FaNAC100调控FaPL1延缓草莓果实软化的机制研究

Strawberry is easy to soften after ripening, resulted in sharp quality losses. Previous study proved that knock down of FaPL1, pectate lyase, led to increase in fruit firmness, as well as delayed exposure of mildew during the storage. We screened the promoter of FaPL1 from strawberry with different firmness, and found a differential NAC binding sites in the key negative regulatory region in strawberry varieties ‘Sweet Charlie’ with high hardness, comparing with ‘Benihoppe’ of low hardness. And we also obtained a NAC transcription factor, FaNAC100, whose expression negativly correlated to FaPL1. It is speculated that it is closely related to inhibiting fruit softening in strawberry, but the exact molecular mechanism has not been elucidated. In this study, transgenic strawberry will be generated via the overexpression and RNAi interference system, thus to illustrate the function of FaNAC100 in softening in strawberry. In addition, the EMSA and yeast one-hybrid system will be employed to analyze the interaction between FaNAC100 and FaPL1. Finally, mRNA expression levels of FaNAC100 and FaPL1 will be analyzed in different transgenic plants，integrated dual luciferase system to elucidate the functional mechanism of FaNAC100 in softening in strawberry. To sum up, this study will devote to elucidate the molecular mechanism of FaNAC100 regulating softening, develop molecular markers for genetic engineering assisted breeding, and improve the storage in strawberry. The study is of both theoretic importance and practical application value.

草莓果实成熟后易软化，造成果实品质急剧下降。课题组前期研究表明干扰FaPL1（果胶裂解酶基因）可提高果实硬度，并在贮藏期推迟霉变的发生。与“红颜”相比，硬度大的“甜查理”的FaPL1启动子关键负调控区段存在一个差异的NAC结合位点，同时通过筛选，课题组已得到一个与FaPL1表达模式负相关的FaNAC100，推测其与抑制草莓果实软化密切相关，但确切的分子机理尚未阐明在此基础上，本项目拟利用超表达和RNAi干扰体系，构建FaNAC100转基因草莓，分析其在果实成熟软化中的功能。其次利用EMSA和酵母单杂交等方法明确二者的互作。最后，以转基因草莓为材料，分析FaNAC100及FaPL1的表达，结合双荧光素酶系统，解析二者的调控模式。本项目研究对于阐明FaNAC100调控草莓果实成熟软化的分子机理，开发分子标记进行草莓基因工程辅助育种以及提高品种的贮运品质，都具有重要理论意义和实际应用价值。

草莓属于典型的非呼吸跃变型果品，而商品货架期短是生产中鲜食草莓面临一个亟需解决的问题。成熟的草莓易软化，果实硬度低，且容易被微生物侵染。果胶裂解酶基因被认为是细胞壁降解、果实软化中的关键基因。前期时空表达和瞬时表达分析，筛选到一个与草莓果实发育相关且保守的果胶裂解酶基因FaPL1。该基因在在红颜（硬度小）和甜查理（硬度大）中表达模式存在差异，通过组织化学染色鉴定出启动子上的差异区段，进行酵母单杂系统的cDNA文库筛选，并利用双荧光素酶系统鉴定与之结合的转录因子分别为FaASIL2、FaHSFB3、FaPHL、FaTTG1A和三个锌指蛋白。通过瞬时表达系统分别将转录因子在草莓果实中超表达，其中FaPHLL、FaTTG1A、FaZF1和FaZF3均可抑制FaPL1的表达和果胶裂解酶酶活，显著地提高果实的硬度，而抑制该基因表达则会降低果实的硬度， 而FaASIL2和FaZF2瞬时表达对果实的影响正好相反。FaHSFB3虽然显著提高了启动子活性，但其过表达或抑制表达后对果实硬度影响无明显差异。本研究从FaPL1转录水平上鉴定出与之结合的转录因子，并明确了转录因子对其表达的调控作用。本项目的开展，在已知成熟软化相关效应分子的基础上，协同其调控基因（转录因子），不仅有助于进一步解析草莓果实成熟软化的分子机理，同时为浆果类植物提高果实硬度、延长货架期寿命的遗传改良提供具有自主知识产权的基因资源，因而具有重要的理论意义和潜在的应用价值。