广西柳树桑寄生内生真菌的分离鉴定与抗肿瘤活性菌株筛选

Isolation, identification and antitumor activity of endophytic fungi in Taxilli herba from Salix babylonica in Guangxi

关键词:  柳树, 桑寄生, 内生真菌, 拟盘多毛孢菌, 抗肿瘤

Key words:  Salix babylonica, Taxilli Herba, endophytic fungi, Pestalotiopsis, antitumor

摘要:

该研究从广西钦州市采集健康的柳树桑寄生的根、茎和叶中分离并纯化内生真菌,对真菌进行形态学鉴定,提取内生真菌的DNA,采用真菌ITS序列对内生真菌进行分子鉴定。利用A549和H460细胞作为抗肿瘤活性指示细胞,采用MTT法测定真菌乙酸乙酯提取物的抗肿瘤活性。经过初步分离分析,从柳树桑寄生中纯化出27株内生真菌,经鉴定它们分别属于7个目9个属15个种。拟盘多毛孢属和间座壳属为优势属,其中拟盘多毛孢属全部定植于寄生根; 其次为新壳梭孢属、拟茎点霉属和球座菌属,各分离到3株; 其他包括青霉属、镰刀菌属、炭疽菌属和派伦霉属,各分离到1株。抗肿瘤活性研究表明,有一株与Pestalotiopsis protearum的ITS序列相似性达100%的拟盘多毛孢属菌株Gen24表现有抑制肿瘤细胞A549和H460生长的特性,在真菌乙酸乙酯浸提物浓度为800 μg·mL^-1时,对A549细胞的抑制率达到了56.92%,对H460细胞的抑制率达到了70.11%。该研究结果表明广西柳树桑寄生内生真菌较丰富,在寄主中的分布表现了一定的组织特异性,而且还存在一些具有抗肿瘤活性的菌株及其活性物质可供进一步深入研究。

Abstract:

Taxilli herba, the branches with leaves of T. chinensis(DC.)Danser, is a traditionally used Chinese medicine. T. herba has been used to cure powerless bones and tendons, cancer, virus infection, rheumatic arthralgia and so on. However, the endophytic fungi of T. herba has never been studied previously. In this article, the diversity of endophytic fungi in T. herba from Salix babylonica and their antitumor activity were researched. Endophytic fungi were isolated and purified from healthy stems, leaves and roots of Taxilli herba from Salix babylonica in Qinzhou and Guangxi, China. They were identified by morphological characterization firstly, and then the DNA of endophytes was extracted and subjected to ITS sequence analysis. The antitumor activity of ethyl acetate extracts from eight endophytic fungi was tested by MTT method using cancer cell A549 and H460. After preliminary separation, 27 strains of endophytes were isolated from Taxilli herba, and they belong to seven orders, nine genera and fifteen species respectively. Two strains, jin2 and jin30, share sequence max identity of ≤97% with available ITS sequences in NCBI database, and they formed a independent clade in Phylogenetic tree of ITS-rDNA sequences of existing fungi. Therefore, jin2 and jin30 maybe identified as novel species. Pestalotiopsis and Diaporthe were the preponderant genera [accounting for colonization frequencies(CF)25.9% for each], and all the strains of Pestalotiopsis were isolated from roots. The isolating frequency of Neofusicoccum, Phomopsis and Guignardia were fewer, with three strains for each respectively. Other rare isolated fungi were Penicillium, Fusarium, Colletotrichum and Peyronellaea, with one strain for each. Tissue specificity of endophytes was also observed. For example, Penicillium sp. and Fusarium sp. colonized roots exclusively, Diaporthe arecae, Colletotrichum sp. can only colonized stems, and Guignardia sp. and Peyronellaea sp. only colonized leaves. The antitumor activity showed that one strain, which most related to Pestalotiopsis protearum with ITS sequence similarity at 100%, inhibited the growth of A549 and H460. The ethyl acetate extracts from fungus Gen24 inhibited the growth of A549 and H460 cell at concentration of 800 μg·mL^-1, with the inhibiting rate to A549 reached 56.92%, and the inhibiting rate to H460 reached 70.11%. Our research showed the diversity of endophytes from Taxilli herba, and the antitumor endophytes and compounds can be further studied.