银杏内生真菌多样性与产黄酮类物质真菌的分离和鉴定

Distribution and Identification of Flavonoid-producing Eendophytic Fungi in Ginkgo biloba L.

采用传统组织培养法从银杏根、叶片和茎中分离内生真菌,以相对频率作为指标分析其多样性,通过显色反应、薄层层析(TLC)、高效液相色谱(HPLC)等方法对内生真菌的发酵产物进行初步分析,筛选能够产黄酮类物质的内生真菌,并通过分光光度法测定总黄酮产量;同时根据菌株的形态学特征,结合真菌r DNA的居间序列(internal transcribed spacer,ITS)的系统进化分析进行菌株鉴定。结果表明:共分离得到217株内生真菌,分属于5纲、8目、10科、13属;经初步筛选,2株分离自根组织的内生真菌GF111和GF521具有产黄酮类化合物的能力,发酵液总黄酮产量分别为(14.50±1.30)mg/L和(21.10±1.30)mg/L;初步鉴定2株产黄酮内生真菌GF111和GF521分别为卷枝毛霉Mucor circinelloides和尖镰孢菌Fusarium oxysporum。可见,内生真菌在银杏不同部位在数量、种群及其组成上存在着较大差异,并且菌株GF111和GF521产黄酮能力较高,可作为生物发酵生产黄酮类物质的备选菌株。本研究为更好地开发和保护银杏资源提供了依据。

In order to reserve the resource of Ginkgo biloba L. and to seek for altematives of flavonoids, the flavonoid- producing endophytes were isolated and selected from the root, stem and leaf samples of Ginkgo biloba. Firstly, endophytic fungus was obtained through the PDA and Martin medium. Secondly, their diversities were analyzed. Thirdly, whether these strains could yield flavonoid were tested by the ways of characteristic color reaction of flavonoid, thin layer chromatography （TLC） and High Performance Liquid Chromatography （HPLC）. In the end, the production of flavonoids was measured by UV-spectrophotometry, and these strains according to the morphological characteristic and the similarity of the nucleotide sequence of internal transcribed spacer （ITS） between rDNAs were identified. The results showed that 217 strains were isolated from the samples, which were classified into 8 orders, 10 families and 13 genera by morphological study. Among the strains, the strains named GF 111 and GF521 were proved to produce flavonoids. The amounts of total flavonoids in GF 111 and GF521 were （14.50 ±1.30） mg/L and （21.10 ± 1.30） mg/L, respectively. The strains named GFlll and GF521 were classified as Mucor circinelloides and Fusarium oxysporum. The results indicated the distribution of the endophytic fungi varied with different parts of Ginkgo biloba. The strains named GF 111 and GF521 had a higher production of flavonoids, so these strains could be used as the alternative fungus for bio-fermentation of flavonoids.