日光温室不同结果枝类型对柑橘果实有机酸含量的影响

1 DOI:10.13925/j.cnki.gsxb.20170426 日光温室不同结果枝类型对柑橘果实有机酸含量的影响 孙晓华，叶丽红，刘杰才，李晓静，张之为，崔世茂，宋 阳* （内蒙古农业大学农学院内蒙古设施园艺工程技术研究中心，呼和浩特 010019） 摘要：【目的】探明日光温室中柑橘结果枝类型对果实中有机酸含量的影响，为温室柑橘结果枝组的培养提供理论依据。【方法】以枳Poncirus trifoliate（L.）Raf.砧南丰蜜橘（Citrus reticulate Blanco cv. Kinokun）有叶结果枝和无叶结果枝果实为试材，对其糖酸含量、有机酸代谢相关酶活性以及基因表达量进行分析。【结果】南丰蜜橘果实发育过程中蔗糖含量呈上升趋势，奎宁酸含量呈下降趋势，葡萄糖和果糖含量为先降后升，柠檬酸含量在果实成熟时与发育初期相近；无叶果和有叶果的糖酸含量变化趋势基本一致，但30 DAF无叶果苹果酸含量显著高于有叶果；两类果实中柠檬酸合成酶（citrate synthase，CS）、磷酸烯醇式丙酮酸羧化酶（phosphoenolpyruvate carboxylase，PEPC）、乌头酸酶（aconitate hydratase，ACO）和苹果酸脱氢酶（malate dehydrogenase，MDH）等4种重要酶活性变化趋势基本一致；两类果实多数基因的表达量都存在显著性差异，然而酶活性以及有机酸含量则未出现相应的显著性变化。【结论】南丰蜜橘果实中糖酸含量以及有机酸代谢相关重要酶活性不受结果枝组类型的影响，因此，在日光温室中进行柑橘树体枝组修剪时，只需考虑其果实产量即可，而不必过多的去考虑其对果实糖酸等内在品质的影响。 关键词：南丰蜜橘；有机酸；基因表达；结果枝；果实品质 中图分类号：S666.2 文献标志码：A 文章编号：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 基金项目：内蒙古自治区自然科学基金项目（2015BS0317） 作者介绍：孙晓华，女，讲师，研究方向设施栽培果树栽培生理。Tel：0471-4301178，E-mail：sungod0819163.com *通信作者 Author for correspondence. Tel：0471-4301178，E-mail：dbmsysina.com 网络出版时间：2018-03-09 11:47:16网络出版地址：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 近几年，我国休闲农业作为一类新型产业悄然兴起，各类主题观光温室及观光、采摘、娱乐兼顾的大型日光温室在很多城市周边不断涌现，成为城市居民休闲娱乐的重要场所。为了满足人们对休闲农业和热带、亚热带水果采摘的需求，许多热带、亚热带果树也引入北方日光温室种植，其中温室柑橘的种植面积呈上升趋势。然而，温室栽培中加温时间长、温度过高以及光照不足等原因都会造成柑橘花芽减少、落花落果加重，最终导致产量下降1。研究表明，种植在大棚内的柑橘，其果实中可溶性固形物、总糖和维生素C含量均低于露地栽培果实，而有机酸含量则高于露地果实2。基于以上栽培中出现的问题，日光温室栽培柑橘果实的品质提高则显得尤为重要。 有机酸是影响柑橘果实风味和营养品质的重要因子，大多数柑橘品种在成熟期的糖酸比较低，严重影响了果实的风味品质和消费者的接受度，很大程度上制约了设施柑橘产业的发展3。相对而言，柑橘果实中可溶性糖含量的变化幅度较小，而有机酸的种类以及含量变化幅度较大，因此，柑橘果实中所含有机酸对糖酸比的贡献更为显著4。 在柑橘设施栽培中，果树生长发育的最适环境因子，如光照、温湿度、土壤、气体条件等均可通过人为控制来实现，研究报道植物光合作用的源库比率、水分供给、矿物质营养和温度等环境因子影响着果实细胞中柠檬酸和苹果酸的积累，进一步阐明这些环境因子和细胞中有机酸代谢和贮藏之间的相互作用5，6。此外，叶片作为植物光合作用的主要部位，为柑橘果实发育提供光合产物，碳水化合物供给的限制对于三羧酸循环（Tricarboxylic Acid Cycle, TCA）中代谢产物的积累是非常不利的7。鉴于此，笔者以日光温室中种植的南丰蜜橘为试材，选取有叶花枝和无叶花枝所结果实（以下称为有叶果和无叶果），测定其糖和有机酸含量，分析有机酸代谢重要酶的活性和相关基因表达量，旨在探明日光温室柑橘中不同结果枝对果实有机酸含量的影响，为研究日光温室中柑橘枝组修剪与果实品质之间的相关性提供了理论依据。 1 材料和方法 1.1 植物材料 试验材料为枳砧南丰蜜橘，种植于内蒙古农业大学教学科研基地日光温室内。试验期间日光温室光照度最低为28000 lx（11-12月），最高为44000 lx（7-8月）；最低温度为8（1-2、11-12月），最高温度为45（7-8月）；3-8月日光温室湿度为34%-87%；而在1-2、9-12月湿度为28%-95%。在南丰蜜橘完全谢花后（2015年4月24日），选取5株生长势较好且挂果量较大的植株挂牌。从2015年5月24日开始，每隔30d对上述挂牌植株进行随机取样，分别采集有叶结果枝所结果实和无叶结果枝所结果实进行分析研究。 1.2 方法 4 1.2.1 果实中糖和有机酸含量的测定 果实中糖酸提取以及含量测定参考孙晓华博士论文8，称取1 g果肉组织于液氮中进行研磨，加入80%甲醇10 ml，将匀浆置于70恒温水浴锅温浴30 min，取出后冷却。上述匀浆于超声波中萃取90 min，4000 g离心10 min，取上清液于10 ml容量瓶中，加入0.2 ml现配的内标液，再加入80%甲醇定容，摇匀。取上述溶液2 ml置于2 ml离心管中，于12000 g离心15 min，取上清液0.5 ml置于真空旋转浓缩仪中，于60干燥至无水状态。对干燥物进行衍生化反应，加入0.8 ml盐酸羟胺溶液，置于70反应1 h，冷却。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下再加热2 h。取上清液0.5 ml于2 ml自动进样瓶中，进行气相色谱火焰离子化检测器（gas chromatography-flame ionization detection， GC-FID）分析，通过各个组分的保留时间进行定性。 1.2.2 有机酸代谢重要酶活性测定 1.2.2.1 酶液的制备 酶液提取方法参照罗安才等9，果实剖开后对称取样，取果肉2 g加入2 ml缓冲液进行研磨，缓冲液为0.2 molL-1Tris-HCl（pH 8.2），0.6 molL-1蔗糖，10 mmolL-1异抗坏血酸，冰浴，4 4000 g离心20 min，取上清液定容至5 ml，其中2 ml 15000 g离心15 min，取上清液用缓冲液提取，缓冲液为0.2 molL-1Tris-HCl（pH 8.2），10 mmolL-1异抗坏血酸，0.1% TritonX-100，定容至4 ml即得细胞质乌头酸酶液（Cyto-Acontiase，EC 4.2.1.3），另外3 ml加入等体积提取缓冲液，即可用于测定苹果酸脱氢酶（MDH，EC 1.1.1.37），取2ml在大量透析液（即提取缓冲液）中4透析过夜，用新鲜透析液定容即得磷酸烯醇式丙酮酸羧化酶（PEPC，EC 4.1.1.31），柠檬酸合成酶（CS，EC 4.1.3.7）酶液。 1.2.2.2 测定方法 上述酶活性测定参照Srene法10,11，略有改动。酶活反应体系设为0.5 ml，加入反应底物后立即用UV-8500型紫外分光光度计测定其吸光度值，以0.5 s为单位读数，共扫描3 min，记录吸光度值变化，重复3次；以1 min吸光度变化0.01作为一个酶单位，酶活性以单位每克鲜果肉每分钟表示（Ug-1FWmin-1）。 1.2.3 有机酸代谢途径中关键酶基因表达 采用Trizol法提取南丰蜜橘果肉总RNA，具体操作参照刘庆博士论文12。cDNA合成使用MBI公司生产的RevertAidTMFirst Strand cDNA Synthesis Kit。 检测所需引物利用Primer Express 2.0软件（Applied Biosystems, CA, USA）设计。以Actin作为内参基因，对目标基因的表达水平进行相对定量，其引物序列为ActinF（5-CCAAGCAGCATGAAGATCAA-3），ActinR（5-ATCTGCTGGAAGGTGCTGAG-3）13。试验检测有机酸代谢相关基因实时定量PCR的引物序列14见表1。 采用ABI 7500实时定量PCR仪（Applied Biosystems, CA, USA）进行qRT-PCR扩增。5 将待检测基因和内参基因的特异引物与SYBER GREEN Master Mix （Applied Biosystems, CA, USA）混合，然后加入到含有模板的反应管中，反应体系为10 l：cDNA模板0.5 l；灭菌蒸馏水3.5 l；Mix 5 l；正反向引物各0.5 l。反应程序为：50 2 min，95 1 min，（95 15 s，60 1 min）40个循环。所产生的数据经Sequence Detector Version 1.3.1软件（Applied Biosystems, CA, USA）转化后在Excel中进行分析。 6 表 1 qRT-PCR引物序列 Table 1 Specific primers used in qRT-PCR 基因号 探针号 引物序列 退火温度 长度 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 统计分析 使用Excel（2013）进行数整理，并用SPSS（19.0, IBM）软件进行显著性分析。 2 结果与分析 2.1 果实中糖和有机酸含量的变化 由图1-A可知，两类果实中蔗糖含量变化趋势一致，即在整个发育时期始终呈上升趋势，且在花后120 DAF表现为积累加速。与之不同的是，葡萄糖（图1-B）和果糖（图1-C）含量在整个发育期内变化趋势一致，但在花后30 DAF急剧下降，60 DAF再缓慢上升。两类果实成熟后的蔗糖含量均远高于葡萄糖和果糖。 由图1-D可知，不论是有叶果还是无叶果中，奎宁酸含量在整个发育期内呈现出下降趋势，且在花后30 DAF至90 DAF下降幅度较大，之后降低幅度较为平缓。两类果实中柠檬酸含量变化趋势大体一致且较为复杂，在成熟时与发育初期的含量相近（图1-E）。有叶果和无叶果中的苹果酸含量变化趋势基本一致，在花后30 DAF至120 DAF波动较明显，120 DAF后的变化趋于平缓。在花后30 DAF时，无叶果果实中苹果酸含量为2.53 mg/g，显著高于有叶果中苹果酸的含量1.51 mg/g，但从90 DAF开始，苹果酸含量平稳降低，且有叶果和无叶果中的苹果酸含量变化趋势一致（图1-F）。结果显示，果实成熟时两类果实中柠檬酸含量均高于苹果酸。 8 图1 南丰蜜橘果实中糖、酸含量变化 Fig. 1 Changes in the concentrations of sugars and organic acids in “Nanfeng”fruit 2.2 有机酸代谢相关酶活性变化 CS、PEPC、ACO和MDH是影响柑橘果实中有机酸代谢的重要酶。所含有机酸类型不同的各类果实中，有机酸代谢酶活性存在很大差异。酶活测定结果见图2，两类果实中MDH的活性呈现出较为平稳的上升趋势；CS和ACO的活性在整个发育期内呈现出局部波动明显，但整体上升的趋势；而PEPC的活性在整体上略有降低，但是局部波动变化较为明显。然而，4种重要酶的活性变化趋势在两类果实中基本一致，且未表现出显著性差异。 9 图2 南丰蜜橘果实中有机酸代谢相关酶活性变化 Fig. 2 Changes in enzyme activities of organic acid metabolism-related in“Nanfeng”fruit 2.3 有机酸代谢相关酶基因表达 有机酸代相关酶基因表达量测定见图3，两类果实中CS（图3-A）和ACO（图3-B）表达量呈现为先降低后升高的趋势，这与柠檬酸含量整体变化趋势相似（柠檬酸含量在个别时期存在波动，但并不显著），但在时间上略有滞后，且二者的基因表达量多数为发育初期有叶果高于无叶果，发育后期则为无叶果高于有叶果，且存在显著性差异。SDH（图3-C）和FH（图3-D）在多数时期为无叶果中的表达量显著高于有叶果，但是变化趋势无明显规律。线粒体中的MDH（图3-E）表达量在花后30 DAF时为有叶果显著高于无叶果，之后为有叶果显著低于无叶果，直至花后210 DAF果实成熟时二者表达量相当；而两类果实细胞质中的MDH（图3-F）表达量只在少数几个发育期内存在显著性差异。结果显示以上几个基因表达量均呈现出先降低后升高的趋势，且有叶果和无叶果果实中基因表达量的变化趋势基本一致。两类果实细胞质中的苹果酸酶基因（Malic enzyme，ME）（图3-G）的表达量在整个发育期内均存在显著性差异，但变化趋势略有不同，有叶果中表现为花后30 DAF至60 DAF略有升高，之后降低直到花后150 DAF开始再次升高至成熟；无叶果在整个发育期内则表现为两次降低、两次升高的波动变化。 10 图3 南丰蜜橘果实中有机酸代谢相关酶基因表达 Fig. 3 Gene expressions of organic acid metabolism-related in “Nanfeng”fruit 3讨论 柑橘果实中糖酸组分、含量及其之间的比例在很大程度上决定着果实的风味特征，这对于评价果实内在品质至关重要15。在对南丰蜜橘果实发育过程中糖酸代谢及其调控机制的研究中发现，果实中蔗糖、果糖和葡萄糖含量随着果实成熟均呈现出逐渐上升的趋势，其中蔗糖含量呈现为典型的“S”型增长趋势，它不仅影响着果实风味，还作为一种信号分子调控果实成熟16,17。本试验结果显示，日光温室中南丰蜜橘两类果实在发育初期均以葡萄糖和果