副干酪乳杆菌TetR家族转录因子调控胆盐胁迫抗性机制的研究

胆盐胁迫影响益生菌在肠道内的存活，而转录因子是调控菌株胆盐胁迫应激反应的关键。前期研究发现，副干酪乳杆菌L9在胆盐胁迫下，TetR家族转录因子（LPL9_1280）及相邻转运蛋白在转录水平显著上调，初步分析发现转录因子和转运蛋白均与胆盐存在相互作用。目前该转录因子参与副干酪乳杆菌胆盐胁迫的机制尚不明晰。本项目拟研究：①构建转录因子及转运蛋白突变体，考察突变株对不同类型胆盐耐受的特异性；②通过体外凝胶迁移技术确定转录因子结合的顺式元件，利用RT-qPCR确定转录因子对转运蛋白的调控方式；③利用分子动力学模拟探究胆盐组分与转录因子和转运蛋白的相互作用，明确转录因子胆盐响应机制，结合等温量热滴定和稳定同位素标记技术确定转运蛋白与胆盐组分的结合及外排作用。本项目将阐明TetR家族转录因子调控的转运蛋白系统在乳酸菌胆盐胁迫抗性机制中的重要作用，为丰富乳酸菌胁迫响应机制及推动益生菌产业化应用提供理论。

Probiotics suffer from bile salts stress when they getting through the human gut，which affect their survival. Transcription factors participate in the sensing of bile stress signals and activating of the stress defense system in bacteria. Our previous study has shown that TetR family transcription factors LPL9_1280 was significantly induced at transcript level under bile salt stress in Lactobacillus paracasei L9, as well as their contiguous genes encoding an transporter system. Meanwhile, LPL9_1280 and the transporter could interact with bile salts components. But the regulation mechanism of LPL9_1280 and the function of the ABC transporter in bile resistance are unknown. Therefore, the following experiments will be carried out: (1) Mutants of TetR transcription factor and the transporter will be constructed, different kind of bile salt tolerance of mutants will be studied;(2) Electrophoretic mobility shift assay and RT-qPCR will be used to investigate the regulation of TetR transcription factor on transporter genes;(3)Molecular dynamics simulation and isothermal titration calorimetry will be used to investigate the interaction between bile salts components and LPL9_1280/ transporter, in order to confirm the efflux of bile salts through transporter.These results will reveal the regulation mechanism of TetR family transcription factors in L. paracasei L9 under bile salt stress, meanwhile, provide the theoretical foundation for the mechanism of bile tolerance response and industrial application of probiotic lactic acid bacteria.