芍药内生真菌的鉴定及产生活性次生代谢产物的评估

Fungal endophytes in Paeonia lactiflora and their secondary metabolites

摘要:

[目的]研究药用植物芍药（Paeonia lactiflora Pall.）内生真菌的种群多样性，同时对其可能存在的聚酮合酶（Polyketide synthase，PKS）和非核糖体多肽合成酶（Non-ribosomal peptide synthetase，NRPS）基因多样性进行评估，预测芍药内生真菌产生活性次生代谢产物的潜力。[方法]采用组织分离法获得芍药根部内生真菌菌株，结合形态学特征和ITS序列分析，进行鉴定；利用兼并性引物对内生真菌中存在的聚酮合酶（PKS）基因和非核糖体多肽合成酶（NRPS）基因进行PCR扩增及序列测定分析，构建系统发育树，明确芍药内真菌PKS基因序列和NRPS基因序列的系统进化地位。[结果]从芍药组织块中共分离得到105株内生分离物，去重复后获得52株内生真菌，菌株ITS基因序列信息显示，52株芍药内生真菌隶属于7目、13科、15属，其中小球腔菌属（Leptosphaeria）、土赤壳属（Ilyonectria）和镰孢属（Fusarium）为优势种群；从52株内生真菌中筛选获得13株含PKS基因片段的菌株，8株含NRPS基因片段的菌株，部分菌株功能基因的氨基酸序列与GenBank中已知化合物的合成序列具有一定的同源性，预示芍药根部内生真菌具有合成丰富多样的次生代谢产物的潜力。[结论]药用植物芍药根部具有丰富的内生真菌资源，且具有产生活性次生代谢产物的潜力，值得进一步开发研究和应用。

Abstract:

[Objective] To explore the biosynthetic potential of endophytic fungi isolated from medicinal plants Paeonia lactiflora, we studied their population diversity and detected richness of their putative polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) gene sequences.[Methods] Culture-dependent method was used to obtain endophytes from the surface-sterilized plant samples. Endophytes were identified based on morphological characteristics and ITS gene phylogenies. PKS and NRPS gene fragments were PCR amplified from genomic DNA with degenerate primers. The biosynthetic potential of endophytes was accessed by bioinformatic and phylogenetic analyses of PKS and NRPS gene sequences.[Results] A total of 105 endophytic isolates were isolated from P. lactiflora root tissues, yielding a total of 52 endophytic fungi through dereplication. The analysis results of endophtytic fungi ITS gene sequences showed that they affiliated to 15 genera of 13 families in seven orders, and Leptosphaeria, Ilyonectria and Fusarium were the predominant genera. Thirteen PKSs and eight NRPSs gene fragments were detected in a total of 52 endophytic fungi. Some fungi amino acid translated fragments were similar to sequences of PKS and NRPS involved in the biosynthesis of the antifungal compound in GenBank. Analysis of the detected endophyte gene fragments afforded consideration of the possible bioactivity of the natural products produced by endophytes from P. lactiflora.[Conclusion] This study demonstrated that the endophytic fungi from P. lactiflora roots had great significant diversity and potential to synthesize bioactive secondary metabolite, therefore, their natural products are worth of further research and development.