日光溫室不同結(jié)果枝類型對柑橘果實有機酸含量的影響

1 DOI:10.13925/j.cnki.gsxb.20170426 日光溫室不同結(jié)果枝類型對柑橘果實有機酸含量的影響 孫曉華，葉麗紅，劉杰才，李曉靜，張之為，崔世茂，宋 陽* （內(nèi)蒙古農(nóng)業(yè)大學(xué)農(nóng)學(xué)院內(nèi)蒙古設(shè)施園藝工程技術(shù)研究中心，呼和浩特 010019） 摘要：【目的】探明日光溫室中柑橘結(jié)果枝類型對果實中有機酸含量的影響，為溫室柑橘結(jié)果枝組的培養(yǎng)提供理論依據(jù)?！痉椒ā恳澡譖oncirus trifoliate（L.）Raf.砧南豐蜜橘（Citrus reticulate Blanco cv. Kinokun）有葉結(jié)果枝和無葉結(jié)果枝果實為試材，對其糖酸含量、有機酸代謝相關(guān)酶活性以及基因表達量進行分析?！窘Y(jié)果】南豐蜜橘果實發(fā)育過程中蔗糖含量呈上升趨勢，奎寧酸含量呈下降趨勢，葡萄糖和果糖含量為先降后升，檸檬酸含量在果實成熟時與發(fā)育初期相近；無葉果和有葉果的糖酸含量變化趨勢基本一致，但30 DAF無葉果蘋果酸含量顯著高于有葉果；兩類果實中檸檬酸合成酶（citrate synthase，CS）、磷酸烯醇式丙酮酸羧化酶（phosphoenolpyruvate carboxylase，PEPC）、烏頭酸酶（aconitate hydratase，ACO）和蘋果酸脫氫酶（malate dehydrogenase，MDH）等4種重要酶活性變化趨勢基本一致；兩類果實多數(shù)基因的表達量都存在顯著性差異，然而酶活性以及有機酸含量則未出現(xiàn)相應(yīng)的顯著性變化。【結(jié)論】南豐蜜橘果實中糖酸含量以及有機酸代謝相關(guān)重要酶活性不受結(jié)果枝組類型的影響，因此，在日光溫室中進行柑橘樹體枝組修剪時，只需考慮其果實產(chǎn)量即可，而不必過多的去考慮其對果實糖酸等內(nèi)在品質(zhì)的影響。 關(guān)鍵詞：南豐蜜橘；有機酸；基因表達；結(jié)果枝；果實品質(zhì) 中圖分類號：S666.2 文獻標志碼：A 文章編號：1009-9980(2018)05-0001-08 Effect of organic acid content in different species of fruiting branches of citrus fruit in solar greenhouse SUN Xiaohua, YE Lihong, LIU Jiecai, LI Xiaojing, ZHANG Zhiwei, CUI Shimao, SONG Yang*(College of Agronomy, Inner Mongolia Agricultural University, Hohhot 010019 China， Inner Mongolia engineering research center of facility horticulture, Hohhot 010019 China) Abstract:【Objective】In order to provide a reference model for pruning of greenhouse citrus fruiting branches, the experiment was carried out to explore the influence of different species of fruiting branches on the content of organic acid in citrus fruit.【Methods】The experiment used “Nanfeng” tangerine (Citrus reticulate Blanco cv. Kinokun) seedlings with stock of trifoliate orange Poncirus trifoliate (L.) Raf. as material, using Gas chromatography to measure the content of sugars and organic acids. The activities of enzymes that included in tricarboxylic acid cycle (TCA) were analyzed through biochemical methods, and key genes related organic acid metabolism were expressed by real-time quantitative reverse transcription polymerase chain reaction. 【Results】The results showed that sucrose contents in the two types fruits showed an upward trend throughout 收稿日期：2017-11-09 接受日期：2018-02-05 基金項目：內(nèi)蒙古自治區(qū)自然科學(xué)基金項目（2015BS0317） 作者介紹：孫曉華，女，講師，研究方向設(shè)施栽培果樹栽培生理。Tel：0471-4301178，E-mail：sungod0819163.com *通信作者 Author for correspondence. Tel：0471-4301178，E-mail：dbmsysina.com 網(wǎng)絡(luò)出版時間：2018-03-09 11:47:16網(wǎng)絡(luò)出版地址：http:/kns.cnki.net/kcms/detail/41.1308.S.20180309.1147.006.html2 the whole development period, and the accumulation of sucrose was accelerated on the 120 DAF (day after flower). Quinic acid contents presented a downward trend during the development period whether in leafy fruit or leafless fruit, and decrease amplitude was outstanding from 30 DAF to 90 DAF, then the change trends of quinic acid content became smooth until the fruit matured. Glucose and fructose levels dropped sharply on the 30 DAF, and began to rises lowly on the 60 DAF. The change trends of glucose and fructose were consistent with slight fluctuation during the whole development period. Sucrose content was much higher than those of glucose and fructose when the fruit reached mature. During the whole development period, the changes of citric acid content in two types fruits were relatively complicated with fluctuation, and the change trends of two types fruits were consistent. The differences in citric acid contents were observed in two types fruits at the individual period, but they were not significantly. The change trends of malic acid content in leafy fruit and leafless fruit were obvious from 30 DAF to 120 DAF, and they was leveling off after the 120 DAF. The change trends of malic acid in the two types fruits were almost the same, and malic acid content in leafless fruit was 2.53 mg/g at 30 DAF, which is significantly higher than that of leafy fruit (1.51 mg/g). Malate dehydrogenase (MDH) activities in two types fruits showed a relatively stable uptrend. The activities of citrate synthase (CS) and aconitase (ACO) showed a significant fluctuation in some regions, but they showed an upward trend during the whole development period. Phosphoenolpyruvate carboxylase (PEPC) activities declined slightly in overall, following by obviously fluctuated at a local. However, the change trends of four important enzyme activities were consistent in the two types fruits and showed no significant difference on the quantities. The expression trends of CS and ACO was similar with that of citric acid content, but it was slightly backward in time. Two genes expressions demonstrated that leafy fruit were higher those of leafless fruit at early stage of development, but it was inverse at late stage of development, and there were significant differences in two types fruits. The expressions of SDH and FH in leafless fruit were significantly higher than those of leafy fruit at most time, but the change trends of gene expression were not obvious regularity. The expression of MDH encoding the mitochondria malate dehydrogenase in leafy fruit was significantly higher than that of leafless fruit on the 30 DAF, after that, the expression of MDH in leafy fruit was significantly lower than leafless fruit until the fruit became mature on the 210 DAF, which the expressions of MDH reached the same levels. The expression of MDH encoding malate dehydrogenase in the cytoplasm existed significant difference at only several developmental period in two types fruits. Above several genes expression patterns presented first reduced and then rose, and the gene expression trends of two types fruits presented consistent basically. The expression of ME (malic enzyme) in the cytoplasm were significant difference in between of leafy fruit and leafless fruit during the whole development period, ME expression in leafy fruit showed slightly increases from 30 DAF to 60 DAF, then reduced until 150 DAF and rose again to mature; the expression of ME in leafless fruit expressed two rise and two decline in the whole development period. 【Conclusion】In the case of “Nanfeng” tangerine grown in the greenhouse, the types of fruiting branches only affects the fruit setting rate and yield, without affects the contents of sugars and organic acids and the activities of key enzymes related organic acid metabolism in fruit. Once the fruit was formed, there was no significant difference in the sugar and organic acid content in fruit whether leafy fruiting branches or leafless fruiting branches. Therefore, when we prune the citrus tree branches, only the yield of fruit should be considered, regardless of its influence on the intrinsic quality about the sugar and organic acid in fruit. 3 Key words: “Nanfeng” tangerine; Organic acid; Gene expression; Fruiting branches; Fruit quality 近幾年，我國休閑農(nóng)業(yè)作為一類新型產(chǎn)業(yè)悄然興起，各類主題觀光溫室及觀光、采摘、娛樂兼顧的大型日光溫室在很多城市周邊不斷涌現(xiàn)，成為城市居民休閑娛樂的重要場所。為了滿足人們對休閑農(nóng)業(yè)和熱帶、亞熱帶水果采摘的需求，許多熱帶、亞熱帶果樹也引入北方日光溫室種植，其中溫室柑橘的種植面積呈上升趨勢。然而，溫室栽培中加溫時間長、溫度過高以及光照不足等原因都會造成柑橘花芽減少、落花落果加重，最終導(dǎo)致產(chǎn)量下降1。研究表明，種植在大棚內(nèi)的柑橘，其果實中可溶性固形物、總糖和維生素C含量均低于露地栽培果實，而有機酸含量則高于露地果實2?；谝陨显耘嘀谐霈F(xiàn)的問題，日光溫室栽培柑橘果實的品質(zhì)提高則顯得尤為重要。 有機酸是影響柑橘果實風(fēng)味和營養(yǎng)品質(zhì)的重要因子，大多數(shù)柑橘品種在成熟期的糖酸比較低，嚴重影響了果實的風(fēng)味品質(zhì)和消費者的接受度，很大程度上制約了設(shè)施柑橘產(chǎn)業(yè)的發(fā)展3。相對而言，柑橘果實中可溶性糖含量的變化幅度較小，而有機酸的種類以及含量變化幅度較大，因此，柑橘果實中所含有機酸對糖酸比的貢獻更為顯著4。 在柑橘設(shè)施栽培中，果樹生長發(fā)育的最適環(huán)境因子，如光照、溫濕度、土壤、氣體條件等均可通過人為控制來實現(xiàn)，研究報道植物光合作用的源庫比率、水分供給、礦物質(zhì)營養(yǎng)和溫度等環(huán)境因子影響著果實細胞中檸檬酸和蘋果酸的積累，進一步闡明這些環(huán)境因子和細胞中有機酸代謝和貯藏之間的相互作用5，6。此外，葉片作為植物光合作用的主要部位，為柑橘果實發(fā)育提供光合產(chǎn)物，碳水化合物供給的限制對于三羧酸循環(huán)（Tricarboxylic Acid Cycle, TCA）中代謝產(chǎn)物的積累是非常不利的7。鑒于此，筆者以日光溫室中種植的南豐蜜橘為試材，選取有葉花枝和無葉花枝所結(jié)果實（以下稱為有葉果和無葉果），測定其糖和有機酸含量，分析有機酸代謝重要酶的活性和相關(guān)基因表達量，旨在探明日光溫室柑橘中不同結(jié)果枝對果實有機酸含量的影響，為研究日光溫室中柑橘枝組修剪與果實品質(zhì)之間的相關(guān)性提供了理論依據(jù)。 1 材料和方法 1.1 植物材料 試驗材料為枳砧南豐蜜橘，種植于內(nèi)蒙古農(nóng)業(yè)大學(xué)教學(xué)科研基地日光溫室內(nèi)。試驗期間日光溫室光照度最低為28000 lx（11-12月），最高為44000 lx（7-8月）；最低溫度為8（1-2、11-12月），最高溫度為45（7-8月）；3-8月日光溫室濕度為34%-87%；而在1-2、9-12月濕度為28%-95%。在南豐蜜橘完全謝花后（2015年4月24日），選取5株生長勢較好且掛果量較大的植株掛牌。從2015年5月24日開始，每隔30d對上述掛牌植株進行隨機取樣，分別采集有葉結(jié)果枝所結(jié)果實和無葉結(jié)果枝所結(jié)果實進行分析研究。 1.2 方法 4 1.2.1 果實中糖和有機酸含量的測定 果實中糖酸提取以及含量測定參考孫曉華博士論文8，稱取1 g果肉組織于液氮中進行研磨，加入80%甲醇10 ml，將勻漿置于70恒溫水浴鍋溫浴30 min，取出后冷卻。上述勻漿于超聲波中萃取90 min，4000 g離心10 min，取上清液于10 ml容量瓶中，加入0.2 ml現(xiàn)配的內(nèi)標液，再加入80%甲醇定容，搖勻。取上述溶液2 ml置于2 ml離心管中，于12000 g離心15 min，取上清液0.5 ml置于真空旋轉(zhuǎn)濃縮儀中，于60干燥至無水狀態(tài)。對干燥物進行衍生化反應(yīng)，加入0.8 ml鹽酸羥胺溶液，置于70反應(yīng)1 h，冷卻。依次迅速加入0.4 ml 六甲基二硅胺烷（Hexamethyldisilazane，HMDS）和0.2 ml三甲基氯硅烷（Trimethylchlorosilane，TMCS），于70下再加熱2 h。取上清液0.5 ml于2 ml自動進樣瓶中，進行氣相色譜火焰離子化檢測器（gas chromatography-flame ionization detection， GC-FID）分析，通過各個組分的保留時間進行定性。 1.2.2 有機酸代謝重要酶活性測定 1.2.2.1 酶液的制備 酶液提取方法參照羅安才等9，果實剖開后對稱取樣，取果肉2 g加入2 ml緩沖液進行研磨，緩沖液為0.2 molL-1Tris-HCl（pH 8.2），0.6 molL-1蔗糖，10 mmolL-1異抗壞血酸，冰浴，4 4000 g離心20 min，取上清液定容至5 ml，其中2 ml 15000 g離心15 min，取上清液用緩沖液提取，緩沖液為0.2 molL-1Tris-HCl（pH 8.2），10 mmolL-1異抗壞血酸，0.1% TritonX-100，定容至4 ml即得細胞質(zhì)烏頭酸酶液（Cyto-Acontiase，EC 4.2.1.3），另外3 ml加入等體積提取緩沖液，即可用于測定蘋果酸脫氫酶（MDH，EC 1.1.1.37），取2ml在大量透析液（即提取緩沖液）中4透析過夜，用新鮮透析液定容即得磷酸烯醇式丙酮酸羧化酶（PEPC，EC 4.1.1.31），檸檬酸合成酶（CS，EC 4.1.3.7）酶液。 1.2.2.2 測定方法 上述酶活性測定參照Srene法10,11，略有改動。酶活反應(yīng)體系設(shè)為0.5 ml，加入反應(yīng)底物后立即用UV-8500型紫外分光光度計測定其吸光度值，以0.5 s為單位讀數(shù)，共掃描3 min，記錄吸光度值變化，重復(fù)3次；以1 min吸光度變化0.01作為一個酶單位，酶活性以單位每克鮮果肉每分鐘表示（Ug-1FWmin-1）。 1.2.3 有機酸代謝途徑中關(guān)鍵酶基因表達 采用Trizol法提取南豐蜜橘果肉總RNA，具體操作參照劉慶博士論文12。cDNA合成使用MBI公司生產(chǎn)的RevertAidTMFirst Strand cDNA Synthesis Kit。 檢測所需引物利用Primer Express 2.0軟件（Applied Biosystems, CA, USA）設(shè)計。以Actin作為內(nèi)參基因，對目標基因的表達水平進行相對定量，其引物序列為ActinF（5-CCAAGCAGCATGAAGATCAA-3），ActinR（5-ATCTGCTGGAAGGTGCTGAG-3）13。試驗檢測有機酸代謝相關(guān)基因?qū)崟r定量PCR的引物序列14見表1。 采用ABI 7500實時定量PCR儀（Applied Biosystems, CA, USA）進行qRT-PCR擴增。5 將待檢測基因和內(nèi)參基因的特異引物與SYBER GREEN Master Mix （Applied Biosystems, CA, USA）混合，然后加入到含有模板的反應(yīng)管中，反應(yīng)體系為10 l：cDNA模板0.5 l；滅菌蒸餾水3.5 l；Mix 5 l；正反向引物各0.5 l。反應(yīng)程序為：50 2 min，95 1 min，（95 15 s，60 1 min）40個循環(huán)。所產(chǎn)生的數(shù)據(jù)經(jīng)Sequence Detector Version 1.3.1軟件（Applied Biosystems, CA, USA）轉(zhuǎn)化后在Excel中進行分析。 6 表 1 qRT-PCR引物序列 Table 1 Specific primers used in qRT-PCR 基因號 探針號 引物序列 退火溫度 長度 Gene name Probe number Primer sequence （53） Annealing ( ) Length (bp) Malate dehydrogenase (cytosolic) CUST_1159_PI402576686 F: TCATAACCACAGTCCAACAACG R: ACAATTGTCCATTCACCGTTGC 59 59 229 Aconitate hydratase 1 CUST_1342_PI402576686 F: TACAGAGGTGGAATTGGCTTACTT 60 92 R: TCTTGCGGAATCATTGTCTCA 60 Malate dehydrogenase (mitochondrial) CUST_199_PI402576686 F: GGTGGGACAGAAGTTGTGGAAGC 60 266 R: GGCTTCAGTTTTTCCAAGCCCTC 60 NADP-malic enzyme 1 (cytosolic) CUST_434_PI402576686 F: GCAAGTGGGAGCCCCTTTGA 60 333 R: GCTCTCAGCATACTTCACCAGGT 60 Citrate synthase 5 CUST_602_PI402576686 F: GCCTGATGATCCATTGTTCCAGC 60 216 R: CGGTCCCATATCAACTGAGAGCA 60 Succinate dehydrogenase CUST_696_PI402576686 F: GTCCGAGCATTCGAGTCAGG 59 376 R: TGGTAATGCAAGCGGTGTTGA 59 Fumarate hydratase 1 CUST_77_PI402576686 F: TCTCTGGATCGCGGGTATTC 60 64 R: CCAAATACACACGCAAAATAAGATG 60 7 1.2.4 統(tǒng)計分析 使用Excel（2013）進行數(shù)整理，并用SPSS（19.0, IBM）軟件進行顯著性分析。 2 結(jié)果與分析 2.1 果實中糖和有機酸含量的變化 由圖1-A可知，兩類果實中蔗糖含量變化趨勢一致，即在整個發(fā)育時期始終呈上升趨勢，且在花后120 DAF表現(xiàn)為積累加速。與之不同的是，葡萄糖（圖1-B）和果糖（圖1-C）含量在整個發(fā)育期內(nèi)變化趨勢一致，但在花后30 DAF急劇下降，60 DAF再緩慢上升。兩類果實成熟后的蔗糖含量均遠高于葡萄糖和果糖。 由圖1-D可知，不論是有葉果還是無葉果中，奎寧酸含量在整個發(fā)育期內(nèi)呈現(xiàn)出下降趨勢，且在花后30 DAF至90 DAF下降幅度較大，之后降低幅度較為平緩。兩類果實中檸檬酸含量變化趨勢大體一致且較為復(fù)雜，在成熟時與發(fā)育初期的含量相近（圖1-E）。有葉果和無葉果中的蘋果酸含量變化趨勢基本一致，在花后30 DAF至120 DAF波動較明顯，120 DAF后的變化趨于平緩。在花后30 DAF時，無葉果果實中蘋果酸含量為2.53 mg/g，顯著高于有葉果中蘋果酸的含量1.51 mg/g，但從90 DAF開始，蘋果酸含量平穩(wěn)降低，且有葉果和無葉果中的蘋果酸含量變化趨勢一致（圖1-F）。結(jié)果顯示，果實成熟時兩類果實中檸檬酸含量均高于蘋果酸。 8 圖1 南豐蜜橘果實中糖、酸含量變化 Fig. 1 Changes in the concentrations of sugars and organic acids in “Nanfeng”fruit 2.2 有機酸代謝相關(guān)酶活性變化 CS、PEPC、ACO和MDH是影響柑橘果實中有機酸代謝的重要酶。所含有機酸類型不同的各類果實中，有機酸代謝酶活性存在很大差異。酶活測定結(jié)果見圖2，兩類果實中MDH的活性呈現(xiàn)出較為平穩(wěn)的上升趨勢；CS和ACO的活性在整個發(fā)育期內(nèi)呈現(xiàn)出局部波動明顯，但整體上升的趨勢；而PEPC的活性在整體上略有降低，但是局部波動變化較為明顯。然而，4種重要酶的活性變化趨勢在兩類果實中基本一致，且未表現(xiàn)出顯著性差異。 9 圖2 南豐蜜橘果實中有機酸代謝相關(guān)酶活性變化 Fig. 2 Changes in enzyme activities of organic acid metabolism-related in“Nanfeng”fruit 2.3 有機酸代謝相關(guān)酶基因表達 有機酸代相關(guān)酶基因表達量測定見圖3，兩類果實中CS（圖3-A）和ACO（圖3-B）表達量呈現(xiàn)為先降低后升高的趨勢，這與檸檬酸含量整體變化趨勢相似（檸檬酸含量在個別時期存在波動，但并不顯著），但在時間上略有滯后，且二者的基因表達量多數(shù)為發(fā)育初期有葉果高于無葉果，發(fā)育后期則為無葉果高于有葉果，且存在顯著性差異。SDH（圖3-C）和FH（圖3-D）在多數(shù)時期為無葉果中的表達量顯著高于有葉果，但是變化趨勢無明顯規(guī)律。線粒體中的MDH（圖3-E）表達量在花后30 DAF時為有葉果顯著高于無葉果，之后為有葉果顯著低于無葉果，直至花后210 DAF果實成熟時二者表達量相當；而兩類果實細胞質(zhì)中的MDH（圖3-F）表達量只在少數(shù)幾個發(fā)育期內(nèi)存在顯著性差異。結(jié)果顯示以上幾個基因表達量均呈現(xiàn)出先降低后升高的趨勢，且有葉果和無葉果果實中基因表達量的變化趨勢基本一致。兩類果實細胞質(zhì)中的蘋果酸酶基因（Malic enzyme，ME）（圖3-G）的表達量在整個發(fā)育期內(nèi)均存在顯著性差異，但變化趨勢略有不同，有葉果中表現(xiàn)為花后30 DAF至60 DAF略有升高，之后降低直到花后150 DAF開始再次升高至成熟；無葉果在整個發(fā)育期內(nèi)則表現(xiàn)為兩次降低、兩次升高的波動變化。 10 圖3 南豐蜜橘果實中有機酸代謝相關(guān)酶基因表達 Fig. 3 Gene expressions of organic acid metabolism-related in “Nanfeng”fruit 3討論 柑橘果實中糖酸組分、含量及其之間的比例在很大程度上決定著果實的風(fēng)味特征，這對于評價果實內(nèi)在品質(zhì)至關(guān)重要15。在對南豐蜜橘果實發(fā)育過程中糖酸代謝及其調(diào)控機制的研究中發(fā)現(xiàn)，果實中蔗糖、果糖和葡萄糖含量隨著果實成熟均呈現(xiàn)出逐漸上升的趨勢，其中蔗糖含量呈現(xiàn)為典型的“S”型增長趨勢，它不僅影響著果實風(fēng)味，還作為一種信號分子調(diào)控果實成熟16,17。本試驗結(jié)果顯示，日光溫室中南豐蜜橘兩類果實在發(fā)育初期均以葡萄糖和果