类黄素单加氧酶YUCCA11在桃果实成熟软化过程中的作用机理研究

Prompt post-harvest softening of fruits is one of the major bottleneck constraints of peach industry development. It's of great significance on studying on peach fruit maturation and softening mechanism, developing new storage-tolerant varieties of peach for prolonging the market supply and reducing post-harvest loss. The fruit of melting-flesh peach cultivars produce high levels of ethylene caused by high concentration of IAA, resulting in rapid fruit softening at the late-ripening stage. In contrast, the fruit of stony hard peach cultivars do not soften and produce little ethylene due to low concentration of IAA. In our previous study, we explored the difference of IAA accumulation between melting-flesh peach and stony hard peach via expression analysis of IAA related genes. YUCCA11 was showed significant difference expression and may play important roles in the difference of IAA accumulation. YUCCA encodes flavin monooxygenase-like enzyme, and converts indole-3-pyruvic acid to IAA. Here the functions of YUCCA11 in fruit maturation and softening were further investigated. The study is mainly presented as follows: investigate the sequence of YUCCA11 gene and promoter in peach with different fruit texture; In vitro activity of prokaryotic expression of the recombinant YUCCA11 protein; the over-expression manipulation of YUCCA11 will be performed using tomato as explants, virus-induced gene silencing and YUCCA inhibitor treatment will be carried out to verify the effects of YUCCA11 on fruit maturation and softening. The study will help clarify the roles of YUCCA11 in fruit ripening and softening, and consequently provide the molecular basis of peach fruit texture valuable for genetic improvement.

果实成熟后迅速软化是长期制约桃产业发展的关键问题，研究桃果实成熟软化机理，培育耐贮运桃新品种，对于延长市场供应期，降低采后损耗具有重大意义。溶质桃果实成熟期产生高浓度IAA，诱导了乙烯的大量释放，导致果实迅速软化；而硬质桃果实缺乏IAA的积累，导致乙烯释放量少，果实不变软。申请人前期通过比较溶质和硬质桃果肉IAA动态平衡相关基因的表达，筛选出导致IAA跃变的候选基因YUCCA11。YUCCA编码类黄素单加氧酶，催化吲哚丙酮酸合成IAA。本项目拟对YUCCA11在果实成熟软化中的作用进行深入探讨。主要包括YUCCA11基因及启动子在不同肉质类型的序列差异分析；原核表达初步验证YUCCA11的功能，并结合转基因功能验证、病毒介导的基因沉默和YUCCA活性抑制剂处理，从正反两个方面探究其对果实成熟软化的影响。旨在明确YUCCA11在果实成熟软化的作用，为桃肉质性状分子改良提供理论依据。

果实成熟后迅速软化是长期制约桃产业发展的关键问题，研究桃果实成熟软化机理，培育耐贮运桃新品种，对于延长市场供应期，降低采后损耗具有重大意义。溶质桃果实成熟期产生高浓度IAA，诱导了乙烯的大量释放，导致果实迅速软化；而硬质桃果实缺乏IAA的积累，导致乙烯释放量少，果实不变软。研究主要包括以下两个方面：.1. 控制硬质桃果实肉质性状基因的挖掘：通过对硬质桃和非硬质桃果实成熟期转录组对比，筛选到差异表达的类黄素单加氧酶基因YUCCA11，YUCCA是生长素合成途径的限速酶，YUCCA11在溶质桃果实成熟期明显上调表达，而在硬质桃中变化不明显。比较YUCCA11基因在多个不同肉质类型品种启动子和编码区域的DNA序列，发现存在三处多态性位点，并针对每个多态性位点分别开发标记并对其在自然群体的分布进行了分析，结果表明第一个内含子区域的微卫星重复区域可作为区分非硬质和硬质类型的候选区域。同时通过对14个不同肉质类型桃品种的研究发现，成熟阶段果肉中YUCCA11表达与IAA（生长素）含量，以及果实乙烯释放量存在协同性变化。这些研究表明PpYUC11是调控桃果实硬质的候选基因。.2. YUCCA11基因在硬质型和非硬质型桃果肉成熟期表达迥异的原因分析：通过对不同品种YUCCA11基因启动子的上游区域进行克隆，结果发现硬质桃YUCCA11基因起始密码子ATG上游区域的2.1 kb处存在ATCTA型DNA转座子插入。对YUCCA11基因预测的启动子元件分析发现，在ATG上游2.2 kb左右存在果实特异性元件，转座子插入可能导致该基因在果实中不表达或表达量很低。同时基于转座子开发的标记相比之前内含子的SSR标记可以更加明显的区分硬质和非硬质性状。该转座子与前人报道的MADS6的插入突变高度相似，表明该转座子在近代事件中可能发生了跳跃，导致桃果实的硬质类型的产生。.本项研究拓展了人们对桃果成熟软化分子机理的认识，开发的硬质分子标记，在培育耐贮运桃新品种方面具有重要的应用价值。