【作者】 孙端方；

【导师】 王嘉福； 冉雪琴；

【作者基本信息】 贵州大学 ， 微生物学， 2008， 硕士

【摘要】 从贵州省喀斯特地区生长的数种罗汉松属(Podocrapus)植物中采用组织块法自罗汉松的根、茎、叶等组织中分离到155株内生真菌。其中地上组织的内生真菌种类与地下组织分别占83.87%,16.13%。155株内生真菌经过扩大培养,采用抽提法抽提内生真菌次生代谢产物,通过杯碟法检测其抑菌活性,显示有47株(30.3%)内生真菌有较高的抑菌活性。其中6株(3.87%)内生真菌粗提物在稀释10~2倍后仍表现出显著抑菌活性;另有5株(3.23%)内生真菌对三种受试菌株均有抗性。同样采用抽提法抽提经过扩大培养155株内生真菌的次生代谢产物,选取Vero、BHK-21细胞应用(Methyl Thiazolyl Tetrazolium,MTT)四唑蓝比色法对抽提内生真菌次生代谢产物的活性进行了分析。结果显示28株内生真菌有较高的抑癌活性。对28株抗肿瘤内生真菌进行的薄层层析(Thin LayerChromatography,TLC)和高效液相色谱(High Performance Liquid Chromatography,HPLC)分析结果证明,菌株A2为罗汉松产紫杉醇内生真菌,将其命名为EPTP-1。对EPTP-1进行了形态学研究,EPTP-1的产孢结构、菌落形态符合曲霉属的分类特征。CTAB法提取EPTP-1菌株全基因组,对其核糖体基因居间序列(ITS)进行Blastn分析,Mega 4.0建系统树后显示与烟曲霉同源性99%。经形态学和分子分类学分析鉴定EPTP-1菌株为烟曲霉(Aspergillus fumigatus)。对EPTP-1在PDB培养基中不同时间段的紫杉醇含量进行测定,结果表明培养第5d EPTP-1产紫杉醇的得率最高,紫杉醇产率为0.5578±0.0294mg/L。将EPTP-1菌株的粗提物进行刮板法纯化紫杉醇得到紫杉醇粗纯品,采用荧光染色法(AO/EB)进行细胞凋亡分析,证实所产紫杉醇的活性与紫杉醇标准品一致;浓度为10μg/L～10~3μg/L的紫杉醇粗纯品作用24h就有明显的致Vero、BHK-21细胞凋亡作用,细胞膜呈泡状,细胞从瓶壁大量脱落;并且EPTP-1紫杉醇粗纯品的作用剂量和时间与细胞的凋亡率呈线性增加关系,菌株EPTP-1所产紫杉醇具有明显的抑癌作用。更多还原

【Abstract】 Endophytic fungus is one of the source producing active products benefited for patients during symbiosis with host plants.Endophytic fungus are also one of the source producing active products benefited for patients during symbiosis with host plants,thus,in the plant the fresh fungus secondary metabolite multiplicity as well as other merits cause the present situation which its hopeful solution partial medicine falls short of demand.To protect the resource of plants and relieve the urgent requirement for taxol,a total of 155 isolates of endophytic fungus were isolated from the Podocrapus in Cartagena territory GuiZhou province in this study.Furthermore, we separated an endophytic fungi strain producing-taxol from Podocrapus.It was resulted that the aerial part endophytic fungus is 83.87%of these 155 isolates of endophytic fungus,and the underground part is 16.13%.Moreover,these funguses were cultured amplifty to PDB,and were conservated.Thereby,the antibacterial activity of 155 strains Podocrapus endophytic fungi was tested through antimicrobial experiments in the exterior body at equal pace,the result showed that 47 strains(30.3%of the total isolated endophytic fungus) are able to produce antibacterial active constituents(inhibition zone≥0.5mm),6 strains are antibacterial activity strongly when their extract was diluted to 10~2 times,and 5 strains are antiboisis activity to experimental baeterias overall on this study.Meanwhile,the anti-tumor activity of endophytic fungi strains was detected by Methyl Thiazolyl Tetrazolium(MTT) method.It was confirmed that one strain produced Taxol by ways of thin layer chromatography(TLC) and high performance liquid chromatography(HPLC).This strain was classified by morphology together with similarity of internal transcribed spacer(ITS) sequence by Clustal W method. The activity of anti-tumor of Taxol produced from the strain was detected by Vero cells.A total of 155 endophytic fungi were isolated from the tissue of Podocrapus.The result showed that twenty-eight strains(18.1%) were able to inhibit the growth of Vero and BHK-21 cell(inhibitory ratio≥10%),and seven strains(4.52%) were detected with high activity(inhibitory ratio≥70%).It was confirmed that one of the seven strain A2 produced Taxol by ways of TLC and HPLC.Therefore,we recognized strain A2 as Endophytic fungi Producing Taxol from Podocrapus-1 (EPTP-1).EPTP-1 was classified as Aspergillus fumigates.Taxol extracted from strain EPTP-1 resulted in significant apoptosis of Vero and BHK-21 cells at concentration of 10μg/L for 24h.And the activity of anti-Vero and anti-BHK-21 growth was not significant between the purchased standard taxol and the taxol extracted from strain EPTP-1(P＞0.05,x~2).It suggested that the taxol extracted from strain EPTP-1 was similar with the standard taxol in anti-tumor activity.The distinction of taxol extracted from strain EPTP-1,furthermore,was not significant between Vero cells and BHK-21 cells(P＞0.05,x~2).It suggested also that the taxol extracted from strain EPTP-1 was activity when it treated a variety of cancer cell.The cultured hypha of the strain EPTP-1 was moved to PDB and cultured at 28℃,200rpm.The maximum yield of Taxol was 0.5578±0.0294mg in per liter liquid fungal culture on day 4,thus,we recognized the strain EPTP-1 output of Taxol was 0.56mg/L.The identified endophytic fungi,strain EPTP-1,might be a candidate for Taxol production and benefit to understand the relationship of ecology and pharmacology between endophytic fungi and the host.更多还原