3个不同类型西瓜品种果实发育过程中游离氨基酸的积累分析.pdf

果树学报2023，40（8）：1651-1665Journal of Fruit ScienceDOI:10.13925/ki.gsxb.202200873个不同类型西瓜品种果实发育过程中游离氨基酸的积累分析杨东东1，袁明2，路绪强1，龚成胜1，赵永1，袁飞2，刘文革1*（1中国农业科学院郑州果树研究所，郑州 450009；2东台国家现代农业产业园，江苏东台 224200）摘要：【目的】探讨3个不同类型西瓜果实中游离氨基酸含量和积累模式的差异及游离氨基酸在西瓜种质分类中的潜在价值。【方法】以3个不同类型的西瓜品种为试材，对不同发育时期果实中游离氨基酸进行测定分析。【结果】共检测到24种游离氨基酸，发现瓜氨酸、谷氨酰胺和甘氨酸在3个类型西瓜中含量（w，后同）较高，为0.10.5 gkg-1，且含量差异较大；精氨酸仅在冰糖脆中含量高，为0.12 gkg-1；其余游离氨基酸含量均低于0.05 gkg-1。在游离氨基酸含量的积累模式中，PI532726中17种呈抛物线型，6种呈上升型，1种为其他形式；宁夏红籽瓜中13种呈上升型，6种呈抛物线型，5种为其他形式；冰糖脆中11种呈“N”型，13种为其他形式。PI532726和宁夏红籽瓜中游离氨基酸含量和积累模式相似。【结论】3个类型西瓜果实中瓜氨酸、甘氨酸和谷氨酰胺含量高，且差异显著，精氨酸仅在冰糖脆中含量高；PI532726和宁夏红籽瓜中游离氨基酸含量和积累模式相似，与冰糖脆差异显著；对多种游离氨基酸含量和积累模式的结合分析可能在西瓜种质分类中发挥作用。关键词：西瓜；发育时期；游离氨基酸；积累模式；高效液相色谱中图分类号：S651文献标志码：A文章编号：1009-9980(2023)08-1651-15收稿日期：2022-03-17接受日期：2023-02-23基金项目：中国农业科学院科技创新工程专项经费项目（CAAS-ASTIP-2021-ZFRI）；国家现代农业产业技术体系（CARS-25-03）；国家重点研发计划（2018YFD0100704）；“科技兴蒙”行动重点专项（NMMKJX202114）作者简介：杨东东，男，在读硕士研究生，研究方向为代谢组学。Tel：[phone]，E-mail：*通信作者Author for correspondence.Tel：[phone]，E-mail：Analysis of free amino acids accumulation during fruit development inthree different types of watermelonsYANG Dongdong1,YUAN Ming2,LU Xuqiang1,GONG Chengsheng1,ZHAO Yong1,YUAN Fei2,LIUWenge1*(1Zhengzhou Fruit Research Institute,CAAS,Zhengzhou 450009,Henan,China;2Dongtai National Modern Agricultural Industrial Park,Dongtai 224200,Jiangsu,China)Abstract:【Objective】Watermelon belongs to the genus Watermelon in the Cucurbitaceae family.Ithas 7 species,and there are many varieties within the genus.Among them,egusi watermelon(Citrullusmucosospermus),seed edible watermelon(C.lanatus var.megalospermus)and common watermelon(C.lanatus var.vulgaris)have been cultivated at commercial scales.Common watermelon is a sweet-flesh watermelon for daily consumption.Egusi watermelon is an important economic crop in West Afri-can countries,and the seed edible watermelon is the main economic crop in the arid area of northwestChina.Free amino acids are not only important nutrients in watermelon fruit,but also an important ni-trogen source.They participate in fruit development and perform their respective functions,which areconnected to each other.Studies have shown that there are significant differences in the accumulationand content of some free amino acids such as citrulline in different varieties of watermelons.The differ-ences in metabolites can be used for distinguishing varieties.This study determined amino acids infruits at four development stages(10,18,26,34 d)in three watermelon varieties,PI532726(egusi wa-termelon),Ningxia Red Seed(seed edible watermelon)and Bing Tang Cui(common watermelon)with果树学报第40卷HPLC.The accumulation and metabolism of 24 kinds of free amino acids was analyzed.【Methods】Ac-cording to the development characteristics of different varieties,four watermelon fruit with the samegrowth and good development status were sampled for each variety and quickly frozen with liquid nitro-gen.The watermelon pulp was completely freeze-dried in a freeze dryer and then ground into fine pow-der.A sample of 1.00 g of freeze-dried pulp was taken and suspended in 90 mL boiling water,andbathed in 95 water for 10 minutes after full mixed.The suspension was forced through a 0.22 m wa-ter-phase filter,and the filtrate was cooled to room temperature and set to a volume of 125 mL.10 L ofthe filtrate was pipetted into a glass-lined tube,added with 20 L of derivatization reagent and 70 L ofderivatization buffer reagent,vortexed for 10 s,oven heated at 55 for 10 min,and used for HPLCanalysis,which was repeated for three times for each measurement.The standard curves of 24 free ami-no acids were constructed with four concentration gradients for HPLC determination,so as to quantifyamino acids in the watermelon sample filtrate.【Results】It was found that the contents of citrulline,gly-cine and glutamine were among the highest in the three watermelon varieties.For example,citrullinewas 0.11 gkg-1in PI532726,0.12 gkg-1in Ningxia Red Seed,and 0.39 gkg-1in Bing Tang Cui.The con-tent of arginine(0.1-0.2 gkg-1)in Bing Tang Cui(0.12 gkg-1)was higher than in PI532726 and Ningx-ia Red Seed(0.018 gkg-1and 0.068 gkg-1,respectively).The contents of-aminobutyric acid,glutam-ic acid,valine,isoleucine,asparagine and phenylalanine in the three watermelons ranged from 0.01 to0.06 g kg-1.The contents of theanine,hydroxyproline,tyrosine,sarcosine,alanine,leucine,ornithineand cysteine were all lower than 0.01 gkg-1.The contents of the other free amino acids differed signifi-cantly in the three watermelon varieties.For example,the contents of proline and methionine in BingTang Cui were 0.028 gkg-1and 0.013 gkg-1,respectively,while those in PI532726 and Ningxia RedSeed were all lower than 0.01 gkg-1.The content of serine in Ningxia Red Seed and Bing Tang Cui was0.011 gkg-1and 0.015 gkg-1,respectively,while that in PI532726 was lower than 0.01 gkg-1.Principalcomponent analysis was carried out on the 24 amino acids in four developmental stages of the three wa-termelon varieties after standardization,and two principal components with eigenvalue1 were extract-ed.The eigenvalues of the two principal components for PI532726 were 14.503 and 5.511;those forNingxia Red Seed were 19.006 and 3.368;and for Bing Tang Cui were 10.73 and 9.79.The cumulativevariance contribution rates were all greater than 83%,showing strong information representability.PCAloading plot can be used to find the differential variables.The greater the distance from the origin,thegreater the contribution rate to the samples.From the loading plot,it could be seen that the free aminoacids that contributed more to PI532726 were glycine and glutamine.The free amino acids that contrib-uted more to Ningxia Red Seed were glycine,citrulline and glutamine,and those to Ningxia Red Seedwere glycine,citrulline,arginine and glutamine.Clustering heat map analysis showed significant differ-ences in the amounts and patterns of glycine,citrulline,arginine and glutamine among the three water-melon varieties,and the free amino acid evolutionary tree also showed differences among the three vari-eties.The metabolic characteristics of free amino acids in PI532726 and Ningxia Red Seed were moresimilar.In conclusion,there were significant difference in contents of free amino acids in the three wa-termelon varieties,which may be used as the signature differential metabolites to distinguish watermel-on varieties.It was found that glycine(26%-34%),glutamine(12%-24%),citrulline(8%-23%)and-aminobutyric acid(4%-9%)were dominant in the fruit of PI532726.In Ningxia Red Seed,glycine(20%-35%),glutamine(12%-28%),citrulline(9%-15%),glutamic acid(4%-12%)and arginine(4%-9%)were the major amino acids.Citrulline(23%-45%),glutamine(21%-35%),glycine(4%-19%)andarginine(5%-11%)were dominant in Bing Tang Cui.In addition,there were significant differences in1652，等：游离氨基酸的积累分析第8期西瓜（Citrullus lanatus）属葫芦科1年生蔓生植物，原产非洲，是我国重要的经济作物，在我国的栽培面积和产量均居世界第一1。西瓜有7个种，且属内栽培类型繁多2，具有一定栽培规模的有黏籽西瓜、籽用西瓜和普通（栽培）西瓜。黏籽西瓜是西非国家的重要经济作物3，果肉白色、质地硬且味苦不可食，以食用种子为主。籽用西瓜是我国西北旱区的主要经济作物4，籽瓜籽具有较高的营养价值和经济价值，瓜瓤有利尿、润肺、益肝和健脾等功效5。普通西瓜即鲜食甜西瓜，果实甘甜可口，富含多种营养物质。西瓜果实中含有多种游离氨基酸6，它们在西瓜的生长发育中发挥重要作用7-8；部分游离氨基酸自身为呈味氨基酸或参与风味物质的形成，且在人体中发挥重要作用，极大提高了西瓜的营养和风味价值9。对不同类型西瓜果实在动态发育过程中的游离氨基酸进行研究，既可以全面了解其含量和积累模式的差异，也可以针对特定游离氨基酸进行遗传改良以选育出高目标游离氨基酸含量的西瓜新品种，同时the proportion of various free amino acids among varieties and developmental stages.The resultsshowed that there were always a large proportion of free amino acids in fruits of the three varieties,andthere were significant differences.The presence of these free amino acids in fruit development may berelated to genotype of watermelon.The changes in the total 24 detected free amino acids in PI532726were in a parabolic(up-down)pattern.In Ningxia Red seed,the change pattern was an ascending type,while that in Bing Tang Cui watermelon showed an“N”pattern(up-down-up).The total free amino ac-id content in Bing Tang Cui was higher than that in PI532726 and Ningxia Red Seed at the four develop-mental stages.The overlap of total free amino acid accumulation curves of PI532726 and Ningxia RedSeed was high.The change patterns of each free amino acid were analyzed respectively.It was foundthat citrulline,-aminobutyric acid,aspartic acid and ornithine in PI532726 were ascending;cysteinewas in a descending pattern,and the change patterns of glutamine,glycine,arginine,glutamic acid,phe-nylalanine,aspartic acid and isoleucine were parabolic.The contents of alanine and cysteine in NingxiaRed Seed descended constantly;glutamic acid showed a“V”pattern(down-up);glycine,leucine,ser-ine and tyrosine showed a parabolic pattern;citrulline,glutamine,arginine,phenylalanine,-aminobu-tyric acid,aspartic acid,asparagine,lysine,valine,methionine,ornithine and theanine showed ascendedconstantly.In Bing Tang Cui,glycine and alanine were constantly decreasing;citrulline and ornithinewere increasing;aspartic acid and-aminobutyric acid were parabolic;and glutamine,arginine,phenyl-alanine,glutamic acid,leucine,isoleucine,lysine,proline,serine,valine and methionine changed in an“N”pattern(up-down-up).Threonine displayed an inverted“N”pattern(down-up-down).The changecurves in PI532726 and Ningxia Red seed were similar in various free amino acids,such as glycine,leu-cine,asparagine,threonine,isoleucine,serine,-aminobutyric acid and tyrosine,while the changecurves of free amino acids in PI532726 and Bing Tang Cui had high similarities.The coincidence de-gree of cysteine was high only in Ningxia Red Seed and Bing Tang Cui.PCA analysis of free amino ac-id data of all samples showed that principal component 1 clustered PI532726 and Ningxia Red Seed to-gether,and Bing Tang Cui was grouped into a single class.The results indicate that the free amino acidmetabolic network and metabolic pattern in the fruits of PI532726 and Ningxia Red Seed may be moresimilar.【Conclusion】The contents of citrulline,glycine and glutamine were high in fruits of the threewatermelon varieties,and the difference was significant.The content of arginine was high only in BingTang Cui.The contents and change patterns of free amino acids in PI532726 and Ningxia Red Seedwere similar,and significantly different from Bing Tang Cui.The combination analysis of the contentsand change patterns of various free amino acids may be used in the classification of watermelon germ-plasm.Key words:Watermelon;Developmental stages;Free amino acid;Accumulation mode;HPLC杨东东，等：3个不同类型西瓜品种果实发育过程中游离氨基酸的积累分析1653果树学报第40卷还能在代谢组协助种质分类中呈现潜在价值。代谢组学主要研究分子质量小于1 ku的代谢物10。目前，代谢组学检测技术已经在水稻11、黄瓜12、番茄13-15和辣椒16等多种作物的研究中发挥重要作用，为研究植株生长、代谢物合成、品种驯化改良和遗传机制揭示等提供了新的手段。游离氨基酸代谢物参与植物生长发育、细胞生长和分化、能量代谢、次生代谢、转录和翻译、调控及抗性等形成过程。通过代谢组学检测技术鉴定出多种游离氨基酸，有助于了解各游离氨基酸之间的差异，同时有助于发掘植物中游离氨基酸的营养价值9。对植物果实游离氨基酸代谢物的研究也有助于种质分类。袁平丽等6,17研究表明代谢组差异和表型差异均可以作为区分西瓜品种的重要依据，有助于充分了解和利用西瓜种质资源；刘圆等18表明不同甜瓜品种特有的香气物质及特征性酯类种类和含量均不同，这些差异构成了各品种甜瓜特有的典型性香气。西瓜果实中含有丰富的游离氨基酸，但目前相关研究主要集中在含量较其他水果或蔬菜更高的瓜氨酸和精氨酸方面19-20。李蒙蒙等21研究了嫁接对西瓜果实中瓜氨酸含量的影响；Davis等22通过测定多个西瓜品种中瓜氨酸含量，选择出高瓜氨酸含量的品种；Assefa等23研究了瓜氨酸、精氨酸含量与可溶性固形物含量及瓤色的相关性。目前，对西瓜果实中其他游离氨基酸的研究报道较少。虽然对西瓜果实中含量较高的游离氨基酸的研究已经存在一定的基础，但不同类型西瓜果实中多种游离氨基酸的组分和含量及发育过程中的积累模式并未被完全揭示和详细报道。笔者在本研究中采用果实表型差异明显的黏籽西瓜、籽用西瓜和普通西瓜3个类型的西瓜为试材，系统测定和分析了3个类型西瓜果实中多种游离氨基酸的组分及含量和积累模式的差异，明确3个类型西瓜果实中差异积累的游离氨基酸组分和含量及积累模式的差异，并结合前人研究将游离氨基酸含量和积累模式特征与品种类型联系在一起进行分析。通过对PI532726、宁夏红籽瓜和冰糖脆3个不同类型的西瓜果实在生长发育过程中游离氨基酸的含量构成及积累模式差异情况的探究，为西瓜营养、西瓜风味、抗逆评价和西瓜种质分类提供数据支撑和参考，并为西瓜遗传育种和西瓜中游离氨基酸的遗传机制研究提供参考。1材料和方法1.1试验材料供试材料种子由中国农业科学院郑州果树研究所国家西甜瓜中期库和多倍体西瓜遗传育种课题组提供。根据种质特点选取PI532726（黏籽品种）、宁夏红籽瓜（籽瓜品种）及冰糖脆（栽培品种）3个种质类型的西瓜品种作为试验材料（表1）。表 1试验材料基础信息Table 1Basic information of experimental materials品种名称Variety namePI532726宁夏红籽瓜 Ningxia Red Seed冰糖脆 Bing Tang Cui品种类型Variety type黏籽西瓜 Egusi籽用西瓜 Edible seed栽培西瓜Common果肉颜色Pulp color白色 White黄色 Yellow白色 Whitew（可溶性固形物）Soluble solid content/%3.04.79.5果实质量Fruit mass/kg1.72.43.5果皮颜色Rind color绿色 Green绿色 Green绿色 Green果皮厚度Rind thickness/cm1.41.10.9供试材料于2019年春季种植于中国农业科学院新乡综合试验基地大棚中，种子统一破壳处理，清水浸泡2.5 h，置于培养箱中32 条件下催芽，芽长0.5 cm时播种于营养钵中，苗期统一管理。实生苗播种后35 d移栽至大棚，随机区组设计，双蔓整枝，行间距1.5 m，株间距0.5 m，田间管理一致。花期人工自交授粉，在第二雌花节位留瓜，并悬挂吊牌标记授粉日期。根据授粉日期，在果实分别发育至10、18、26、34 d时根据品种发育特征，每个品种选取长势良好、均匀一致的6个西瓜，纵切后对每2个西瓜果实的果肉进行5点混合取样作为1个生物学重复，共3个生物学重复。对样品进行液氮速冻，随后使用冷冻干燥机进行冷冻干燥。1.2游离氨基酸含量的检测1.2.1标准溶液与样品溶液的配制氨基酸标准品：L-丙氨酸（Ala）、L-精氨酸（Arg）、L-瓜氨酸（Cit）、L-鸟氨酸（Orn）、L-天冬酰胺（Asn）、L-天冬氨酸（Asp）、L-胱氨酸（Cys）、-氨基丁酸（GABA）、L-谷氨酰胺（Gln）、L-谷氨酸（Glu）、L-甘氨酸（Gly）、L-组氨酸（His）、L-异亮氨酸（ILe）、L-亮氨酸（Leu）、L-赖氨酸（Lys）、L-甲硫氨酸（Met）、L-苯丙氨酸（Phe）、L-脯氨酸（Pro）、L-羟脯氨酸（HYP）、L-丝氨酸（Ser）、L-苏1654，等：游离氨基酸的积累分析第8期氨酸（Thr）、L-酪氨酸（Tyr）、L-缬氨酸（Val）、肌氨酸（Sar）和L-茶氨酸（The），纯度均98%，购自郑州盛百欣生物科技有限公司。将各标准品分别配制成5000、2500、1250、625、312.5 mmolL-15 个浓度梯度，于-20 保存待测。取1.00 g冷冻干燥的果肉样品研磨成细粉状，使用100 mL的沸水冲泡，随后放入95 水浴锅水浴10 min，期间搅拌混匀5次，水浴结束趁热使用0.22 m水相滤膜进行抽滤，冷却至室温后定容至125 mL，置于-20 保存待测。1.2.2高效液相色谱条件采用岛津公司的高效液相色谱仪对游离氨基酸进行测定，色谱仪型号：LC-20AD；色谱条件，荧光检测器 A Ex：250 nm，Em：395 nm；色谱柱：C18，5 m，4.6 mm 250 mm。流动相A（95 mL 800 mmolL-1磷酸二氢钾溶液+5 mL800 mmolL-1磷酸氢二钠溶液+10 mL 1 molL-1盐酸三乙胺溶液+55 mL乙腈+835 mL水）；流动相B（100 mL 800 mmolL-1磷酸二氢钾溶液+500 mL乙腈+400 mL超纯水）；流速 1 mLmin-1；二元梯度高压：泵A+泵B总浓度为100%，洗脱梯度为泵A浓度：025 min（100%），2540 min（97%），4055 min（81%），5568 min（74%），6888 min（0%）。1.2.3游离氨基酸含量的测定取10 L样品溶液于玻璃内衬管中，并加入20 L衍生试剂和70 L衍生用缓冲试剂，放入进样瓶涡旋混合10 s，转移至55 烘箱中加热10 min，取出样品瓶放置于高效液相色谱样品瓶架上，开启检测按钮，重复测定3次。1.3数据处理以3次生物学重复测定结果的平均值作为最终含量值。采用Excel对试验数据进行平均数、标准差和相对标准偏差分析处理和作图。使用TBtools软件进行热图绘制，利用IBM SPSS Statistics 26软件的因子模型对数据进行主成分分析，使用Origin2018 64 Bit绘制主成分分析图。2结果与分析2.13个类型西瓜果实发育过程中游离氨基酸含量分析参考4个发育时期中游离氨基酸的最高含量，发现瓜氨酸、甘氨酸和谷氨酰胺在3个类型西瓜果实中含量均较高，且存在显著差异；瓜氨酸在PI532726中为0.11 gkg-1，在宁夏红籽瓜中为0.12 gkg-1，而在冰糖脆中为0.39 gkg-1。精氨酸（0.10.2 gkg-1）仅在冰糖脆中含量较高（0.12 gkg-1），而在PI532726和宁夏红籽瓜中分别为0.018和0.068 gkg-1。-氨基丁酸、谷氨酸、缬氨酸、异亮氨酸、天冬酰胺和苯丙氨酸在3个不同类型西瓜果实中含量均在0.010.06 gkg-1之间，茶氨酸、羟脯氨酸、酪氨酸、肌氨酸、丙氨酸、亮氨酸、鸟氨酸和半胱氨酸在3个类型西瓜果实中含量均低于0.01 gkg-1。其余几种游离氨基酸在3个不同类型西瓜中含量差异显著，脯氨酸和甲硫氨酸在冰糖脆中含量分别为0.028和0.013 gkg-1，而在PI532726和宁夏红籽瓜中含量均低于0.01 gkg-1。丝氨酸在宁夏红籽瓜和冰糖脆中含量为 0.011 和0.015 gkg-1，而在PI532726中含量均低于0.01 gkg-1（表2，图1）。对3个类型品种西瓜4个发育时期果实中的24个指标标准化后进行主成分分析，提取特征值1的 2 个主成分。其中，PI532726 特征值分别为14.503、5.511，宁夏红籽瓜特征值分别为 19.006、3.368，冰糖脆特征值分别为10.73、9.79，累计方差贡献率均大于83%，具有较强信息代表性，达到分析要求。PCA 载荷图（loading plot）可用来寻找差异变量，距离原点越远表示对样本贡献率越大。从载荷图（图2）可以看出，对PI532726贡献率较大的游离氨基酸是甘氨酸和谷氨酰胺，对宁夏红籽瓜贡献率较大的游离氨基酸是甘氨酸、瓜氨酸和谷氨酰胺，对冰糖脆贡献率较大的游离氨基酸是甘氨酸、瓜氨酸、精氨酸和谷氨酰胺。聚类热图（图3）分析能够清晰地看出3个品种西瓜果实中的甘氨酸、瓜氨酸、精氨酸和谷氨酰胺存在显著差异的积累量与积累模式，而游离氨基酸进化树也能清晰地呈现出3个类型品种之间的差异，其中，PI532726和宁夏红籽瓜中游离氨基酸的代谢特征更相似。综上可以看出，在3个类型的西瓜中存在含量显著差异的游离氨基酸，可能在不同种质类型的西瓜品种中作为区分种质的标志性差异代谢物存在。将检测到的24种游离氨基酸作为总游离氨基酸。PI532726发育过程中甘氨酸（26%34%）、谷氨酰胺（12%24%）、瓜氨酸（8%23%）和-氨基丁酸（4%9%）含量占比较大。宁夏红籽瓜中甘氨酸（20%35%）、谷氨酰胺（12%28%）、瓜氨酸（9%15%）、谷氨酸（4%12%）和精氨酸（4%9%）含量占比较大。冰糖脆中瓜氨酸（23%45%）、谷氨酰胺杨东东，等：3个不同类型西瓜品种果实发育过程中游离氨基酸的积累分析1655果树学报第40卷表 23 个不同类型品种 4 个发育时期果肉游离氨基酸含量Table 2Contents of free amino acids in pulp of three watermelon varieties at four developmental stages（gkg-1）游离氨基酸Free amino acidL-天冬氨酸AspL-天冬酰胺AsnL-丝氨酸SerL-谷氨酸GluL-谷氨酰胺GlnL-甘氨酸GlyL-精氨酸ArgL-瓜氨酸Cit-氨基丁酸GABAL-缬氨酸ValL-甲硫氨酸MetL-鸟氨酸OrnL-赖氨酸LysL-异亮氨酸IleL-亮氨酸LeuL-苏氨酸ThrL-羟脯氨酸HYPL-脯氨酸ProL-酪氨酸Tyr肌氨酸SarL-茶氨酸TheL-丙氨酸AlaL-苯丙氨酸PheL-半胱氨酸CysPI53272610 d0.005 40.008 40.006 00.015 00.032 00.067 00.006 80.044 00.017 00.005 50.00