纽荷尔脐橙突变体“赣南一号”果实表面脂质组和CsHD-ZIP转录因子功能分析

Citrus is one of the most important fruit crops worldwide, and its fruit and leaf surfaces are rich in wax and essential oils. Lipid composition is the biochemical basis of the formation of color, aroma, and the cuticular waxy layer. The cuticular waxes of citrus trees and fruits provide a protection against drought, water loss and pathogen invasion, and play an important role in extending the shelf life and improving fruit glossiness. ‘Newhall’ navel orange is the most commercially important navel orange variety worldwide. We have previously studied the appearance and intrinsic quality of the ripe fruit of the ‘Newhall’ navel orange (wild type, WT) and its glossy mutant ‘Gannan No.1’ (MT), and analyzed the changes of surface cuticle of WT and MT during fruit development and ripening. On the basis of previous results, we will conduct parallel lipidomics analysis on the fruit surfaces of both the wild type and mutant. The dynamic changes during fruit development as well as lipidomic differences between the wild type and mutant will be investigated. By integrating the lipidomic and transcriptomic data, the candidate genes related to lipid metabolism and cuticular wax synthesis of citrus fruit will be identified, which will further aid in revealing the key factors in the formation and regulation of the surface cuticle during fruit development and ripening. In addition, we will conduct functional characterization and regulatory mechanism analysis of CsHD-ZIP, a candidate gene selected from our previous study. To this end, CsHD-ZIP-overexpressing tomato lines and Hongkong kumquat lines with suppression of CsHD-ZIP will be generated by using the well-developed gene transformation system in our group. The results of this study is fundamental for citrus germplasm innovation and the improvement of citrus fruit quality during postharvest storage and transportation.

柑橘是世界上最重要果树之一，其果实和叶片表面富含蜡质和精油。脂质成分是色泽和香气性状形成的生化基础，且由脂质成分构成的蜡质层对柑橘树体和果实抵御干旱、防治水分过度散失、阻止病原生物入侵、延长贮运期限、提高果实光泽度等具有十分重要的作用。纽荷尔脐橙是全球最重要的脐橙品种。本项目在对普通纽荷尔脐橙（野生型对照）和其蜡质缺失突变体“赣南一号”成熟果实的外观和内在品质、果实表面角质膜动态变化初步研究的基础上，对其多个发育时期果实表面脂质成分和含量进行测定，分析野生型和突变体果面脂质组差异，并整合脂质组和转录组数据，挖掘与柑橘果实表面脂代谢和蜡质合成相关的候选基因，试图揭示果实发育和成熟过程表面角质膜的形成规律及调控机制；利用本项目组已构建好的遗传转化体系对转录因子CsHD-ZIP进行番茄和山金柑的遗传转化，进而验证其功能。本研究成果可望为柑橘新品种培育及贮运性能改良提供重要理论及实践基础。

脂质是果实品质形成的基础，影响果实的色泽，香味以及表面光泽度。本项目研究材料为前期报道的一例纽荷尔甜橙光亮突变体（MT）及其野生型（WT）。MT具有与WT极为相似的遗传背景，且果实集合光亮外观，红艳色泽，浓郁香味以及强抗病性等多优良性状。前人研究结果显示，两材料脂质代谢差异明显。为探究MT多优良性状形成的联系，以及挖掘影响MT性状形成的关键调控因子，本项目对WT与MT四个关键发育时间点进行转录组和代谢组测定，并以脂质及其相关关键基因为分析线索展开研究。首先发现MT发育期蜡质含量的降低伴随甘油磷脂含量的增加，因此，我们认为MT中本应该流向蜡质合成的碳代谢流受阻，转而流向了叶绿体膜脂的合成，增高的叶绿体脂质又激活了MT茉莉酸（JA）合成途径，最终使得MT表现出前人报道的依赖于JA的高抗病性状。该代谢水平的发现与转录组水平蜡质关键基因Cscer3显著下调，脂质与茉莉酸途径关键基因CsFAD2和CsDAD1等显著上调是一致的。代谢水平分析还发现，MT中糖酵解和柠檬酸裂解途径被显著激活，为脂质合成提供了比WT多的碳源，这些过量的碳源还有一部分流向了异戊二烯代谢途径，使得MT中高积累挥发性的倍半萜烯类物质，因此，MT表现出浓香的性状。为挖掘关键调控因子，对转录组进一步分析，发现CsESE3转录因子和蜡质、JA途径关键基因共表达。通过柑橘愈伤组织转化，金柑果实瞬时注射证实该基因能够促进柑橘蜡质和JA积累。异源转化番茄后，植株抗旱能力增强，果实表面黯淡无光泽，蜡质和JA含量同时增多。结合Luc，酵母单杂交以及EMSA实验证实，CsESE3间接调控蜡质合成，但是能够通过激活PLIP磷脂酶家族成员直接调控JA积累。本研究通过多组学解析，梳理了MT多优良性状形成之间的联系，提升了人们对柑橘品质形成的认知。此外，通过CsESE3的工作模式探究，明确了蜡质和JA在转录调控水平的联系，蜡质是植物防护的外屏障，JA是植物逆境信号传导网络的重要组成部分。因此，本研究为探究植物采取的防护策略提供了新的视角。