基于PCR-DGGE技术的渤海湾滩涂原油降解菌群分析

Analysis in oil-degrading microbial community in Bohai bay tide-flat area based on PCR-DGGE method

采集天津渤海湾滩涂沉积物,以原油为唯一碳源富集原油降解菌群.采用三种不同细菌DNA提取试剂盒提取原油降解菌总DNA,结果显示New Probe细菌基因组DNA小量提取试剂盒提取出的细菌总DNA质量和纯度最理想.通过对PCR体系中引物量、模板量和PCR程序中退火温度、循环数的优化,确定优化的PCR条件为:25μL Master、3μL DNA模板、1μL Primer-1、1μL Primer-2、20μLdd H_2O.扩增程序为:94℃预变性4 min;94℃变性1 min、55℃退火1 min、72℃延伸1 min,25个循环;72℃延伸6 min.原油降解菌的DGGE指纹图谱分析表明原油降解菌群的群落结构相对稳定,没有出现明显的减弱趋势.

As the sole carbon source, crude oil was used to enrich oil-degrading bacteria from the sediment in Tianjin Habor. Total oil-degrading bacteriaDNA was extracted by three kinds of DNA extraction kits. The results demonstrated that the most ideal quality and purity of total DNA were obtained by New Probe bacterial genome DNA extraction kit. Optimized condition of polymerase chain reaction （ PCR ）, which ensured via the optimization of primer quantity, template quantity, annealing temperature,and cycles in PCR program,was as the following. 25μL Master,3 μL DNA template, 1 μL Primer-1, 1 μL Primer-2,20 μLddH20. PCR amplification contain the initial denaturation at 94 ℃ for 4 min, then 25 cycles were executed： denaturation at 94 ℃ for 1 min,anneal at 55 ℃ for 1 min,elongation at 72 ℃ for lmin. After the 25 cycles,a final elongation was excuted at 72 ℃ for 6min. The analysis to PCR-DGGE fingerprintingof oil-degrading bacteria revealed that the community structure of oil-degrading bacteria was relatively stable and showed no significantly reducing tendency.