本文選題：柑橘　切入點：果實貯藏　出處：《華中農(nóng)業(yè)大學》2017年博士論文

【摘要】：有機酸是柑橘果實風味品質(zhì)的重要組成成分,其含量高低與果實衰老進程和鮮果貨架期長短緊密相關(guān)。檸檬酸是柑橘果實中最重要的有機酸,在柑橘采后貯藏過程中,有機酸會隨著呼吸和能量代謝逐漸被消耗,導致果實風味品質(zhì)下降,失去商品價值。不同果實中有機酸含量的高低受遺傳、環(huán)境和栽培技術(shù)等因素共同影響;細胞內(nèi)有機酸水平受其合成、分解、轉(zhuǎn)運和液泡貯藏綜合調(diào)控。前人關(guān)于有機酸代謝的研究大多集中在TCA循環(huán)關(guān)鍵酶的調(diào)控。近年來越來越多的研究者開始關(guān)注有機酸降解和液泡貯藏對其含量的影響。γ-氨基丁酸(GABA)支路是檸檬酸降解的主要途徑之一,同時GABA代謝的最終產(chǎn)物琥珀酸又能回補到TCA循環(huán),為該循環(huán)提供所需的碳骨架。因而細胞質(zhì)中合成的GABA能否進入線粒體進一步代謝不僅影響檸檬酸的降解,同時還可影響TCA循環(huán)的底物供應。線粒體GABA透性酶(GABP)控制著GABA向線粒體的轉(zhuǎn)運,是連接GABA代謝和TCA循環(huán)的閥門,但有關(guān)其對檸檬酸代謝的具體調(diào)控機制尚不清楚,以及其對GABA的轉(zhuǎn)運引起的線粒體內(nèi)外具體的生理生化反應也缺乏系統(tǒng)的研究。本研究以不同來源的高酸和低酸柑橘果實為材料,研究GABA代謝支路對貯藏期檸檬酸含量的影響,并以柑橘GABP為切入點,對其調(diào)控檸檬酸代謝的機制展開深入研究,主要結(jié)果如下:1.外源GABA處理可以顯著提高采后貯藏期間夏橙(Citrus sinensis Osbeck‘Valencia’)和紐荷爾(Citrus sinensis Osbeck‘Newhall’)果實中檸檬酸的含量。實驗結(jié)果表明,外源GABA處理柑橘果實,可誘導內(nèi)源GABA積累。基因表達分析顯示,處理可抑制GABA上游谷氨酸脫羧酶(GAD)的表達,同時在貯藏初期還降低了GABP的表達,從而抑制檸檬酸的降解。此外,與檸檬酸代謝相關(guān)的氨基酸如谷氨酸、天冬氨酸、絲氨酸、纈氨酸以及脯氨酸在處理的果實中也出現(xiàn)不同程度的積累。GABA處理提高了果實中抗氧化酶如POD的酶活性和ATP的含量;同時顯著降低了貯藏期果實的腐爛率,提高了果實的貯藏性能。與處理夏橙不同的是,GABA處理加速了紐荷爾果實果皮顏色由黃轉(zhuǎn)紅,提高了果實的貯藏品質(zhì)。GABA作為GRAS(Generally Recognized As Safe)類化合物,本研究為柑橘采后生產(chǎn)提供了一種高效且安全的采后處理方法,用以提高果實的貯藏品質(zhì)和性能。2.GABA支路基因的表達活性參與決定HB(Citrus grandis Osbeck‘Hirado Buntan’)×Fairchild(Citrus reticulata×Citrus grandis‘Fairchild’)雜交群體高酸和低酸果實中的酸水平。通過連續(xù)三年對雜交群體果實進行品質(zhì)分析,發(fā)現(xiàn)可滴定酸(TA)含量穩(wěn)定地表現(xiàn)出高于親本的特征。選取雜交群體中穩(wěn)定表現(xiàn)出最高酸和最低酸的果實進行貯藏實驗。結(jié)果表明,在貯藏過程中TA含量下降明顯,而可溶性固形物(TSS)含量變化不大。高酸果實中即使TA含量逐漸下降,卻仍能維持顯著高于低酸果實的特征。初生代謝物含量檢測發(fā)現(xiàn)雜交群體TA含量的超親特征主要歸因于檸檬酸水平,而與蘋果酸無關(guān);除了天冬氨酸和天冬酰胺之外,與檸檬酸代謝密切相關(guān)的其他氨基酸沒有表現(xiàn)出和檸檬酸含量類似的變化特征。值得注意的是,即使這些代謝物的含量在果實貯藏過程中會出現(xiàn)波動變化,卻不改變其在高酸和低酸果實中的相對高低,說明果實發(fā)育成熟過程中積累的代謝物含量水平?jīng)Q定了其在貯藏過程中的含量高低。檸檬酸代謝相關(guān)基因的表達分析表明大部分GABA代謝支路基因以及順烏頭酸酶(Aco)的表達在高酸果實中顯著低于低酸果實,是高酸果實維持高酸含量的主要原因。對果實的呼吸測定分析發(fā)現(xiàn)高酸果實的呼吸速率比低酸果實低,說明高酸果實中通過呼吸消耗的有機酸少。果實失水率測定結(jié)果表明在貯藏過程中失水率呈現(xiàn)逐漸上升的趨勢,并且與TA含量呈現(xiàn)顯著負相關(guān)關(guān)系,即高酸果實的失水率比低酸果實低,說明有機酸與水分之間存在密切聯(lián)系。本研究從遺傳背景對貯藏柑橘果實有機酸含量的影響著手,突出了GABA代謝支路活性對酸積累的決定作用。3.柑橘GABA轉(zhuǎn)運蛋白基因(CgGABP)通過調(diào)控檸檬酸代謝相關(guān)基因的表達參與調(diào)控檸檬酸代謝,且轉(zhuǎn)錄因子Cgb HLH13和CgBBX32能夠通過負調(diào)控GABP的表達參與調(diào)控檸檬酸水平。以檸檬酸含量差異較大的普通高酸柑橘品種HB柚(Citrus grandis Osbeck‘Hirado Buntan’)和低酸品種無酸柚(Citrus grandis Osbeck)為材料,同樣發(fā)現(xiàn)在高酸HB柚果實中GABA代謝支路所有基因的表達都顯著低于無酸柚,說明GABA代謝支路基因同樣參與決定HB柚和無酸柚果實中的酸水平。因而以CgGABP基因為切入點對其進行了功能分析及對檸檬酸代謝的調(diào)控研究。聚類分析表明CgGABP蛋白屬于氨基酸/多胺/有機陽離子(APC)超家族中雙向作用的氨基酸轉(zhuǎn)運蛋白(BAT)亞家族成員;拓撲結(jié)構(gòu)分析表明CgGABP與已報道的AtGABP的結(jié)構(gòu)非常相似,擁有12個跨膜結(jié)構(gòu)域,并有很多保守的氨基酸殘基。亞細胞定位結(jié)果顯示CgGABP定位在線粒體中,屬于線粒體GABA轉(zhuǎn)運蛋白。將CgGABP超量表達轉(zhuǎn)化番茄,成功獲得轉(zhuǎn)基因番茄植株。初生代謝產(chǎn)物分析發(fā)現(xiàn),轉(zhuǎn)基因番茄果實中檸檬酸含量明顯增加。并且轉(zhuǎn)基因番茄果實表現(xiàn)出高含量的谷氨酸、谷氨酰胺、棕櫚酸和硬脂酸,以及低含量的脯氨酸、丙氨酸、纈氨酸、天冬氨酸、天冬酰胺、琥珀酸及蔗糖。進一步對檸檬酸代謝相關(guān)基因的表達分析顯示超量表達CgGABP可以抑制番茄內(nèi)源SlAco3a和SlAco3b的表達,誘導檸檬酸合酶(SlCS)和磷酸烯醇式丙酮酸羧化酶(SlPEPC)基因的表達,因而促進了檸檬酸的積累。酵母單雜交文庫篩選獲得與CgGABP啟動子互作的上游調(diào)控因子,并對其蛋白結(jié)構(gòu)進行生物信息學分析,發(fā)現(xiàn)其中一個為bHLH家族成員,命名為CgbHLH13;另一個為BBX家族成員,命名為CgBBX32。亞細胞定位實驗進一步確定轉(zhuǎn)錄因子CgbHLH13和CgBBX32定位于細胞核中,符合轉(zhuǎn)錄因子定位特征。酵母點對點實驗進一步證實了轉(zhuǎn)錄因子CgbHLH13和CgBBX32與CgGABP啟動子存在互作。煙草雙熒光素酶分析發(fā)現(xiàn)轉(zhuǎn)錄因子CgbHLH13和CgBBX32負調(diào)控CgGABP的表達。超量表達轉(zhuǎn)錄因子CgbHLH13和CgBBX32轉(zhuǎn)化柑橘愈傷組織,得到2個基因的轉(zhuǎn)基因愈傷系。轉(zhuǎn)錄水平分析表明超量表達愈傷組織中GABP的表達顯著低于野生型,符合負調(diào)控特征。同時檸檬酸含量在轉(zhuǎn)基因愈傷中顯著低于野生型,并且脯氨酸、鳥氨酸、GABA、丙氨酸、纈氨酸、甘氨酸以及蔗糖含量在轉(zhuǎn)基因愈傷組織中顯著高于野生型,而谷氨酸、棕櫚酸和硬脂酸的含量顯著低于野生型,這些結(jié)果正好與超量表達CgGABP轉(zhuǎn)基因番茄獲得的結(jié)果相反,符合GABP被抑制的表型。另外轉(zhuǎn)基因愈傷組織中Aco的上調(diào)表達及PEPC和CS的下調(diào)表達同樣能夠解釋檸檬酸積累的下降。此外,超量表達CgGABP激活了GABA代謝支路,而谷氨酸分解代謝主要轉(zhuǎn)向GABA代謝;相反,超量表達轉(zhuǎn)錄抑制子阻滯了GABA代謝,谷氨酸代謝主要向脯氨酸、鳥氨酸等其他路徑進行分解代謝。本研究中,通過超量表達CgGABP基因以及超量表達CgGABP負調(diào)控因子抑制GABP表達的方法,充分證實了GABP基因?qū)幟仕岽x的調(diào)控作用,且中心C/N的重排參與了GABP調(diào)控的檸檬酸積累過程;并第一次從上游調(diào)控的角度研究有機酸代謝,證實了轉(zhuǎn)錄因子CgbHLH13和CgBBX32通過負調(diào)控GABP的方式參與檸檬酸代謝。這些結(jié)果為調(diào)控柑橘果實有機酸代謝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.

【學位授予單位】：華中農(nóng)業(yè)大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：S666

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