Abstract: Endophytic fungi were first isolated from surface-sterilized plant tissues of Coptis chinensis using agar-block method. Antibacterial activities of isolated endophytic fungi against Staphylococcus aureus and Escherichia coli were evaluated. After shake flask fermentation, thin layer chromatography (TLC) and high performance liquid chromatography (HPLC) were employed to determine whether the metabolic substances contained berberine or not. Berberine-producing endophytic fungi were screened and identified by morphology and molecular biology methods. These results showed that a total of 12 endophytic fungi were isolated from the tissue of C. chinensis, of which HL-BC strain expressed antibacterial activity against Staphylococcus aureus. TLC and HPLC results indicated that there was berberine in the metabolic substances of HL-BC strain, and the production was 0.664 mg·L^-1. According to the morphological characteristic and 5.8S rDNA-ITS sequence analysis, the HL-BC strain was classified as Phomopsis. Therefore, there was berberine-producing endophytic fungus existing in C. chinensis. This study laid a good foundation for the fermentation production of berberine, also provided reference and theory basis for the integrated development of C. chinensis.

Key words: Endophytic fungi, Coptis chinensis Franch., Berberine

摘要： 为探索黄连中是否存在产小檗碱的内生真菌,采用组织块法对黄连(Coptis chinensis Franch.)中的内生真菌进行分离,以金黄色葡萄球菌(Staphylococcus aureus)和大肠埃希菌(Escherichia coli)为指示菌,用琼脂块法对分离的内生真菌进行抑菌活性试验,经摇瓶发酵培养后,用薄层层析法(TLC)和高效液相色谱法(HPLC)检测其代谢产物中的小檗碱,并对其产量进行分析,采用形态学和分子生物学相结合的方法对产小檗碱内生真菌进行鉴定。结果表明:共分离获得12株内生真菌,其中菌株HL-BC对金黄色葡萄球菌具有较强的抑菌活性,TLC和HPLC检测结果显示,内生真菌HL-BC的发酵液中含有小檗碱,含量为0.664 mg·L^-1,根据形态学特征和5.8S rDNA-ITS序列分析,将其确定为拟茎点霉属(Phomopsis)。这表明黄连中存在能产生小檗碱的内生真菌,为发酵生产小檗碱及黄连的综合开发奠定了理论基础。

关键词: 内生真菌, 黄连, 小檗碱