树舌菌株的分离、鉴定及培养研究

【作者】 臧金平；

【导师】 连宾； 袁生；

【作者基本信息】 南京师范大学 ， 微生物学， 2005， 硕士

【摘要】 本文以南京树舌（G.applanatum）子实体及其分离培养菌丝体为试验材料,研究内容包括:树舌子实体多糖提取、纯化及其单糖组分分析,树舌液体发酵培养基及培养条件的筛选,袋栽培养,树舌子实体和树舌菌丝体ITS序列分析与分子鉴定,树舌子实体与菌丝体扫描电镜观察等,主要研究结果如下: 1.经过实地考察,证实南京很多地区（如南京师范大学、紫金山、玄武湖等）均有树舌分布,于南京师范大学金陵女子学院香樟树树干基部采到一树舌子实体,采用组织法进行菌种分离、鉴定和培养。并且采用扫描电镜观察树舌子实体、菌丝体及其担孢子的显微形态,以期为树舌药材鉴定提供一种形象直观的新依据。 2.运用PCR技术扩增了树舌子实体和菌丝体的ITS区段序列,所得序列碱基组成:T为30.2%,C为22.8%,A为21.7%,G为25.3%。将本研究测得序列与GenBank上树舌序列（登录号为AJ608709）进行比对,相似性为99.81%,从而证明分离培养所得到的菌丝体确为树舌菌丝体。与灵芝属（Ganoderma）内各菌种ITS序列进行比较和分析,并作了基于ITS的初步系统学研究,为今后进一步开发利用野生树舌种质新资源奠定基础。 3.进行野生树舌人工驯化栽培试验,经过对比试验,选出较为理想的培养基配方为:棉籽壳78%,麦麸20%,蔗糖1%,石膏粉1%,含水量60%—65%;并对菌丝体进行液体培养条件的研究,由氮源、碳源、温度、转速四个单因子实验所得出的初步试验结果按照正交表L₉（3⁴）设计正交试验,进一步考察多因素综合时对树舌灵芝菌丝体发酵的影响,得到其发酵培养条件的最佳组合为:糖蜜9%,蛋白胨0.4%,温度34℃,转速115r/min。 3.树舌子实体经热水浸提、醇析、脱蛋白等工艺得到粗多糖,经薄层层析法确定其单糖组分为:甘露糖,木糖、葡萄糖等。在本研究中选用子实体和水之比为1:5:试验发现,当浸提液体积一定时,分多次浸提比一次浸提的多糖浸提率高,这与许多文献的报道是一致的:提取过程中长时间的高温浓缩难免会影响所提多糖的活性和增加多糖中的杂质,故提取液低于70℃下旋转蒸发浓缩;当浸提时间多于5h后,大部分多糖已溶出,故无需过长的提取时间,本研究中浸提时间选择在2.5h。本项实验采用Sevage法（氯仿:异戊醇=3:1）进行脱蛋白,更多还原

【Abstract】 This article elaborately studied the identification, analyzing the ITS sequence of Ganoderma applanatum, separating, purification and structural analyzing of polysaccharide from the fruiting body of G applanatum from the City of NanJing, JiangSu Province and its cultured mycelia were studied by means of tissue isolation and culture, which include the result showed as below:1. G applanatum was discovered widely by our investigation in Nanjing, Jiangsu. The fruiting body applied in this study was collected from the lower trunk of camphor in Ginling college, Nanjing normal university. The strain was sepreated, identified and cultivated with tissue separating. The fruiting body, mycelia and basidiospores were observed by scanning electron microscope for providing intuitionistic evidence to identify G applanatum as medicinal materials.2. The internal transcribed spacer (including ITS1, ITS2 and 5.8S rDNA gene) of nuclear ribosomal DNA from the fruiting body and mycelia of G applanatum were amplified by means of PCR technology. The internal transcribed spacer(ITS) of Ganoderma applanatum fruiting body and mycelia were determined. T, C, A and G were 30.2%, 22.8%, 21.7% and 25.3% respectively in these sequences. The comparability between the sequence determined and the sequence in GenBank(access number is AJ608709) is 99.81% by aligment. This indicated the isolated mycelia must be G applanatum. Phylogenetic relationship species among Ganoderma was analyzed elementary based on ITS sequences. It lay foundation for the application of G applanatum from wild source.3. The optimum culture medium of G applanatum fruiting body was 78% cottonseed shell, 20% wheat bran, 1% sucrose, 1% gesso, 60%—65% of water. The optimum liquid fermentation culture medium of G applanatum was 9% molasses and 0.4% peptone and optimum culture conditions of G applanatum was investigated by the orthogonal experiment in three factors and four levels, the optimum temperature was 34℃ and the optimum rotation rate was 115 rpm.4. Polysaccharide was obtained from the fruiting body of Ganodermaapplanatum by extraction with hot water and precipitated by alcohol. The protein in it was removed by sevage method(chloroform:isoamyl alcohol was 3:1). The best ratio of hot water and fruiting body was 5: Land the extraction effect for adding water time after time was better than only adding water once under the condition of same volume water. To protect the activity of polysaccharide, We choosed to extract polysaccharide under 70°C for 2.5 hours once. The monosaccharide compositions of Ganoderma applanatum polysaccharide by means of TLC analysis were xylose, mannose, glucose and et al.更多还原