贵州优质高产茯苓菌株分离纯化筛选及应用研究

【作者】 屈直；

【导师】 刘作易；

【作者基本信息】 贵州大学 ， 微生物学， 2007， 硕士

【摘要】 真菌茯苓(Wolfiporia cocos(Schwein．)Ryvarden et Gilbn．)的干燥菌核，是我国珍贵的传统中药材。茯苓的药用价值和营养价值极高，保健作用独特，经济价值可观，应用前景广阔。本试验对采自贵州的10个野生茯苓菌核和3个人工栽培茯苓菌核进行组织分离，对所得分离菌株进行菌丝形态及荧光核染色显微观察；筛选适合其生长的最适母种、原种培养基。以13个茯苓野生及人工栽培菌核分离株、中国微生物菌种保管委员会收录菌株(6株)以及国内主栽品种(4株)作为试验对象，对其进行菌丝生长初筛、原种培养复筛，筛选出黎13、台17、剑19、剑20号4个优质高产菌株，这4个菌株在野外栽培生产试验中发菌情况良好，采收后测定菌核总重／蔸、结苓个数／蔸、最大个苓重、单蔸最高产量等产量指标，结果发现各项指标均优于对照中5号菌株，结苓率为100％，4株菌株个苓形状规则，肉质纯白，无砂苓，无跑苓现象，结实程度(比重)较好，具有结苓性能好、产量高、质量好等特点。对上述4个优质高产菌株进行遗传特性鉴定，结果显示其遗传稳定。在上述试验基础上建立优质高产菌株筛选模型，为今后茯苓菌株选优及生产研究等提供现实依据。测定供试菌株在木屑培养基上的生长速率及其向木屑培养基中分泌的CMC酶活力，并对两者进行相关性分析，相关系数为0.951977，表明两者间具有一定的相关性。但测定滤纸酶活力为阴性。本试验利用RAPD技术对23株供试菌株进行引物筛选，从41条引物中筛选了17条对供试菌株扩增重复性好、谱带清晰，具有一定多态性的引物，进而对10株外来菌株和贵州道地茯苓菌核分离株进行遗传分析和鉴定，通过数据分析建立无根树，探讨其亲缘关系。RAPD分析结果显示不同来源的茯苓菌株亲缘关系非常相近，但在某些引物的扩增物上存在较小的差异，所得聚类结果与23株菌株形态特征的鉴定结果基本一致。这表明采用RAPD技术对茯苓菌株的聚类分析有助于这些菌株亲缘关系及同源性的研究，可为道地茯苓的检测、鉴定提供分子生物学依据。通过比较两种保藏方法、不同保藏时间茯苓菌种的菌落形态、菌丝体生物量及其液体培养基和固体培养基上的生长规律，发现甘油菌种保藏法和斜面菌种保藏法对茯苓菌种的保藏效果均较理想。更多还原

【Abstract】 The drying sclerotium of fungus Wolfiporia cocos (Wolfiporia cocos(Schwein.)Ryvarden et Gilbn.) is a traditional costful medicine in China. Wolfiporia cocos is promissing because of its high medical value, excellent nutritional value, unique healthy Function and considerable economic value.Organisation separation of sclerotium of wild Wolfiporia cocos from guizhou and Wolfiporia cocos of artificial cultivation were done, micro-morphological observation and observation by fluorescent HOECHST 33258 Stain wrere done in this paper. The optimum medium of female parent and mother seed were screened out in this experiment. We screened out the 4 trains with excellent quality and high yield by primary screeningtest of hyphae growth and secondary screening of protospecies cultivation from 13 trains of wild Wolfiporia cocos from guizhou and Wolfiporia cocos of Artificial cultivation, 6 trains collected by CCTCC and 4 main cultivated trains in our country. In outdoo rexamination cultivation, mycelial germination and growth of the 4 trains is good, the percentage of sclerotium forming from hyphae of wolfiporia cocos is 100%, all target aims were superior to the control strain, No.5. The results of identification for emphytic character showed that emphytic characters were stabile. We propose the screening model of the high yielding and good quality of Wolfiporia cocos on the base of above-mentioned test to offer the realistic basis for the selectiond and production study of Wolfiporia cocos.We determined the growth rate of test organisms on boxwood dust culture medium and the activity of CMCase secreted to boxwood dust culture medium and analyze the correlation between growth rate and activity of CMCase. The experimental results indicated there was positive correlation between growth rate and activity of CMCase (r =0.951977)and the activity of FPA is negative.17 primers with clear bands ,good polymorphism and repeating were screened from 41 primers through analysis of 23 strains by PCR. Furthermore genetic analysis and identification about 10 exotic tuckahoe strains and separation strains of tuckahoe in guizhou were done in this study according to RAPD method, building unrooted tree and analysising of data to analyse genetic relationship primarily. The result show that there are extremely close genetic relationship between wolfiporia cocos from different area. The cluster results were basically in consistence with the identification results of morphological character of 23 trains. This part show the clustering analysisis of wild Wolfiporia cocos is conducive to the research of genetic relationship and nucleotide homology and offer molecular biology evidence for the classification and identification of Wolfiporia cocos.Results of inoculum preservation show that the culture preservation with glycerol and culture preservation with bevel have same perfect effect through comparison of colonial morphology,mycelium biomass and growth rhythm on fluid nutrient medium and solid culture medium. This conclusion accords with results of morphological character identification of 23 strains.更多还原