兰咏哲,李启瑞,黄劲,赵亮,董宇华,阚雨,王迪,廖万清,康颖倩
LAN Yongzhe,LI Qirui,HUANG Jin,ZHAO Liang,DONG Yuhua,KAN Yu,WANG Di,LIAO Wanqing,KANG Yingqian

• 1:贵州医科大学基础医学院微生物学教研室
• 2:贵州省微生物与人类健康关系研究人才基地&贵州省普通高校病原生物学特色重点实验室
• 3:贵州医科大学
• 4:贵州医科大学基础医学院生物化学与分子生物学教研室

摘要(Abstract)：

目的:了解贵州关岭余甘子内生真菌的多样性,探究余甘子果汁抗氧化及抑菌功能的相关性。方法:取贵州省关岭县10月份野生健康成熟新鲜的余甘子果实,采用研磨法获得余甘子果汁;采用组织分离法对果实进行内生真菌分离,按照菌丝尖端切割法接种至马铃薯葡萄糖琼脂(PDA)培养基纯化3次并保存;取本校真菌室提供的标准菌株,采用Ezup柱式真菌基因组DNA抽提试剂盒提取分离获得的真菌菌株DNA、并通过聚合酶链式反应(PCR)扩增其内转录间隔区(ITS)基因序列后测序,采用分子系统学方法对分离获得的真菌菌株进行鉴定、归类及多样性评估,采用邻接法(N-J)基于ITS序列构建系统发育树;摇床培养提取内生菌发酵液,采用纸片扩散法对发酵液和余甘子果汁进行抑菌活性测试,并绘制维生素C(Vc)标准曲线测定菌株的总抗氧化能力。结果:余甘子果实中分离出内生真菌43株,其中青霉属19株、横梗霉属9株、间座壳属11株、叶点霉属1株、葡萄座腔菌属3株,青霉属为优势属,其中Penicillium chrysogenum分离出15株,数量最多,为优势菌种;ITS序列比对结果显示,分离株43株、标准株5株,构建N-J系统发育树;43株内生真菌对大肠杆菌、金黄色葡萄球菌及白念珠菌均无抑制效果,余甘子果汁对金黄色葡萄球菌和大肠杆菌有较明显效果,但对白念珠菌无抑制效果;Vc标准曲线回归方程为■=0.809 5 x-0.082 8,R~2=0.993 7,经样品测试,43株菌的发酵液总抗氧化能力明显高于空白培养基,相对于Vc的抗氧化能力范围104.76～156.64 mg/L。结论:分离出的余甘子内生真菌中,青霉属为优势菌属,P.chrysogenum为优势菌种;2株内生真菌具有较强的抗氧化作用,分离出的43株内生真菌对金黄色葡萄球菌、大肠杆菌及白色念珠菌均无明显抑制效果。
Objective: To explore the diversity of endophytic fungi from Phyllanthus emblica in Guanling area of Guizhou province and analyze the correlation between the antioxidative activity of P. emblica juice and its inhibitory effect on fungi. Methods: The wild,healthy,ripe and fresh fruit of P. emblica was harvested from Guanling area,Guizhou province in October. P. emblica juice was obtained by grinding its fresh fruit. The endophytic fungi were isolated from the fruit samples by tissue separation method,purified through re-inoculating mycelial tip into potato dextrose agar( PDA)medium three times and then preserved. The standard strains were provided by the fungus laboratory of our school. Fungal genomic DNA was isolated using the Ezup column fungal genomic DNA extraction kit. Internal Transcribed Spacer( ITS) of fungal genomic DNA was amplified by polymerase chain reaction( PCR) and sequenced. Molecular systematics was used to identify,classify and assess the diversity of isolated fungal strains. The phylogenetic tree was constructed based on ITS sequence by Neighbor-Joining method( N-J). The fermentation broth was isolated from endophytic fungi cultured using shaker. The antibacterial activity of the fermentation broth and fruit juice was assayed by disc diffusion method( K-B method). Vitamin C( Vc) standard curve was set to determine the total antioxidant capacity. Results: Forty-three strains of endophytic fungi were isolated from the fruit of P.emblica,including 19 strains of Penicillium,9 strains of Lichtheimia,11 strains of Diaporthe,1 strain of Phyllosticta, and 3 strains of Neofusicoccum. Penicillium was a dominant genus. Penicillium chrysogenum was dominant species since 15 fungi strains were isolated from it. Moreover, ITS sequences were aligned through the NCBI BLAST( https://blast. ncbi. nlm. nih. gov),and N-J phylogenetic tree was constructed by 43 isolated strains and 5 standard strains. The endophytic fungi of43 strains didn't have inhibitory effect on Escherichia coli,Staphylococcus aureus or Candida albicans,while the fruit juice had obvious inhibitory effect on S. aureus and Escherichia coli,but not on Candida albicans. Moreover,the regression equation of Vc standard curve is ^y = 0. 809 5 x-0. 082 8,R2=0. 993 7. The total antioxidant capacity of the fermentation broth of 43 strains was significantly higher than that of the blank control,and antioxidant capacity relative to Vc was ranged from 104. 76 to156. 64 mg/L. Conclusion: Penicillium was a dominant genus and Penicillium chrysogenum was a dominant species. Two endophytic fungi had strong antioxidant effect( P < 0. 05). Endophytic fungi of43 isolated strains had no significant inhibitory effect on S. aureus,E. coli and C. albicans.

关键词(KeyWords)： 余甘子;内生真菌;贵州;多样性;抗氧化;抑菌活性
Phyllanthus emblica;neotyphodium;guizhou;diversity;antioxidation;antibacterial activity

Abstract:

Keywords:

基金项目(Foundation): 国家科技部及教育部“111计划”[国科发专(2019)475];; 贵州省第六批人才基地项目[RCJD2018-22];; 贵州省科技厅社会发展攻关支撑项目[黔科合支撑(2019)2873];贵州省科技厅优秀青年科技人才培养计划项目[黔科合(20175639)];; 贵州省科学技术厅国际科技合作基地[黔科合平台人才(2020)4101]

作者(Author): 兰咏哲,李启瑞,黄劲,赵亮,董宇华,阚雨,王迪,廖万清,康颖倩
LAN Yongzhe,LI Qirui,HUANG Jin,ZHAO Liang,DONG Yuhua,KAN Yu,WANG Di,LIAO Wanqing,KANG Yingqian

DOI: 10.19367/j.cnki.2096-8388.2020.09.004

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