产苦马豆素疯草内生真菌实时荧光定量PCR检测方法的建立

Establishment of Real-time Fluorescence Quantitative PCR Detection Method for Swainsonine-producing Endophytic Fungi in Locoweed

根据疯草内生真菌苦马豆素合成基因簇中swnK基因KS区域设计引物,构建SYGR Green I实时荧光定量PCR检测方法,初步分析黄花棘豆植物样品中内生真菌生物量与苦马豆素含量的关系。结果表明,基于swnK基因KS区域构建的实时荧光定量PCR法特异性较好,灵敏性较高,当真菌起始DNA浓度在0.009~90ng/μL范围时,内生真菌起始DNA浓度的对数值与Ct值呈负相关,内生真菌DNA的最低检出限为0.009ng/μL。在此基础上对黄花棘豆内生真菌生物量的分析表明,苦马豆素含量与内生真菌生物量呈正比。用于疯草内生真菌生物量检测时,基于swnK基因KS区域构建的实时荧光定量PCR方法特异性要高于基于ITS序列构建的方法,其灵敏度、检测限与后者相当,可用于疯草中产苦马豆素内生真菌的定量分析。

The primers were designed based on the KS sequence of swnK gene in swainsonine biosynthesis gene cluster of locoweed endophytic fungus,and a real-time PCR based on SYBR-Green I fluorescence of KS was established for quantitative testing of fungal endophytes from locoweeds.The relationship between the amount of the fungi and concentration of swainsonine in a species of locoweed,Oxytropis ochrocephala,were analyzed by the above-mentioned methods.The results indicated that the real-time fluorescent quantitative PCR method based on the KS sequence of the swnK gene had good specificity and sensitivity.When the initial DNA concentration of fungi was in the range of 0.009~90ng/μL,the logarithm of initial DNA concentration of endophytic fungi was negatively correlated with Ct value,and the minimum detection limit of endophytic fungal DNA was 0.009ng/μL.On this basis,the analysis of endophytic fungi amounts of O.ochrocephala showed that swainsonine content was in direct proportion to the endophyte fungi amounts.The specificity of the real-time fluorescent quantitative PCR method constructed based on KS sequence of the swnK gene was higher than that based on ITS sequence reported method.There was no significant difference in sensitivity and detection limit of two methods.The real-time fluorescent quantitative PCR based on KS sequence of swainsonine-producing endophytic fungi in locoweed.