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松口蘑菌丝体的分离和RAPD-PCR分析
引用本文:曾东方,罗信昌,傅伟杰.松口蘑菌丝体的分离和RAPD-PCR分析[J].微生物学报,2001,41(3):278-286.
作者姓名:曾东方  罗信昌  傅伟杰
作者单位:1. 华中农业大学应用真菌室
2. 延边大学农学院
基金项目:国家自然科学基金资助项目(39770025)和高等学校博士学科点专项科研基金资助课题(970403)
摘    要:针对松口蘑 Tricholomamatsutake(S .ItoetImai)Sing .]菌丝体分离培养困难和各种相关分离物目前难以用出菇试验鉴定的现实 ,采用 8种培养基配方 ,对 9个不同来源的松口蘑子实体的不同部位及菌根、菌土进行组织分离 ,计接种试管 81 0多支 ,结果从菌褶部位获得 94支慢生型的菌丝体分离菌株 ,从菌柄部位仅获得 1支快生型的菌丝体分离菌株。以马铃薯葡萄糖土壤滤液培养基 (PDAS)、马铃薯葡萄糖麦麸滤液培养基 (PDAW )、BM培养基、马铃薯葡萄糖琼脂培养基 (PDA)对菌褶进行组织分离 ,获慢生型菌丝体的成功率依次为 74.4%、35.5%、156%和 8.9%。以各分离菌株的来源松口蘑子实体和中日两国松口蘑研究者提供的分离菌株作为DNA参照样品 ,对从供试子实体、菌根、菌土进行组织分离获得的各种相关纯培养物进行亲菌鉴定。采用筛选的 1 7个随机引物介导 2 5个供试松口蘑子实体及其分离菌体的RAPD(RandomAmplifiedPolymorphicDNA) PCR反应 ,全部获得了清晰而稳定的DNA指纹图谱 ,结果一致表明 :每个松口蘑子实体的菌盖 (含菌褶…

关 键 词:松口蘑    子实体    分离菌株    亲菌鉴定    RAPD-PCR分析
文章编号:0001-6209(2001)03-0278-09
修稿时间:2000年7月19日

STUDIES ON THE ISOLATION, CULTURE AND DNA IDENTIFICATION OF MYCELIA OF TRICHOLOMA MATSUTAKE
D Zeng,X Luo,W Fu.STUDIES ON THE ISOLATION, CULTURE AND DNA IDENTIFICATION OF MYCELIA OF TRICHOLOMA MATSUTAKE[J].Acta Microbiologica Sinica,2001,41(3):278-286.
Authors:D Zeng  X Luo  W Fu
Institution:Institute of Applied Mycology, Huazhong Agricultural University, Wuhan 430070, China.
Abstract:The tissue isolation for Tricholom matsutake(S Ito et Imai) Sing were made with 8 media in 810 test tubes from different positions of 9 basidiocarps of different source and from mycorrhizae, and soil with the fungi in the studies. The results showed that 94 test tubes of slow-growing mycelia were isolated from lamellae and their success percentages of isolation with media PDAS, PDAW, BM, PDA were 74.4%, 355%, 15.6% and 8.9% respectively. The fast-growing mycelia were easily got from the mycorrhizae and soil related to matsutake. The isolates with different culture characteristics were appraised through DNA fingerprinting comparison with matsutake basidiocarps collected from Jilin province, China and reference isolates presented by matsutake research workers of China and Japan, in which RAPD (Random Amplified Polymorphic DNA)-PCR patterns were sharply prepared using 17 arbitrary decamer nucleotide primers screened. The statistical data indicated that all slow-growing mycelia isolated from lamellae had the same DNA fingerprinting patterns as their origin basidiocarps tissues such as pileus (containing lamellae) and stipe, whose similarity coeffecients all were 1.000, and were therefore identified as true Tricholoma matsutake. However, the fast-growing mycelia or yeast colony were identified as not matsutake. The results suggested that matsutake and its own mycelia have DNA homogeneity, and there exists no any other microbe in the basidiocarps. The results also demonstrated that all matsutake from east China and reference isolates of matsutake from southwest China and Japan were one same species Tricholoma matsutake, whose DNA similarity coeffecients varied from 0.934 to 0.994.
Keywords:Tricholoma matsutake\%  Basidiocarps  Isolates  DNA identification  RAPD\|PCR
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