转基因小麦根际土壤镰刀菌Real-time QPCR方法的建立及应用

摘要

对转基因抗病小麦根际土壤中镰刀菌含量进行绝对定量,建立了实时荧光定量PCR(Real-time QPCR)检测体系.应用该反应体系,对转基因小麦各个生长时期的镰刀菌拷贝数进行绝对定量.结果表明,镰刀菌通用引物具有较好的特异性;Real-time QPCR反应的扩增曲线中各梯度标准质粒的循环阈值间隔均匀,溶解曲线峰值较突出,该标准曲线的相关系数(r)=0.99920,斜率为-3.203,计算其扩增效率为105％,符合荧光定量PCR方法中对各项指标的要求.应用试验结果表明,在转基因小麦的播种前期、灌浆期、成熟期镰刀菌数量较低,在苗期、返青期、拔节期镰刀菌数量较高.%Fusarium sp. is the major group of soil fungi. The effect of transgenic crop on soil rhizosphere microorganism is an important aspect of environmental biosafety research. In this paper, a real-time quantitative PCR detection system for Fusarium was established. The primer showed good specificity, and all indexes met the demands of normal real-time QPCR method. The spacing of cycle threshold of standard substance at each concentration gradient was uniform. The melting curve showed a simple peak, with the correlation coefficient (r) and slope being 0. 999 20 and -3. 203, respectively. The amplification efficiency was 105%. Using this method, the Fusarium in transgenic wheat rhizosphere soil at various growth stages was quantified low at early seeding, grain filling, and maturity, and high at seedling, turning green and jointing stages of wheat.