【作者】 杨珠英；

【导师】 王剑文； 丁建忠；

【作者基本信息】 苏州大学 ， 微生物与生化药学（专业学位）， 2013， 硕士

【摘要】 竹黄菌（Shiraia bambusicola P. Hennigs）是一种稀有的寄生性药用真菌，其子实体是传统的中药-竹黄资源，近期研究发现竹黄苝醌类有效成分-竹红菌素具有抗炎、抗菌、镇痛、局麻及抗肿瘤的作用，而且已成为抗菌和抗肿瘤的临床光疗药物。但由于野生竹黄采收期短、产量低，竹红菌素、竹黄多糖及其他活性成分的研究和生产都受到了极大的限制。为了综合开发利用竹黄菌，本文开展了竹黄菌的无性系培养，并通过物理（紫外线）和化学（甲基磺酸乙酯）处理技术，对竹黄菌开展诱变育种研究，以期选育得到遗传稳定的竹红菌甲素高产菌株，为竹黄菌的生物技术生产和竹红菌素药物资源的开发提供技术和参考，同时竹黄菌的生物技术培养也有利于对野生竹黄菌的资源保护。本研究首先开展了竹黄菌Shiraia sp. S8菌株培养、菌丝中竹红菌甲素的提取分离和竹黄菌的液体发酵培养，通过筛选和优化了竹黄菌产竹红菌素培养基碳源、氮源及不同培养时间、起始pH值及装液量条件，筛选出优化的培养基和发酵条件，培养基为马铃薯20%，葡萄糖2%，KH2PO40.3%，MgSO40.15%，维生素B10.001%，酵母膏0.5%。最佳发酵条件为：起始pH值为6.0，装液量200mL/500mL，并在培养10d后收获竹黄菌菌丝体。其次开展了竹黄菌Shiraia sp. S8菌株的原生质体形成条件的研究，探索出竹黄菌原生质体形成的最适条件：液体培养菌丝体菌龄50h，5mg/mL纤维素酶和10mg/mL蜗牛酶（终浓度）混合酶系，酶解时间2h，酶解温度30℃。在此条件下原生质体产量可以达到3.24×106个/mL，再生率可以达到3.5%。竹黄菌Shiraia sp. S8菌株原生质体经紫外线（距离15W紫外灯30cm处）照射处理0.5-2min，原生质体再生后经筛选得到一株竹红菌甲素高产诱变菌株C6，其遗传稳定性良好，产量与S8菌株相比提高53.7%，达到28.1mg/L。本研究以S8菌株为出发菌株，用0.5-2.5%甲基磺酸乙酯（EMS）处理竹黄菌悬浮孢子10-30min，结合平板色素圈大小初筛和高效液相色谱（HPLC）分析进行复筛，经诱变筛选得到高产菌株10-5，竹红菌甲素产量达到26.8mg/L，与原始出发菌株提高了46.4%，且经过连续传代5次，生长性状和竹红菌甲素产量稳定。更多还原

【Abstract】 The fruit bodies of Shiraia bambusicola P. Hennigs, a rare parasitic medicinal fungus, havebeen used as a valuable traditional Chinese medicine. Hypocrellins, perylenequinoid pigments inS. bambusicola are of excellent anti-inflammatory, antibacterial, analgesic, local anesthetic, andantitumor activities. Due to its light-induced activities of antitumor, antibiosis, hypocrellin hasbeen applied as a clinical drug in photodynamic therapy. At present, S. bambusicola fruit bodiesare collected from the natural world. The production and quality of hyprocrellins, S. bambusicolapolysaccharide and other active ingredients are greatly influenced by climate, distribution andcollecting time. In order to fully use of S. bambusicola resourses, we cultured the anamorph of S.bambusicola and then bred Shiraia sp. mutant strains with higher yield of hypocrellin A throughphysical (ultraviolet irradiation) and chemical (ethyl methane sulphonate) mutagenesis. This canbe helpful to the production and application of hypocrellins and the protection of wild S.bambusicola resourses.In this research, we cultured Shiraia sp. S8as the start strain, then extracted and purified thehypocrellin A from the mycelia. The liquid fermentation medium of S8strain was screened andthe carbon source and nitrogen source were optimized. The optional medium for the hypocrellinproduction was made up of20%potato,2%glucose,0.3%KH2PO4,0.15%MgSO4,0.001%VB1,0.5%yeast extract, and the initial pH was6.0. The mycelia were harvested in ten days afterinoculation.Then the optimum conditions for protoplast preparation were determined as follows. After50h-cultivation the hypha were collected and digested with the mixing enzymes includingcellulase (5mg/mL) and snailase (10mg/mL) for2h under30°C. The protoplast productionreached3.24×106/mL and the regeneration rate was3.5%.The C6mutant with higher hypocrellin A yield was obtained via ultraviolet irradiation (at adistance of30cm from a15W UV lamp) on the isolated protoplasts. The production of themutant was verified to be genetically stable. The hyprocrellin A yield of mutant C6wasincreased by53.7%than that of the start strain S8, which reached28.1mg/L.Ethyl methane sulphonate (EMS) mutation was also applied to the S8strain for higherproduction of hypocrellin A. EMS at0.5-2.5%was used to treat the suspension spores for10-30min. Through the combined methods with the preliminary screening on the diameter of the redpigment ring and HPLC, the10-5mutant with higher HA yield, which reached26.8mg/L, was obtained and the production was verified to be genetically stable. HA yield by mutant10-5wasincreased by46.4%than that of the original strain.更多还原