【作者】 张渊；

【导师】 王谦；

【作者基本信息】 河北大学 ， 微生物学， 2004， 硕士

【摘要】 原生质体技术作为食用菌菌种选育的方法之一，具有操作简便、周期短、易筛选等特点，已有不少成功的例子，但在珍稀食药用真菌中的应用还鲜见报道。 本实验以茶薪菇、刺芹侧耳、白灵侧耳这几种珍稀食(药)用真菌为对象，对其原生质体制备及再生的适宜条件进行了研究，并结合紫外线诱变技术开展了珍稀食药用真菌优化菌株的选育工作。 实验结果表明茶薪菇原生质体的最佳制备条件是：用培养5天的菌丝体和2％的溶壁酶或1％纤维素酶与1％蜗牛酶的组合酶，以甘露醇作为渗稳剂，在30℃下酶解3小时。原生质体再生的渗稳剂仍以甘露醇为最佳，原生质体再生率达到了5.5％。 通过对茶薪菇原生质体的制备再生及紫外诱变，分离出大量生长快、长势强的再生菌株，经筛选得到速生高产菌株C304、C280、C1509，分别比出发菌株产量提高了17.9％、14.3％和8.08％，方差分析达到显著差异。同时筛选到一株高抗逆性菌株C11，在对高温的耐受性及对杂菌的抗性方面均优于出发菌株。 通过对刺芹侧耳原生质体再生菌株的筛选，得到速生优质菌株X1，其长速优于出发菌株，达到显著差异，且菇形好，商品性高。同时得到高产菌株X401，比出发菌株产量提高了10％左右。 通过对白灵侧耳原生质体再生菌株的筛选，得到一株高木质素、纤维素降解菌株B203，其木质素酶和纤维素酶活力以及对木质素、纤维素的降解能力均优于出发菌株。其中羧甲基纤维素酶活力在营养生长期和子实体形成期分别是出发菌株的2.2倍和2.47倍，滤纸酶活力分别是出发菌株的1.75倍和1.68倍，漆酶、愈创木酚氧化酶和邻苯二酚氧化酶活力在营养生长期分别是出发菌株的1.54倍、1.21倍和1.25倍；在现蕾期分别是出发菌株的1.49倍、1.81倍和1.15倍，对木质素、纤维素的降解率分别比出发菌株提高了6.87％和5.65％。 经酯酶同工酶分析，所筛选的优化菌株与出发菌株同工酶谱存在差异，说明其遗传基因有所改变，是不同于原出发菌株的变异菌株。更多还原

【Abstract】 As one of breeding method of edible and medicinal fungi, protoplast technology has the characteristics of easy to operate and select,and of being done within short period,ect.There are a lot of successful examples in this aspect,but few in rare edible and medicinal fungi.This experiment was studied on the optimum conditions of protoplast preparation and regeneration in Agrocybe chaxinggu, Pleurotus erygnii, Pleurotus nebrodensis, and the breeding of rare edible fungi by UV induced mutagenesis.The result showed that the optimum protoplast preparation was determined by using mycelia being cultivated for 5 days and 2% driselase or 1% cellulase+1% helicase as lytic enzymes at 25 癈 for 3 hours, annitol as osmotic stabilizer can give higher protoplast yield and regeneration ratio,the protoplast regenerating ratio was about 5.5%.By preparing and regenerating of Agrocybe chaxinggu plotoplast with UV induced mutagenesis method,among a lot of fast and strong growth strains being selected, the strain C304 ,C280 and C1509 showed rapid mycelial growth rate and increased in yield by 17.9 %,14.3% and 8.08% ,the strain Cll showed good endurance to high temperature and mildew, compared with the original strain.By breeding Pleurotus erygnii plotoplast regenerating strains, the strain X1 showed rapid mycelial growth rate and good fruitbody quality, the strain X401 showed increase in yield by 10%, compared with the original strain.By breeding Pleurotus nebrodensis plotoplast regenerating strains.the strain B203 showed high efficiency of degradation of lignin,cellulose whose activities of lignose enzyme and cellulose enzyme are superior to that of the original strain.Co.mpared with the original strain,the activities of CMCase and FPAase of B203 were 2.2 times and 1.75 times higher in mycelial stage , 2.47 times and 1.68 times higher in fruitbody stage, the activities of laccase, guaiacol oxidase and polyphenol oxidase were 1.54 times,!. 21 times and 1.25 times higher in mycelial stage, 1.49 times,, 1.81 times and 1.15 times in fruitbody stage,the degreadate rate of the lignin and cellulose increased by 6.87% and 5.65%.Study on esterase isoenzyme showed that there existed some difference between the esterase isoenzyme pattern of screened strains and that of the original strain. Whichthe original strain ,they were new variant strain.更多还原