Analysis of the mitochondrial and nuclear DNA of basidiomycete heterokaryon.

研究了影响黑木耳和毛木耳菌丝体原生质体分离及原生质体再生和再生的因素。原生质体的分离受酶系统和菌丝龄的影响。用Novozym2341%和几丁质酶0.1%的混合菌丝体培养4天的黑木耳菌丝体和6天培养的黑曲霉菌丝体获得了最高的原生质体转化率；用碘乙酸钠(TAS)和二乙基碳酸酯(DP)两种代谢抑制剂处理黑木耳菌丝体4天和多毛曲霉菌丝体6天，获得了较高的原生质体转化率。从原生质体破碎率和返原率的结果可以看出，每个单核菌株(MoA11和MoP16)的IAS和DP浓度均为4%，最高融合频率的直流脉冲为10/。对275次间隔约为I See，电场强度为3KV/Em的电融合子进行了研究。在单核融合子(MoA11和MoP16)与5个融合子的配对培养中，所有融合子都形成了对MoA11和MoP16的拮抗线。通过同工酶分析，所有5个融合子都具有单核菌株所具有的两条特异性条带。经限制性内切酶分析，确定FU-9的线粒体DNA分子大小为85.8kBP。5株融合子均含有线粒体质粒状DNA，脉冲场电泳法检测到亲本、单核和融合子均有4条染色体DNA，表明5株融合子均为异核体。

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.