【作者】 许智勇；

【导师】 林芳灿；

【作者基本信息】 华中农业大学 ， 微生物学， 2007， 硕士

【摘要】 以3个不同的金针菇菌株为材料，研究了其担孢子的核相及遗传属性。荧光染色观察显示，担孢子核相以双核为主，双核孢子、单核孢子和无核孢子分别占80.2％、7.5％和12.3％。源于单孢分离物的菌丝为有隔膜、无锁状联合的多核菌丝。研究了3种不同荧光染料对担孢子及其萌发菌丝的适宜染色条件。试验结果显示，担孢子的适宜染色条件为Hoechest33258浓度60μg／mL～80μg／mL、染色时间3min～5min，DAPI浓度6μg／mL、染色时间3min～5min；单孢萌发菌丝的适宜双重染色条件为，先用70μg／mL的Hoechest33258染色4min，再用3μg／mL的FluorescentBrightener28染色4min。对单孢萌发菌丝原生质体化条件进行了初步研究，当使用的溶壁酶浓度为2％，用量为每克菌丝2mL时，综合考虑原生质体产量和再生率，确定单孢萌发菌丝最适宜的酶解温度为30℃，酶解时间为3h。在交配试验中，源于不同菌株单孢分离物的菌丝原生质体的配对形成具锁状联合的菌落，而源于同一单孢分离物的菌丝原生质体的配对则形成无锁状联合的菌落，暗示担孢子中的两个核具有相同的交配型。RAPD分析显示，源于同一单孢分离物的菌丝原生质体为10个随机引物所扩增的图谱彼此完全相同，印证了担孢子中的双核是同质的。此外，观察表明，一个担子上着生有4个担孢子。因此，金针菇是一种具4个含同质双核担孢子的四极性蕈菌。更多还原

【Abstract】 Nuclear phase and genetic attribute of the basidiospores were investigated for 3 strains of Flammutina velutipes. Fluorescence staining observation displayed that the majority of basidiospores were dikaryotic and the ratio of dikaryotic spores, monokaryotic ones and nuclear-free ones was 80.2%, 7.5% and 12.3% respectively. The mycelia derived from single spore isolates were confirmed as polykaryotic septate ones without clamp connection.The suitable dyeing conditions of 3 kinds of different fluorescent dyes for the basidiospore and mycelia were investigated. The results indicated that for basidiospore, the suitable dyeing density of Hoechest33258 was 60μg/mL～80μg/mL when the dyeing time was 3min-5min. Similarly, the suitable dyeing density of DAPI was 6μg/mL when the dyeing time was 3min～5min. The suitable dual dyeing condition of mycelia was that the mycelia was dyed for 4 min by 70μg/mL Hoechst33258, before dyed for 4 min by 3μg/mL Fluorescent Brightener28.The suitable protoplasting conditions were preliminary studied. The results indicated that the suitable enzyme dissolve temperature and time was 30℃and 3h respectively, when the Lywallzyme concentration was 2% and the applied amound was 2mL/1g mucelium.In the mating test, mycelial protoplast pairs between single spore isolates of different strains formed colonies with clamp connection, while mycelial protoplast pairs between the same single spore isolates formed colonies without clamp connection, suggesting that the two nuclei of the spores possessed identical mating type factors.RAPD analysis indicated that the profiles of mycelial protoplasts obtained from same single spore isolates amplified by 10 random primers were entirely identical, corroborating that the two nuclei of a basidiospore were homogeneous.In addition, it is observed that there are 4 basidiospores growing on a basidium.Consequently,F. velutipes is a tetrapolar fungus possessing 4 basidiospores where there are two homogeneous nuclei.更多还原