利用PCR扩增快速筛选天麻萌发真菌小菇属Mycena sp.的研究  

Rapid Screening of the Germination Fungus Mycena sp. of Gastrodia elata by PCR Amplification

作　　者：杨志平 秦丽媛 王玉川 陈林[1,3,4] 杨明挚[1] 张汉波[1,4] YANG Zhi-ping;QIN Li-yuan;WANG Yu-chuan;CHEN Lin;YANG Ming-zhi;ZHANG Han-bo(School of Life Sciences,Yunnan University,Kunming 650091,China;Gastrodia Tuber Research Institute of Zhaotong, Zhaotong 657000,China;School of Ecology and Environmental Science,Yunnan University,Kunming 650091,China;State Key Laboratory for Conservation and Utilization of Bio-Resourees in Yunnan,Yunnan University,Kunming 650091,China)

机构地区：[1]云南大学生命科学学院,云南昆明650091 [2]昭通市天麻研究院,云南昭通657000 [3]云南大学生态与环境科学学院,云南昆明650091 [4]云南大学省部共建云南生物资源保护与利用国家重点实验室,云南昆明650091

出　　处：《中国食用菌》2019年第1期51-57,共7页Edible Fungi of China

基　　金：国家自然科学基金(31360153);2015年度昭通天麻共生优良"两菌"选育项目(201501)

摘　　要：设计能够鉴定小菇属真菌(Mycena sp.)的引物,可准确而快速发现萌发菌分离材料,从而分离小菇属真菌,对提高天麻种子萌发率及天麻产量具有重要现实价值。同时对全面了解小菇属真菌在自然环境中的物种分布情况具有一定生态学意义。根据适用于检测"环境DNA"中某物种的引物设计验证框架,运用Primer Premier v5.0软件,在ITS序列区间内设计了适用于小菇属真菌DNA快速检测的5对引物,并对他们的特异性、灵敏度和实际运用能力进行了评估。其中,特异性较好的4对引物分别为M52-2、M22-3、M11-4和BZZ-5。通过对来自3个门、125个属的125株真菌进行特异性测试,能测出的菌株数量分别为2.4%、6.4%、4.0%和7.2%。灵敏度较好的M22-3和BZZ-5两对引物能检测到小菇属真菌菌丝在土壤中的最小浓度均为0.033%,且能成功从天麻原球茎和种子萌发菌塘土壤样本总DNA中检测出小菇属真菌的存在。结果表明,经过利用2对和多对引物,可在实际运用中快速筛选和发现小菇属真菌。It is significant for discovering and locating Mycena sp., accurately and rapidly, by using specific DNA primers. This kind of work is important not only for improving the germination rate and production of the G. elata, but also for understanding the distribution of Mycena sp. in wild. In this study, based on the validation framework for the design of primers for the detection of a species in"environmental DNA"and the use of software Primer Premier v5.0, five pairs of primers within the range of ITS gene were designed and their specificity, sensitivity and practical application ability for rapid detection of Mycena sp. were evaluated. Among them, the four pairs of primers, including M52-2, M22-3, M11-4 and BZZ-5, showed a good specificity and the percentage of targeting on non-Mycena sp. was 2.4%, 6.4%, 4.0% and 7.2%, respectively, when they were used in the test of 125 fungal strains from 125 genus, 3 phyla. Moreover, primers M22-3 and BZZ-5 showed a good sensitivity,with a minimum hyphae concentration of 0.033% when detecting Mycena sp. in the soils. Moreover, they can successfully detect the presence of the Mycena sp. in the total DNA obtained from the protocorms of G. elata and surround soils in the cultivating holes. Our results showed that it could be achieved in practical applications for rapid screening and discovery of Mycena sp. in environments by combination of two and multiple pairs of primers.

关 键 词：天麻萌发菌 兰科植物 ITS基因 灵敏度 快速筛选 

分 类 号：S646.9[农业科学—蔬菜学]