植物乳杆菌MarR家族新转录因子在氧胁迫中调控机制的研究

The loss of vitality and activity caused by oxidative stress in lactic acid bacteria (LAB) is the main challenge in industrial application of LAB. Transcription factors play key roles in the sensing of reactive oxygen species signals and activating of the protection systems against oxidative stress in bacteria. However, few studies have been carried out to investigate the functional role of transcription factors in response to oxidative stress in LAB. Notably, our previous study has shown that the inactivation of marR-1, a gene encoding a MarR family oxidation-sensing transcription factor, leads to the reduction in resistance to H2O2 of Lactobacillus plantarum. The result suggests the involvements of MarR family transcription factors in oxidative stress resistance in LAB. Therefore, the following experiments will be carried out to investigate the regulation mechanism of novel transcription factors of MarR family in L. plantarum CAUH2 in response to oxidative stress. Firstly, the L. plantarum CAUH2 mutants of MarR_1, MarR_2 and MarR_3 will be constructed by using homologous recombination, and the oxidative stress tolerance of mutants will be evaluated to confirm the involvement of MarR transcription factors in oxidative stress resistance. Then, bacterial one-hybrid and EMSA will be employed to determine the target genes regulated by individual MarR transcription factors, and their regulation models will be revealed by the difference in expression levels of target genes between L. plantarum CAUH2 wild type strain and the mutants. Furthermore, the functional role of target genes will be identified, which will contribute to understanding the antioxidant regulation mechanism of MarR transcription factors in LAB. These results will provide a a theoretical basis for revealing the regulation mechanisms underlying oxidative stress response in LAB, and also provide new insights for solving the problems in the industrial application of LAB raised by oxidative stress.

氧胁迫导致乳酸菌活力下降和活性衰减，是乳酸菌发酵生产和益生应用中面临的重要问题。转录因子在细菌感受氧胁迫信号，激活自身应激防御系统中发挥关键调控作用。目前国内外关于乳酸菌中参与抗氧化反应转录因子的研究刚刚起步，本课题组前期研究显示，植物乳杆菌中MarR家族氧还敏感型转录因子MarR_1突变株耐受H2O2能力明显降低，表明其在菌株抗氧化反应中的重要作用。因此，本课题拟探究MarR家族系列新转录因子在抗氧胁迫中的作用机制。内容包括：构建植物乳杆菌CAUH2的MarR_1、MarR_2和MarR_3突变体，通过抗氧化性能评价鉴定关键MarR转录因子；利用细菌单杂交和凝胶迁移实验确定转录因子调控的靶基因，并根据靶基因在突变体中的差异表达明确MarR的调控方式；通过靶基因功能分析阐明MarR系列转录因子在植物乳杆菌抗氧胁迫反应中的作用机制。研究结果将为全面揭示乳酸菌的抗氧化反应调控机制奠定基础。

氧胁迫导致乳酸菌活力下降和活性衰减,是乳酸菌发酵生产和益生应用中面临的重要问题。MarR型转录因子在细菌感受氧胁迫信号,激活自身应激防御系统中发挥关键调控作用。本项目旨在探究植物乳杆菌中参与抗氧胁迫的MarR型转录因子及其作用机制。首先，通过植物乳杆菌CAUH2全基因组序列分析，定位了24个MarR型转录因子编码基因，进一步根据编码蛋白的结构同源性分析和蛋白序列中半胱氨酸残基的存在情况，筛选到5个可能参与氧胁迫应激的MarR型转录因子；分别构建各转录因子突变株，并进行氧胁迫耐受实验，明确了转录因子A1F92_03745是CAUH2中参与无机过氧化物胁迫的关键转录因子，不同于枯草芽孢杆菌中其同源物对有机过氧化物胁迫的特异性响应；进一步利用转录组学分析了过氧化氢胁迫下A1F92_03745突变体中的差异转录基因，结合实验室前期的CAUH2过氧化氢转录组图谱，筛选到7个可能的靶基因，分别编码谷胱甘肽还原酶、NADH过氧化物酶、过氧化氢酶、2个甲硫氨酸亚枫还原酶和2个铁硫簇合成蛋白。这些蛋白质可通过清除活性氧、维持胞内氧化还原平衡、修复氧胁迫受损蛋白等方式提高CAUH2对过氧化氢胁迫的抗性。这些功能基因及转录因子A1F92_03745可作为耐氧胁迫的标志物应用于高耐氧益生菌的筛选。

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