基于Yap5转录因子的金属/氧响应解析酿酒酵母OLE1基因的镉胁迫分子响应机制

The protein-binding complex is the main form of cadmium in cadmium-contaminated rice and other food products, which makes it difficult to remove the cadmium from the foods with the available physical or chemical methods. Living Saccharomyces cerevisiae can be used in the cleavage of the free cadmium from the protein-cadmium complex. However, living S. cerevisiae shows a low cadmium tolerance to the free cadmium. In our previous study, a novel cadmium tolerance gene OLE1 will be up-regulated in response to cadmium stress, and confer high cadmium tolerance to living S. cerevisiae. Therefore, the promise and base for the promotion and application of the living S. cerevisiae in the field of cadmium removal from foods is to reveal the molecular mechanism of OLE1 in response to cadmium stress. Based on the theory of cadmium inducing oxygen-free radicals, the phenotype of OLE1 in response to cadmium stress and the potential role of Yap5 in regulation of OLE1, this study will firstly ascertain the effects of cadmium, oxygen-free radicals and YAP5 deletion on OLE1 expression by RT-PCR in wild type and yap5∆ strains. Secondly, the effects of cadmium, oxygen-free radicals on Yap5 will be detected by the subcellular localization of GFP-protein method in GFP-YAP5 strains. Thirdly, the plasmid for the OLE1 promoter analysis will be constructed from basic plasmid with a firefly luciferase reporter through gene cloning method, and the response YRE element in OLE1 promoter to cadmium and Yap5 will be determined. The molecular mechanism of OLE1 in response to cadmium stress will be revealed finally.

蛋白结合态镉是大米等食品镉污染物的主要形式，难以通过物理、化学方法脱除。活体酿酒酵母能分割蛋白质结合态镉，是解决该问题的重要突破口。蛋白结合态镉在酵母的作用下能够分解产生游离态镉。但酿酒酵母并不耐受游离态镉。前期发现酿酒酵母OLE1能响应镉胁迫，提高酿酒酵母对游离态镉的耐受性。因此，探明OLE1镉胁迫的分子响应机制，是实现其在大米等食品镉脱除领域中推广应用的前提。从镉诱发氧自由基的理论出发，在明确OLE1响应镉胁迫的表型，及Yap5对OLE1潜在的调控作用，本项目拟利用野生型和yap5∆株及RT-PCR技术，检测镉、氧自由基、YAP5缺失对OLE1表达的影响；利用GFP-YAP5菌株及蛋白亚细胞定位技术，检测镉、氧自由基对Yap5的调控作用；再利用荧光素酶报告基因表达载体及基因克隆技术，构建OLE1启动子分析质粒，证实YRE元件对镉及Yap5的响应。最终揭示OLE1的镉胁迫分子响应机制。

活体酵母作为重要的食品脱镉微生物，在分割蛋白结合态镉为游离态时，难以避免的会遭受镉毒胁迫而影响脱镉效果。针对酵母镉耐受性差的问题，本项目以前期发现的耐镉基因OLE1镉应激行为为突破口，从镉氧胁迫理论出发，基于Yap5对OLE1的潜在调控作用，利用多项分子生物学技术，在转录因子筛选、调控通路挖掘、应激行为分析等方面开展大量的研究工作。在研究过程中，基于OLE1镉应激行为无法被抗氧化剂消除的表型，提出了镉、氧胁迫独立作用于OLE1的观点；基于NCBI、SGD数据的信息学推算，从11个候选转录因子筛选出了介导OLE1镉应激的关键转录因子Yap5；基于Yap5-Aft1的上下游调控关系，探索了线粒体Isu1和Isa1介导的Cd-S簇合成，明确了Yap5感应Cd-S簇并介导OLE1镉应激的分子机理；基于Yap家族入核调控理论，利用anti-GFP原位杂交技术，解决了Yap5-GFP表达量低无法利用GFP荧光定位的问题，发现了镉显著提高Yap5在核内聚集水平；基于Yap5入核调控OLE1的行为，利用Dap-Seq技术，探明了Yap5结合OLE1启动子区的关键元件ARE（序列：ATTACGTA）。本项目从Yap5介导的OLE1镉应激行为上，深入剖析微生物与镉之间的互作行为，发现了微生物识别、感知、应对镉的重要方式。这些发现，为全面揭示生物体应对镉胁迫的机理奠定了基础，为解决镉污染、镉残留问题提供了重要的理论支撑。