应用16S rDNA-RFLP方法分析宁夏地区稻田土壤细菌的多样性

Bacteria diversity in paddy field soil by 16S rDNA-RFLP analysis in Ningxia

水稻是宁夏地区主要粮食作物,水稻种植也具有维持生态系统平衡,防止土地荒漠化等重要的生态功能。而稻田土壤细菌是维持土壤生态功能的基础。但长期以来缺乏对干旱地区稻田土壤细菌多样性的认识。本研究采用非培养技术提取稻田土壤样品总DNA,构建其16S rDNA克隆文库,用PCR-RFLP分析进一步测序后聚类分析细菌群落的多样性。从稻田土样中分离获得了大于23kb的DNA片段。PCR-RFLP共得到74种酶切带型,序列分析发现77.3%的克隆序列与环境中未培养细菌的16S rDNA序列有较高的相似性,仅有22.7%的克隆序列与数据库中可培养细菌有较高的相似性,表明宁夏稻区土壤中的多数细菌尚未培养。系统发育研究发现74个序列分属于12个类群,其中变形细菌所占比例最大(37.8%),依次为酸杆菌(16.2%)、放线菌(12.2%)、拟杆菌(10.8%)、绿屈挠菌(10.8%)、浮游霉菌(8.1%),另外有少量厚壁菌门、芽单胞菌门和疣微菌门细菌克隆。在变形细菌的序列中包括α、β、γ和δ4个类型,比例分别为13.5%、5.4%、12.2%和6.8%。表明宁夏稻区土壤中优势细菌类群为变形杆菌和酸杆菌,且土壤细菌类群具有丰富的多样性。

Rice is one of the most important crops in the Ningxia region of China, and rice planting helps to maintain ecosystem balance and prevent land desertification. Soil microbial diversity provides basic functions for rice field soil ecosystems. To better understand bacterial diversity and community composition in Ningxia paddy soil, the total bacterial DNA was extracted from paddy soil collected from a typical rice field of Ningxia using the culture independent method. A 16S ribosomal DNA （16S rDNA） clone library of soil bacteria was constructed. The 16S rDNA fragments were analyzed by PCR-RFLP. Further sequencing and cluster analysis were conducted to elucidate the bacterial diversity. Over 23 kb DNA fragments were obtained from the paddy soil and 74 MspI restriction endonuclease types were detected by PCR-RFLP analysis. Sequence analysis revealed that 77.3% of clone sequences were similar to those of uncultured bacteria in the environment, while only 22.7% clone sequences were most closely related to those of cultured bacteria in GenBank, suggesting great potential for undeveloped bacterial resources was available in paddy fields. Our phylogenetic analysis found that the sequenced clones fell into 12 major lineages within the domain bacteria. Among them, members of the Proteobacteria were the dominant group, accounting for 37.8%, including α-Proteobacteria （13.5%）, γ-Proteobacteria （12.2%）, δ-Proteobacteria （6.8%） and β-Proteobacteria （5.4%）, followed by Acidobacteria, Actinobacteria, Bacteroidetes, Chloroflexi and Planctomycetes division with 16.2%, 12.2%, 10.8%, 10.8%, 8.1%, respectively. Firmicutes, Gemmatimonadetes, and Verrucomicrobia were less well represented. Our study revealed an extensive diversity of soil bacteria in a paddy field in Ningxia.