宿根甘蔗根际土壤细菌多样性分析中培养法与非培养法比较研究

Comparison of Culture-dependent and-independent Approaches for Diversity Analysis of Soil Bacteria in the Rhizosphere of Sugarcane

采用培养法和非培养法提取甘蔗根际土壤微生物的总DNA,基于通用引物PCR扩增构建两种方法的细菌16SrDNA文库,并进行核糖体DNA扩增片段限制性内切酶分析(ARDRA),通过部分克隆序列测定构建细菌克隆文库的系统发育树,进而对基于传统的细菌平板培养法和直接提取总细菌DNA的非培养法进行比较分析.结果显示,非培养法文库的香侬–威纳指数、辛普森指数、丰富度分别为4.94、0.998、28.91,均高于培养法文库中相应的多样性参数(分别为4.27、0.996、16.79),均一度数值都>0.95.结合统计学数据和序列测定信息,反映出甘蔗根际土壤存在着丰富的细菌多样性,但平板培养法所展现的多样性低于直接提取法,表明前者存在着很大的局限性,而非培养法文库中主要是未培养微生物的16S rDNA序列,这从一个侧面反映了土壤样品中未培养微生物占很大比例.因此,在土壤微生物群落研究中,必须结合新的分子生态学技术手段,如采用直接提取总DNA的方法进行研究,才能比较全面地认识土壤微生物多样性.

The total DNA of soil microorganisms in the rhizosphere of sugarcane was extracted by the cultivation method or the uncultivation method.Based on general primer PCR,the 16S rDNA library was constructed and the Amplified Ribosomal DNA Restriction Analysis（ARDRA） was conducted.Ultimately the phylogenetic tree was constructed based on the cloning sequences and the comparison between the cultivation and the uncultivation approaches was investigated.For the uncultivation method,the diversity indexes of Shannon-Wienner（H）,Simpson（D） and Richness（R） were 4.94,0.998 and 28.91,respectively,all higher than those of the cultivation method（H： 4.27,D： 0.996,R： 16.79）.The evenness indexes（E） of both approaches were higher than 0.95.With the analysis of ARDRA and sequences of 44 clones,the results revealed that the microbes in the rhizospheric soil of sugarcane had huge structural,functional and genetic diversity.Because of the limitations of the cultivation method,the uncultivation method along with the techniques of molecular ecology would more appropriate for analyzing the diversity of the microbial community in the soil.Fig 3,Tab 3,Ref 18