苇状羊茅与多年生黑麦草内生真菌菌丝基因组DNA提取的优化及PCR初步检测

Optimization of extraction of mycelial genomic DNA of Neotyphodium coenophialum and N.lolii and their preliminary detection based on PCR

感染内生真菌的禾草在牧草和草坪业上具有重要的生态和经济意义，家畜采食感染Neotyphodium coenophialum和N．lolii的苇状羊茅Festuca arundinacea和多年生黑麦草Lolium perenne会发生中毒。研究收集天津口岸1998年以来进境的部分苇状羊茅和多年生黑麦草种子，对经镜检确认带有内生真菌的种子进行分离培养，对疑似菌株的茵丝用改进的Moiler等方法进行基因组DNA抽提，测定浓度及纯度，对照原方法，DNA的纯度有较大提高，浓度略有上升。Tubulin2基因的引物IS1～IS3扩增结果显示为单一的条带，结合形态学和序列比对，分离培养得到的茵株可以基本确定为N．coenophialum和N．lolii。根据Genbank中N．coenophialum和N．lolii的NC25基因序列设计出引物F1～R1，扩增得到能区分开N．coenophialum和N．lolii的单一条带（相差160bp），建立了N．coenophialum和N．lolii的PCR检测方法，结果准确可靠。

Endophytes-infected grasses have an extraordinary impact on the ecology and economy of pasture and turf. Festuca arundinacea and Lolium perenne infected Neotyphodiurn coenophialurn and N. lolii are toxic to livestock fed on them. N. coenophialurn and N. lolii in the imported grass seeds of Festuca arundinacea and Lolium perenne by Tianjin Port since 1998 were microscopically affirmed to take endophytes and were separated and cultivated. The purity and concentration of the genomic DNA extracted from hyphaes of 12 suspected strains with improved Moller, et al. method was measured by ultraviolet spectroscopy. The purity and the concentration increased slightly in contrast with the Moiler, et al. method. PCR products with primers IS1--IS3 according to Tubulin2 appeared to be a single band respectively through gel electrophoresis, and sequence analysis united with morphologic proofs showed that the suspected strains could be confirmed to be N. coenophialurn and N. lolii re- spectively. The primer F1-R1 of NC25 gene had designed according to sequences from Genbank, and the PCR result showed that N. coenophialurn was evidently different from N. lolii by gel electrophoresis, since the product of N. coenophialum was 160 bp larger than that＇of N. lolii by amplification with the same primer F1-R1. The detection based on PCR is accurate and credible.