工与生物工程学院 教育部生物冶金重点实验室，长沙 410083)

摘 要：嗜酸氧化亚铁硫杆菌(Acidithiobacillus ferrooxidans，At. ferrooxidans)广泛应用于铜、锌、锰和镉等金属离子的生物浸出，浸出液中含有大量的金属离子。研究At. ferrooxidans DC菌株在金属离子胁迫下金属转运基因的相对表达，测定At. ferrooxidans DC对Mn²⁺、Zn²⁺和Cd²⁺的最大耐受浓度。应用RT-qPCR技术分析At. ferrooxidans DC中4个金属转运基因(afe_0671、afe_0674、afe_1143和afe_1144)在Mn²⁺、Zn²⁺和Cd²⁺胁迫下的差异表达。利用BLAST等生物学软件对各基因及其编码的蛋白进行生物信息学分析。结果表明：At. ferrooxidans DC菌株对Mn²⁺、Zn²⁺和Cd²⁺的最大耐受浓度分别是0.38、0.18和0.08 mol/L；在Mn²⁺、Zn²⁺和Cd²⁺胁迫下，目标基因的表达均上调，且随着金属离子浓度的增加，上调倍数升高；生物信息学分析表明目标基因编码为与金属转运相关的膜蛋白；在At. ferrooxidans DC中，目标基因编码的蛋白与Mn²⁺、Zn²⁺和Cd²⁺的转运密切相关。

关键词：嗜酸氧化亚铁硫杆菌；金属转运基因；Zn²⁺；Cd²⁺；Mn²⁺；RT-qPCR；差异表达

Clone and differential expression of metal transport genes in Acidithiobacillus ferrooxidans

WU Xue-ling, ZHANG Yun-jing, LIU Dai-gang, DUAN Hong, FAN Hong-wei, LIU Xue-duan

(Key Laboratory of Biometallurgy of Ministry, Education, School of Resources Processing and Bioengineering,
Central South University, Changsha 410083, China)

Abstract:At. ferrooxidans were widely used in bioleaching of copper, zinc, manganese, cadmium, and so on. There were large quantities of metal ions in the bioleaching extract. The relative expression of metal transport genes was investigated when At. ferrooxidans were cultured in the presence of metal ions, the foundation for studying the molecular mechanism of At. ferrooxidans tolerated high concentration of metal ions was established. The tolerance levels of At. ferrooxidans DC to Mn²⁺, Zn²⁺ and Cd²⁺ were determined. RT-qPCR were employed to study the differential expression of four metal transport genes (afe_0671, afe_0674, afe_1143 and afe_1144) in At. ferrooxidans strain DC cultured in the presence of Zn²⁺, Mn²⁺ and Cd²⁺. And then, the target genes and proteins encoded by the four genes were analyzed by BLAST bioinformatics software. The results show that the tolerance levels of At. ferrooxidans DC to the stress of Mn²⁺, Zn²⁺ and Cd²⁺ are 0.38, 0.18 and 0.08 mol/L, respectively. The differential expressions of the target genes increase under the stress of Mn²⁺, Zn²⁺ and Cd²⁺. The bioinformatics analysis shows that the proteins encoded by target genes are membrane proteins involving in metal transport. These results suggest that the proteins encoded by target genes may be involved in Mn²⁺, Zn²⁺ and Cd²⁺ transport in At. ferrooxidans DC.

Key words:At. ferrooxidans; metal tranport genes; Zn²⁺; Mn²⁺; Cd²⁺; RT-qPCR; differential expression