大豆内生细菌种群多样性PCR-DGGE分析方法的优化研究

Optimization of PCR-DGGE diversity analysis of soybean endogenous bacteria

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6 h最为合适,上样量为15μL时可获得高分别率的DGGE条带。

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 pattenas 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 15μL the high-resolution DGGE bands could be achieved. The diversity of soybean endogenous bacterial community was analyzed with a PCR-DGGE method.