火龙果色素合成代谢组分析及分子调控机制研究

The red flesh pitaya in Guizhou characterizes in superior quality, so as to be generally accepted by customers. Previously, the metabolomic and transcriptomic analysis were carried out using the flesh with different development stage of red (Zihonglong) and white (Jinghonglong) flesh cultivars, and the guide sample of betacyanin was extracted. The pigment-related metabolites were quantified in different fruit ages, and differentially expressed genes (DEGs) and transcription factors (TFs) were identified, among which CYP76AD1, DODA andMYB1etc were involved in pigment metabolism. Currently, the metabolomics of key development stages of red and white flesh were further analyzed, so as to unravel the characters of pigment metabolite. Together with the morphological and cytological investigation during flesh colorization, the relationship between metabolites and flesh colorization will be illuminated. Based on the preliminarily discovery in DEGs and TFs, the key genes and TFs which regulated the differentiation of flesh color will be further identified and cloned, whose functions will be verified. Subsequently, the molecular mechanism determining the divergence of flesh color will be elucidated. Further, the key genes/ TFs related to betacyanin metabolism will be screened and identified, and the expressions during different development stage of red flesh will be profiled. Combined with the result of pigment metabolomic analysis, the regulation of betacyanin metabolism will be elucidated in red flesh pitaya.

贵州红肉火龙果品质优、效益好，申请团队在前期工作中，对红肉和白肉品种果实不同发育期色素代谢组及转录组分析，分离提纯到甜菜苷标样，测定了着色相关代谢物的种类和含量，筛选到差异表达基因/转录因子，其中CYP76AD1、DODA及MYB1等与色素代谢相关。本项目拟在此基础上，进一步阐明果实不同发育期代谢物变化规律，结合果肉着色形态学及组织细胞学特征观测，寻找代谢物与着色的对应关系；继续发掘和克隆调控果肉颜色分化的关键基因/转录因子，特别针对CYP76AD1、DODA及MYB1等基因的功能开展深入研究，揭示火龙果肉色分化的分子调控机制；筛选和鉴定红肉品种甜菜苷代谢相关的重要基因/转录因子，并分析其在果实不同发育期的表达水平，结合色素代谢组分析结果，解析火龙果甜菜苷代谢的调控机制。本研究能为深入、全面认识火龙果着色的分子调控机理提供新信息，也能为通过分子育种策略培育高色素含量新种质提供新思路。

色泽是果实主要品质性状，甜菜色素是火龙果果肉主要呈色物质，由于甜菜色素具有抗氧化功能，且在食品领域应用前景广泛。本项目以‘紫红龙’（红皮红肉）和‘晶红龙’（红皮白肉）两个火龙果品种为研究材料，比较果肉在表型、代谢组、蛋白质组和转录组水平上的差异。结果表明，火龙果果实组织色素细胞数量的增加促进了果实呈色，红肉品种果肉破色期可观察到红色色素积累，甜菜红色素在细胞间不存在相互转运情况；不同类型或不同组织间存在差异化的色泽参数特征，色泽参数变化早于色素含量变化；筛选到酪氨酸、柠檬酸、色氨酸和色胺等4个果皮和果肉中共有的差异代谢物；发现红、白肉火龙果差异代谢物主要富集在苯丙氨酸和酪氨酸等代谢，主要涉及酪氨酸代谢、氨酰生物合成、异喹啉生物碱生物合成和氮代谢；通过转录组结合蛋白质组学技术，筛选到78个差异蛋白，主要参与植物抗逆、色素合成、产物代谢和脂肪酸代谢等过程；采用SMRT+ RNA-Seq技术进行转录组测序，分别从‘紫红龙’和‘晶红龙’果肉中获得65,312和91,638个unigenes，平均长度是分别为1,175 nt和1,337 nt，分别鉴定到111,650和11,113个lncRNA，建立了火龙果三代全长转录组数据；找到2个火龙果甜菜色素合成相关新基因(HpCYP76AD4和HpDODA)，其表达水平与甜菜色素累积含量一致，在果肉甜菜色素合成中发挥了重要作用，在白色果肉中主要缺少DODA基因，导致不能合成甜菜醛氨酸，从而导致甜菜色素的缺失；克隆了火龙果色素合成相关基因全长序列6个，其中HpPEAMT基因还参与抗旱应答调控；初步建立了火龙果遗传转化体系。研究结果为全面认识火龙果色素合成的分子调控机理提供了重要基础数据。项目发表相关论文14篇，其中SCI收录7篇；授权发明专利1件；资助研究生9名，其中毕业博士2名、硕士5名；有4人职称得到提升。