谷氧还蛋白PsGrx在南极海冰细菌极端生境适应中的功能研究

Glutaredoxin (Grx), as an important regulatory protein in antioxidant system, plays an important role in adjusting the intracellular redox status by reducing the intermolecular disulfide bond and forming an oxidative stress regulation network, which has been demonstrated in higher plants. However, the Grx gene from sea-ice microbes has not been reported. Based on our previous studies, extreme environment adaptation mechanism of Antarctic sea-ice microbes was closely related to its antioxidant system. On the basis of the cloning and expression of PsGrx gene and the identification of biological functions, it was found that PsGrx may play an important regulatory role in extreme environment adaptation mechanism. In view of this, the biological activity, structure and function of the PsGrx gene will be studied using the methods of site-directed mutagenesis and spectral anlysis, which will expect understanding the functions of PsGrx, and the relations between active sites and functions systematically from the aspects of the qualitative and quantitative, the appearances and mechanisms, the functions and activities. In this study, we will identify and verify some target proteins that interact with Grx using the yeast two-hybrid and transcriptomics methods, which will help to explore the PsGrx regulatory network triggered oxidative stress by response the extreme environment. This project will further reveal the extreme environmental adaptation mechanisms of Antarctic sea-ice microorganisms with independent intellectual property rights, and provide new stress resistance genes for the biological genetic breeding, which have both the theoretical significance and practical significance.

谷氧还蛋白（Grx）作为抗氧化系统中一种重要调控蛋白，通过还原蛋白分子间二硫键以调节细胞内氧化还原态势，在逆境胁迫中形成氧化应激调控网络发挥着重要功能，目前在高等植物中已经得到证实，而有关海冰微生物Grx基因相关研究还未见报道。我们前期研究发现，海冰微生物的生境极端适应机制与其抗氧化系统密切相关；在完成PsGrx的基因克隆表达与生物学功能鉴定基础上，发现PsGrx在其生境适应性中可能起着重要的调控作用。鉴于此，本项目拟采用基因突变和光谱分析等手段，从定性及定量、表象及机制、功能及活性等方面系统认识海冰细菌PsGrx的功能、活性位点与功能之间的关系；采用酵母双杂交和转录组学等技术鉴定与验证Grx作用靶蛋白，解析逆境下PsGrx及其作用靶蛋白形成的氧化应激调控网络。本项目将从全新的角度和更深的层次完善海冰细菌极端生境适应机制，为生物遗传育种提供新的抗逆基因资源，都有着重要的理论意义和实际意义。

谷氧还蛋白（Grx）作为抗氧化系统中一种重要调控蛋白，在细胞对氧化应在逆境胁迫中形成氧化应激调控网络发挥着重要功能，目前在高等植物中已经开展大量研究，而有关南极海冰细菌Grx基因相关研究还未见报道。本项目首先挖掘了南极海冰微生物来源的新型Grx基因，鉴定菌株ANT206中PsGrx的Cys13、Pro14和Cys16为催化关键位点；利用同源建模和分子动力学模拟阐明了PsGrx蛋白活性、稳定性以及构象柔性三者关系，揭示了PsGrx蛋白低温催化活性及结构特征。在利用两次同源重组法获得PsGrx缺失突变菌株基础上，结合酵母双杂交技术构建了低温生境下PsGrx及其作用靶蛋白形成的氧化应激调控网络，涉及应激反应、蛋白质分泌、RNA代谢和蛋白质合成等；进一步利用BiFC和Co-IP验证等手段，提出了PsGrx参与低温下谷胱甘肽代谢和ROS清除的氧化应激调控新途径，揭示菌株ANT206的低温生境适应机制。以上研究结果从PsGrx调控代谢途径的全新角度为深入理解南极海冰细菌的低温生境适应机制提供新的理论依据。在项目资助下正式发表SCI收录论文9篇，申请国家发明专利5项，其中3项获得授权，培养硕博研究生7名。