Virgibacillus halodenitrificans PDB-F2转运四氢嘧啶类物质的机制及全基因测序分析

发布时间：2018-04-04 01:01

本文选题：中度嗜盐菌　切入点：相容性溶质　出处：《华东理工大学》2017年硕士论文

【摘要】：Virgibacillus halodenitrificans PDB-F2是一株可以在高盐条件下高效降解苯酚的中度嗜盐菌,可用于强化高盐有机废水的生物处理。中度嗜盐菌在遇到外界渗透压变化时,会通过积累相容性溶质来进行耐盐,而目前对于V.halodenitrificans PDB-F2利用相容性溶质进行耐盐的机制研究还十分欠缺。研究该菌利用相容性溶质的耐盐机制,尤其是利用典型相容性溶质的耐盐机制,对于全面阐明该菌的耐盐机制、指导该菌在废水处理中的应用以及为微生物相容性溶质耐盐机制提供基础性研究数据等方面具有重要意义。本研究首先探究了不同相容性溶质对该菌在高盐条件下的渗透保护效果,结果表明不同相容性溶质对该菌的渗透保护作用依次是羟基四氢嘧啶四氢嘧啶脯氨酸海藻糖谷氨酸甜菜碱。利用氢谱核磁共振检测了外源添加相容性溶质时该菌体内积累相容性溶质的情况,结果表明任何一种单一的相容性溶质都不能提供该菌在高盐条件下所需的渗透压,该菌会在体内合成多种相容性溶质进行耐盐,但四氢嘧啶类物质是其积累的主要相容性溶质。利用全基因组测序和基因预测分析技术,鉴定了该菌体内和以上相容性溶质有关的合成基因和转运基因,并对四氢嘧啶类物质的转运基因ehuABCD进行了结构分析。ehuC、ehuD、ehuA三个基因按照同一方向依次串联,与ehuB之间相隔156bp,基因翻译方向为ehuB-ehuC-ehuD-ehuA。利用异源表达功能互补实验进一步验证了ehuABCD和另一个全基因组测序分析鉴定出来的转运基因ehuE的功能,证明了ehuABCD和ehuE基因确实是四氢嘧啶类物质的转运基因。利用高效液相色谱(HPLC)和实时荧光定量PCR技术探究了渗透冲击条件下该菌对四氢嘧啶类物质的表观转运过程和转运基因表达情况。结果发现从8%(w/v)NaCl冲击到12%(w/v)NaCl时,该菌对羟基四氢嘧啶的最大转运速率为7.41 ± 0.37 mg/g细胞干重· min,约是四氢嘧啶最大转运速率(1.65 ± 0.01 mg/g细胞干重· min)的4.5倍;四氢嘧啶转运速率的变化与转运基因表达量的变化趋势一致,都是先增高再降低;盐度对四氢嘧啶的转运过程具有明显的抑制作用(低渗冲击的最大转运速率是高渗冲击时的近20倍);四氢嘧啶的转运过程受到转运蛋白活性和转运基因转录水平的共同调控。
[Abstract]:Virgibacillus halodenitrificans PDB-F2 is a moderately halophilic bacterium that can efficiently degrade phenol under high salt conditions. It can be used to enhance the biological treatment of high salt organic wastewater.When the external osmotic pressure changes, moderately halophilic bacteria will accumulate compatible solutes to carry out salt tolerance. However, the mechanism of V.halodenitrificans PDB-F2 using compatible solutes for salt tolerance is still lacking.In order to elucidate the salt tolerance mechanism of this bacterium, the mechanism of salt tolerance by using compatible solute, especially the salt tolerance mechanism of typical compatible solute, was studied.It is of great significance to direct the application of this bacterium in wastewater treatment and to provide basic research data for the mechanism of salt tolerance of microbial compatibility solute.In this study, we first investigated the osmotic protection effect of different compatibility solutes on the bacteria under high salt condition.The results showed that hydroxytetrahydropyrimidine tetrahydropyrimidine proline trehalose glutamate betaine was the order of osmotic protection of different compatibility solutes.Hydrogen nuclear magnetic resonance (HNMR) was used to detect the accumulation of compatible solute in the bacteria with exogenous compatible solute. The results showed that no single compatible solute could provide the osmotic pressure required by the strain under high salt conditions.A variety of compatible solutes are synthesized in vivo for salt tolerance, but tetrahydropyrimidines are the main compatible solutes.The biosynthesis and transport genes related to solute in vivo and above were identified by whole genome sequencing and gene prediction analysis.The structural analysis of the tetrahydropyrimidine transporter gene ehuABCD was carried out. The three genes were sequentially connected in the same direction and separated from ehuB by 156bp.The direction of gene translation was ehuB-ehuC-ehuD-ehuA.The function of ehuABCD and another transporter gene ehuE identified by whole genome sequencing was further verified by heterologous functional complementation experiment. It was proved that ehuABCD and ehuE genes were indeed the transporter genes of tetrahydropyrimidines.High performance liquid chromatography (HPLC) and real-time fluorescence quantitative PCR (PCR) were used to investigate the apparent transport process and gene expression of tetrahydropyrimidines.The results showed that the maximum transport rate of hydroxy tetrahydropyrimidine from 8%(w/v)NaCl to 12%(w/v)NaCl was 7.41 卤0.37 mg/g cell dry weight, about 4.5 times of that of tetrahydropyrimidine cell line (1.65 卤0. 01 mg/g cell dry weight).The change of the transport rate of tetrahydropyrimidine was consistent with that of the expression of the transporter gene, which increased first and then decreased.Salinity has a significant inhibitory effect on the transport of tetrahydropyrimidine (the maximum transport rate of hypotonic shock is nearly 20 times higher than that of hypertonic shock, and the transport process of tetrahydropyrimidine is regulated by the transporter activity and transporter transcription level.
【学位授予单位】：华东理工大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：X172

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