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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.图3表3参18
The total DNA of soil microorganisms in the rhizosphere soil of sugarcane was extracted by culture and non-culturing methods. The bacterial 16S rDNA libraries of two methods were amplified by universal primer PCR. The restriction endonuclease analysis (ARDRA) The phylogenetic tree of bacterial clone library was constructed by partial cloning sequence analysis, and then the non-culture method based on traditional bacterial plate culture method and direct extraction of total bacterial DNA were compared and analyzed.The results showed that Shannon- Index, Simpson index and richness were 4.94, 0.998 and 28.91, respectively, which were all higher than the corresponding diversity parameters (4.27, 0.996 and 16.79, respectively) in the culture library and the average values were all> 0.95. Combining statistical data and sequence The results showed that there was abundant bacterial diversity in the rhizosphere soil of sugarcane, but the diversity exhibited by the plate culture method was lower than that of the direct extraction method, indicating that the former had great limitations. However, 16S rDNA sequence of uncultured microorganisms, which reflects a large proportion of uncultured microorganisms in soil samples from one side.Therefore, in the study of soil microbial community, New molecular ecology be binding techniques, such as by direct DNA extraction were studied, in order to more comprehensive understanding of soil microbes. FIG. 3 Table 3 Reference 18