【作者】 历丽；

【导师】 夏金兰；

【作者基本信息】 中南大学 ， 生物工程， 2010， 硕士

【摘要】 嗜酸氧化亚铁硫杆菌(Acidithiobacillus ferrooxidans)是一种重要的浸矿功能菌,同时也是目前研究最多的浸矿菌之一。Acidiphilium cryptum是一个浸矿辅助菌,它可以还原Fe(Ⅲ)并利用A. ferrooxidans合成的有机物。通过这种方式,它可以消除这些有机物的抑制和毒害作用并为A. ferrooxidans提供Fe(Ⅱ)作为能源。由于两种菌均生长于酸性矿坑水,环境特殊恶劣,因此能够耐受高浓度的重金属如铜、砷等。Acidiphilium cryptumDX1-1在促进A. ferrooxidans生长的同时是否也对A. ferrooxidans砷抗性相关基因的转录表达也产生一定的影响是本文研究的重点。本文研究了两株菌A. ferrooxidans CMS和Acidiphilium cryptumDXl-1对Cu2+、As3+两种重金属离子的最高耐受能力。结果显示CMS菌对Cu2+的最高耐受浓度高于DX1-1菌,而DX1-1菌对As3+的最高耐受浓度高于CMS菌。实验表明DX1-1菌的加入对CMS菌的亚铁氧化活性以及As3+抗性有显著促进作用。研究了在重金属胁迫下A. ferrooxidans菌株CMS和Acidiphilium cryptumDX1-1单独及混合浸矿作用,结果发现DX1-1菌本身浸矿效果较差,但能够明显提高CMS菌的浸矿效率。重金属离子的加入导致浸矿率下降,且重金属离子浓度越高,浸矿率越低。通过Real-timePCR研究了在不同砷环境中以及Acidiphilium cryptumDX1-1作用下抗砷基因arsH在A. ferrooxidans ATCC23270中的差异表达。结果表明抗砷基因arsH对As3+的胁迫较为敏感,在细菌砷的抗性机制中很可能起着重要作用。单独培养时,随着生长环境中As3+浓度的增加,ATCC23270菌株中的arsH表达量上调。砷离子浓度越高,上调越明显。ATCC23270菌株与DX1-1菌株混合培养时,与单独培养较为相似,随生长环境中As3+浓度的增加,ATCC23270菌株中的arsH基因表达量上调。砷离子浓度越高,上调越明显。但上调倍数低于相同砷离子浓度下ATCC23270菌株单独培养时的上调倍数。DX1-1菌株的加入一定程度上影响了ATCC23270菌株中arsH基因的转录表达。生物信息学分析表明,水平基因转移不仅在同纲不同种的菌株之间发生,也发生在不同纲的种之间。稀释曲线显示环境中仍然存在大量的含arsH基因抗砷细菌有待于发现。arsH基因对微生物砷抗性起重要作用,然而作用机制仍有待于进一步研究证明。同时分析发现含arsH基因抗砷细菌的特殊功能十分广泛,包括感染病原、植物共生、分解高分子聚合物、产叶绿素以及抗重金属。综合以上结果表明抗砷基因arsH在细菌砷抗性中发挥着重要的作用,Acidiphilium cryptumDX1-1能够促进A. ferrooxidans的生长,同时提高砷抗性。这些研究为进一步研究抗砷基因arsH的功能,以及Acidiphilium cryptumDX1-1与A. ferrooxidans的相互作用奠定了基础。更多还原

【Abstract】 Acidithiobacillus ferrooxidans, an important microorganism in bioleaching, is also one of the most studied organisms in the bioleaching microbial community. While Acidiphilium cryptum is an assistant bioleaching bacterium, which can reduce Fe(Ⅲ) and utilize organic matter formed by A. ferrooxidans. In this way, it can eliminate the inhibition and toxicity effect of these organic matters and produce Fe(Ⅱ) as energy for A. ferrooxidans. Because of the rude living environment, they are resistant to many heavy metals including arsenite and copper. Both of them have arsH gene, which is not common and its function is not very clear so far. The aim of this paper is to investigate the influence of Acidiphilium cryptum DX1-1on As resistance gene arsH expression of A. ferrooxidans.In this paper, the tolerance on two heavy metal ions Cu2+、As3+ of A. ferrooxidans CMS and Acidiphilium cryptum DX1-1 was studied. The results showed that strain CMS’s tolerance on Cu2+ was higher than strain DX1-1, while strain DX1-1’s tolerance on As3+ was higher than strain CMS. Meanwhile strain DX1-1 can promote the living activity and As resistance of strain CMS.Bioleaching research of A. ferrooxidans CMS and Acidiphilium cryptum DX1-1 showed that the leaching efficiency of strain DX1-1 was worse than strain CMS, but when mixed culture together the leaching efficiency of strain CMS is much better. Adding heavey metal caused the leaching efficiency decline. And the higher concentration of heavey metal, the lower leaching efficiency.Real-time PCR was used to detect the differential expression of arsH in A. ferrooxidans ATCC23270 in different concentrations of As3+ and differential expression of arsH when culture with Acidiphilium cryptum DX1-1 or not. The results showed that arsH gene expression was sensitive to As3+, and it may have important role in As resistance. The arsH gene expression was enhanced while the As3+ concentration increased. When strain ATCC23270 was cultured with strain DX1-1, the arsH gene expression was lower than that when strain ATCC23270 was cultured itself. To some extent, strain DX1-1 influenced the arsH gene expression of strain ATCC23270.Bioinformatic analysis of the diversity of arsH genes in arsenic-resistant bacteria revealed that the horizontal gene transfer (HGT) is occurred not only among the species within the same phyla but also among the species of different phyla. Rarefaction curves indicate that current efforts on arsenic resistant bacteria isolation and sampling are largely inadequate. Researches indicate that arsH plays an important role in bacteria resistance to arsenite, but there is still much work to do to make clear its exact function. Among these arsH containing species, many have special functions like infecting pathogens, plant symbiosis, macromolecular polymer degradation, chlorophyll production, and heavy metal resistance.All of these results confirmed that arsH plays an important role in As resistance. Acidiphilium cryptum DX1-1 can enhance the living activity and As resistance of A. ferrooxidans. These results lead us to a further step for understanding the mechanism of As resistance in this extremophilic microorganism, and interrelationship of Acidiphilium cryptum DX1-1 and A. ferrooxidans.更多还原