皱边石杉内生真菌DNA快速有效提取的研究

A Rapid and Effective Method for Isolating Total DNA from Huperzia crispata's Endophyte Fungi Mycelia

为了选择一种省时省力、简单有效的皱边石杉内生真菌菌丝体总DNA提取方法,采用氯化苄法提取其总DNA,对比研究石英砂研磨与超声波振荡2种破壁方式对其DNA的提取效能。结果表明,采用石英砂研磨法提取的DNA,其点样孔附近残余杂质较多、拖尾现象严重;超声波破壁法提取的DNA质量总体较好,DNA条带在23.1kb处集中,多糖、蛋白质等杂质污染少,拖尾现象轻微。比较而言,超声波破壁法可快捷、高效提取皱边石杉内生真菌菌丝体DNA,适于植物内生真菌DNA大规模快速提取,尤其对质地坚硬的菌落较石英砂研磨更有效。提取的DNA经琼脂糖凝胶电泳、ITS序列扩增,表明超声波破壁法提取的DNA与石英砂研磨法一样,均可获得良好的PCR扩增结果。

In order to select a time-saving,simple and efficient total DNA extraction method for Huperzia crispata＇s endophyte fungi mycelia which were isolated from Huperzia crispata.In this study,using benzyl chloride method to isolate total DNA of endophyte fungi and comparing quartz sand grind method with ultrasonic break method two break treatments for endophyte fungi mycelia DNA extraction efficiency.Results showed that,the DNA extraction efficiency and quality obtained by ultrasonic break method was superior to quartz sand grind method.Using quartz sand grind this method,the extracted DNA had more impurity and appeared serious tailing.But using ultrasonic break this method,the extracted DNA were concentrated in v 23.1 kb band,little polysaccharides and proteins were polluted near loading well.Ultrasonic break treatment was a fast,efficient and time-saving method for endophyte fungi mycelia DNA extraction,especially suitable for large sacle DNA extraction.The genomic DNA extracted by this two different break treatments were digested by RNase A and amplified with ITS primers,agarose gel electrophoresis checking results showed that this two different break treatments could obtain good quality PCR effects.But,the ultrasonic break method was a faster and more efficient method for DNA extraction from Huperzia crispata＇s endophyte fungi mecylia than quartz sand grind method,especially for large scale samples and firm texture fungi colony.