【作者】 孙晓华；

【导师】 程运江；

【作者基本信息】 华中农业大学 ， 果树学， 2013， 博士

【摘要】 有机酸是影响柑橘果实风味的重要因素之一,在贮藏期间,高浓度的有机酸对于果实保鲜极为重要。柚(Citrus grandis)是我国广泛栽培的重要柑橘类型之一,因其优良的贮藏性能和独特的风味品质备受消费者青睐。本研究以我国主栽的柚品种果实为研究对象,对其果实采后贮藏期间果皮和果肉组织中有机酸和可溶性糖浓度的变化进行分析；并从生理生化、基因表达、代谢物分析等方面着重对HB柚[Hirado Butun Pumelo (Citrus grandis)]采后贮藏期间有机酸代谢以及有机酸与果实衰老之间的联系进行研究,获得的主要结论如下：1、建立了基于高效毛细管电泳法测定柑橘果实中有机酸浓度的技术体系。通过对缓冲溶液、电渗流改性剂、分离电压和检测波长等电泳条件的优化,精确地测定了柚果肉中4种重要有机酸(柠檬酸、苹果酸、乌头酸和延胡索酸)的浓度。优化参数如下：100mM磷酸盐缓冲溶液(0.5mM CTAB, pH7.0),0.5psi (pounds per square inch,压强单位)进样3sec,分离电压12kV,分离温度20℃,检测波长200nm。2、揭示了采后贮藏期间,沙田柚、琯溪蜜柚、HB柚、无酸柚和南康甜柚果肉中有机酸浓度的变化。结果显示,无论哪一个柚品种,有机酸浓度均伴随着波动变化,而并非简单的降低。HB柚果肉中有机酸浓度呈现出明显的波动特性,在78DOS (days of storage)时出现一个有机酸浓度的峰值,随后整体上呈现先升高后降低的趋势。此外,实验中也测定了沙田柚、琯溪蜜柚和HB柚在低温(8—10℃)贮藏条件下果肉中有机酸浓度的变化。沙田柚和HB柚果肉中有机酸浓度的变化趋势不受贮藏温度的影响,琯溪蜜柚果肉中有机酸浓度在室温(16-20℃)和低温(8-10℃)贮藏条件下,呈现出相反的变化趋势。3、揭示了采后贮藏期间,沙田柚、琯溪蜜柚和HB柚果实的不同组织中有机酸和可溶性糖浓度的变化。结果显示,沙田柚和HB柚果肉中有机酸浓度的变化趋势基本相似,有机酸浓度在整体下降的过程中出现一个暂时的峰值,且4种有机酸浓度的变化趋势基本一致；琯溪蜜柚果肉中柠檬酸的浓度在贮藏期间呈现上升趋势,苹果酸、乌头酸和延胡索酸则不同程度地降低。柚果皮(白皮层和黄皮层)中均以奎宁酸为主,且随着贮藏时间的延长,各有机酸浓度均呈现降低趋势；三种可溶性糖的浓度也呈现降低趋势。4、HB柚采后贮藏期间有机酸代谢机制研究生产实践中发现HB柚果实采后存在“返酸”现象。前期的研究证实,HB柚在采后贮藏期间有机酸浓度整体上呈现出先升高后降低的变化趋势,且存在一个有机酸浓度明显高于贮藏初期的时期。因此,我们选取HB柚为实验材料,对其采后贮藏期间有机酸代谢机制进行深入研究。(1)利用毛细管电泳技术精确地测定了HB柚在采后贮藏期间果肉中有机酸的浓度。三年的重复实验证实,HB柚在采后贮藏期间,有机酸浓度呈现规律的波动变化,且在变化过程中存在一个有机酸浓度显著高于贮藏初期的峰值,进一步研究表明,贮藏温度和采后果实自身的呼吸作用不是引起果肉中有机酸浓度波动变化的关键因素。(2)通过qRT-PCR验证已开发的细胞器表达谱芯片中的部分差异表达基因。果肉中柠檬酸的浓度与直接参与柠檬酸合成和代谢的酶(柠檬酸合成酶和乌头酸酶)基因表达之间不存在任何相关性。我们从芯片结果中筛选得到4个参与不同苹果酸代谢途径的酶基因,然而,除了过氧化酶体中的苹果酸脱氢酶基因表达与苹果酸浓度呈现高度的负相关外,其它3个基因均未呈现任何相关性,由此推测,乙醛酸循环可能在HB柚果实采后贮藏期间有机酸代谢中起到了重要的作用。(3) GC/MS测定结果显示,2-酮戊二酸的含量随着贮藏时间的延长呈现降低趋势,琥珀酸的含量显著增加,然而,琥珀酸脱氢酶和2-酮戊二酸脱氢酶基因表达则没有显示出有利于琥珀酸积累的变化趋势。同时,我们也发现γ-氨基丁酸(y-aminobutyric acid, GABA)和谷氨酰胺的含量在果实采后贮藏期间呈现出相似的上升趋势,且正相关于琥珀酸的含量,负相关于2-酮戊二酸的含量。因此,我们推测HB柚果实在采后贮藏期间,除了TCA循环,可能还有其它的代谢路径参与了有机酸的利用。(4)为了进一步研究HB柚在采后贮藏期间果实内部衰老的变化,本研究测定了果肉中ATP和H202的含量,以及抗氧化酶(过氧化物酶和超氧化物歧化酶)的活性。结果显示,HB柚果肉中ATP的含量随着贮藏时间的延长而增加,且变化趋势正相关于有机酸浓度。H202的含量呈现小幅波动变化,整体上呈现上升趋势。此外,分析发现2种抗氧化酶的活性均与H202含量以及有机酸浓度成正相关。本研究发现HB柚果肉中线粒体膜通透性转换孔(MPTP)的开放程度随着贮藏时间的延长持续增加,这表明线粒体膜的损伤程度加剧。综上所述,不同的柚品种因其遗传背景不同,在贮藏期间有机酸浓度变化趋势出现差异,而同一柚品种的不同组织中有机酸种类和浓度也表现出很强的组织特异性。HB柚在采后贮藏期间,存在于有机酸浓度变化中的波动现象在部分细胞器中的基因表达以及代谢物含量变化中也有发现,说明该现象是柑橘类果实采后生命活动固有的特性,随着果实品质劣变,波动特征趋于平缓,甚至消失。柑橘果实在采后贮藏期间,随着2-酮戊二酸含量的降低,GABA和谷氨酰胺的含量呈现升高趋势,推测GABA是调控柑橘果实采后有机酸利用的重要路径。贮藏果实中有机酸浓度与活性氧酶促清除系统中2种抗氧化酶活性的相关性也表明有机酸在缓减果实衰老方面行使功能。更多还原

【Abstract】 Organic acid is one of the important factors which have a great contribution to the overall organoleptic quality of the non-climacteric citrus fruit during post-harvest storage. In stored citrus fruit, a high concentration of organic acids or a low pH value is an indication of delayed fruit senescence. Pumelo (Citrus grandis), a kind of citrus fruit originated and widely cultivated in China, is very popular among consumers for its storability and flavor. The present work focuses on pumelo fruit, aiming to reveal the concentration variation of organic acids and soluble sugars in different tissues among different cultivars. Systemic works were performed at physiological, biochemical and molecular levels in order to explore the organic acid metabolism and the relationships between organic acid and senescence in Hirado Butun pumelo (HBP)(Citrus grandi) during post-harvest storage. The main results are listed below:1. Technological system of organic acid detection in citrus fruit was established based on high performance capillary electrophoresis (HPCE). The method could accurately determine four main organic acids (citric, malic, aconitic and fumaric acid) in pumelo fruit throught optimizing buffer, electroosmotic flow modifier, separation voltage and detection wavelength. Optimized parameters are as follows:buffer solution:100mM phosphate (0.5mM CTAB, pH7.0); the UV detection wavelength:200nm; the injection pressure:0.5psi for3sec; the separation voltage:12kV; column temperature:20℃.2. The changes of organic acids concentration were assayed among the stored fruits of Shatian pumelo (Citrus grandis), Guanxi pumelo (Citrus grandis), HBP (Citrus grandis), acidless pumelo (Citrus grandis) and Nankang pumelo (Citrus grandis). Regardless of cultivars, organic acids concentrations in the pulp fluctuated significantly instead of simply decreasing or increasing. The general trend of organic acids concentration in HBP showed an increase at the prior storage phase and latter decreased, with a peak value occurred at78DOS (days of storage). In addition, organic acids concentrations of Shatian pumelo, Guanxi pumelo and HBP stored at low temperature (8-10℃) were also determined. The results showed that no difference in the changes of organic acids concentration in Shatian and HBP at both ambient temperature (16-20℃) and low temperature (8-10℃), while Guanxi pumelo revealed an inverse trend at the two storage temperatures.3. The changes in concentration of organic acids and soluble sugars were identified in the albedo and flavedo among Shatian pumelo, Guanxi pumelo and HBP. There are similar change tendencies of organic acids concentration in the pulp between Shatian pumelo and HBP, i.e. organic acid concentration shows a temporary peak value during the decreasing process and four organic acids concentrations display basic consistent change tendencies, while the concentration of citric acid in the pulp of Guanxi pumelo increased, the other organic acids (malic, aconitic and fumaric acid) decreased to different extent. Quinic acid takes over the dominate position in the peel of pumelo. Each organic acid decreased with storage duration. Regardless of albedo or flavedo, soluble sugars concentrations declined among the three pumelo varieties.4. Research on organic acid metabolism in HBP during post-harvest storageIt had been reported that HBP showed’returned acidity’phenomenon in the production practice. Previous researches revealed that organic acid concentration increased at the prior storage phase and latter decreased. However, there is an obvious fluctuation during storage, at which the organic acid concentration was higher than that at the earlier storage phase. Therefore, we selected HBP as material to study the organic acid metabolism in post-harvest storage phase.(1) The concentrations of organic acids in HBP pulp during post-harvest storage were determined precisely by HPCE. Reduplicative experiments of three years confirmed that organic acids concentrations changed with fluctuation during post-harvest storage, and a peak value was observed, with a significantly higher abundance than that at the earlier stage of storage. Further research revealed that storage temperature and respiration might not be the critical factors to cause the fluctuation of organic acids concentrations.(2) Differentially expressed genes which were detected by subcellular organelle expression microarray were then verified by qRT-PCR. No correlation was found between citric acid concentration and the expression of genes which participated directly in citrate biosynthesis and degradation in TCA cycle, including citrate synthase and aconitate hydratase. We screened four genes which were involved in different malate metabolic pathways from microarray, but no correlation was found between malic acid concentration and the expressions of genes except malate dehydrogenase (peroxisome), which was obviously negatively correlated with malic acid concentration and was down-regulated with storage prolonged. The result indicated that the glyoxylate cycle might play an important role in organic acid metabolisn of HBP fruit during post-harvest storage.(3) GC/MS analysis showed that2-oxoglutaric acid content decreased with the storage time, while that of succinic acid increased. However, the gene expressions of succinate dehydrogenase and2-oxoglutarate dehydrogenase E1were not favorable for the accumulation of succinic acid. It was also noticed that the contents of both GABA and glutamine increased during post-harvest storage, and were positively correlated with the content of succinic acid and negatively with2-oxoglutaric acid. Besides TCA cycle, we inferred that other metabolic pathways might be also involved in the conversion of2-oxoglutaric acid into succinic acid.(4) For the further research on internal senescence of HBP fruit during post-harvest storage, we determined the contents of ATP and H2O2and the activities of antioxidases (POD and SOD). ATP content in HBP increased with prolonging of storage time, and its changes was positively correlated with that of organic acids concentrations. H2O2content slightly fluctuated while increasing. It was found that the activities of both antioxidases were positively correlated with H2O2content and organic acids concentrations. The opening of mitochondrial permeability transition pore (MPTP) in HBP pulp rose persistently during post-harvest storage, which indicated that the mitochondrial membrane was damaged.Taken together, different pumelo cultivars showed diverse changes in organic acids concentration during post-harvest storage due to their specific genetic backgrounds, while different tissues of pumelo possessed dissimilar types and concentrations of organic acids due to their distinct function. Our research showed that not only the concentrations of organic acids, but also the gene expressions in some subcellular organelles, as well as metabolite contents fluctuated during post-harvest storage in HBP fruit. These fluctuations are intrinsic features of the life activities of harvested citrus fruit. As flavor and commercial quality declined, these fluctuations became irregular and disordered and gradually faded away. As2-oxoglutaric acid content in the harvested fruit declined, that of GABA and glutamine rose. So, we speculated that GABA shunt is an important pathway in regulating the utilization of organic acids in post-harvest citrus fruit. Moreover, the correlation of organic acids concentrations with the enzymatic systems of scavenging ROS indicated that organic acids play an important role in delaying fruit senescence.更多还原