鱼腥草内生微生物的分布及其发酵液抑菌活性分析

Distribution of Endophytic Microorganisms in Houttuynia cordata and Analysis on Antibacterial Activity of Fermentation Broth

【目的】探明鱼腥草内生菌的类群组成,筛选对幽门螺杆菌和金黄色葡萄球菌具有抑菌活性的菌株,为进一步开发抗生素类药物先导化合物提供物质基础。【方法】采用组织培养法对贵州地区鱼腥草全植株进行内生菌分离培养和纯化,提取内生菌株基因组DNA,应用PCR技术获得细菌16S rDNA和真菌ITS区扩增产物,经测序并进行BLAST比对确定内生菌属。用摇瓶发酵法制备内生菌发酵液,将发酵液用旋转蒸发仪浓缩,通过24孔板抑菌试验检测发酵液对幽门螺杆菌的抑菌活性;通过打孔法测定抑菌圈直径试验检测发酵液对金黄色葡萄球菌的抑菌活性。【结果】分离到内生细菌60株和内生真菌27株。其中,芽胞杆菌属(Bacillus)和刺盘孢属(Colletotrichum)分别为细菌和真菌的优势菌群,分别占内生细菌和内生真菌的78%和41%。检测到9株内生菌(Kg_(3)、Tg_(23)、Kg_(9)、Tg_(3)、Zg_(3)、Kj_(2)、Ty_(9)、Ty_(2)、Zy_(3))发酵液对幽门螺杆菌有抑菌活性,其中内生菌Kg_(3)发酵液对金黄色葡萄球菌也有抑菌活性。【结论】鱼腥草具有丰富的内生菌资源,其中芽胞杆菌属和刺盘孢属的含量较丰富。

【Objective】The group composition of endophytes of Houttuynia cordata was explored,and the strains with antibacterial activity against Helicobacter pylori and Staphylococcus aureus were screened,which provided material foundation for further developing antibiotics lead compound.【Method】The endophytes were isolated,cultured and purified from whole plants of H.cordata in Guizhou by tissue culture method,and the genomic DNA of endophytic strain was extracted,and the amplified products of bacterial 16S rDNA and fungal ITS region were obtained by PCR,finally the endophytes genera were determined by sequencing and BLAST aligning.Endophytes fermentation liquid was prepared by shaking flask fermentation method.The fermentation liquid was concentrated by rotary evaporation apparatus.The antibacterial activity of the fermentation liquid against H.pylori was tested by_(2)4-hole plate antibacterial test.The bacteriostatic activity of fermentation broth against S.aureus was measured by the diameter test of bacteriostatic zone.【Result】A total of 60 strains of endophytic bacteria and 27 strains of endophytic fungi were isolated.Bacillus and Colletotrichum were the dominant groups of bacteria and fungi,accounting for 78%of endophytic bacteria and 41%of endophytic fungi,respectively.9 strains of endophytes(Kg_(3),Tg_(23),Kg_(9),Tg_(3),Zg_(3),Kj_(2),Ty_(9),Ty_(2)and Zy_(3))fermentation broth had antibacterial activity against H.pylori,among which endophyte Kg_(3)fermentation broth had antibacterial activity against S.aureus.【Conclusion】H.cordata has abundant endophytes resources,among which the contents of Bacillus and Colletotrichum in endophytes of H.cordata are relatively rich.