维生素B12在细菌与真核微藻共生耐热中的功能与调控机制研究

The photosynthetic eukaryotes known as algae are the key contributors to primary production. Current evidences indicate that algal growth and stress-tolerance are greatly influenced by Vitamin B12(VB12)-producing bacteria, while the VB12-involved physiological functions and genetic regulations are poorly understood. We have identified that certain VB12-producing bacteria is able to enhance thermal tolerance of the microalga, Chlamydomonas reinhardtii, by the supply of VB12 revealing new lights on the interactions between VB12-producing bacteria and algae. In this project, we will focus on the functional roles and regulatroy mechanisms of VB12 in the thermotolerance of bacteria-microalgae symbiosis. The main objectives are: elucidate the function and regulation of VB12-dependent methionine synthase METH with subcellular localization identification, interacting protein capture and overexpression studies; identify the metabolite from C. reinhardtii which is able to activate the expression of VB12 biosynthesis genes in Sinorhizobium meliloti, by the use of a reporter systerm of bluB-luxAB; uncover the mechanisms underlying how bluB’s promoter activity and its VB12 riboswitch structure are influenced by the inducer metabolite; and reveal the effects on the global gene expression pattern of VB12 biosynthesis, with a metatranscriptomic approach. Our research will not only advance the understanding of the molecular mechnism involved in the VB12-producing bacteria-algal interaction, but also assiast to enhance their thermal tolerance in other applications.

近期研究表明，作为初级生产力的重要贡献者，许多真核藻类的生长和抗逆能力会得益于与其共生的产维生素B12(VB12)细菌，然而具体分子机制并不清楚。本项目将基于根瘤菌属产VB12细菌显著增强莱茵衣藻耐热能力的前期发现，开展VB12在细菌与真核微藻共生耐热中的功能与调控机制研究：通过亚细胞定位、互作蛋白钓取、过量表达等研究，揭示衣藻中关键酶VB12依赖型甲硫氨酸合成酶METH的功能和调控；通过检测报告基因bluB-luxAB确定可诱导苜蓿根瘤菌大量合成VB12的衣藻诱导物；基于诱导物对VB12合成基因bluB启动子活性与5-UTR核糖开关结构的影响，阐明VB12合成基因的诱导表达机制；并通过对共生体系的宏转录组分析，揭示VB12的合成对共生双方全局基因表达的影响。本研究不仅对深入了解产VB12细菌与藻类相互作用的分子机制有重要意义，还将为提高相关物种在实际应用中的耐热能力提供科学依据。

本研究利用产VB12细菌-莱茵衣藻相互作用体系，综合利用分子生物学、生物化学、细胞生物学和基因组学方法，开展VB12在增强微藻耐热过程中的功能与调控机制研究。本研究探索了该过程中METH等关键基因的功能和调控方式，基于蛋白质组学阐明细菌与微藻相互作用的全局蛋白表达模式，揭示出该过程中VB12可能参与调控的途径，并通过遗传操作和表型鉴定取得多个新的候选重要参与基因信息，有望在后期的工作中取得新的突破。本研究也在菌藻复杂相互作用体系的延伸构建及其受环境因素的调控、相关种类微生物的环境效应与潜在应用等研究中取得进展。综上所述，本研究更加深入地认识了产VB12细菌增强藻类耐热能力的分子机制，为细菌与藻类相互作用的研究提供了新的科学基础。