对虾养殖体系中细菌群体感应抑制菌株的筛选及其特性初步研究

Preliminary study of screening and characteristics of strains with Quorum sensing inhibition activity in shrimp culture environment

  • 摘要: 从对虾养殖环境及肠道内筛选能够抑制细菌群体感应的活性菌株,并探讨了影响菌株活性的环境因素及其对病原坎氏弧菌(Vibrio campbellii)毒力基因表达的影响。采用滤纸片法和高通量法对分离出的纯化菌株进行筛选,获得活性菌株。评估不同培养时间和盐度对活性菌株降解N-酰基高丝氨酸内酯类(AHLs)信号分子能力的影响,并检测活性菌株粗提物对坎氏弧菌毒力基因toxRtlh mRNA表达的影响。分离得到2株活性菌株DC13和RS5,经16S rRNA基因序列分析,初步鉴定为枯草芽孢杆菌(Bacillus subtilis)和巨大芽孢杆菌(B. megaterium)。AHLs信号分子对活性菌株生长无显著性影响(P>0.05)。菌株DC13培养24 h后降解AHLs信号分子能力达到最大值,菌株RS5培养12~48 h降解AHLs信号分子能力无显著变化。盐度10~30时对菌株DC13降解AHLs信号分子能力无显著性影响,但是对菌株RS5降解AHLs信号分子能力影响显著(P<0.05)。活性菌株粗提物的添加使坎氏弧菌毒力基因toxR的表达上调,而毒力基因tlh的表达下调。

     

    Abstract: We screened the active strains with Quorum sensing inhibition activity in shrimp gut and their culture environment, to investigate the environmental factors that affect the activity of strains and their effects on the virulence gene expression of Vibrio campbellii. The methods of filter paper and high-throughput were applied to screen active strains. The effects of AHLs signal molecule on active strains growth with different culture time and salinities were estimated. The effects of crude extract products of active strains on the virulence genes toxR and tlh mRNA expression levels of the pathogenic bacteria were also investigated. Two active strains named as DC13 and RS5 were obtained and preliminarily identified as Bacillus subtilis and B.megaterium by 16S rRNA gene sequence analysis. No significant difference was observed in the active strains growth treated with AHLs signaling molecule (P>0.05). The AHLs signal molecule degradation ability of strain DC13 reached the maximum value at 24th hour. However, after a 12—48 h treatment, no significant difference was observed in AHLs signal molecule degradation ability by strain RS5. When the strains were cultured at salinity of 10–30, there was no significant difference in AHLs signal molecule degradation ability by strain DC13, while significant difference was observed by AHLs signal molecule degradation ability of strain RS5 (P<0.05). It is demonstrated that addition of crude extract products of active strains can increase virulence gene toxR expression but decrease virulence gene tlh expression level.

     

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