太湖地区典型菜地土壤微生物16S rDNA的PCR-RFLP分析

PCR-RFLP analysis of bacterial 16S rDNA from a typical garden soil in Taihu region

土壤微生物多样性是土壤生态功能的基础,但长期以来缺乏对高强度土地利用条件下的土壤微生物多样性的认识。作者采用间接法提取了江苏省太湖地区典型菜地土壤微生物的总DNA,以细菌的通用引物27F和1492R扩增16SrDNA片段,将扩增产物与T-载体酶连,转化大肠杆菌,建立土壤微生物16SrDNA克隆文库。PCR扩增基因文库中插入的16SrDNA外源片段,用两种限制性内切酶HhaI和RsaI分别酶切,获得该土壤173个克隆的酶切指纹图谱。结果表明,HhaI和RsaI联合酶切产生了63个基因分型,文库的覆盖度达76.30%,单一酶切产生的基因分型少,但文库的覆盖度高;克隆文库中存在两种优势类群,分别占总克隆的16%和12%。16SrDNA测序结果表明,太湖地区菜地土壤细菌在分类方面主要属于-和-变形杆菌亚门。以上结果为进一步研究太湖地区菜地土壤微生物生态功能提供了基础资料。

Soil microbial diversity provides basic function of a soil ecosystem. In this study, the total DNA of microorganisms was extracted by an indirect method from a typical garden soil of Taihu region, Jiangsu Province. The 16S rDNAs of the extracted DNA were amplified using bacterial universal primers 27F and 1492R. PCR products were ligated into the pMD 18-T Vector and transformed into Escherichia coli DH5α to construct a 16S rDNA clone library of the soil microbes. A total of 173 clones from the library were screened and their 16S rDNA fragments were reamplified. The PCR products were digested by Rsa I and Hha I, respectively, and their fingerprints were analyzed. The results indicated that the library includes 63 Hha I and Rsa I restriction endonuclease types and the coverage （C value） of the clone library is 76.30%. The number of genotypes digested either by Hha I or Rsa I is only 40 and 27 although it has a high coverage. There were two main restriction types accounting for 16% and 12% of the total 16S rDNA clones, respectively. Phylogenetic analysis suggests that the dominant bacteria in this garden soil belong to α-proteobacteria and γ-proteobacteria.