蓝细菌蔗糖合成关键基因spsA转录调控研究

Sucrose, which is a kind of compatible solutes synthesized and accumulated under salt stress, plays an important role on responding and acclimation to salt stress in cyanobacteria. The spsA gene, encoding sucrose-phosphate synthase which catalyzes the key step of sucrose biosynthesis, is salt-induced in unicellular cyanobacteria. Recently, we have identified a key cis-acting element from the upstream region of the spsA gene which controls the spsA transcription under salt stress. However, the molecular mechanisms of spsA transcriptional regulation is still unclear. In this study, the transcription factors which could interact with this cis-acting element and also control the transcription of spsA will be firstly screened and identified from the cyanobacterial genome. Then the molecular mechanism on interaction between this cis-acting element and the identified trancription factors will be elucidated by electrophoretic mobility shift assay experiments. And the dynamic transcription of spsA and the identified transcription factors as well as the sucrose content in cyanobacteria under the normal culture condition and salt stress will be anaylzed by Real-Time PCR and ion chromatography analysis. By performing these researches, detailed mechanisms on transcriptional regulation of spsA will be interpreted. And this study will be helpful for the further elucidation of mechanisms of salt responding and acclimation in cyanobacteria, improvement of salt tolerance in crop species, and construction of novel resource platform providing sugar feedstocks.

蔗糖是蓝细菌响应盐胁迫而合成并积累的一种渗透调节物质，在蓝细菌响应和适应盐胁迫的过程中发挥了重要的作用。spsA基因编码单细胞蓝细菌蔗糖合成途径关键酶蔗糖-磷酸合成酶，该基因受到盐胁迫诱导转录，但是其转录调控的具体分子机制仍不明确。本项目将在前期工作已鉴定了spsA启动子区域一个顺式作用元件的基础上，以蓝细菌全基因组随机突变和酵母单杂交等技术，筛选和鉴定与该顺式作用元件相互作用并控制spsA基因转录的转录因子，再通过凝胶阻滞实验体外研究鉴定得到的转录因子与顺式作用元件的相互作用机制，以及通过实时定量PCR和离子色谱分析等技术，动态分析盐胁迫条件下转录因子和spsA的转录以及蔗糖合成情况，解析spsA转录调控的具体机制，探索盐胁迫诱导蔗糖合成的信号通路和具体分子机理，这对于进一步解析蓝细菌盐胁迫响应和适应机制以及阐明光合生物高盐适应的普遍规律具有重要的意义，也有助于建立新一代糖资源平台。

蔗糖作为相容性物质在蓝细菌响应和适应盐胁迫的过程中发挥了重要的作用。spsA基因编码蔗糖合成关键酶蔗糖-磷酸合成酶，其转录受到盐胁迫诱导，但是其转录调控的具体分子机制在本项目执行前并不清楚。本项目执行之初，有文献报道称Slr1588能够结合集胞藻PCC6803 spsA基因启动子区域，而缺失slr1588会导致盐敏感的表型和spsA基因的转录下调。因此，本项目通过基因转录分析实验证明了ggpP的转录起始位点位于slr1588基因ORF的第1008到1156 bp之间,而缺失slr1588基因整个开放阅读框（ORF）时不仅缺失了slr1588基因也抑制了下游ggpP基因盐胁迫下的转录，而这正是slr1588缺失突变株盐敏感的原因。通过逐段缺失slr1588基因ORF构建系列突变株，并通过后续盐胁迫耐受力评价和ggpP转录水平分析，最终确定缺失slr1588基因前976 bp既造成Slr1588功能失活又不影响下游ggpP正常转录。进一步分析该突变株和slr1588基因过表达株发现，slr1588能够抑制spsA盐胁迫条件下的转录，和蔗糖分解酶基因invA在普通培养条件下的转录，而且该基因的失活或过表达会抑制蔗糖的合成。本研究指出了前人研究中存在的问题以及产生的原因，证明了调控因子Slr1588对蔗糖代谢关键基因的转录调控，为进一步解析蓝细菌盐胁迫适应的分子机制和代谢工程改造提高蓝细菌蔗糖合成能力奠定了基础，对于建立基于光合蓝细菌的糖资源平台具有一定的意义。