参与速生蓝细菌盐胁迫调控的非编码RNA的鉴定和功能表征

Cyanobacteria are able to grow in water habitats with various and changing salt concentrations. Researches on stress responding mechanisms of cyanobacteria are important for uncovering the common mechanism of photosynthetic organisms and improving their stress tolerances. Although physiological responses to salt stress and some key genes for salt acclimation have been identified, it is still not clear that how cyanobacteria regulate the expression of downstream genes in response to salt shock. Especially, it is totally unknown which and how non-coding RNA (ncRNA) participates in the post-transcriptional regulation during salt acclimation of cyanobacteria. In this project, a fast-growing cyanobacterium, Synechococcus elongatus UTEX 2973 will be used as a research model. Based on the pre-established approaches for ncRNA identification, we will compare and screen differentially transcribed ncRNAs in response to salt stress by the differential RNA-seq (dRNA-seq) technique. Then, bioinformatic and differential transcriptomic tools will be used for the target prediction of ncRNA. And the interactions between ncRNAs and their candidate targets will be confirmed by an E. coli reporting system. The results generated from this project will be helpful for further clarifying the stress responding mechanisms of cyanobacteria in future.

光合蓝细菌能够适应陆地和海洋不同盐度的水体环境，研究其盐胁迫适应机制对于理解光合生物盐适应普遍规律和提高其耐盐性具有重要的意义。虽然相关盐胁迫生理和盐适应关键基因已得到系统鉴定，不过蓝细菌响应盐胁迫信号调控下游基因表达的调控机制仍有待研究，特别是非编码RNA（ncRNA）参与的转录后调控机制研究目前尚属空白。在前期初步建立蓝细菌ncRNA鉴定技术体系的基础上，本研究以一株速生蓝细菌聚球藻UTEX 2973为模式，拟通过dRNA-seq技术比较普通和盐胁迫条件下聚球藻UTEX 2973转录组差异，系统筛选响应盐胁迫信号差异转录的ncRNA，并结合生物信息学分析和异源报告系统预测和验证ncRNA与靶标的相互作用，初步解析ncRNA调控靶标转录水平的分子机制，以进一步揭示蓝细菌盐胁迫调控机制。

研究光合蓝细菌盐胁迫适应机制对于理解光合生物盐胁迫适应普遍规律和提高其抗逆性具有重要的意义。蓝细菌响应盐胁迫信号调控下游基因表达的分子机制特别是小调控RNA（sRNA）参与的转录后调控机制仍有待研究。本研究通过dRNA-seq比较了聚球藻UTEX2973在普通和盐胁迫条件下的初始转录组，首次鉴定了响应盐胁迫大幅上调表达的sRNA——PsrR1，基于生物信息学分析和E. coli荧光报告系统预测和初步验证了PsrR1对光系统和色素合成基因的调控作用；而且发现体内过表达PsrR1也能够降低细胞光合色素含量和显著提高聚球藻UTEX2973对高盐环境的耐受能力。虽然前人研究证明了PsrR1在高光照和缺铁转录调控中的重要作用，本研究则首次证实了PsrR1在蓝细菌盐胁迫适应中的重要转录调控作用，为进一步解析基于PsrR1的蓝细菌盐胁迫转录后调控机制奠定了基础。此外，针对聚球藻UTEX2973缺乏高效基因敲除工具的问题，本研究建立了基于自杀载体的基因靶向敲除方法，大幅提高了聚球藻UTEX2973基因敲除的效率，不仅支撑了本项目通过体内实验研究sRNA生物学功能的工作，也为以聚球藻UTEX2973为研究模式的遗传学和代谢工程研究奠定了基础。