【作者】 张洁；

【导师】 高锦明； 王亮生；

【作者基本信息】 西北农林科技大学 ， 化学生物学， 2011， 博士

【摘要】 花色是观赏植物重要的观赏性状之一,扩大花色变异和选育新奇花色品种一直是花卉育种家的努力目标。花色素的研究是花色改良的基础,花色与花色素关系的阐明使花色育种工作更具有方向性。然而,自然界花色多样性、花色素结构复杂性以及二者之间存在密切的关系,使得观赏植物的花青苷成分分析及其花色形成机制的研究具有一定的难度,为观赏植物的花色定向改良提出的切实可行方案也不鲜见。为了探讨观赏植物花色形成机制的一些共性问题或者一般规律,本文选取了几种代表性植物类群—丁香(Syringa spp.)、贴梗海棠(Chaenomeles speciosa)、早开堇菜(V. yedoensis)、紫花地丁(V. prionantha)和圆叶牵牛(Ipomoea purpurea)(丁香和贴梗海棠为木本灌木类,堇菜为草本地被类,圆叶牵牛为草本藤本类)为研究对象,采用国际通用的CIEL*a*b*表色系统、高效液相色谱-质谱联用技术(HPLC-DAD-ESI/MS)及多元线性逐步回归分析方法,系统地开展了不同结构修饰的花青苷成分分析鉴定及其花色形成化学机制研究。在探明几种代表性植物类群的花色形成化学机制的基础上,对观赏花卉的色素结构与花色的关系的一般规律进行总结。此外,推定了丁香的花青苷代谢途径,对至今稀有的红色丁香育种提出了具体的思路,并推及其他观赏植物的蓝色系及红色系花卉的育种策略。主要结果如下:1.花青苷分析方法的建立和优化通过筛选流动相体系,调节洗脱梯度、温度、流速等参数,建立了一套微量、快速、高效、定性、定量分析早开堇菜和紫花地丁花青苷成分的HPLC方法。线性(R = 0.9997)及精密度(RSD < 3.67%)实验证明HPLC方法稳定,可靠。2.花青苷成分的多样性利用HPLC-DAD/ESI-MS技术,在四种观赏植物中共分析鉴定出29种结构多样的花青苷成分,包括多种花青苷元、糖苷化及酰基化的花青苷。其中丁香、早开堇菜和紫花地丁花瓣中的色素成分为首次报道。丁香野生种与栽培品种的花瓣中鉴定出2种花青苷和2种黄酮醇苷,分别是:飞燕草素-3-O-芸香糖苷(Dp3Ru)、矢车菊素-3-O-芸香糖苷(Cy3Ru)、芦丁(Rutin)和山奈酚-3-O-鼠李糖-7-O-葡萄糖苷(Km3R7G)。贴梗海棠品种中含有6种花青苷,分别是:矢车菊素-3-O-(半乳糖葡萄糖苷)[Cy3 (Ga-G)]、矢车菊素-3-O-半乳糖苷(Cy3Ga)、天竺葵素-3-O-(半乳糖葡萄糖苷)[Pg3 (Ga-G)]、矢车菊素-3-O-葡萄糖苷(Cy3G)、天竺葵素-3-O-半乳糖苷(Pg3Ga)和矢车菊素-3-O-琥珀酰阿拉伯糖苷(Cy3SucAra)。Cy3SucAra首次在贴梗海棠花瓣中鉴定。早开堇菜和紫花地丁花瓣中含有14种花青苷,分别是:飞燕草素-3-O-芸香糖-5-O-葡萄糖苷(Dp3Ru5G)、矢车菊素-3-O-芸香糖-5-O-葡萄糖苷(Cy3Ru5G)、矮牵牛素-3-O-芸香糖-5-O-葡萄糖苷(Pt3Ru5G)、Dp-3-O-乙酰芸香糖-5-O-葡萄糖苷(Dp3acRu5G)、Cy-3-O-乙酰芸香糖-5-O-葡萄糖苷(Cy3acRu5G)、Pt-3-O-乙酰芸香糖-5-O-葡萄糖苷(Pt3acRu5G)、Dp-3-O-顺式-香豆酰芸香糖-5-O-葡萄糖苷(Dp3cispCRu5G)、Pt-3-O-顺式-香豆酰芸香糖-5-O-葡萄糖苷(Pt3cispCRu5G)、锦葵素-3-O-顺式-香豆酰芸香糖-5-O-葡萄糖苷( Mv3cispCRu5G )、Dp-3-O-反式-香豆酰芸香糖-5-O-葡萄糖苷(Dp3transpCRu5G)、Pt-3-O-反式-香豆酰芸香糖-5-O-葡萄糖苷(Pt3transpCRu5G)、Mv-3-O-反式-香豆酰芸香糖-5-O-葡萄糖苷(Mv3transpCRu5G)、芍药花素-3-O-香豆酰芸香糖-5-葡萄糖苷(Pn3pCRu5G)和Cy-3-O-香豆酰芸香糖-5-葡萄糖苷(Cy3pCRu5G)。圆叶牵牛花瓣中共鉴定出7种花青苷成分,为高度糖苷化及酰基化的矢车菊素和天竺葵素苷的衍生物,根据它们所含的咖啡酸和葡萄糖基的个数将它们简写为Cy2C3G、Cy2C4G、Cy3C5G、Pg3C4G、Pg3C5G、Pg2C4G和Pg2C3G。3.花青苷组成及含量的比较丁香花瓣中花青苷成分单一,只含有Dp3Ru和Cy3Ru两种花青苷,白色和黄色品种不含花青苷,并且Dp3Ru占绝对优势,平均含量达到88.1%。Cy3Ga和Cy3 (Ga-G)是贴梗海棠花瓣中的主要色素成分,两种花青苷的平均含量占总花青苷含量的94.1%。其它4种花青苷为微量成分。早开堇菜及紫花地丁花瓣中含有的花青苷成分在苷元类型及结构特征两方面表现出显著差异。紫花地丁含有Dp(22.0%),Cy(10.7%),Pt(29.8%),Mv(30.5%)和Pn(7.0%),而早开堇菜中主要以Dp(61.0%)和Cy(28.3%)为主,还有少量的Pt(10.8%)。紫花地丁的花青苷结构绝大多数为3pCRu5G型(96.9%),余下3.0%为3acRu5G。而早开堇菜的花青苷主要以3acRu5G为主(82.1%),3pCRu5G只有11.2%,其它为3Ru5G(6.8%)。蓝紫色圆叶牵牛中含有Cy2C3G、Cy2C4G和Cy3C5G,其中Cy3C5G为主要的花青苷成分,其相对含量平均值为79.6%。紫红色及粉色圆叶牵牛中含有相同的花青苷成分,Pg3C4G、Pg3C5G、Pg2C4G和Pg2C3G,但其相对含量不同。Pg3C5G为紫红色和粉色圆叶牵牛中的主要花青苷成分。Pg3C5G在紫红色圆叶牵牛中的相对含量为68.8%,粉色中为86.5%。白色圆叶牵牛不含花青苷。4.花色形成化学机制利用多元线性逐步回归分析,首次在花青苷水平上阐明四种观赏植物花色形成的化学机制。花青苷和花黄素苷共同决定丁香的花色。Dp3Ru是丁香花瓣的主要花青素类色素成分,这一色素决定了丁香花色主要以紫色—蓝紫色色调为主。回归结果表明Dp3Ru和Rutin是影响花色的主要色素。单纯提高Dp3Ru含量可使丁香花色趋向蓝色,而在减少Rutin含量并同时提高Dp3Ru含量时可使丁香花色的红色程度增强。花青苷是影响贴梗海棠花色的主要色素。Cy3 (Ga-G)和Cy3Ga的含量差异是影响贴梗海棠粉色和红色的重要色素。回归结果表明,Cy3Ga、Pg3 (Ga-G)和Cy3SucAra是决定贴梗海棠花色的最主要色素,这3种色素的含量增加有利于花色变得更红。Dp和Pt型的花青苷是决定早开堇菜和紫花地丁花色的重要花青苷。Dp花青苷含量增加可促进花色趋向蓝色,而Pt花青苷含量增加可促进花色更红。Pg3C5G、Cy3C5G和TF的含量决定了圆叶牵牛的花色变化。Pg3C5G含量增加,同时Cy3C5G和TF含量的减少,可促进花瓣红色的程度增大;Pg3C5G含量降低、Cy3C5G的含量增加促进花瓣趋向蓝色。5.花色育种选育或创制红色丁香是丁香花色育种的目标,建议选取Cy3Ru相对含量为78.7%的丁香‘5’作为母本进行杂交育种;或通过基因共抑制方法,在丁香花瓣中导入F3’5’H基因,抑制F3’5’H的活性,促进Cy3Ru的积累来实现这一目标。总之,从上述四种植物花色形成机制中凸现的一般规律可概括为,Dp型花青苷并不是植物花瓣形成蓝色的唯一色素基础,还应充分考虑Dp型花青苷与TF的含量共调节、花青苷与其它辅助色素(copigment)形成共色效应(copigmentation effect)等关键因素。更多还原

【Abstract】 Flower color is one of the most important ornamental characteristics. The object of a prolonged endeavor for flower breeders is to expand the flower color range and breed cultivars with novel color. However, due to the diversity of flower color, complexity of anthocyanins structures and their intensive relations, it is difficulty to analyze anthocyanins and find out the coloration mechanism of ornamental plants. In order to explore the general law of coloration mechanism, the representative ornamental plants namely Syringa spp., Chaenomeles speciosa, Viola (V. yedoensis, V. prionantha) and Ipomoea purpurea were chosen as the study objects in the present paper, in which Syringa spp. and Chaenomeles speciosa belong to the woody shrub, Viola belongs to the herbaceous cover plant, and Ipomoea purpurea belongs to the herbaceous climber. The identification of anthocyanin constitutions and clarification of petal coloration mechanism of these representative plants were systematically studied. Color were measured by CIEL*a*b* scale and anthocyanins compositions were determined by high-performance liquid chromatography coupled to photodiode array detection and electrospray ionization mass spectrometry (HPLC-DAD/ESI-MS). Multiple linear regression analysis (MLR) was used to explore the relationship of petal color and anthocyanin contents. Based on the clarification of petal coloration mechanism of representative plants, the universal law for the relationship between anthocyanin structures and petal color were summarized. In addition, the biosynthetic pathway of anthocyanins from Syringa was deduced, and the specific methods of breeding rare red Syringa flowers were reported.Main results were as follows:1. Optimization of HPLC separation for anthocyaninsA fast and reliable HPLC method was developed for separation of anthocyanins in V. yedoensis and V. prionantha petals by optimizing solvent system and adjustment of gradient elution, temperature and flow rate. All data from linearity (R = 0.9997) and precision (RSD < 3.67%) experiments revealed that the optimized HPLC method was precise and reliable.2. Complexity of anthocyanin structures29 anthocyanins were characterized in petals of four ornamental plants by HPLC-DAD/ESI-MS, which derivated from various anthocyanidins, glycosylations and acylations. Anthocyanins from Syringa, V. yedoensis and V. prionantha were firstly reported.There were two anthocyanins and two flavonols identified in Syringa spp., which were delphinidin 3-O-rutinoside (Dp3Ru), cyanidin 3-O-rutinoside (Cy3Ru), quercetin 3- O-rutinoside (Rutin) and kaempferol-3-O-rhamnoside-7-O-glucoside (Km3R7G).Six anthocyanins were found in petals of C. speciosa, which were Cy-3-O-galactoside (Cy3Ga), Cy-3-O-glucoside (Cy3G), Pg-3-O-galactoside (Pg3Ga), Pg-3-O-(galactose-glucose) [Pg3 (Ga-G)], Cy-3-O-(galactose-glucose) [Cy3 (Ga-G)] and Cy-3-O-succinylarabinoside (Cy3SucAra). Cy3SucAra was identified firstly in C. speciosa.14 anthocyanins were found in flowers of V. yedoensis and V. prionantha, which were Dp-3-O-rutinoside-5-glucoside (Dp3Ru5G), Cy3Ru5G and Pt3Ru5G; Dp-3-O-(acetyl)-rutinoside-5-glucoside (Dp3acRu5G), Cy3acRu5G and Pt3acRu5G; Dp-3-O-(p-cis-coumaroyl)-rutinoside-5-glucosid (Dp3cispCRu5G), Dp-3-(p-tans- coumaroyl)-rutinoside-5-glucoside (Dp3transpCRu5G), Pt3cispCRu5G, Pt3transpCRu5G, Mv3cispCRu5G, Mv3transpCRu5G, Cy3pCRu5G and Pn3pCRu5G.Seven anthocyanins were analyzed in petals of I. purpurea. They were Pg and Cy derivatives intensively glycosylated and acylated by glucoses and caffeic acids. According to the numbers of glucoses and caffeic acids, they are abbreviated as Cy3C5G, Pg3C5G, Cy2C3G, Cy2C4G, Pg3C4G, Pg2C4G and Pg2C3G.3. Comparison of anthocyanin contentsDp3Ru and Cy3Ru were identified in petals of Syringa. Dp3R is predominant (88.1%). There were no anthocyanins found in white and yellow Syringa flowers.Cy3Ga and Cy3 (Ga-G) are the main anthocyanins in C. speciosa flowers, the average content of which accounts for 94.1%. Other four anthocyanins are trace amounts.There are remarkable differences in anthocyanins between V. yedoensis and V. prionantha. In V. yedoensis, anthocyanins derived from Dp (22.0%), Cy (10.7%), Pt (29.8%), Mv (30.5%) and Pn (7.0%). In V. prionantha, anthocyanins derived from Dp (61.0%), Cy (28.3%) and Pt (10.8%). In V. yedoensis, anthocyanins are mostly of pC3Ru5G type (96.9%), and the rest are ac3Ru5G (3.0%). In contrast, in V. prionantha, predominant anthocyanins belong to ac3Ru5G type (82.1%), and the rest are pC3Ru5G (11.2%) and 3Ru5G (6.8%).Cy2C3G, Cy2C4G and Cy3C5G were characterized in purple-blue I. purpurea flowers. Cy3C5G is major (79.6%). Purple-blue and pink petals of I. purpurea comprise the same anthocyanins, Pg3C4G, Pg3C5G, Pg2C4G and Pg2C3G, but their contents are different. Pg3C5G is the major anthocyanin in purple-blue and pink petals, which account for 68.8% and 86.5%, respectively.4. Clarification of petal coloration mechanismIt was the first time to explore the relationships between petal color and anthocyanin contents in the levels of anthocyanin glycosides of four ornamental plants by multiple linear regression analysis (MLR).Dp3Ru and Rutin determine the Syringa petal color together. Dp3Ru is the main anthocyanin, which gives blue to purple-blue to Syringa petal colors. Increase of Dp3Ru content can shift flower color towards bluer, while in the condition of decrease of Rutin content and increase of Dp3Ru content can enhance the red extent.The different content of Cy3 (Ga-G) and Cy3Ga influences the pink and red color of C. speciosa. MLR showed that Cy3Ga, Pg3 (Ga-G) and Cy3SucAra are important components, high contents of which significantly enhance the red extent of C. speciosa petals.Dp and Pt derivatives are vital anthocyanins impacting on the flower color of V. prionantha and V. yedoensis. Increase of Dp derivatives contents can facilitate flower color bluer, while increase of Pt derivatives contents can shift flower color towards redder.The contents of Pg3C5G, Cy3C5G and TF impact on the flower color of I. purpurea. Increase of Pg3C5G, decrease of Cy3C5G and TF can enhance the red extent, while decrease of Pg3C5G and increase of Cy3C5G can make the flower color shift to bluer.5. Guidance for flower color breedingSelection or creation of red petals is the aim of flower color breeding in Syringa. It is advised to hybrid with the parent of‘5’which comprises high amount of Cy3Ru (78.7%) or to utilize the co-suppression technique. Introduction of F3’5’H can inhibit F3’5’H, interfere or reduce the synthesis of Dp3Ru, and then Cy3Ru amount increases, the petal shift to redder.In brief, we can conclude that Dp derivatives are not the only anthocyanin base in the formation of blue flowers. We should fully consider other key factors such as the co-adjustment of contents of Dp derivatives and TF, the copigmentation effects between anthocyanins and copigments and so on.更多还原