Analysis of the mitochondrial and nuclear DNA of basidiomycete heterokaryon.

担子菌异核体的线粒体和核 DNA 分析。

• 批准号: 02660164
• 负责人: MIURA Kiyoshi
• 金额: $ 1.34万
• 依托单位: Hokkaido University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1990
• 资助国家: 日本
• 起止时间: 1990 至 1991
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-02660164/
• 关键词: basidiomycete; mitochondrial DNA; pulsed field electrophoresis; protoplast; pulse time; chromosome; restriction endonuclease; fusant; プラスミド株DNA; 融合株; 一株菌糸; 核DNA; パルスフィ-ルド; 電気泳動

Some factors affecting the isolation of protoplasts from mycelia and the regeneration and reversion of isolated protoplasts for A. auricula-judae and A. polytricha were investigated. Protoplast isolation was affected by enzyme system and mycelial age. Maximum yield of protoplasts was obtained by a combination of Novozym 234 1%, and chitinase 0.1%, and by using mycelia of 4-day-old culture for A. auricula-judae and that of 6-day-old culture for A. 'polytricha.High protoplast reversion was obtained by using protoplasts isolated from mycelia of 4-day-old culture for A. auricula-judae and that of 6-day-old culture for A. polytricha.In order to select hybrid cells by protoplast fusion, the protoplasts of both monokaryotic strains were treated with two different metabolic inhibitors, iodoacetic acid sodium salt(TAS)and diethylpy rocarbonate(DP). From the results of broken and reversion frequencies of protoplasts, the concentrations of IAS and DP were found to be 4% for each monokaryotic strain(Mo A 11 and Mo P 16).The condition which gave the largest fusion frequency of protoplasts was a DC pulse of 10 /. tsee, 275 times with intervals of approximately I see, and a field strength 3 kv/em.Fusants obtained by electrical protoplast fusion, were investigated in order to clarify their characteristics.In paired cultures between monokaryotic strains(Mo A 11 and Mo P 16)and 5 fusants, all the fusants formed antagonistic lines against both Mo A 11 and Mo P 16.All 5 fusants were found, by analyzing the electrophoretic isozyme, to possess both specific bands which were present in the monokaryotic strains. By using restriction endonuclease analysis, the molecular size of mitochondrial DNA of Fu 9 was confirmed to be 85.8 kbp. All 5 fusants had a mitochondrial plasmid-like DNA.The result of pulsed field electrophoresis indicated that parent, monokaryotic, and fusant strains had 4 chromosomal DNA.From these evidences, it is concluded that all 5 fusants are heterokaryon.

研究了影响木耳和多毛木耳从菌丝中分离原生质体及原生质体再生和还原的因素。原生质体的分离受酶系统和菌丝年龄的影响。以Novozym 234,1 %和几丁质酶0.1%的组合，以及以4日龄木耳菌丝体和6日龄木耳菌丝体为原料，获得了最大的原生质体产量。“polytricha。利用4日龄木耳菌丝和6日龄多毛木耳菌丝分离的原生质体获得了较高的原生质体还原率。为了通过原生质体融合选择杂交细胞，用两种不同的代谢抑制剂碘乙酸钠盐（TAS）和乙酸二乙酯（DP）处理这两种单核菌株的原生质体。从原生质体破碎和还原频率的结果来看，每个单核菌株（Mo a11和Mo p16）的IAS和DP浓度均为4%。原生质体融合频率最高的条件是10 / DC脉冲。看，275次，间隔大约为1次，场强为3kv /em。对原生质体电融合获得的融合体进行了研究，以阐明其特性。在单核菌株（Mo a11和Mo p16）与5个融合体的配对培养中，所有的融合体都对Mo a11和Mo p16形成拮抗系。通过电泳同工酶分析，发现所有5个融合体都具有单核菌株中存在的两个特定带。通过限制性内切酶分析，证实Fu 9线粒体DNA的分子大小为85.8 kbp。所有5个融合体均具有线粒体质粒样DNA。脉冲场电泳结果表明，亲本菌株、单核菌株和融合菌株具有4条染色体DNA。结果表明，5个融合体均为异核体。