直接、快速地从罹病小麦叶片中提取条锈菌基因组DNA的方法

Direct DNA Extraction Method of Puccinia striiformis f. sp. tritici from Infected Wheat Leaves

小麦条锈菌(Puccinia striiformis f.sp.tritici)是专性程度很高的活体寄生菌,通过分子技术对其进行群体遗传结构研究的过程中,基因组DNA提取是基础之一。本研究利用Chelex-100法和CTAB法直接提取罹条锈病的小麦病叶片上的小麦条锈病菌的基因组DNA,使用PCR对所得到的DNA进行真菌核糖体rDNA-ITS和小麦条锈病菌特异性引物CYR33进行检测,对2种基因组DNA的提取方法进行比较分析,结果表明Chel-ex-100法和CTAB法都能够适合于从罹病组织中直接提取小麦条锈病菌的基因组DNA,但是Chelex-100法较CTAB法所需的样品量少,操作步骤少,操作简单,并且整个过程无需使用有机溶剂进行抽提,对人体比较安全。通过PCR检测DNA提取情况,发现Chelex-100法提取的DNA的PCR结果优于CTAB法,且能够满足基于PCR的群体遗传结构的分析等研究。

Puccinia striiformis f. sp. tritici is an obligate parasitic fungus. Molecular technology is used to study their population genetic structure and genomic DNA extraction is one of basic methods. In this research, two different DNA extraction methods, Chelex-100 and CTAB, were used to directly extract DNA of P. striiformis f. sp. tritici from infected leaves. The PCR detections were carried out by a race-specific-marker CYR33 of P. striiformis f. sp. tritici and rDNA-ITS. Both extracted DNA meth- ods were compared and analyzed. The results showed that both Chelex-100 and CTAB were fit to ex- tract genomic DNA of P. striiformis f. sp. tritici from wheat leaves infected by wheat stripe rust fungi. But in the progress of DNA extraction, required samples using Chelex-100 was less than using CTAB method, and operation steps of Chelex-100 method was less and simpler than CTAB. And organic sol- vent was not needed to use in the DNA extraction process of Chelex-100. Chelex-100 method was safe to people. Checked the DNA quality by PCR, The results showed that genomic DNA of P. striiformis f. sp. tritici extracted by Chelex-100 method was used to analyze the population genetic structure based on PCR.