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花色是观赏植物最重要的观赏性状之一,是植物进化史上最具有适应意义的表型[1]。植物的花色是多种因子协同作用的结果,其中,色素的组成和含量以及不同色素物质的时空组合起着决定性作用[2-4]。花色素类物质主要有类黄酮、类胡萝卜素和花青素等,类胡萝卜素是一类呈黄色、橙色和红色的脂溶性化合物,目前分离和鉴定出的类胡萝卜素多达750余种,是自然界中存在最广的色素[4-8]。
色彩鲜艳的黄色花以其明朗和高贵的感觉而广受喜爱,纯正的黄色花也是石斛兰花色育种的重要目标[8-10]。目前,市场上的黄色石斛兰多为乳白、乳黄和黄绿色,而石斛兰原生种中不乏纯正明黄色种质[9-11],研究黄色花石斛兰原生种花瓣中的类胡萝卜素成分变化,对于明确花色呈色机理及育种具有重要意义。
目前,有关石斛属(Dendrobium Lindl.)花瓣中类胡萝卜素的研究鲜见报道。Kanchit[12]于1984年利用HPLC对部分开黄色花的石斛兰杂交种盛花期花瓣进行了初步研究[12],分析结果表明,类胡萝卜素和叶绿素是使石斛属呈现黄色和绿色的重要色素。Mudalige等[13]调查了石斛兰花瓣中的色素分布,发现黄色花瓣中主要含类胡萝卜素,而其上的红色、深红棕色或褐色印记则是类胡萝卜素和花青素共同作用的结果。关于石斛属类黄酮和花青素合成相关基因的研究已逐渐深入[14],而关于类胡萝卜素合成相关基因的研究还鲜见报道。
鼓槌石斛(Den. chrysotoxum Lindl.)花瓣明黄,唇瓣上常有U型栗色斑块;球花石斛(Den. thyrsiflorum Rchb.f.)和密花石斛(Den. densiflorum Lindl.)唇瓣皆呈金黄色,球花石斛花瓣白色,而密花石斛花瓣淡黄[11];它们均花量大,群体花期长,是著名的春石斛观赏种,也是重要的育种亲本[11]。本研究以鼓槌石斛、密花石斛和球花石斛原生种为材料,定性、定量分析它们盛开期花瓣中的类胡萝卜素,为探讨石斛属黄色花呈色机理和花色育种提供参考。
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将鼓槌石斛花瓣、密花石斛和球花石斛花瓣和唇瓣与英国皇家园艺比色卡(RHSCC)进行比对,结果(图 1)显示:球花石斛和密花石斛唇瓣皆为金黄色,比色卡代码分别为24A和23A;球花石斛花瓣为白色,比色卡代码为NN155B,基部偶带黄斑;密花石斛花瓣为淡黄色,比色卡代码为11A;鼓槌石斛花瓣为明黄色,比色卡代码为15A。
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类胡萝卜素分子通常由8个异戊二烯单位连接而成,根据其含氧与否常分为碳水化合物型类胡萝卜素(胡萝卜素)和氧化型类胡萝卜素(叶黄质)。常见的胡萝卜素有α-胡萝卜素、β-胡萝卜素和番茄红素等,极性稍弱。常见的叶黄质有叶黄素、玉米黄质和类胡萝卜素酯等,极性稍强[16]。
通常类胡萝卜素在正离子ESI源模式下电离,产生特征母离子和碎片离子,这些组分在450 nm附近都有强吸收,会产生特征吸收峰[17-18],根据这些吸收峰可以初步推定样品中是否有类胡萝卜素存在。类胡萝卜素分离时,洗脱顺序往往与所测组分的极性密切相关,组分中的羟基数目越多,往往极性越大,如β-胡萝卜素(无-OH)会较早的洗脱,之后是β-隐黄质(1个-OH),随后是玉米黄质(2个-OH)和紫黄质(2个-OH),角黄质(无-OH)也会早于虾青素(2个-OH)洗脱[15]。根据以上推定原则,结合标准品,对3种石斛属植物花瓣和唇瓣中检测到的类胡萝卜素组分进行推定。合相色谱串联三重四极杆质谱仪(UPC2-MS/MS)分析检测到9个类胡萝卜素化合物吸收峰,可以推定的类胡萝卜素化合物结构有8种(表 1),分别是β-胡萝卜素(β-carotene)、α-隐黄质(α-cryptoxanthin)、β-隐黄质(β-cryptoxanthin)、紫黄质(Violaxanthin)、叶黄素(Lutein)、花药黄质(Antheraxanthin)、玉米黄质(Zeaxanthin)和叶黄素酯化物(Lutein-5, 6-epoxide)。
表 1 3种石斛属植物花瓣和唇瓣中类胡萝卜素结构推定
Table 1. Analysis of carotenoids in petals and labellums of three species of Dendrobium
保留时间
Retention time/min检测波长
Detection waudio-video length λmax/nm母离子
Parent ion二级离子(MS2)
Second ion推定结果
Tentative Identification推定依据
Constructive basis2.52 435,461 536 444 β-胡萝卜素β-carotene 标品 4.33 431,458 552 460 α-隐黄素α-cryptoxanthin [15] 4.50 436,462 552 - β-隐黄素β-cryptoxanthin [15] 4.99 431,458 613 335 叶黄素酯化物Lutein-5, 6-epoxide 标品 5.13 429,456 601 - 紫黄质Violaxanthin [15] 5.26 432,459 568 - 叶黄素Lutein 标品 5.33 432,457 584 568 花药黄质Antheraxanthin [15] 5.55 438,463 568 - 玉米黄质Zeaxanthin 标品 注: “-”:未检测到或不存在。Notes: “-”:Not detected or not existed。 -
对推定结构的8种类胡萝卜素进行定量分析,结果(表 2、图 2)表明:球花石斛白色花瓣中的类胡萝卜素总量最低,约为52.26 μg·g-1,随着黄色加深,类胡萝卜素总量逐渐增加,球花石斛金黄色唇瓣中的类胡萝卜素总量最高,达到3 810.89 μg·g-1。
表 2 3种石斛属植物类胡萝卜素组分的定性定量分析
Table 2. Quantitative analysis of carotenoids in petals and labellums of three species of Dendrobium
μg·g-1 组分
Component密花石斛-唇瓣
Den. densiflorum labellums密花石斛-花瓣
Den. densiflorum petals球花石斛-唇瓣
Den. thyrsiflorum labellums球花石斛-花瓣
Den. thyrsiflorum petals鼓槌石斛-花瓣
Den. chrysotoxum petalsβ-胡萝卜素β-carotene 14.32±1.38 2.99±0.28 20.06±0.56 0.64±0.05 30.03±2.46 α-隐黄素α-cryptoxanthin 315.61±28.94 29.02±5.35 2 056.96±62.44 - 20.80±1.92 β-隐黄素β-cryptoxanthin 4.18±0.09 - 13.78±1.04 0.05±0.01 23.04±0.51 叶黄素酯化物Lutein-5, 6-epoxide 265.56±24.43 23.30±4.78 431.48±31.12 - 3.89±0.24 紫黄质Violaxanthin 37.17±4.55 2.84±0.46 64.94±0.85 - 10.44±0.08 叶黄素Lutein 247.82±18.34 103.66±10.72 812.20±33.77 51.57±0.72 166.09±3.74 花药黄质Antheraxanthin 41.20±6.21 1.20±0.06 265.57±4.04 - 108.20±4.63 玉米黄质Zeaxanthin 24.36±3.02 - 145.90±3.80 - 451.00±12.41 总计Total 950.22 163.01 3 810.89 52.26 813.49 注:“-”:未检测到或不存在。Notes:“-”:Not detected or not existed. 图 2 3种石斛属植物花瓣和唇瓣中类胡萝卜素组分的UPC2图谱(检测波长450 nm)
Figure 2. UPC2 chromatograme of carotenoids compounds in petals and labellums of three species of Dendrobium(Detection waudio-videoelength 450 nm)
球花石斛白色花瓣中的类胡萝卜素主要是叶黄素,金黄色唇瓣中α-隐黄素含量最高,约占54%;其次是叶黄素,约占21%,叶黄素酯化物约占11%。密花石斛淡黄色花瓣中叶黄素含量最高,约占64%,其次是α-隐黄素,约占18%,而叶黄素酯化物约占14%;金黄色唇瓣中α-隐黄素含量最高,约占33%,其次是叶黄素酯化物,约占28%,叶黄素约占26%。明黄色鼓槌石斛花瓣中,玉米黄质含量最高,约占55%,其次是叶黄素,约占20%,花药黄质约占13%。
3种石斛属植物类胡萝卜素成分及代谢途径分析
Analysis of Carotenoids Compounds and Their Biosynthesis Pathways in Flowers of Three Dendrobium Species
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摘要:
目的 分析黄色花石斛兰盛开期花瓣中的类胡萝卜素种类和含量,推定石斛属类胡萝卜素合成途径,为探讨黄色花石斛的呈色机理和黄色花育种提供参考。 方法 以花色为黄色的鼓槌石斛、密花石斛和球花石斛的盛开期花瓣和唇瓣为试验材料,采用英国皇家园艺学会比色卡进行花色描述,利用合相色谱串联三重四极杆质谱仪(UPC2-MS/MS)进行类胡萝卜素定性、定量分析,推测石斛属类胡萝卜素代谢途径。 结果 表明:3种石斛属植物花瓣和唇瓣色系可分为白色系和淡黄-明黄-金黄色系,花瓣和唇瓣中共鉴定出8个类胡萝卜素组分,分别是β-胡萝卜素、α-隐黄质、β-隐黄质、紫黄质、叶黄素、花药黄质、玉米黄质和叶黄素酯化物。定量分析显示:球花石斛白色花瓣中类胡萝卜素含量极少,总量约为52.26 μg·g-1,随着黄色加深,类胡萝卜素总量逐渐增加,球花石斛金黄色唇瓣中的类胡萝卜素总量最高,达到3 810.89 μg·g-1。密花石斛淡黄色花瓣中以叶黄素为主;球花石斛和密花石斛金黄色唇瓣中主要是α-隐黄素、叶黄素和叶黄素酯化物;明黄色鼓槌石斛花瓣中以玉米黄质、花药黄质和叶黄素为主。 结论 α-胡萝卜素及其衍生物是淡黄色和金黄色石斛花瓣中的主要类胡萝卜素成分,而β-胡萝卜素及其衍生物是明黄色石斛花瓣的主要类胡萝卜素成分。结合类胡萝卜素代谢途径,推测通过调控LCYE基因的表达,可实现黄色花石斛的花色定向改良。 Abstract:Objective To investigate the composition content and structure of carotenoids in yellow flower of native Dendrobium species and their biosynthesis pathways, thus proving references for further investigation of the floral pigment mechanism and molecular breeding of Dendrobium. Method The flower color in yellow petals and labellum of Den. chrysotoxum, Den. densiflorum and Den. thyrsiflorum were observed according to the Royal Horticultural Society Color Card (RHSCC). Besides, the carotenoids in the petals and labellum in 3 Dendrobium species at full opening stages were determined using UPC2-MS/MS. Based on the results of UPC2-MS/MS, the biosynthesis pathways of carotenoids in Dendrobium were proposed. Result The result indicated that the floral color of the 3 Dendrobium species could be divided into white group and pale yellow-yellow-golden yellow group. Totally, 8 kinds of carotenoids were detected from the petals and labellum in the 3 Dendrobium species, respectively, including β-carotene, α-cryptoxanthin, β-cryptoxanthin, violaxanthin, lutein, antheraxanthin, zeaxanthin and lutein-5, 6-epoxide. According to the results of quantitative analysis, white petals of Den. thyrsiflorum contained the lowest carotenoids (52.26 μg·g-1). The content of carotenoid increased with the enhancement of yellow color and the carotenoid compounds in the golden yellow labellum of Den. thyrsiflorum was the highest, reaching 3 810.89 μg·g-1. Among 3 carotenoids contained cultivars, the lutein was majorly contained in pale yellow petals of Den. thyrsiflorum, while golden yellow labellum of Den. densiflorum and Den. thyrsiflorum majorly contained α-cryptoxanthin, lutein and lutein-5, 6-epoxide. Yellow petals of Den. chrysotoxum contained violaxanthin, lutein and antheraxanthin. Conclusion The carotenoids in pale yellow and golden yellow petals are mainly α-carotene and its derivatives, while that in bright yellow petals are mainly β-carotene and its derivatives. Regulation of the LCYE genes which may cause carotenoid biosynthesis will result in flower color change in yellow flower of Dendrobium. -
Key words:
- Dendrobium
- / yellow flower
- / carotenoids
- / carotenoids pathway
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表 1 3种石斛属植物花瓣和唇瓣中类胡萝卜素结构推定
Table 1. Analysis of carotenoids in petals and labellums of three species of Dendrobium
保留时间
Retention time/min检测波长
Detection waudio-video length λmax/nm母离子
Parent ion二级离子(MS2)
Second ion推定结果
Tentative Identification推定依据
Constructive basis2.52 435,461 536 444 β-胡萝卜素β-carotene 标品 4.33 431,458 552 460 α-隐黄素α-cryptoxanthin [15] 4.50 436,462 552 - β-隐黄素β-cryptoxanthin [15] 4.99 431,458 613 335 叶黄素酯化物Lutein-5, 6-epoxide 标品 5.13 429,456 601 - 紫黄质Violaxanthin [15] 5.26 432,459 568 - 叶黄素Lutein 标品 5.33 432,457 584 568 花药黄质Antheraxanthin [15] 5.55 438,463 568 - 玉米黄质Zeaxanthin 标品 注: “-”:未检测到或不存在。Notes: “-”:Not detected or not existed。 表 2 3种石斛属植物类胡萝卜素组分的定性定量分析
Table 2. Quantitative analysis of carotenoids in petals and labellums of three species of Dendrobium
μg·g-1 组分
Component密花石斛-唇瓣
Den. densiflorum labellums密花石斛-花瓣
Den. densiflorum petals球花石斛-唇瓣
Den. thyrsiflorum labellums球花石斛-花瓣
Den. thyrsiflorum petals鼓槌石斛-花瓣
Den. chrysotoxum petalsβ-胡萝卜素β-carotene 14.32±1.38 2.99±0.28 20.06±0.56 0.64±0.05 30.03±2.46 α-隐黄素α-cryptoxanthin 315.61±28.94 29.02±5.35 2 056.96±62.44 - 20.80±1.92 β-隐黄素β-cryptoxanthin 4.18±0.09 - 13.78±1.04 0.05±0.01 23.04±0.51 叶黄素酯化物Lutein-5, 6-epoxide 265.56±24.43 23.30±4.78 431.48±31.12 - 3.89±0.24 紫黄质Violaxanthin 37.17±4.55 2.84±0.46 64.94±0.85 - 10.44±0.08 叶黄素Lutein 247.82±18.34 103.66±10.72 812.20±33.77 51.57±0.72 166.09±3.74 花药黄质Antheraxanthin 41.20±6.21 1.20±0.06 265.57±4.04 - 108.20±4.63 玉米黄质Zeaxanthin 24.36±3.02 - 145.90±3.80 - 451.00±12.41 总计Total 950.22 163.01 3 810.89 52.26 813.49 注:“-”:未检测到或不存在。Notes:“-”:Not detected or not existed. -
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