内生真菌链格孢菌醋酸乙酯提取物对大肠杆菌抑菌机制的研究  

Antibacterial mechanisms of endophytic fungi Alternaria alternata of ethyl acetate extract on Escherichia coli

作　　者：王永刚[1,4] 杨光瑞[1] 马雪青[2] 王鸣刚[1] 冷非凡[1] 王帆 马建忠[1] 陈吉祥 

机构地区：[1]兰州理工大学生命科学与工程学院,甘肃兰州730050 [2]中国农业科学院兰州兽医研究所,家畜疫病病原生物学国家重点实验室,国家口蹄疫参考实验室,农业部畜禽病毒学重点开放实验室,甘肃兰州730046 [3]中国科学院青岛生物能源与过程研究所,山东青岛266101 [4]兰州理工大学能源与动力工程学院,甘肃兰州730050

出　　处：《中草药》2018年第2期374-381,共8页Chinese Traditional and Herbal Drugs

基　　金：国家自然科学基金资助项目(31460032,8166058t)

摘　　要：目的研究分离自药用植物白毛蛇Humata tyermanni的内生真菌链格孢菌发酵产物醋酸乙酯提取物(B06e)对大肠杆菌的抑菌机制。方法采用倍比稀释法测定了B06e对大肠杆菌的最低抑菌浓度(MIC);测定了B06e作用前后大肠杆菌培养液电导率、核酸和蛋白质的变化;结合流式细胞仪、扫描电子显微镜、凝胶阻滞实验、圆二色谱和荧光实时定量PCR的分析方法研究了B06e对大肠杆菌细胞膜结构、形态、DNA的影响。结果 B06e对大肠杆菌的MIC为25μg/m L,3×MIC处理组的电导率是对照组的1.01倍;3×MIC处理组的β-半乳糖苷酶活性是对照组的11.6倍;流式细胞仪分析表明3×MIC处理组被碘化丙啶(PI)染色的阳性细胞比是对照组的286.5倍;扫描电子显微镜结果显示,B06e处理后菌体表面变得粗糙,细胞膜大量塌陷;以上结果说明B06e能增大细胞膜的通透性,破坏细胞膜的完整性。凝胶阻滞、圆二色谱的结果表明,B06e能够以嵌插的方式与DNA结合,但不能降解DNA;实时定量PCR结果表明,经B06e处理后rec A和rec N基因的表达量分别是对照组的2.9和3.7倍,说明B06e能破坏DNA的结构,迫使大肠杆菌启动了SOS修复。结论 B06e通过破坏大肠杆菌细胞膜完整性和DNA的结构,使得细菌代谢紊乱,最终导致细菌丧失了生长繁殖的能力。Objective To study the antibacterial mechanisms of ethyl acetate extract （B06c） from the fermentation liquid of an endophytic fungus Alternaria spp. Alternaria spp isolated from the medicinal plant Humata tyermanni. Methods Double dilution method was adopted to measure the minimum inhibitory concentration （MIC） of B06e against Escherchia coll. Then, the changes of electric conductivity of bacterial culture, nucleic acid and protein concentration before and after treated by B06e were analysed, respectively. Besides, flow cytometry, scanning electron microscopy, gel retardation experiments, circular dichroism spectrum and Real-time quantitative PCR were introduced to study the antimicrobial mechanisms of B06e against E. coli. Results The results showed that MIC value of B06e against E. coli was 25 μg/mL. The electric conductivity of 3 × MIC treatment group was 1.01 times the value of the control group. The β-galactosidase activity of 3 × MIC treatment group was 11.6 times more than the value of the control group. Flow cytometry analysis showed that PI positive cells ratio of 3 × MIC treatment group was 286.5 times the value of the control group. Scanning electron microscopy showed that cell surface becomes rough after the treatment of B06e, a large number of cell membrane collapse. These results suggested that B06e can increase the permeability of cell membrane, destroy the integrity of cell membrane. The results of gel retardation experiments and circular dichroism spectrum applied that B06e can be inserted into DNA structure at particular position, however, can not cause DNA degradation. Real-time quantitative PCR results showed that the expressions of recA and recN genes were both up-regulated with the values of 2.9 and 3.7 times the value of the control group, respectively. This result suggested that B06e can destroy the DNA structure, which force E. coli to initiate SOS repair. Conclusion B06e can kill E. coli cell by destroying the cell membrane and damaging DNA structure.

关 键 词：大肠杆菌 白毛蛇 内生真菌 链格孢菌 抗菌机制 细胞膜通透性 细胞膜结构 DNA损伤 

分 类 号：R285.5[医药卫生—中药学]