【摘要】 应用普通细胞裂解法提取 3株实验菌株 (Escherichia coli DH5α,Staphylococcus aureus SA- 1和 A-grobacterium tumerfaciens 1 31 2 9)的基因组 DNA和应用基于高盐和长时高热的细胞裂解法提取 7种不同土壤样品中的微生物的基因组 DNA,两组不同结构的引物 F3 57GC,R51 8(在正向引物的 5′端有 GC发卡结构 )和 F3 57,R51 8,分别对实验菌株和土壤样品中微生物的 1 6Sr RNA基因 V3区进行扩增 ,均得到了目的片段。比较了不同引物扩增的 1 6S r DNA片段在 DGGE中的不同电泳行为 ,结果表明 ,含 GC发卡结构的PCR扩增产物在 DGGE中能够得到很好的分离 ,而无 GC发卡结构的 PCR产物则不能在 DGGE中获得满意分离。引入 GC发卡结构 ,使得对不同微生物的定性和分类更深入细致更多还原

【Abstract】 As a new DNA fingerprinting technique, denaturing g radient gel electrophoresis (DGGE) was used to analyze the microbial diversity i n different environmental samples, which has given people new ideas of studying the microbial community in soil. This technique is based on the direct extractio n of genomic DNA from soil samples, the amplification of 16S rRNA genes (V3 regi on) by using the specific primers and the separation of the PCR products by DGGE . Three strains (Escherichia coli DH 5α, Staphylococcus aureus SA 1 an d Agroba cterium tumerfaciens 13129) and seven soils sampled in different places and di ff erent depths were used in this study to evaluate the influence of GC clamp on th e result of bacterial diversity by PCR DGGE. The genomic DNA of three strains a nd the soil samples were extracted by a traditional cell lysis method and a cell lysis method based on the high NaCl (1 5mol/L) and long time high temperture (65℃ for 2 hours)respectively. Then, 16S rDNA fragments (16S rRNA gene V3 regio n) were amplified by using two sets of specific primers F 357 GC, R 518 with a GC clamp in 5′end of the forward primer and F 357 , R 518 with out a GC clamp. The PCR products amplified by two sets primers and separated by DGGE respectively. The results showed that the 16S rDNA fragments with a GC cla mp (amplified by primers F 357 GC, R 518 ) could be clearly separated, b ut those DNA fragments without a GC clamp (amplified by primers F 357 , R 518 ) couldn’t be separated well in DGGE. In conclusion, the GC clamp was av ailable for separation of PCR products of the microbial community in DGGE,and i t is more helpful for identification and classification of the microbial communi ty in environmental soil samples.更多还原