禾本科植物内生真菌研究13禾本科植物内生真菌的分离鉴定及基因组DNA的快速提取Reviewed.docVIP

禾本科植物内生真菌研究1DNA的快速提取 摘要：7省12个不同地区的禾本科植物的内生真菌，经分离、培养后鉴定有Epichlo?属1种和Neotyphodium属5种内生真菌。提取真菌基因组DNA主要是SDS法，但提取步骤繁琐且耗时，难以用于大量样品的处理。本研究采用改良的QS（quick and safe）法进行禾本科植物内生真菌基因组DNA的提取，并通过PCR进行验证。以生长在平板上的菌丝为起始材料，仪器要求简单、操作简便，快速安全、省时省力，整个提取过程大约40 min。新方法和常规SDS法相比，时间缩短到2周，为原来的1/4。之后以采用改良QS法提取的DNA为模板，检测禾本科植物内生真菌中的NRPS基因，并在NCBI上BLAST比对。这种快速提取法所获得的基因组DNA质量高，可用于后续PCR、基因克隆及其它高通量测试，特别适合获取大量真菌菌株基因组DNA时使用。 关键词：禾本科植物内生真菌；DNA的提取；NRPS；PCR扩增；序列比对Grass endophyte researches 13： isolation and rapid extraction of genomic DNA for grass endophytes Abstract: Endophytic fungi were surveyed in 12 different regions of 7 provinces in China, 1 species of Epichlo? and 5 species of Neotyphodium were identified. SDS procedure is mainly used for extraction of fungal genomic DNA. But they are too sophisticated, and time-consuming to deal with large scale samples. In this study, we used improved method for genomic DNA extraction from slow-growing fungal endophytes. The improved QS method was achieved by using plate-grown mycelia as the starting material and using glass rod as a tool for smashing the cell wall. This new method was in virtue of simple equipment, easy operation, rapid and safe extraction, and low cost. The whole extraction process could be accomplished in 40 minutes. It took 2 weeks which was 1/4 of SDS procedures. Then DNA was used as template to detect the NRPS genes of the endophytes. The resulted sequences were blasted in NCBI. The quality of DNA samples from this method could be used for subsequent PCR, cloning, and other high-throughput testing requirements, particularly suitable for a large number of fungal strains. Keyword: Grass endophytes; Extraction of genomic DNA; PCR amplification; Alignment 禾本科植物内生真菌是生活于宿主植物体内的特异性真菌，一般在植物体内完成其生活史的部分或全部但不引起任何病症[1,2]。目前Epichlo?属有11个种，Neotyphodium属有23个种[3,4]。我国地域广阔，禾本科植物资源丰富，从2004年至今，相继有多个新种被报道[3]。Epichlo?属和Neotyphodium属的各种与各自的宿主植物之间具有比较严格的宿主特异性[5]。由于内生真菌对宿主植物具有强烈的生存依赖性，在离体培养条件下菌株的生长非常缓慢，这大大的限制了对其生物学特性的研究。近年来，随着分子生物学技术的发展，内生真菌已经进入了利用其DNA片段进行基因水平的研究