棘托竹荪提取液对单增李斯特菌的抑菌机理初步研究

Preliminary research on the antimicrobial mechanism of Dictyophora echinovolvata extracts against Listeria monocytogenes

在细胞超微结构观察的基础上,采用实时荧光定量PCR技术初步分析棘托竹荪提取液对单增李斯特菌的抑菌机理。使用最小抑菌浓度(15.0mg/m L)的棘托竹荪提取液分别对单增李斯特菌菌液进行0.5、1.0、1.5h处理,提取对照组与处理组菌株的总RNA,通过反转录获得各自c DNA,并选择相关基因合成引物作Real-time PCR分析。以本菌的16S r RNA作为内参基因,以prf A、Act A、Iap为转录过程与表达蛋白基因,分析提取液对单增李斯特菌相关基因表达量的影响。由细胞超微结构观察可知,单增李斯特菌经提取液处理后,菌体细胞的细胞壁变薄,细胞膜的完整性发生改变,内容物从胞内释放出来。由荧光定量PCR法对不同处理时间下的相关基因相对表达量变化情况分析得出:处理组的prf A基因在0.5h时表达量有所上调,后期表达量上调速度减慢。1h后表达量基本停止上调,1.5h后,表达量转为下调,说明提取液能在转录阶段对单增李斯特菌的生命活动构成影响。Act A和Iap基因在处理0.5h后基因表达量上调,处理1h后上调量最大,Act A基因在1.5h后表达量上调量有所降低,Iap基因表达量出现下调,说明提取液能在转录水平上调控单增李斯特菌的生命活动,特别是多种毒力因子的表达过程。由此可知,棘托竹荪提取液能对单增李斯特菌细胞的转录过程和抗性产生显著影响,使菌体的细胞膜受损,细胞质溶出,从而导致菌体细胞死亡。

Based on the observation of cell ultrastructure,the method of real-time fluorescent quantitative PCR was used to evaluate the antimicrobial mechanism of Dictyophora echinovolvata extracts against Listeria monocytogenes. The strain was treated with 15.0mg/mL（minimum inhibitory concentration,MIC） of Dictyophora echinovolvata extracts under 0.5,1.0 and 1.5h. Total RNA of the control and treated group strains were extracted, cDNA were obtained by reverse transcription and the relevant gene primers were selected for Real-time PCR method respectively. The strains of 16S rRNA as reference gene,prfA^ActA and lap as feature genes. It was showed that the cell wall become thinner,the integrity of cell membrane was changed and the contents released from intracellular when treated with Dictyophora echinovolvata extracts by the ultrastructure of cell observations. Compared with the control group,the level of gene expression in prfA was up-regulated at 0.5h, the speed of up-regulated became slower at lh,the trend of up-regulation stopped at 1.5h with the prolong of treatment time,which proved that extracts could affect the activities of Listeria monocytogenes in transcription. While the level of gene expression in ActA and lap were up-regulated in 0.5h,the amount of up-regulated expression was increased fast at lh. The level of gene expression in ActA was decreased at 1.5h and that in lap was down-regulated,which showed that extracts could regulate the level of transcription in Listeria monocytogenes,especially on the expression of virulence factors. Dictyophora echinovolvata extracts had a significant impact on Listeria monocytogenes for the process of transcription and the resistant of bacteria,which could make the damage of cell membrane and the dissolution of cytoplasm leading to the death of cell.