大豆内生细菌种群多样性PCR-DGGE分析方法的优化研究

摘要

By screening primers of soybean endogenous bacteria,we obtained V6,V7 and V8 three hypervariable regions from endogenous bacterial 16S rDNA using a nested PCR method.The optimized maps of DGGE band patterns were obtained by changing the DGGE parameters including the denaturing gradient range,electrophoresis time and sample quantity.Through the optimization,968F-1378R was determined as the primer for the soybean endophytic bacteria; the optimal denaturing gradient range was 40％ ～ 60％ ; the optimal electrophoresis time was 6 h with a voltage of 180 V; and when the sample quantity was 15 μ L the high-resolution DGGE bands could be achieved.The diversity of soybean endogenous bacterial community was analyzed with a PCR-DGGE method.%建立了一种适合研究大豆内生细菌种群结构的PCR-DGGE分析方法,筛选适合分析大豆内生细菌的引物,采用巢氏PCR方法获得内生细菌16S rDNA的V6、V7、V8三个高变区,通过优化DGGE技术的变性梯度范围、电泳时间和上样量的大小来获得最佳的DGGE条带图谱.经过优化确定了适合大豆内生细菌研究的引物为968F-1378R,变性剂梯度为40％～60％,且在电压180 V时电泳时间6h最为合适,上样量为15 μL时可获得高分别率的DGGE条带.