转录因子SlbZIP06负调控番茄耐热性的分子机制研究

Our previous study found that tomato transcription factor gene SlbZIP06 could be induced intensively by heat stress, and its over-expression led tomato to be sensitive to high temperature, which means this gene can regulate negatively the thermotolerance of tomato. Its mechanism, however, is not yet clear. In this study, we try to screen and identify its downstream target genes and interacting-protein genes by RNA-seq, ChIP-seq, and Yeast Two-Hybrid (Y2H) analysis. To dissect the signal pathways it involved in heat response by association analysis of quantitative RT-PCT. To analyze the regulation function of some key target genes and interacting-protein genes via over-expression and CRISPR/Cas9 knockout (or RNAi silence). Finally, we integrate all the results to construct gene regulation network of SlbZIP06 during the heat shock reaction, to explore its function on negative regulation of heat tolerance, and to lay the foundation for tolerance mechanism analysis and heat tolerance improvement of tomato.

前期研究发现，番茄转录因子基因SlbZIP06可被热胁迫强烈诱导，超表达导致番茄对高温敏感，表明该基因对番茄的耐热性具有负调控作用，但其中的机制尚不清楚。因此，本申请拟通过RNA-seq、ChIP-seq和酵母双杂等技术，筛选与鉴别该基因的下游靶基因和互作蛋白基因；通过qRT-PCT进行关联分析，明确该基因参与的热应答信号途径，并对关键的靶基因和互作蛋白基因进行超表达和CRISPR/Cas9敲除（或RNAi沉默），分析对番茄热适应的调控功能，从而明确SlbZIP06在番茄热激反应过程中的基因调控网络，阐明它对番茄耐热性负调控的生物学功能，为番茄耐适应机理解析和耐热性改良奠定基础。

前期研究发现，番茄转录因子基因SlbZIP06可被热胁迫强烈诱导，过表达导致番茄对高温敏感，表明该基因对番茄的耐热性具有负调控作用，但其中的机制尚不清楚。因此，本项目采用RNA-seq、DAP-seq和qRT-PCR表达关联分析等技术，筛选与鉴别该基因的下游靶基因；通过酵母双杂等技术，鉴别该基因的互作蛋白基因；通过酵母单杂等技术，鉴别该基因的上游调控基因；从而解析SlbZIP06负调控番茄耐热性的分子机制。结果显示，SlbZIP06过表达诱导了番茄叶片中22个差异表达基因上调表达，101个差异表达基因下调表达，且GO和KEEP富集分析表明，这些基因富集在代谢进程、物质运输、应激响应、调控活性、信号转导等生物学进程并参与其中的调控通路；通过DAP-seq分析发现，SlbZIP06主要与DNA上的“TGCCACGTCAGC”和“CAKCA”序列结合,并结合RNA-seq结果、qRT-PCR关联分析和双荧光素酶检测，鉴别出了转录因子SlbZIP06的靶标基因SlLBD1和SlDHN；通过酵母双杂技术，并经Y2H点对点和BiFC检测，鉴别到了SlbZIP06的互作蛋白基因SlZIP39；通过酵母单杂筛库，并经Y1H点对点和表达关联分析，鉴别出SlbZIP06的上游调控转录因子基因SlMYB59，再经双荧光素酶测定，确定SlMYB59能负调控SlbZIP06的表达。建立了SlbZIP06负调控番茄热适应的基因作用网络，即SlbZIP06通过与SlZIP39蛋白互作，调控下游SlLBD1等靶基因的表达，负调控番茄叶片的热激响应，而转录因子SlMYB59可以负调控SlbZIP06的表达。该研究结果补充了番茄高温胁迫响应的调控网络，为解析番茄耐热机制奠定了基础，并可为番茄的精准育种提供了参考。