NAC转录因子家族SlNAM1基因调控番茄果实成熟的分子机制研究

Fruit ripening is a fine transcription regulation, mining ripening-related transcription factors is very helpful for us to understand the molecular mechanism of fruit ripening. Our previous study found that silencing of SlNAM1 gene, a member of NAC transcription factor family, inhibited the ripening process including lycopene accumulation of tomato fruit, but the mechanism is not clear. The purpose of this project is to obtain the ripening-related target gene regulated by SlNAM1 and protein interaction with SlNAM1 by using RNA-seq, ChIP-seq and the screening of yeast cDNA library from tomato fruit, these target genes and proteins were functional analysis through the VIGS technology. Through studying the intermediate product of lycopene synthesis pathway, expression and EMSA of its key genes in SlNAM1 transgenic tomato fruit, the regulation mechanism of SlNAM1 gene on lycopene synthesis was explained in tomato fruit. The functional identification of SlNAM1 can enrich the fruit ripening theory, provides the resources of candidate genes to obtain ripening-control tomato by transgenic method.

果实成熟是个精细的转录调控过程，成熟相关转录因子的挖掘对了解果实成熟的分子机理意义重大。前期研究发现NAC转录因子家族SlNAM1基因沉默影响了番茄果实的成熟过程，其中包括对番茄红素的合成抑制，但具体机制不清。本项目旨在利用RNA-seq, ChIP-seq和番茄果实酵母cDNA文库的筛选获得SlNAM1调控成熟相关靶基因和互作蛋白，并通过VIGS技术对其进行功能验证，解析SlNAM1调控番茄果实成熟的分子机理；通过转基因SlNAM1番茄果实番茄红素合成中间产物的含量测定，关键基因表达和EMSA分析，解析SlNAM1基因调控番茄红素合成的分子机理。SlNAM1的转录调控的功能鉴定能丰富番茄果实的成熟理论，为今后通过转基因手段获得控制成熟过程的转基因番茄提供候选基因资源。

成熟是果实品质形成的关键阶段，受到转录因子的精细调控，挖掘成熟相关转录因子对于理解果实成熟和品质形成具有重大的科学意义。课题组前期通过VIGS筛选多个在果实成熟过程中表达量较高的NAC转录因子家族成员时发现SlNAM1基因沉默影响了果实成熟，但是机理不清楚。本研究首先构建了SlNAM1的基因编辑和超表达转基因材料，发现其影响了果实成熟过程。以此材料为基础，开展果实成熟相关生理指标的测定，如乙烯，果实硬度和色素合成，同时进行RNA-seq和靶基因挖掘，结合代谢组学分析，发现SlNAM1调控果实乙烯生物合成和色素积累的靶基因，并加以验证，另外，发现该基因编辑影响了番茄的营养素合成，最终发现SlNAM1是通过直接调控番茄果实成熟过程中系统II乙烯生物合成关键酶基因SlACS2和SlACS4来调控果实的成熟进程。与对照果实相比，SlNAM1基因编辑果实延迟成熟，而SlNAM1超表达果实促进成熟，SlNAM1是果实成熟的正向调控因子。该结果丰富了番茄果实的转录调控理论，为将来通过生物技术方法获得耐贮藏的果实品种提供理论依据。