一株产紫杉醇罗汉松内生真菌的分离和鉴定

Isolation and identification of a taxol-producing endophytic fungus from Podocrapus

【目的】紫杉醇是重要的抗癌药物,主要从罗汉松等植物中提取,为了保护罗汉松等种质资源,本文从罗汉松植株中分离产紫杉醇内生真菌,并对内生真菌所产紫杉醇的抗肿瘤活性进行了分析。【方法】采用组织块法自罗汉松的根、茎、叶等组织中分离内生真菌;通过四唑蓝(Methyl Thiazolyl Tetrazolium,MTT)比色法筛选有抗肿瘤活性的内生真菌菌株,通过薄层层析(Thin Layer Chromatography,TLC)和高效液相色谱(High Performance Liquid Chromatography,HPLC)对内生真菌所产活性物质进行鉴定;采用抽提法抽提内生真菌所产紫杉醇,应用Vero细胞对抽提的紫杉醇的活性进行了分析。【结果】从罗汉松属(Podocrapus)植物中分离到155株内生真菌,其中28株内生真菌具有较高的抑癌活性。将其中一株菌株A2命名为EPTP-1,经形态学和分子分类学分析鉴定为烟曲霉(Aspergillus fumigatus)。菌株EPTP-1中抽提的紫杉醇5.553μg/L～555.3μg/L作用24h表现出明显的致细胞凋亡作用。菌株EPTP-1发酵5天时紫杉醇的产率为0.5578±0.0294mg/L。【结论】从罗汉松中分离到了一株产紫杉醇内生真菌EPTP-1,可作为紫杉醇类药物工业化生产的候选菌株。

[Objective] Endophytic fungi can produce beneficial active components during symbiosis with host plants. We isolated a taxol-producing endophytic fungus strain from Podocrapus. [Methods] The anti-tumor activity of the endophytic funguswas detected by Methyl Thiazolyl Tetrazolium （MTT） method with Veto cells. The production of taxol by one fungus was confirmed by 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 deduced apoptosis of taxol produced from the strain was detected by fluorescent staining method with Veto cells. [Results] A total of 155 endophytic fungi were isolated from the tissue of Podocrapus. The result showed that 28 strains inhibited the growth of Vero cell （inhibitory ratio ≥ 10%）, and 7 strains had high activity （inhibitory ratio ≥ 70%）. The taxol-producing ability of strain A2 was confirmed by TLC and HPLC. Therefore, we recognized strain A2 as an endophytic fungus capable of producing taxol from Podocrapus-1 and named it EPTP-I. Its output of taxol was 0.56 mg/L when growing in liquid potato dextrose medium. EPTP-1 was classified as Aspergillus fumigates. Taxol extracted from strain EPTP-1 resulted in significant apoptosis of Veto cells at concentration of 5.553 μg/L for 24h. The activity of anti-Veto growth by extracts from strain EPTP-1 was similar to that of the purchased standard taxol （P〉0.05）. [Conclusion] The identified endophytic fungus, strain EPTP-1, can be a candidate for taxol production.