猴头菌原生质体制备及单核体鉴定研究  

Study on the preparation of protoplast and mononuclear identification of Hericium erinaceus

作　　者：张琪[1] 刘宏伟[2] 陈娟[3] 郭顺星[3] 

机构地区：[1]山东中医药大学药学院,济南250000 [2]中国科学院微生物研究所,北京100101 [3]中国医学科学院北京协和医学院药用植物研究所,北京100193

出　　处：《中国医药生物技术》2015年第2期113-118,共6页Chinese Medicinal Biotechnology

基　　金：国家重点基础研究发展计划(973计划)(2014CB138304)

摘　　要：目的通过优化猴头菌原生质体的制备条件,结合荧光染色鉴定及分子鉴定获得高纯度的单核体,为进一步开展猴头菌基因组水平的研究提供材料。方法对三类酶系统、溶壁酶浓度、无机和有机两类稳渗剂、pH 3.5~6.5、酶解温度20~40℃、培养基成分以及菌龄为48~144 h菌丝的原生质体制备条件进行考察。其中三类酶系统分别为：1.5%的单一酶系统（溶壁酶、纤维素酶、蜗牛酶）,1.5%的纤维素酶和蜗牛酶的混合酶系统,2%的溶壁酶系统;溶壁酶浓度：1%、1.5%、2%、3%、4%;培养基成分的考察：碳源种类筛选及综合培养基中添加成分的筛选。将制得的原生质体稀释后涂板再生及鉴定,以选取单核菌丝。单核菌丝的鉴定方法包括：性状、显微及分子鉴定。其中显微鉴定的观察方法包括一般显微观察和DAPI荧光染色观察;分子鉴定扩增的是单核菌丝的核糖体内转录间隔区（ITS）序列。结果在马铃薯培养基中静置培养72 h的猴头菌丝,用2%的溶壁酶液,以0.6 mol/L KCl为渗透压稳定剂,在p H5.5、温度30℃的条件下酶解3 h,原生质体的释放量最大;再生的单核体经形态及分子生物学鉴定证明是猴头菌。结论通过原生质体制备及再生方法获得的猴头菌的单核菌丝可以为猴头菌活性次生代谢产物相关基因的发掘奠定基础。Objective To obtain the mononuclear mycelia for further genomic research. Methods The preparation conditions of Hericium erinaceus protoplast were studied systematically. These conditions included three types of enzyme systems; concentrations of lywallzyme; inorganic and organic osmotic stabilizers; p H 3.5 ~ 6.5; decomposition temperature between 20 ~ 40 ℃, and medium component and mycelium age. Three types of enzyme systems were 1.5% single enzyme system（lywallzyme, cellulose, snailase）, 1.5% mixed enzyme system consisting of cellulase and snailase and 2% lywallzyme system. The concentrations of lywallzyme were 1%, 1.5%, 2%, 3% and 4%. The medium components included type of carbon source and additive ingredients in synthetic medium. Mycelial age changed from 48 h to 144 h. The protoplast was diluted and inoculated on the regeneration plate. The regenerative mononuclear body is selected by morphological identification, observation of general and fluorescence microscope, and molecular identification by ITS analysis. Results The highest yield of protoplast can be acquired by using the liquid fermentation mycelium which were cultured in potato medium for 72 h. The optimal treatment conditions consisted of 2% lywallzyme in 0.6 mol/L KCl, p H 5.5 and 30 ℃. Conclusion The mononuclear body which is regenerated from protoplast can lay a foundation for genomic study of the Hericium erinaceus secondary metabolism.

关 键 词：猴头菌 原生质体 单核体 荧光染色 

分 类 号：S567.39[农业科学—中草药栽培]