DNA提取方法对盐环境放线菌多样性分析的影响

Evaluation outcome of actinobacteria diversity in saline environment influenced by different DNA extraction methods

【目的】研究DNA提取方法对盐环境中放线菌的16S rDNA RFLP(Restriction Fragment LengthPolymorphism,限制性片段长度多态性)多样性分析结果的影响。【方法】采用CTAB-SDS法、玻璃珠法和反复冻融法3种方法提取焉耆盐场土壤样品DNA,利用放线菌特异性引物扩增16S rDNA并分别构建文库。采用HaeⅢ限制性内切酶对阳性克隆进行RFLP分析,选取不同的克隆进行测序、比对并构建16S rDNA序列系统发育树。【结果】3种DNA提取方法构建的16S rDNA克隆文库OTUs-(Operational Taxonomic Unit)数不同,其中CTAB-SDS法获得35个OTUs,玻璃珠法获得19个OTUs,反复冻融法获得14个OTUs。3种方法中有52%的OTUs序列与已知可培养的放线菌或未发表的克隆子序列相似性较低,可能代表着放线菌新的类群;3种方法得到的放线菌均分布于放线菌纲(Actinobacteria)的放线菌亚纲(Actinobacteridae)、酸微菌亚纲(Acidimicrobidae)和红色杆菌亚纲(Rubrobacteridae)。【结论】DNA提取方法是影响评价放线菌多样性的重要因素。本研究所采用的DNA提取方法各自存在一些优缺点,因此评估盐环境中的微生物多样性时建议采用几种方法组合。而焉耆盐场这一盐环境可能存在丰富的放线菌物种多样性,并蕴藏着新的分类单元有待进一步研究。

[ Objective] To evaluate the influence of DNA extraction methods on the actinobacteria diversity analysis in saline environment via 16S rDNA Restriction Fragment Length Polymorphism. E MethodsJ CTAB-SDS method, glass bead beating method and repeated freezing and thawing method were used to extract total DNA in soil samples from the Yanqi Salten. The 16S rDNA clone libraries were constructed by using the purified 16S rDNA PCR amplicons to transform the E. coli DH5a. The transformants in the library were further analyzed by RFLP. The unique 16S rDNA clones were sequenced and further used for phylogenetic analysis. E Results] Different Operational Taxonomic Units （OTU） were obtained from DNA extracts and total 35 OTUs were obtained from CTAB-SDS method, 19 OTUs from galss bead beating method and 14 OTUs from repeated freezing and thawing methods. Up to 52% OTUs in the three libraries constructed displayed lower similarity with the published sequence, perhaps representing novel taxons. The total OTUs belong to Actinobacteridae, Acidimicrobidae and Rubrobacteridae subclasses. [ Conclusion] DNA extraction methods influence the actinobacterial diversity. Each of the DNA extraction method in our study has some drawbacks and biases, so it is better to use combined DNA extracts from different DNA methods to evaluate the microbial diversity in salty environments.