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馬家柚果實(shí)常溫貯藏期間檸檬酸含量變化及相關(guān)基因的表達(dá)分析

2024-04-30 14:57:10樊友富胡威楊莉張王妮劉德春宋杰劉勇匡柳青
果樹學(xué)報(bào) 2024年3期
關(guān)鍵詞:基因表達(dá)檸檬酸

樊友富 胡威 楊莉 張王妮 劉德春 宋杰 劉勇 匡柳青

摘? ? 要:【目的】明確江西省特色品種馬家柚果實(shí)貯藏期間有機(jī)酸含量的變化規(guī)律及檸檬酸積累調(diào)控相關(guān)基因的表達(dá)特征,為篩選出調(diào)控貯藏早期檸檬酸含量顯著增加的關(guān)鍵基因以及提高貯藏品質(zhì)提供理論依據(jù)?!痉椒ā恳再A藏0~150 d的馬家柚果實(shí)為試材,采用高效液相色譜法(HPLC)測(cè)定果實(shí)內(nèi)有機(jī)酸的含量,利用實(shí)時(shí)熒光定量PCR(qRT-PCR)技術(shù)測(cè)定檸檬酸合成、轉(zhuǎn)運(yùn)和降解相關(guān)基因的相對(duì)表達(dá)量?!窘Y(jié)果】馬家柚果實(shí)有機(jī)酸含量在貯藏初期(0~40 d)顯著上升,并在貯藏40 d時(shí)達(dá)到最大值,貯藏40~70 d時(shí)下降,貯藏70~80 d短暫上升后直至貯藏結(jié)束沒有明顯變化。檸檬酸為果實(shí)中最主要的有機(jī)酸,且其變化趨勢(shì)與有機(jī)酸變化動(dòng)態(tài)基本一致,而蘋果酸、奎寧酸、酒石酸含量極低,并在整個(gè)貯藏期變化不明顯。對(duì)檸檬酸含量顯著變化的貯藏期(0~70 d)進(jìn)行檸檬酸積累調(diào)控相關(guān)基因的表達(dá)分析,發(fā)現(xiàn)合成基因CmPEPC1和CmCS1/2表達(dá)量在貯藏0~40 d增加,CmPEPC1/2和CmCS2在貯藏40~70 d減少;轉(zhuǎn)運(yùn)基因CmVHA-c4表達(dá)量呈先上升(貯藏0~40 d)后下降(貯藏40~70 d)的趨勢(shì),而CmVHP2只在貯藏40~70 d顯著下降,CmDIC的變化趨勢(shì)與二者相反,在貯藏0~40 d逐漸下降。降解基因CmGS2和CmGAD5在貯藏0~40 d期間表達(dá)量逐漸減少。【結(jié)論】馬家柚果實(shí)有機(jī)酸含量在貯藏期間主要受檸檬酸含量變化的影響,貯藏早期檸檬酸含量顯著變化可能受其合成(CmPEPC1/2、CmCS1/2)、轉(zhuǎn)運(yùn)(CmDIC、CmVHP2、CmVHA-c4)和降解(CmGS2、CmGAD5)相關(guān)基因的共同調(diào)控。

關(guān)鍵詞:馬家柚;果實(shí)貯藏;檸檬酸;基因表達(dá)

中圖分類號(hào):S666.3 文獻(xiàn)標(biāo)志碼:A 文章編號(hào):1009-9980(2024)03-0448-11

Changes of citrate acid contents and expression of related genes in Majiayou pomelo fruit during room temperature storage

FAN Youfu, HU Wei#, YANG Li, ZHANG Wangni, LIU Dechun, SONG Jie, LIU Yong, KUANG Liuqing*

(School of Agricultural Sciences, Jiangxi Agricultural University, Nanchang 330045, Jiangxi, China)

Abstract: 【Objective】 Majiayou pomelo [Citrus grandis (L.) Osbeck] is a local characteristic variety in Guangfeng district, Shangrao City, Jiangxi province. Majiayou pomelo is popular among consumers because of its large fruit size, high nutritional value, unique favor and easy storability. Organic acid is an important component to determine the intrinsic quality of citrus fruit. Excessive or low acid contents will affect the favor and storage performance of citrus fruit. At present, the research on organic acids of Majiayou pomelo fruit mainly focuses on the development process, but the changes of its organic acid and molecular regulation mechanism during postharvest storage are less studied. In this experiment, the contents of organic acid components in Majiayou pomelo pulp during a long storage period (150 d) were determined, and the expression levels of genes regulating citrate acid that was dramatically accumulated at early storage period (0-70 d) were analyzed. The purpose of this study was to clarify the changes of the content and molecular regulation mechanism of citrate acid accumulation at early storage period, so as to provide theoretical basis for identifying key genes regulating citrate acid accumulation and improving storage quality of Majiayou pomelo. 【Methods】 The experiment material was Majiayou pomelo fruit harvested from 12 years old trees. Fruits with the same maturity and size, and without diseases and pests as well as mechanical surface damage were selected for storage at room temperature for 150 days. Samples were taken every 10 days during storage period, and three biological replicates were set for each sample. Three fruits were taken from each replicate for pulp separation, and then quickly frozen in liquid nitrogen. The contents of citric acid, malic acid, quinic acid and tartaric acid in pulp were determined by the high performance liquid chromatography (HPLC). The HPLC test was performed on Shimadzu SPD-M20A with a C18 column (4.6 mm×250 mm, 5 μm) at 30 ℃, the mobile phase was 0.01 mol·L-1 H2SO4 and flow rate was 0.5 mL·min-1. The diode array detector was used for detection at wavelength of 210 nm. Malic acid, citric acid, quinic acid and tartaric acid standards were chromatographically pure. Real-time quantitative fluorescent PCR (qRT-PCR) was used to analyze the relative expression levels of genes related to citric acid synthesis, transport and degradation in Majiayou pomelo pulp after the sample fruits were stored for 0, 20, 40 and 70 days. RNA was extracted with the kit, and the integrity of RNA was detected by the agarose gel electrophoresis. The first cDNA strand was synthesized using the reverse transcription test kit (Cat.#RR047A). RT-qPCR gene expression level was analyzed using SYBR?Preminx Ex TaqTM kit. Three biological replicates were set up for each sample, and the data were analyzed by 2-??Ct method. Finally, the relationship between the change pattern of citric acid content and the expression levels of related genes was analyzed. 【Results】 The organic acids in the pulp of Majiayou pomelo mainly included citric acid, malic acid, tartaric acid and quinic acid, among which citric acid was the most important component, accounting for 73.4% of the total organic acids. With the increase of storage time, the content of total organic acids showed a trend of first increasing and then decreasing, that is, it gradually increased to a peak value (7.0 mg·g-1) at 0-40 d, then showed a significant downward trend at 40-70 d, reached a minimum value (3.8 mg·g-1) at 70 d, and then recovered to a stable level at 80 d. The change pattern of citric acid contents was basically consistent with that of the total organic acid contents, which decreased to the lowest value of 2.7 mg·g-1 at 70 d, and did not change significantly until the end of storage (150 d) after a short increase from 70 d to 80 d. The contents of malic acid, quinic acid and tartaric acid changed steadily throughout the storage period. The expression of genes related to citric acid metabolism in the pulp of Majiayou pomelo fruits stored for 0 d, 20 d, 40 d and 70 d was analyzed. The results showed that the expressions of citric acid synthetic genes CmPEPC1 and CmCS1/2 increased at 0 d to 40 d, while CmPEPC1/2 and CmCS2 decreased at 40 d to 70 d. The relative expression of proton pump gene CmVHA-c4 first increased (0 d to 40 d) and then decreased (40 d to 70 d). However, the relative expression of CmVHP2 decreased significantly only from 40 d to 70 d. The relative expression of mitochondrial dicarboxylic acid carrier gene CmDIC decreased gradually from 0 d to 40 d after storage, and there was no significant difference between 40 d and 70 d. The relative expression levels of CmGS2 and CmGAD5 decreased gradually during 0-40 d storage, which was opposite to that of citric acid content, and the difference was not significant at 40 d to 70 d. The expression levels of other genes related to citric acid degradation, such as CmACL1, CmACL3, CmACO3, CmNAD-IDH2, CmNAD-IDH3 and CmGABA-T, were inconsistent with the trend of citric acid degradation. 【Conclusion】 The content of organic acid in Majiayou pomelo pulp was mainly affected by the change of citric acid content during storage, and the significant change of citrate acid content in early storage period may be co-regulated by genes related to its synthesis (CmPEPC1/2 and CmCS1/2), transport (CmDIC, CmVHP2 and CmVHA-c4) and degradation (CmGS2 and CmGAD5).

Key words: Majiayou pomelo; Fruit storage; Citric acid; Gene expression

馬家柚[Citrus grandis (L.) Osbeck]是江西省上饒市廣豐區(qū)的地方特色品種,因其果大、營養(yǎng)價(jià)值高、獨(dú)特的香味和耐貯性而廣受消費(fèi)者歡迎,它也是中國首個(gè)通過臨床驗(yàn)證治療糖尿病的保健食品[1]。有機(jī)酸是決定柑橘類水果內(nèi)在品質(zhì)的重要成分。其含量過高或過低都會(huì)影響水果的正常風(fēng)味。檸檬酸是柑橘類水果中的主要有機(jī)酸,不同種類的柑橘類水果的檸檬酸含量及其積累模式在貯藏期間存在很大差異[2]。楊嵐琪等[3]研究發(fā)現(xiàn)湖南新引進(jìn)的4個(gè)寬皮柑橘品種(春見、早蜜椪柑、金秋砂糖橘、春香)和4個(gè)甜橙品種(錦紅、橘湘瓏、錦秀、橘湘元)果實(shí)的有機(jī)酸含量在整個(gè)貯藏過程中均呈下降趨勢(shì);李永杰等[4]也發(fā)現(xiàn)紅美人雜柑果實(shí)的有機(jī)酸含量在貯藏過程中下降;但汪妮娜等[5]發(fā)現(xiàn)三紅蜜柚、紅肉蜜柚和桂紅柚1號(hào)在貯藏過程中有機(jī)酸含量逐漸上升。

柑橘中的檸檬酸首先在線粒體中由磷酸烯醇式丙酮酸(PEP)與CO2結(jié)合,在磷酸烯醇式丙酮酸羧化酶(PEPC)催化下生成草酰乙酸(OAA),然后在檸檬酸合成酶(CS)的作用下由OAA和乙酰-CoA合成,隨后檸檬酸通過跨膜轉(zhuǎn)運(yùn)方式進(jìn)入液泡中積累,并依賴轉(zhuǎn)運(yùn)載體和離子通道進(jìn)入細(xì)胞質(zhì)中被降解利用。趙淼等[6]發(fā)現(xiàn)在不同早、晚熟柑橘品種果實(shí)發(fā)育過程中PEPC和CS活性都呈現(xiàn)降低-升高-降低的表達(dá)模式,與檸檬酸含量變化的趨勢(shì)呈顯著正相關(guān);而在靖安椪柑果實(shí)發(fā)育過程中CitCS2和CitPEPC1相對(duì)表達(dá)先增加后減少,檸檬酸含量呈現(xiàn)相同變化趨勢(shì)[7];檸檬酸合成后多余部分在液泡膜V型質(zhì)子泵和P型質(zhì)子泵的參與下,通過特異轉(zhuǎn)運(yùn)蛋白運(yùn)輸?shù)揭号葜羞M(jìn)行儲(chǔ)存;Li等[8]通過對(duì)高橙(高酸)和溫州蜜柑(低酸)兩個(gè)柑橘品種研究,發(fā)現(xiàn)VHA-c4通過與轉(zhuǎn)錄因子ERF13發(fā)生蛋白互作促進(jìn)檸檬酸的積累;而溫州蜜柑果實(shí)中V-PPase和V-ATPase被報(bào)道促進(jìn)檸檬酸在液泡中的積累[9]。

當(dāng)果實(shí)中的有機(jī)酸含量達(dá)到一定水平時(shí),檸檬酸開始在細(xì)胞質(zhì)中降解和被利用。檸檬酸的分解主要有三種途徑:GABA、谷氨酰胺和ACL途徑。其中,GABA和谷氨酰胺途徑是果實(shí)中最重要的檸檬酸降解途徑。在GABA途徑中,細(xì)胞質(zhì)中的檸檬酸被順烏頭酸酶(Cyt-Aco)分解成異檸檬酸,異檸檬酸被異檸檬酸脫氫酶(NADP-IDH)分解成α-酮戊二酸(α-KG),隨后在谷氨酸脫氫酶、大冬氨酸轉(zhuǎn)氨酶或丙氨酸轉(zhuǎn)氨酶的作用下生成谷氨酸。谷氨酸一方面被谷氨酸脫羧酶(GAD)催化,生成γ-氨基丁酸(GABA)進(jìn)入GABA途徑,隨后GABA在GABA轉(zhuǎn)氨酶(GABA-T)的作用下生成琥珀酸半醛,最后在琥珀酸半醛脫氫酶的作用下生成琥珀酸;另一方面在谷氨酰胺合成酶(GS)的催化下形成谷氨酰胺。張規(guī)富等[10]發(fā)現(xiàn)水分脅迫處理下的椪柑果實(shí)中有機(jī)酸含量顯著增加,而檸檬酸降解基因CitAco3、CitIDH3和CitGAD5表達(dá)量顯著下調(diào),說明這些基因可能與檸檬酸的降解有關(guān);Chen等[11]研究發(fā)現(xiàn)早熟椪柑果實(shí)中檸檬酸降解相關(guān)基因CitAco3、CitIDH1/3、CitGAD4/5、CitGS2的表達(dá)水平均顯著高于普通椪柑,推測(cè)可能是早熟椪柑果實(shí)中檸檬酸含量顯著低于普通椪柑的重要原因。在ACL途徑中,檸檬酸由ATP-檸檬酸裂解酶(ACL)催化生成草酰乙酸和乙酰輔酶A,紐荷爾、溫州蜜柑果實(shí)汁胞中CitACL基因表達(dá)隨著果實(shí)成熟顯著升高,檸檬酸含量則顯著降低[12];紐荷爾幼果經(jīng)過熱處理后,主要通過ACL途徑降解檸檬酸[13]。

目前關(guān)于馬家柚果實(shí)中有機(jī)酸的研究主要集中在果實(shí)發(fā)育過程中,而針對(duì)馬家柚果實(shí)在采后貯藏過程中有機(jī)酸含量的變化規(guī)律以及分子機(jī)制研究較少[1,14-15]。筆者在本研究中以馬家柚為試材,研究果實(shí)有機(jī)酸組分在貯藏期間含量及相關(guān)基因表達(dá)的變化,探明貯藏早期檸檬酸含量顯著變化的調(diào)控機(jī)制,為提高馬家柚貯藏品質(zhì)提供理論依據(jù)。

1 材料和方法

1.1 試驗(yàn)材料

馬家柚果實(shí)采自江西省上饒市廣豐區(qū)蘆林鎮(zhèn)果園。選取6株生長勢(shì)相當(dāng)、樹齡為12年生的枳砧成年結(jié)果樹,2株為1個(gè)生物重復(fù)。果實(shí)采收時(shí),選擇成熟度和大小一致、無病蟲害、表面無機(jī)械損傷的果實(shí)在室溫(20±2 ℃)下貯藏,每10 d每個(gè)重復(fù)取3個(gè)果實(shí),果肉用液氮速凍后貯藏于-80 ℃的超低溫冰箱中進(jìn)行后續(xù)研究。

1.2 測(cè)定指標(biāo)及方法

1.2.1 有機(jī)酸的提取與含量測(cè)定 果實(shí)中的有機(jī)酸的提取參考趙淼等[6]的方法,略作改動(dòng)。稱取4 g左右的果肉樣品,加入5.0 mL的80%乙醇,35 ℃水浴20 min,室溫下10 000 r·min-1離心15 min,取上清液至25 mL容量瓶,重復(fù)提取3次后定容。取1 mL提取液,旋轉(zhuǎn)蒸干后加入1 mL抽濾排氣的超純水溶解,使用一次性注射器吸取后用直徑13 mm、孔徑0.44 ?m的水系濾頭過濾,濾液用于HPLC測(cè)定酸組分及含量。

色譜條件為Shim-pack VP-ODS 反相色譜柱(4.6 mm×250 mm,5 μm),柱溫為30 ℃,流動(dòng)相為0.01 mol·L-1 H2SO4(0.543 mL濃硫酸定容至1 L,用氫氧化鉀調(diào)節(jié)pH=2.6,抽濾排氣后使用),流速為0.5 mL·min-1;進(jìn)樣體積為20 μL。用二極管陣列檢器檢測(cè)PDA(島津SPD-M20A)(只需氘燈,需要把鎢燈關(guān)閉);檢測(cè)波長為210 nm。

1.2.2 RNA的提取和逆轉(zhuǎn)錄 采用華越洋試劑盒提取馬家柚果肉的RNA,采用瓊脂糖凝膠電泳法檢測(cè)RNA完整性;采用TaKaRa公司的反轉(zhuǎn)錄試劑盒(Cat.#RR047A)合成cDNA第一鏈用于熒光定量分析。反轉(zhuǎn)錄體系為5 × PrimeScript Buffer 4 ?L,PrimeScript RT Enzyme MIX Ⅰ 1 ?L,Oligo dT Primer(50 ?mol·L-1)1 ?L,Random 6 mers(100 ?mol·L-1)2 ?L,Total RNA(1 ?g·?L-1)1 ?L,RNase Free ddH2O補(bǔ)齊至20 ?L。cDNA儲(chǔ)存于-20 ℃冰箱用于后續(xù)試驗(yàn)。

1.2.3 實(shí)時(shí)熒光定量PCR 相關(guān)基因表達(dá)水平分析在Bio-Rad CFX 96-PCR儀上進(jìn)行,試劑來自TaKaRa公司的SYBR?Preminx Ex TaqTM試劑盒,體系為:ddH2O(滅菌蒸餾水)8.0 ?L,SYBR?Preminx Ex Taq 10.0 ?L,PCR Forward Primer(10 ?mol·L-1)0.5 ?L,PCR Reverse Primer(10 ?mol·L-1)0.5 ?L,cDNA 1.0 ?L,總體積20 ?L。內(nèi)參基因與檸檬酸合成、轉(zhuǎn)運(yùn)和降解相關(guān)基因序列來自馬家柚基因組數(shù)據(jù)庫(http://citrus.hzau.edu.cn)[16],引物用Primer 5設(shè)計(jì),引物序列見表1。每個(gè)樣品設(shè)置3次生物學(xué)重復(fù),利用2-??Ct方法進(jìn)行數(shù)據(jù)分析。

1.3 數(shù)據(jù)統(tǒng)計(jì)與分析

使用Microsoft Excel 2010和SPSS Statistic 17.0軟件對(duì)數(shù)據(jù)進(jìn)行整理,顯著性分析采用ANOVA式方差分析和Duncan差異顯著性檢驗(yàn)(p<0.05),使用Origin2018進(jìn)行圖表繪制。

2 結(jié)果與分析

2.1 馬家柚果實(shí)貯藏期間有機(jī)酸含量的變化

馬家柚果實(shí)中有機(jī)酸包括檸檬酸、蘋果酸、酒石酸和奎寧酸等,其中檸檬酸為最主要的有機(jī)酸組分,采收時(shí)占總有機(jī)酸的73.4%。總有機(jī)酸含量(w,后同)隨貯藏時(shí)間的增加呈現(xiàn)先上升后下降,最后趨于平穩(wěn)的變化趨勢(shì)(圖1),在貯藏0~40 d(貯藏初期)逐漸上升至峰值(7.0 mg·g-1),隨后在貯藏40~70 d時(shí)呈明顯下降趨勢(shì),并在貯藏70 d時(shí)為最低值(3.8 mg·g-1),在貯藏80 d時(shí)回升至平穩(wěn);檸檬酸含量變化趨勢(shì)與總有機(jī)酸含量變化基本保持一致,在貯藏70 d時(shí)下降至最低值2.7 mg·g-1,貯藏70~80 d短暫上升后直至貯藏結(jié)束(150 d)沒有明顯變化;而蘋果酸、奎寧酸、酒石酸的含量在整個(gè)貯藏期變化較為平穩(wěn)。

2.2 馬家柚果實(shí)貯藏期檸檬酸合成相關(guān)基因表達(dá)分析

選擇檸檬酸含量顯著變化的貯藏0、20、40、70 d馬家柚果實(shí)進(jìn)行檸檬酸代謝相關(guān)基因的表達(dá)分析(圖2)。在馬家柚果實(shí)貯藏過程中,CmPEPC1基因相對(duì)表達(dá)量在貯藏0~40 d基本呈上升趨勢(shì),在貯藏40~70 d時(shí)顯著降低;CmPEPC2基因相對(duì)表達(dá)量在貯藏0~40 d 時(shí)變化不明顯,但在貯藏40~70 d時(shí)顯著降低;CmCS1基因相對(duì)表達(dá)量在貯藏開始20 d基本保持不變,在貯藏40 d時(shí)顯著上升,貯藏40~70 d時(shí)沒有顯著變化;CmCS1在整個(gè)貯藏過程中呈現(xiàn)先上升后下降趨勢(shì):CmCS2在貯藏第40天時(shí)上升至峰值,在貯藏40~70 d時(shí)顯著下降。以上結(jié)果可推測(cè),CmPEPC1和CmCS1/2與貯藏0~40 d檸檬酸含量增加有關(guān),而CmPEPC1/2和CmCS2可能調(diào)控了貯藏40~70 d期間檸檬酸和含量的減少。

2.3 馬家柚果實(shí)貯藏期檸檬酸轉(zhuǎn)運(yùn)相關(guān)基因表達(dá)分析

檸檬酸通過多種轉(zhuǎn)運(yùn)體和質(zhì)子泵協(xié)同作用向液泡中轉(zhuǎn)運(yùn)。對(duì)貯藏0、20、40、70 d馬家柚果實(shí)進(jìn)行檸檬酸轉(zhuǎn)運(yùn)相關(guān)基因的表達(dá)分析(圖3),陽離子質(zhì)子轉(zhuǎn)運(yùn)體基因CmCHX的相對(duì)表達(dá)量在貯藏0~40 d時(shí)顯著上升,并在貯藏第40天時(shí)顯著上升至最大值,貯藏40~70 d沒有顯著差異;線粒體二羧酸載體基因CmDIC相對(duì)表達(dá)量在貯藏0~40 d逐漸下降,貯藏40~70 d沒有顯著差異;質(zhì)子泵基因CmVHA-c4相對(duì)表達(dá)量呈先上升(貯藏0~40 d)后下降(貯藏40~70 d)的趨勢(shì);而CmVHP2基因相對(duì)表達(dá)量在貯藏0~40 d沒有顯著差異,貯藏40~70 d顯著下降。由以上結(jié)果可知,CmCHX、CmVHA-c4和CmVHP2基因表達(dá)量與此期間檸檬酸含量變化趨勢(shì)相似,而CmDIC基因表達(dá)量則與之相反。

2.4 馬家柚果實(shí)貯藏期間檸檬酸降解相關(guān)基因表達(dá)分析

對(duì)貯藏0、20、40、70 d馬家柚果實(shí)進(jìn)行檸檬酸降解相關(guān)基因的表達(dá)分析(圖4)。在馬家柚貯藏前期,CmACL1基因相對(duì)表達(dá)量持續(xù)顯著上升,CmACL3基因在貯藏40 d時(shí)顯著上升至最大值,貯藏40~70 d時(shí)顯著下降;CmAco3基因表達(dá)量在貯藏20 d時(shí)達(dá)到最大值,隨后趨于平穩(wěn);CmNAD-IDH2基因在貯藏0~40 d時(shí)沒有顯著差異,貯藏40~70 d時(shí)顯著下降;CmNAD-IDH3基因呈先下降后上升再下降的波動(dòng)趨勢(shì);CmGS2和CmGAD5基因相對(duì)表達(dá)量在貯藏40 d之前均呈現(xiàn)下降趨勢(shì),貯藏40~70 d時(shí)差異不顯著。γ-氨基丁酸轉(zhuǎn)氨酶基因CmGABA-T的相對(duì)表達(dá)量在貯藏0~40 d的時(shí)候沒有顯著的變化,在貯藏40~70 d顯著上升,貯藏70 d時(shí)檸檬酸含量最低。由以上結(jié)果可知,只有檸檬酸降解基因CmGS2和CmGAD5在貯藏0~40 d期間與檸檬酸含量呈現(xiàn)相反情況,推測(cè)與此期間果實(shí)檸檬酸含量上升有關(guān)。

3 討 論

檸檬酸是柑橘類水果中的主要有機(jī)酸,占有機(jī)酸總量的63.7%~96.7%。不同柑橘品種在貯藏期間果實(shí)中有機(jī)酸的組成和含量變化趨勢(shì)各不相同。一般來說,貯藏期間柑橘類果實(shí)中的有機(jī)酸含量總體呈下降趨勢(shì)[17-19];但也有部分品種呈現(xiàn)先上升,再下降,所謂“返酸”的現(xiàn)象,如泰國柚在貯藏過程中總酸及檸檬酸含量逐漸增加[20],六月早柚果實(shí)在貯藏20 d出現(xiàn)低程度的“返酸”現(xiàn)象[21];水晶蜜柚以及琯溪蜜柚果實(shí)在貯藏過程中有機(jī)酸含量在貯藏45~60 d時(shí)出現(xiàn)高峰之后逐漸減少[22];李宏祥等[23]研究發(fā)現(xiàn)三個(gè)不同采收期的桃溪蜜柚果實(shí)在貯藏0~60 d有機(jī)酸含量都出現(xiàn)了增加;徐世榮等[24]研究發(fā)現(xiàn),琯溪蜜柚果實(shí)在貯藏過程中檸檬酸含量逐漸增加。筆者在本研究中發(fā)現(xiàn)馬家柚果實(shí)在貯藏40 d前也會(huì)出現(xiàn)“返酸”現(xiàn)象,檸檬酸含量在貯藏初期(0~40 d)顯著增加,且在貯藏40 d時(shí)達(dá)到最大值,在貯藏70 d下降至最低值。因此,為了解馬家柚果實(shí)貯藏前期檸檬酸顯著積累的原因,筆者在本研究中對(duì)此期間檸檬酸代謝相關(guān)基因的相對(duì)表達(dá)量進(jìn)行了分析。

柑橘果實(shí)內(nèi)檸檬酸的含量是其合成、轉(zhuǎn)運(yùn)和降解相關(guān)基因協(xié)同調(diào)控的結(jié)果。檸檬酸的合成主要與PEPC和CS基因相關(guān),有大量研究表明,PEPC和CS基因表達(dá)量或者酶活性的增加與琯溪蜜柚[25]、臍橙[26]、砂梨[27]、菠蘿[28]、蜂糖李[29]、檸檬[30]等果實(shí)發(fā)育前期或貯藏期檸檬酸或蘋果酸積累增加呈顯著正相關(guān)。筆者在本研究中通過對(duì)馬家柚果實(shí)貯藏早期檸檬酸合成相關(guān)基因表達(dá)分析,發(fā)現(xiàn)PEPC1/2和CS1/2基因相對(duì)表達(dá)量變化趨勢(shì)與檸檬酸含量基本動(dòng)態(tài)一致,均在貯藏后40 d時(shí)達(dá)到最大值,與前人研究結(jié)果相符,因此PEPC1/2和CS1/2可能參與馬家柚果實(shí)內(nèi)檸檬酸在貯藏前期的合成。

在植物細(xì)胞中,有機(jī)酸在線粒體中合成,主要儲(chǔ)存在液泡中,并在細(xì)胞質(zhì)中進(jìn)行降解,其在細(xì)胞內(nèi)膜系統(tǒng)中的運(yùn)輸主要通過轉(zhuǎn)運(yùn)蛋白或通道實(shí)現(xiàn),而質(zhì)子泵蛋白為有機(jī)酸相關(guān)次級(jí)轉(zhuǎn)運(yùn)蛋白提供必需的電化學(xué)勢(shì)梯度和能量,因此,質(zhì)子泵蛋白在有機(jī)酸的轉(zhuǎn)運(yùn)過程中發(fā)揮著重要作用。液泡膜上的質(zhì)子泵有3種類型:V-ATPase,V-PPase和P-type ATPase。擬南芥中AtVHA-a2和AtVHA-a3基因突變,會(huì)導(dǎo)致植物中有機(jī)酸含量降低[31];而檸檬果實(shí)VHA蛋白C亞基的激活會(huì)導(dǎo)致檸檬酸的積累增加[32];同樣VHP的活性與葡萄[33]、番茄[34]、檸檬[35-36]果實(shí)中有機(jī)酸含量呈正相關(guān)。筆者在本研究中發(fā)現(xiàn),CmVHA-c4和CmVHP2基因的表達(dá)量變化趨勢(shì)與馬家柚果實(shí)內(nèi)檸檬酸含量變化的趨勢(shì)一致,由此推測(cè)CmVHA-c4和CmVHP2在調(diào)控果實(shí)中檸檬酸在液泡中的積累方面可能發(fā)揮了重要作用,后期可對(duì)二者的生物功能進(jìn)行進(jìn)一步的研究。線粒體二羧酸轉(zhuǎn)運(yùn)蛋白DIC能促進(jìn)線粒體與細(xì)胞質(zhì)中三羧酸鹽陰離子與二羧酸鹽陰離子的交換,并負(fù)責(zé)擬南芥葉片液泡內(nèi)檸檬酸以及蘋果酸選擇性地轉(zhuǎn)運(yùn)[37-38];在柑橘上,CHX和DIC轉(zhuǎn)運(yùn)蛋白可能參與果實(shí)液泡中的檸檬酸向細(xì)胞質(zhì)的轉(zhuǎn)運(yùn)[39]。筆者在本研究中發(fā)現(xiàn)馬家柚果實(shí)在貯藏前期,即檸檬酸含量持續(xù)增加時(shí),CmDIC基因表達(dá)顯著下調(diào),推測(cè)可能是CmDIC的下調(diào)表達(dá),抑制了有機(jī)酸向線粒體的轉(zhuǎn)運(yùn)和代謝,從而增加了液泡和細(xì)胞質(zhì)中檸檬酸的含量。

檸檬酸運(yùn)輸?shù)郊?xì)胞質(zhì)后,主要通過GABA和谷氨酰胺途徑降解。在谷氨酰胺途徑中,谷氨酰胺合成酶(GS)起主導(dǎo)作用。有研究表明在果實(shí)發(fā)育后期,CsGS1基因的高表達(dá),導(dǎo)致奉節(jié)72-1[40]、紅橘砧臍橙果實(shí)[41]、贛州臍橙果實(shí)[42]檸檬酸含量降低。在GABA途徑中,谷氨酸在GAD的催化下生成GABA。易明亮等[14]、Liu等[43]和宋江濤等[44]的研究表明,CsGAD1、CsGAD2和GAD5基因表達(dá)上調(diào)會(huì)導(dǎo)致果實(shí)檸檬酸含量顯著下降;Sheng等[45]通過外源噴施GABA抑制果實(shí)中GAD基因表達(dá),從而導(dǎo)致果實(shí)中檸檬酸含量顯著增加;筆者在本研究中發(fā)現(xiàn),CmGS2和CmGAD5基因表達(dá)都是在貯藏40 d前顯著下調(diào),延緩了檸檬酸的降解速率,從而使得該貯藏階段馬家柚果實(shí)檸檬酸含量上升。因此推測(cè),馬家柚果實(shí)中檸檬酸可能主要通過谷氨酰胺和GABA途徑進(jìn)行降解。

4 結(jié) 論

筆者在本研究中通過對(duì)馬家柚貯藏過程中的有機(jī)酸含量變化以及貯藏早期檸檬酸積累調(diào)控相關(guān)基因表達(dá)分析,明確了在馬家柚整個(gè)貯藏階段有機(jī)酸的組成以及含量變化規(guī)律,并得出馬家柚果實(shí)在貯藏期間的主要有機(jī)酸為檸檬酸,在貯藏40 d時(shí)出現(xiàn)明顯返酸現(xiàn)象;這種變化可能主要由檸檬酸合成基因PEPC1/2和CS1/2、有機(jī)酸轉(zhuǎn)運(yùn)相關(guān)基因CmDIC、VHA-c4和VHP2以及降解基因GS2、GAD5表達(dá)調(diào)控;為進(jìn)一步研究柑橘果實(shí)貯藏期間檸檬酸積累調(diào)控機(jī)制奠定了理論基礎(chǔ)。

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