凯特芒果实成熟的生理与分子机理研究

	凯特芒果实成熟的生理与分子机理研究
	武士波
导师	许建初、刘爱忠
关键词	芒果，果实成熟，生理变化，代谢组和转录组，ABA Mango, Fruit Ripening, Physiological Changes, Metabolome and transcriptome, ABA
摘要	芒果（Mangifera indica L.）是典型的多年生果树作物，因其果实美味多汁，并富含多种矿物质、氨基酸、糖、维生素、多酚、类黄酮和类胡萝卜素等营养成分，被誉为“热带水果之王”。果实成熟通常伴随着风味、硬度和颜色等一系列生理生化的变化过程，并且受到内外因素的共同调控。解析果实成熟过程的生理和分子调控机理对提高果实品质、培育不同成熟期品种以及果实的贮藏保鲜具有重要的指导意义。目前，虽然芒果的生产已经进入规模化、商品化的发展阶段，有关芒果果实的研究也开展多年，主要集中在营养成分、风味成分、抗氧化活性成分以及芒果的采后生理变化过程，而对于芒果发育成熟调控的分子机理研究严重不足，尚处于非常基础的初级阶段，仍然远远落后于苹果和香蕉等众多的经济果树。由于芒果果实成熟后期乙烯含量显著累积，长期以来芒果一直被认为是典型的跃变型果实。然而，我们在调查芒果成熟过程的生理变化时，发现芒果成熟期经历的时间差异很大，且内源激素乙烯在不同品种中差异明显，特别是早熟和晚熟品种之间的这种现象更加明显。这些乙烯含量低的果实，其成熟过程在生理和分子上是如何介导的？早熟和晚熟特性是如何形成的？这些问题一直缺乏应有的认识。本研究以产量高、抗病强、口感好且种植面积特别广的市场主流大宗品种凯特芒（Keitt）作为研究对象，在对果实全生命周期的多项生理指标进行精细观察测定的基础上，选择果实发育成熟的五个关键阶段，综合运用生理指标测定、广谱靶向代谢组检测、绝对定量转录组测序（UID RNA-seq）以及多种分子生物学实验，较为系统的解析了芒果果实成熟的生理及分子机理。主要研究结果如下： 1. 通过每15天对凯特芒果实大小和重量进行综合分析发现，凯特芒果发育成熟过程可分为幼果期（MS1，45DAB）、膨大期（MS2，105DAB）、绿熟期（MS3，165DAB）、成熟中期（MS4，195DAB）和完熟期（MS5，240DAB）。总可溶性固形物随着果实的成熟逐渐上升，在MS5达到最大值18.5%；总酸度和果实硬度逐渐下降，在MS5最小值，分别为0.55%和0.5 N；内源生长素的含量呈下降趋势，在MS1阶段含量最高，MS5含量最低，分别为32 ng/g FW和 2 ng/g FW左右，显示生长素可能是芒果后熟相关的拮抗因子。以上生理指标均在MS4-MS5阶段发生最为显著的变化，表明MS4到MS5是芒果果实成熟的关键相变过程。值得注意的是，凯特芒果实乙烯产量释放量呈下降趋势，处于较低水平，且在果实成熟期间未发现有明显的跃变爆发，表明乙烯对凯特芒果实的成熟影响可能比较有限。而脱落酸（ABA）含量变化与芒果果实的成熟密切相关，说明ABA在凯特芒果实成熟过程中发挥重要作用。 2. 通过对五个阶段果肉进行广谱靶向代谢组测定分析，共检测出617种代谢物质，其中，检测到最多的物质是多酚类物质（126种）。通过对550种差异代谢物（DAMs）进行k-means聚类及KEGG功能富集分析，初步掌握了五个关键阶段芒果果实所发生的主要生物学过程。其中，酚酸、黄酮、生物碱和单宁类代谢物在MS1阶段含量最高，而糖、醇和脂类等这些与芒果风味相关的代谢物质在MS5阶段高累积。进一步通过对芒果主要的糖（蔗糖）、主要的酸（苹果酸）及细胞壁主要代谢产物半乳糖醛酸等成熟标志物的变化模式分析发现，这些物质含量的变化与果实的甜度（TSS）、酸度（TA）及果实的硬度（Firmness）等表型变化一致。表明这些物质代谢通路可以作为调控芒果果实成熟分子机理分析研究的靶标。 3. 通过应用绝对定量转录组测序技术，分析了凯特芒果实五个关键发育成熟阶段的高分辨率转录本特征。通过对18398个差异表达基因（DEGs）进行k-means聚类和GO功能富集分析，进一步确定了MS1-MS5所对应的主要的生物学过程，结果与代谢组分析结果具有较好一致性。通过乙烯和ABA合成通路所有的DEGs分析，鉴定到两个差异表达的乙烯合成关键限速酶基因MiACS2和MiACS6在芒果发育早期MS1阶段高表达，而MiPSY、MiBCH1/2、MiZEP和MiNCED1/2等ABA合成关键基因在MS5阶段特异性高表达，这与乙烯和ABA的含量一致。通过ABA及其抑制剂（Fluridon）外源施加实验，共同印证了ABA可能在凯特芒果果实成熟中发挥更重要作用的推测。通过WGCNA分析发现，ABA与成熟标志物TSS、蔗糖含量、苹果酸含量以及果实硬度等显著关联到Mebrown模块基因集，进一步佐证了我们的推测，同时我们还鉴定到MiSWEET12/15，MiPLY8，MiPSY，MiHXK1及MiBAM9等六个典型的成熟标志基因，以及MiHY5，MiGBF4，MiABI5-like和MibZIP9等四个潜在调控芒果成熟的转录因子。通过RT-qPCR分析发现，六个成熟标志基因和四个转录因子均受到ABA的诱导和Fluridon的抑制。为了进一步解析ABA调控芒果果实成熟的内在机制，我们通过酵母单杂交实验和双荧光素酶激活实验，证实了ABA介导的转录因子MiHY5可以直接靶向并激活上述六个成熟标志基因的表达。综上所述，本研究依据凯特芒果实不同发育成熟阶段的内源乙烯和ABA含量变化、成熟标志物和成熟标志基因变化模式，提出了ABA可能对芒果果实成熟具有重要作用的假设，并通过一系列生化实验初步验证和解析了ABA调控凯特芒果实成熟的内在分子机理，为芒果果实成熟调控网络构建提供了新见解。; Mango (Mangifera indica L.), is a typical tropical perennial fruit, known as “The King of Fruits” for its attractive color, pleasant aroma, delicate taste and diverse nutrient composition, containing vitamins, minerals and antioxidants. Fruit ripening is usually accompanied by a series of physiological and biochemical events, such as changes in flavour, firmness and color, which are regulated by internal and external factors. Dissecting the physiological and molecular mechanisms behind the fruit ripening process will not only be significant for improving fruit quality and breeding new cultivars with different maturity periods, but also indispensable for fruit fresh-keeping. Although the production of mango has entered the large-scale and commercialized developmental stage, and the fruit has been in the focus of research for many years. Most of research has been mainly focuses on the nutrition, flavor, antioxidant compounds and the postharvest physiological changes. However, the research on the molecular mechanism underlying mango fruit development and ripening is seriously insufficient, which is still in a very basic initial stage, and still lags far behind many economic fruits such as apple and banana. Due to the burst of ethylene during the ripening process, mango has been regarded as a typical climacteric fruit. However, when we investigated the physiological changes in the ripening process of mango, we found that the ripening time and the endogenous ethylene content among different mango cultivars were various, and the phenomena between early- and late-ripening cultivars were more obvious. It is still largely unknown how the ripening process of fruits with low ethylene is mediated and how the characteristics of early- and late-ripening are formed. In this study, we selected ‘Keitt’ as our material, it is one of the most widely cultivated commercial variety with less fiber, high-yield, and good disease resistance. Based on the intensive observation and measurement of a series of physiological parameters in the fruit whole life cycle, five key developmental and ripening stages were selected for further analysis. Combined with widely targeted metabolomics, UID RNA sequencing and a series of biochemical experiments, the physiological and molecular mechanism underlying mango fruit ripening was systematically analyzed. The main findings are as follows: 1. Through continuous recording and observation of ‘Keitt’ fruit size and weight every 15 days, it was found that the development and ripening process could be divided into five key stages: early fruitlet stage (MS1, 45 DAB), fast-enlarging stage (MS2, 105 DAB), mature green stage (MS3, 165 DAB), the middle stage of fruit ripening (MS4, 195 DAB) and late stage of fruit ripening (MS5, 240 DAB). The total soluble solids gradually increased with the fruit ripening, reaching the maximum value of 18.5% at MS5; the total acidity and fruit firmness gradually decreased, reaching the minimum value of 0.55% an
语种	中文
	2022-05
学位授予单位	中国科学院大学
文献类型	学位论文
条目标识符	http://ir.kib.ac.cn/handle/151853/75112
专题	昆明植物所硕博研究生毕业学位论文
推荐引用方式 GB/T 7714	武士波. 凯特芒果实成熟的生理与分子机理研究[D]. 中国科学院大学,2022.

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