【作者】 张畅；

【导师】 叶邦策；

【作者基本信息】 华东理工大学 ， 生物化学与分子生物学， 2014， 博士

【摘要】 胞内小分子代谢物在调控以及识别细胞各种生命活动中起到至关重要的作用。在过去的二十年里,研究者们通过不懈的努力提升遗传编码传感器的性能以达到有效监测细胞内小分子的目的。目前已经成功开发出大部分细胞内代谢相关信号的传感器,包括ATP, cAMP, cGMP, NADH, ROS,糖类,CO, NO等。当前,内源性遗传编码传感器已经成为单细胞内分子检测强有力的工具,它们具有实时监测,活细胞监测,高通量分析,高时空分辨率,对细胞本身的活动影响小等优势。本文的主要研究内容如下：(1)α-酮戊二酸(2OG)是表征细胞内碳氮营养状态的重要信号分子。α-酮戊二酸作为三羧酸循环中一个高度保守的代谢物,不仅在代谢过程中起到关键作用,同时也是多种生物体内的信号分子。追踪α-酮戊二酸对相关的细胞代谢和信号传导研究有重要意义。我们针对具有重要生理活性的α-酮戊二酸的原位分析,构建遗传编码荧光传感器,开展细胞内活性小分子原位分析及生物成像新方法研究。我们采用α-酮戊二酸特异性结合多肽(棕色固氮菌NifA蛋白的GAF结构域)作为敏感元件设计可用于实时动态监测细胞内α-酮戊二酸的荧光蛋白分子传感器。基于荧光共振能量转移的传感器能够响应α-酮戊二酸,动力学范围为100μM到10mM,符合活细胞体内的α-酮戊二酸浓度范围,可用于体内检测。通过结构域优化,得到了最大荧光比值为0.95的传感器,同未经优化的传感器比较信号提高了6倍。我们尝试将优化后的传感器表达到大肠杆菌中进行体内α-酮戊二酸代谢的研究,验证了该传感器在体外以及体内对α-酮戊二酸进行实时监测的可行性。(2)最新的研究表明,α-酮戊二酸不仅是三羧酸循环的重要代谢物,在癌细胞异常代谢,脑神经信号传导中也起到重要的作用。我们尝试基于黄色荧光蛋白YFP的传感器设计研究,进行了大量经验式的优化工作,并且借助生物信息学的方法对实验设计进行有指导性的摸索,得到的单荧光传感器也能很好的响应2OG,该传感器被应用于大肠杆菌细胞对不同营养环境响应的研究。(3)群体感应(简称QS)是存在于不同种细菌之间的普遍现象,它是指细菌通过生产和调节信号分子的方式来调节基因的表达以及种群密度的现象。QS调节系统在很多生物学功能中起到了重要的作用,例如生物发光,抗生素生产以及生物被膜的生成。信号分子3OC6HSL是自诱导分子酰基高斯氨酸内酯(AHL)的一种,它是海洋费氏弧菌的QS系统的调控分子。追踪监测3OC6HSL在QS系统的相关研究中有重要意义。我们采用海洋费氏弧菌QS系统的调控蛋白LuxR来设计针对3OC6HSL检测的传感器。并且验证该传感器在体外以及体内运行的可行性。更多还原

【Abstract】 Intracellular metabolites play a crucial role in characterizing and regulating corresponding cellular activities. Tracking intracellular metabolites in real time by traditional means was difficult until the powerful toolkit genetically encoded biosensor was developed. In the last decades, iterative improvements of these biosensors have been made for effectively monitoring metabolites such as ATP, cAMP, cGMP, NADH, ROS, sugar, carbon monoxide, nitric oxide and so on. Endogenous genetically encoded biosensors have become powerful tools in intracellular metabolites detection in single living cells, based no the adventages such as real-time monitor, live cells analysis, high-throughput analysis, high temporal-spatial resolution, minimum interfere to cell activity.2-Oxoglutarate (2OG) is a metabolite from the highly conserved Krebs cycle and not only plays a critical role in metabolism but also acts as a signaling molecule in a variety of organisms. Environmental inorganic nitrogen is reduced to ammonium by microorganisms, whose metabolic pathways involve the conversion of2OG to glutamate and glutamine. Tracking of2OG in real-time would be useful for studies on cell metabolism and signal transduction. Here, we developed a genetically encoded2OG biosensor based on fluorescent resonance energy transfer. The dynamic range of the sensors is100uM to10mM, appeared identical to the physiological range observed in E. coli. We optimized the peptide lengths of the binding domain to obtain a sensor with a maximal ratio change of0.95upon2OG binding and demonstrated the feasibility of this sensor for the visualization of metabolites both in vitro and in vivo. We also developed a novel sensor by inserting the functional2OG-binding domain GAF of the NifA protein into YFP. This sensor was found to be highly specific to2OG Following binding of2OG, fluorescence intensity of the sensor increased with increasing2OG concentration and reached a1.5-fold maximum fluorescence signal change, kinetics of fluorescence signal upon2OG association with sensor was fast, the dynamic response range of the mOGsor sensors was100uM-100mM. This sensor reported cellular2OG dynamics in E. coli cells in real time upon different nutrition challenges and manifested the differences in2OG pool accumulation and depletion velocity.Quorum sensing (QS) is a universal phenomenon that exists in various bacterial species and produces and monitors signaling molecules to regulate specific sets of genes in a population density-dependent manner. The QS system is involved in many important biological functions such as luminescence, antibiotic production, and biofilm formation. The autoinducer N-(3-oxo-hexanoyl)-L-homoserine lactone (3OC6HSL), an N-acylhomoserine lactone (AHL), plays a significant role in the QS system of the marine bacterium Vibrio fischeri. Tracing3OC6HSL would be significant in studies related to QS signal transduction. Traditional detection of QS signaling molecules has relied primarily on bacterial reporter strains and high-performance liquid chromatography, which are time consuming and have low sensitivity. Because3OC6HSL binding to LuxR from V. fischeri causes a conformational change, we developed a genetically encoded biosensor based on Forster resonance energy transfer (FRET) by inserting LuxR between the FRET pair YFP/CFP and demonstrated the feasibility of this sensor for visualizing3OC6HSL both in vitro and in vivo.更多还原