土壤中耐砷细菌的筛选和砷还原基因多样性分析

摘要

采用琼脂平板培养法从湖南富砷土壤中筛选出43株耐砷细菌.16S rRNA序列分析结果表明所筛选菌分属于四个门:Actinobacteria、Proteobacteria、Firmicutes、Bacteroidetes,其中71.1％为革兰氏阳性菌.通过PCR和克隆测序等方法检测耐砷菌的砷还原相关基因(arrA、arsC、arsB/ACR3)及其基因多样性.检测结果显示:43株菌中,6.9％含异化砷还原基因arrA,30.2％含细胞质砷还原基因arsC,27.9％含As (Ⅲ)运载蛋白基因arsB/ACR3,这些基因在细菌中的出现频率较低.通过Mega软件构建系统发育树发现arrA基因的多样性可能受一定的地域差异影响,arsC基因在一些菌株中存在着基因水平转移现象,同时表明a变形菌可能更倾向于拥有Acr3型As(Ⅲ)载体蛋白,而arsB则多出现在芽孢杆菌中.%Forty-three arsenic-resistant bacteria were isolated by agar plates from arsenic-contaminated soil from Hunan Province. The 16S rRNA sequence analysis results showed that these strains belonged to four phyla: Actinobacteria, Proteobacteria, Firmicutes and Bacteroidetes, and 71.1 percent of them were gram-positive bacteria. PCR amplification and sequencing methods were used for detecting the occurrence of some functional genes related to arsenate reduction (arrA, arsC, arsB/ACR3) and the gene diversity. The results showed 6.9% of 43 strains owned arsenate respiratory reductase gene arrA, 30.2% contained cytoplasmic arsenate reductase gene arsC, and 27.9% strains were positive of arsenit transporter gene arsBIACRi. The occurrence of these genes among the strains was in low frequency. Phylogenetic trees constructed by MEGA exhibited the diversity of arrA gene may be influenced by area variation. The horizontal transfer of arsC gene was found in some strains. It also suggested that a-proteobacteria might prefer the arsenite transporter of Acr3, while arsB were mostly present in Bacillus.