GABA支路调控柑橘果实柠檬酸代谢的机理研究

发布时间：2018-03-25 19:50

本文选题：柑橘　切入点：果实贮藏　出处：《华中农业大学》2017年博士论文

【摘要】：有机酸是柑橘果实风味品质的重要组成成分,其含量高低与果实衰老进程和鲜果货架期长短紧密相关。柠檬酸是柑橘果实中最重要的有机酸,在柑橘采后贮藏过程中,有机酸会随着呼吸和能量代谢逐渐被消耗,导致果实风味品质下降,失去商品价值。不同果实中有机酸含量的高低受遗传、环境和栽培技术等因素共同影响;细胞内有机酸水平受其合成、分解、转运和液泡贮藏综合调控。前人关于有机酸代谢的研究大多集中在TCA循环关键酶的调控。近年来越来越多的研究者开始关注有机酸降解和液泡贮藏对其含量的影响。γ-氨基丁酸(GABA)支路是柠檬酸降解的主要途径之一,同时GABA代谢的最终产物琥珀酸又能回补到TCA循环,为该循环提供所需的碳骨架。因而细胞质中合成的GABA能否进入线粒体进一步代谢不仅影响柠檬酸的降解,同时还可影响TCA循环的底物供应。线粒体GABA透性酶(GABP)控制着GABA向线粒体的转运,是连接GABA代谢和TCA循环的阀门,但有关其对柠檬酸代谢的具体调控机制尚不清楚,以及其对GABA的转运引起的线粒体内外具体的生理生化反应也缺乏系统的研究。本研究以不同来源的高酸和低酸柑橘果实为材料,研究GABA代谢支路对贮藏期柠檬酸含量的影响,并以柑橘GABP为切入点,对其调控柠檬酸代谢的机制展开深入研究,主要结果如下:1.外源GABA处理可以显著提高采后贮藏期间夏橙(Citrus sinensis Osbeck‘Valencia’)和纽荷尔(Citrus sinensis Osbeck‘Newhall’)果实中柠檬酸的含量。实验结果表明,外源GABA处理柑橘果实,可诱导内源GABA积累。基因表达分析显示,处理可抑制GABA上游谷氨酸脱羧酶(GAD)的表达,同时在贮藏初期还降低了GABP的表达,从而抑制柠檬酸的降解。此外,与柠檬酸代谢相关的氨基酸如谷氨酸、天冬氨酸、丝氨酸、缬氨酸以及脯氨酸在处理的果实中也出现不同程度的积累。GABA处理提高了果实中抗氧化酶如POD的酶活性和ATP的含量;同时显著降低了贮藏期果实的腐烂率,提高了果实的贮藏性能。与处理夏橙不同的是,GABA处理加速了纽荷尔果实果皮颜色由黄转红,提高了果实的贮藏品质。GABA作为GRAS(Generally Recognized As Safe)类化合物,本研究为柑橘采后生产提供了一种高效且安全的采后处理方法,用以提高果实的贮藏品质和性能。2.GABA支路基因的表达活性参与决定HB(Citrus grandis Osbeck‘Hirado Buntan’)×Fairchild(Citrus reticulata×Citrus grandis‘Fairchild’)杂交群体高酸和低酸果实中的酸水平。通过连续三年对杂交群体果实进行品质分析,发现可滴定酸(TA)含量稳定地表现出高于亲本的特征。选取杂交群体中稳定表现出最高酸和最低酸的果实进行贮藏实验。结果表明,在贮藏过程中TA含量下降明显,而可溶性固形物(TSS)含量变化不大。高酸果实中即使TA含量逐渐下降,却仍能维持显著高于低酸果实的特征。初生代谢物含量检测发现杂交群体TA含量的超亲特征主要归因于柠檬酸水平,而与苹果酸无关;除了天冬氨酸和天冬酰胺之外,与柠檬酸代谢密切相关的其他氨基酸没有表现出和柠檬酸含量类似的变化特征。值得注意的是,即使这些代谢物的含量在果实贮藏过程中会出现波动变化,却不改变其在高酸和低酸果实中的相对高低,说明果实发育成熟过程中积累的代谢物含量水平决定了其在贮藏过程中的含量高低。柠檬酸代谢相关基因的表达分析表明大部分GABA代谢支路基因以及顺乌头酸酶(Aco)的表达在高酸果实中显著低于低酸果实,是高酸果实维持高酸含量的主要原因。对果实的呼吸测定分析发现高酸果实的呼吸速率比低酸果实低,说明高酸果实中通过呼吸消耗的有机酸少。果实失水率测定结果表明在贮藏过程中失水率呈现逐渐上升的趋势,并且与TA含量呈现显著负相关关系,即高酸果实的失水率比低酸果实低,说明有机酸与水分之间存在密切联系。本研究从遗传背景对贮藏柑橘果实有机酸含量的影响着手,突出了GABA代谢支路活性对酸积累的决定作用。3.柑橘GABA转运蛋白基因(CgGABP)通过调控柠檬酸代谢相关基因的表达参与调控柠檬酸代谢,且转录因子Cgb HLH13和CgBBX32能够通过负调控GABP的表达参与调控柠檬酸水平。以柠檬酸含量差异较大的普通高酸柑橘品种HB柚(Citrus grandis Osbeck‘Hirado Buntan’)和低酸品种无酸柚(Citrus grandis Osbeck)为材料,同样发现在高酸HB柚果实中GABA代谢支路所有基因的表达都显著低于无酸柚,说明GABA代谢支路基因同样参与决定HB柚和无酸柚果实中的酸水平。因而以CgGABP基因为切入点对其进行了功能分析及对柠檬酸代谢的调控研究。聚类分析表明CgGABP蛋白属于氨基酸/多胺/有机阳离子(APC)超家族中双向作用的氨基酸转运蛋白(BAT)亚家族成员;拓扑结构分析表明CgGABP与已报道的AtGABP的结构非常相似,拥有12个跨膜结构域,并有很多保守的氨基酸残基。亚细胞定位结果显示CgGABP定位在线粒体中,属于线粒体GABA转运蛋白。将CgGABP超量表达转化番茄,成功获得转基因番茄植株。初生代谢产物分析发现,转基因番茄果实中柠檬酸含量明显增加。并且转基因番茄果实表现出高含量的谷氨酸、谷氨酰胺、棕榈酸和硬脂酸,以及低含量的脯氨酸、丙氨酸、缬氨酸、天冬氨酸、天冬酰胺、琥珀酸及蔗糖。进一步对柠檬酸代谢相关基因的表达分析显示超量表达CgGABP可以抑制番茄内源SlAco3a和SlAco3b的表达,诱导柠檬酸合酶(SlCS)和磷酸烯醇式丙酮酸羧化酶(SlPEPC)基因的表达,因而促进了柠檬酸的积累。酵母单杂交文库筛选获得与CgGABP启动子互作的上游调控因子,并对其蛋白结构进行生物信息学分析,发现其中一个为bHLH家族成员,命名为CgbHLH13;另一个为BBX家族成员,命名为CgBBX32。亚细胞定位实验进一步确定转录因子CgbHLH13和CgBBX32定位于细胞核中,符合转录因子定位特征。酵母点对点实验进一步证实了转录因子CgbHLH13和CgBBX32与CgGABP启动子存在互作。烟草双荧光素酶分析发现转录因子CgbHLH13和CgBBX32负调控CgGABP的表达。超量表达转录因子CgbHLH13和CgBBX32转化柑橘愈伤组织,得到2个基因的转基因愈伤系。转录水平分析表明超量表达愈伤组织中GABP的表达显著低于野生型,符合负调控特征。同时柠檬酸含量在转基因愈伤中显著低于野生型,并且脯氨酸、鸟氨酸、GABA、丙氨酸、缬氨酸、甘氨酸以及蔗糖含量在转基因愈伤组织中显著高于野生型,而谷氨酸、棕榈酸和硬脂酸的含量显著低于野生型,这些结果正好与超量表达CgGABP转基因番茄获得的结果相反,符合GABP被抑制的表型。另外转基因愈伤组织中Aco的上调表达及PEPC和CS的下调表达同样能够解释柠檬酸积累的下降。此外,超量表达CgGABP激活了GABA代谢支路,而谷氨酸分解代谢主要转向GABA代谢;相反,超量表达转录抑制子阻滞了GABA代谢,谷氨酸代谢主要向脯氨酸、鸟氨酸等其他路径进行分解代谢。本研究中,通过超量表达CgGABP基因以及超量表达CgGABP负调控因子抑制GABP表达的方法,充分证实了GABP基因对柠檬酸代谢的调控作用,且中心C/N的重排参与了GABP调控的柠檬酸积累过程;并第一次从上游调控的角度研究有机酸代谢,证实了转录因子CgbHLH13和CgBBX32通过负调控GABP的方式参与柠檬酸代谢。这些结果为调控柑橘果实有机酸代谢及其含量提供了新的思路。
[Abstract]:Organic acid is an important component of the flavor quality of citrus fruit, its content and fruit senescence and fruit shelf life are closely related. Citric acid is the most important organic acids in citrus fruit, citrus postharvest storage, organic acid with respiration and energy metabolism is gradually consumed, resulting in a decline in flavor quality fruit, lose the value of the goods. The content of organic acid in fruits was affected by different genetic effects, common environment and cultivation techniques and other factors; intracellular levels of organic acids by the synthesis, decomposition, transport and storage of comprehensive regulation. Regulation of vacuole on organic acid metabolism before people are mostly concentrated in the TCA cycle. The key enzyme of more and more in recent years began to focus on the effects of organic acid degradation and vacuolar storage on its content. The gamma aminobutyric acid (GABA) branch is one of the main ways of citric acid degradation, and GABA metabolism The final product can be recovered to TCA acid cycle, the cycle is required to provide carbon skeleton. Thus synthesized in the cytoplasm of GABA can enter the mitochondrial metabolism further affects not only the degradation of citric acid, also can affect the TCA cycle substrate supply. Mitochondrial GABA permease (GABP) control GABA to mitochondrial translocation that is to connect to the GABA metabolism and TCA cycle valve, but its of citric acid metabolism and specific regulation mechanism is not clear, and the physiological and biochemical responses of mitochondria inside and outside the transport of GABA caused by the lack of systematic research. In this study, high and low acid citrus fruits from different sources as materials, effects study on the metabolism of GABA branch to the storage period of citric acid content, and citrus GABP as the starting point, study deeply the mechanism of the metabolism of citric acid, the main results are as follows: 1.. Exogenous GABA treatment can significantly Increase during the postharvest storage of Valencia Orange (Citrus sinensis Osbeck 'Valencia') and Newhall (Citrus sinensis Osbeck 'Newhall') citric acid content in fruit. The experimental results show that exogenous GABA treatment of citrus fruit, can induce endogenous GABA accumulation. Gene expression analysis revealed that treatment could inhibit GABA upstream of glutamic acid decarboxylase (GAD) the expression also reduced the expression of GABP in the early stage of storage, thereby inhibiting the degradation of citric acid. In addition, amino acids such as glutamic acid and citric acid metabolism, aspartic acid, serine, valine and proline accumulation also appeared.GABA the extent of the increase in the content of fruits such as antioxidant enzyme activity and ATP POD in the processing of fruit; while significantly reducing the rate of decay of fruits in storage, improve the Storability of fruits. Unlike summer orange, GABA accelerated the Newhall Erguo The real fruit color from yellow to red, improve the storage quality of fruits.GABA as GRAS (Generally Recognized As Safe) compounds, this study provides a method for treatment of efficient and safe for postharvest citrus production, in order to improve the performance of.2.GABA expression activity and storage quality of fruit branch gene involved in deciding (HB Citrus grandis' Osbeck Hirado Buntan '(Citrus) * Fairchild reticulata * Citrus grandis' Fairchild') hybrid groups high acid level and low acid fruit. By three consecutive years of hybrid population fruit quality analysis, found that the titratable acid (TA) content and stable features were higher. Selection of hybrid in the stable group showed the highest and lowest acid acid fruit storage experiment. The results showed that during the storage of TA content decreased significantly, and the total soluble solids (TSS) content changes little. Even if the content of TA gradually decreased high acid fruit, still can maintain the characteristics of fruit was significantly higher than that of low acid content of primary metabolites were found. The detection of TA content in hybrid population transgressive characteristics are mainly attributed to the citric acid level, and has nothing to do with malic acid; except for aspartic acid and asparagine outside, other amino acids associated with citric acid the metabolism and the content of citric acid showed no similar changes. It is worth noting that, even if the content of these metabolites will fluctuate change during the storage of fruits, but do not change the relative level of high and low acid fruits, accumulation during fruit development and maturation in the metabolite content determines the level of the in the process of storage. The content of citric acid metabolism related gene expression analysis showed that most of GABA metabolism genes and aconitase (Aco) expression in high acid fruit in reality The fruit was significantly lower than that in low acid, high acid content is the main reason for maintaining a high acid fruit. Determination of high acid fruit respiration rate lower than the low acid fruit on fruit respiration, organic acid consumption by respiration in high acid fruit. The fruit water loss rate determination results show that the storage loss rate during the show a gradual upward trend, and has a significant negative correlation with the content of TA, namely high acid fruit water loss rate than the low acid fruit is low, that there is a close relationship between organic acid and water. This study from the genetic background effect on the content of organic acids in citrus fruit storage to highlight the decisive role of citrus GABA transporter gene.3. the metabolism of GABA branch activity on acid accumulation (CgGABP) is involved in the regulation of citrate metabolism by regulating the expression of citric acid metabolism related genes, transcription factor Cgb and HLH13 and CgBBX32 through the negative regulation of GABP The expression is involved in the regulation of citric acid level. The difference in the content of citric acid of ordinary high acid Citrus Cultivars (Citrus grandis Osbeck HB pummelo 'Hirado Buntan') and low acid varieties (Citrus grandis Osbeck) grapefruit acid as material, also found expression in high acid in HB pummelo GABA metabolism all genes were significantly no less than sour pummelo, GABA metabolism genes are also involved in the decision level of HB acid and non acid in grapefruit citrus fruit. So using CgGABP gene as a starting point for the analysis and Research on the regulation of citric acid metabolism. Cluster analysis showed that CgGABP protein belongs to the amino acid / polyamine / organic cationic amino acid (APC) bidirectional transporter superfamily of sub family members; (BAT) analysis showed that the structure of CgGABP and AtGABP has been reported very similar topology with 12 transmembrane domains, and there are a lot of conserved amino acids Residues. Subcellular localization results showed that CgGABP was located in the mitochondria, which belongs to the mitochondrial GABA transporter. The over expression of CgGABP in tomato transformation, successfully obtained transgenic tomato plants. Analysis of primary metabolites, significantly increased the content of citric acid in transgenic tomatoes. And transgenic tomato fruits showed high levels of glutamine, glutamic acid, palmitic acid and stearic acid, and low content of proline, valine, alanine, aspartic acid, asparagine, succinic acid and citric acid on the expression of sucrose. Further metabolism related genes analysis showed that overexpression of CgGABP can inhibit the expression of endogenous SlAco3a and SlAco3b in tomato, induced citrate synthase (SlCS) and phosphoenolpyruvate carboxylase (SlPEPC) gene expression, thus promoting the accumulation of citric acid. Yeast one hybrid library was screened with CgGABP and upstream mover interaction Regulatory factor, and the structure of protein bioinformatics analysis, found that one is a member of the bHLH family, named CgbHLH13; the other is a member of the BBX family, named CgBBX32. subcellular localization experiments to further determine the transcription factor CgbHLH13 and CgBBX32 localized in the cell nucleus, with transcription factors. Yeast point-to-point positioning feature the experiment further confirmed the existence of the interaction of transcription factors CgbHLH13 and CgBBX32 and CgGABP promoter. Tobacco dual luciferase analysis showed that the expression of transcription factor CgbHLH13 and CgBBX32 negative regulation of CgGABP. Overexpression of the transcription factor CgbHLH13 and CgBBX32 transformation of citrus callus, obtained 2 transgenic callus lines. RT-PCR analysis indicated that overexpression of GABP expression in callus significantly lower than that in wild type, with negative regulation characteristics. At the same time, the content of citric acid in transgenic callus was significantly lower than that of the wild type, And ornithine, proline, GABA, alanine, valine, glycine and sucrose content in transgenic callus was significantly higher than that of the wild type, while the content of glutamic acid, palmitic acid and stearic acid were significantly lower than the wild type. These results coincided with the results of CgGABP overexpression transgenic tomato obtained on the contrary, consistent with the phenotype of GABP suppressed the other. Down regulated expression was up-regulated in transgenic calli in Aco and PEPC and CS can also explain the decline in citric acid accumulation. In addition, the expression of CgGABP activated GABA metabolism and excess, glutamate catabolism mainly to GABA metabolism; conversely, overexpression of transcriptional repressor block GABA metabolism, glutamate metabolism mainly to ornithine proline, etc. the other path of catabolism. In this study, the CgGABP gene and overexpression of CgGABP negative regulatory factor inhibiting the expression of GABP by overexpression Law, fully confirmed the regulatory effect of GABP gene on the metabolism of citric acid, citric acid and the accumulation of C/N in the GABP rearrangement center regulation; metabolism of organic acids and for the first time from the upstream regulation angle, confirmed that the transcription factor CgbHLH13 and CgBBX32 are involved in the metabolism of citric acid through the negative regulation of GABP provides a new way. The idea of these results for the control of citrus fruit organic acid metabolism and its content.

【学位授予单位】：华中农业大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S666

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