Molecular Identification of Gold-Mineralization Indicating Bacteria
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摘要: 近年来, 土壤微生物蜡样芽孢杆菌(Bacillus cereus)芽孢计数对下伏金矿化的指示作用已被不同国家的地质工作者所证实, 但由于鉴别手段的限制, 对金矿化指示菌株的鉴定一直停留在传统的微生物分类鉴定上, 本研究将分子生物学的技术引入到金矿化指示菌株的鉴定中, 从而将微生物找矿中的微生物鉴定手段提高到分子水平.从湖北省嘉鱼县蛇屋山金矿区分离到的矿化指示菌株中随机挑取2株菌Y1和Y2, PCR扩增并测定其16SrDNA和16S-23SrDNA区间序列.经系统发育和生理生化试验综合分析, 表明Y1为蕈状芽孢杆菌(B.mycoides), Y2为蜡样芽孢杆菌, 两者都属于蜡样芽孢杆菌群, 证明蜡样芽孢杆菌群是金矿化指示菌, 同时也证明了传统微生物找矿法利用选择性培养基对蜡样芽孢杆菌群菌芽孢计数方法的可靠性和利用分子生物学技术进行采矿的可行性.菌株16SrDNA和16S-23SrDNA区间核苷酸序列的确定也为下一步微生物找矿中的基因探针试剂盒的制作奠定了基础.
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关键词:
- 16SrDNA /
- 16S-23SrDNA间区 /
- 菌种鉴定 /
- 金矿化指示菌 /
- 微生物找矿
Abstract: Recently the microbial mineral exploration method, spore counting of Bacillus cereus in soil samples which is indicative for blind gold ore deposit, has been confirmed by many geologists in different countries. However, the identification of bacterial strains, indicators for gold mineralization, was still done through the culturing method in previous studies. This paper employed molecular biology technique to microbial identification in mineralized areas. Two bacterial strains as indicators of gold mineralization were randomly isolated from the gold deposit of Shewu mountain in Jiayu county, Hubei Province. On the basis of biochemical and physiological identification, phylogenetic analysis dedrived from 16S rDNA and 16S-23S rDNA sequence, strains Y1 and Y2 were identified as Bacillus mycoides and B. cereus respectively, both of which belong to "B. cereus group". So it is "B. cereus group" that is the indicator for gold mineralization instead of B. ceresus species. The results assured the reliability of the traditional selective culturing method of B. cereus group, and the feasibility of molecular biology techniques on mineral exploration. In addition, 16S rDNA and 16S-23S rDNA sequence analyses were helpful to make fluorogenic-probe reagent box in future work. -
图 1 PCR扩增16SrDNA(a)和ITS(b)的琼脂糖凝胶电泳检测结果
(a)：1.Y1；2.Y2；3.marker；(b)：1.Y1；2.Y2；3.marker
Fig. 1. Gel electrophoresis showing the PCR-amplification products of 16SrDNA (a) and ITS (b)
图 2 基于16SrDNA部分序列的系统发育树
基于16S rDNA部分序列用邻接法完成, 分支上的数字是1 000次重复抽样检测的bootstrap值, 标尺尺度表示10 %的核苷酸取代
Fig. 2. Unrooted phylogenetic tree based on the 16SrDNA sequence of two isolates and sequences of relating species
图 3 基于ITS序列的系统发育树
基于ITS序列用邻接法完成, 分支上的数字是1 000次重复抽样检测的bootstrap值, 标尺尺度表示10 %的核苷酸取代
Fig. 3. Unrooted phylogenetic tree based on the ITS sequence of two isolates and sequences of relating species
表 1 16SrDNA基因序列来源和数据库存取号
Table 1. 16SrDNA sources and accession number
表 2 ITS基因序列来源和数据库存取号
Table 2. ITS sources and accession number
表 3 供试菌株的几项生理生化特性
Table 3. Some physiological characteristics of strains Y1 and Y2
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