滇牡丹花瓣着色过程中类胡萝卜素成分变化和相关基因表达分析

doi:10.16420/j.issn.0513-353x.2021-0613

摘要/Abstract

摘要：

滇牡丹（Paeonia delavayi）中的黄色花类群是十分重要的育种资源。为厘清其花瓣着色过程中类胡萝卜素成分及含量变化,以花发育过程中4个时期的花瓣为材料进行类胡萝卜素定性及定量分析,并对类胡萝卜素生物合成途径中的关键结构基因PdPSY、PdZDS、PdLCYB、PdCHY、PdNXS、PdCRISTO、PdCHYB和降解途径中关键结构基因PdCCD的转录水平进行实时荧光定量PCR分析。首次在滇牡丹花瓣中鉴定出14种类胡萝卜素类物质,分别是α-胡萝卜素、花药黄质、番茄红素、玉米黄质、紫黄质、γ-胡萝卜素、新黄质、β-胡萝卜素、叶黄素、β-隐黄质、β-阿朴胡萝卜素醛、八氢番茄红素、ε-胡萝卜素和α-隐黄质,其中含量最高的为叶黄素和八氢番茄红素。随着花发育,八氢番茄红素、番茄红素和γ-胡萝卜素的积累量不断上升;α-胡萝卜素、β-胡萝卜素、α-隐黄质、叶黄素、紫黄质、新黄质的积累量则不断下降。荧光定量结果显示PdPSY、PdCRISTO、PdZDS、PdLCYB和PdCCD表达量呈上升趋势,PdCHYE、PdCHYB和PdNXS表达量呈下降趋势,其中PdCHYB和PdNXS在透色期开始表达量明显降低;PdCHYE持续降低,该趋势与色素含量变化基本一致。

关键词: 滇牡丹, 花色, 类胡萝卜素, 基因表达

Abstract:

The yellow flower group in Paeonia delavayi is an important breeding resource. In order to clarify the changes of carotenoid components and content during petal coloring,carotenoids in the petals from four stages of flower development were analyzed qualitatively and quantitatively,and the key structural genes of PdPSY,PdZDS,PdLCYB,PdCHY,PdNXS,PdCRISTO,PdCHYB in carotenoid biosynthesis pathway and of PdCCD in degradation pathway were analyzed by Real-time fluorescence quantitative PCR. In this study,14 carotenoids were identified in the petals of Paeonia delavayi for the first time,and they were α-carotene,antheraxanthin,lycopene,zeaxanthin,violaxanthin,γ-carotene, neoxanthin,β-carotene,lutein,β-cryptoxanthin,apocarotenal,(E/Z)-phytoene,ε-carotene and α-cryptoxanthin. Among them,lutein and (E/Z)-phytoene had the highest content. With the development of flowers,the accumulation of (E/Z)-phytoene,lycopene and γ-carotene increased continuously,while the accumulation of α-carotene,β-carotene,α-cryptoxanthin,lutein,violaxanthin and neoxanthin decreased continuously. The results of Real-time quantitative PCR showed that the expression of PdPSY,PdCRISTO,PdZDS,PdLCYB and PdCCD presented an upward trend,the expression of PdCHYE,PdCHYB and PdNXS showed a downward trend,among which the expression of PdCHYB and PdNXS decreased significantly at slightly pigmented soft bud stage,and the expression of PdCHYE decreased gradually during petal coloring,which was basically consistent with the change of pigment content.

Key words: Paeonia delavayi, flower color, carotenoid, gene expression

中图分类号:

S685.11

邹红竹, 周琳, 韩璐璐, 吕纪杭, 王雁. 滇牡丹花瓣着色过程中类胡萝卜素成分变化和相关基因表达分析[J]. 园艺学报, 2021, 48(10): 1934-1944.

ZOU Hongzhu, ZHOU Lin, HAN Lulu, LÜ Jihang, WANG Yan. Changes of Carotenoid Components and Expression of the Related Genes During Petal Coloring of Paeonia delavayi[J]. Acta Horticulturae Sinica, 2021, 48(10): 1934-1944.

图/表 7

参考文献 37

[1]	An Tian-qi. 1989. Physiological and biochemistry of flower colors. Beijing: China Forestry Publishing House.(in Chinese)
	安田齐. 1989. 花色的生理生物化学. 北京: 中国林业出版社.
[2]	Cao H, Wang J, Dong X, Yan H, Deng X. 2015. Carotenoid accumulation affects redox status,starch metabolism,and flavonoid/anthocyanin accumulation in citrus. BMC Plant Biology, 15 (1):27. doi: 10.1186/s12870-015-0426-4 URL
[3]	Cheng Meng-lin, Zhang Yan-yan, Zhao Da-qiu, Tao Jun. 2018. Composition analysis of flavonols and their glycosides in yellow flowers of Herbaceous Peony(Paeonia lactiflora Pall.)cultivars. Natural Product Research and Development,(8):1340-1345.(in Chinese)
	成梦琳, 张艳艳, 赵大球, 陶俊. 2018. 芍药黄色花瓣中黄酮醇及其糖苷类化合物组成分析. 天然产物研究与开发,(8):1340-1345.
[4]	Domonkos I, Kis M, Gombos Z, Ughy B. 2013. Carotenoids,versatile components of oxygenic photosynthesis. Progress in Lipid Research, 52 (4):539-561 doi: 10.1016/j.plipres.2013.07.001 pmid: 23896007
[5]	Fattore M, Montesano D, Pagano E, Teta R, Borrelli F, Mangoni A, Seccia S, Aibrizio S. 2016. Carotenoid and flavonoid profile and antioxidant activity in“Pomodorino Vesuviano”tomatoes. Journal of Food Composition and Analysis, 53:61-68. doi: 10.1016/j.jfca.2016.08.008 URL
[6]	Gamsjaeger S, Baranska M, Schulz H, Heiselmayer P, Musso M. 2011. Discrimination of carotenoid and flavonoid content in petals of pansy cultivars(Viola × wittrockiana)by FT-Raman spectroscopy. Journal of Raman Spectroscopy, 42 (6):1240-1247. doi: 10.1002/jrs.2860 URL
[7]	García-Limones C, Schnäbele K, Blanco-Portales R, Bellido M L, Caballero J L, Schwab W, Muñoz-Blanco J. 2008. Functional characterization of FaCCD1:a carotenoid cleavage dioxygenase from strawberry involved in lutein degradation during fruit ripening. Journal of Agricultural and Food Chemistry, 56 (19):9277-9285. doi: 10.1021/jf801096t pmid: 18778069
[8]	Ge Yu-xuan, Wang Liang-sheng, Xu Yan-jun, Liu Zheng-an, Li Chong-hui, Jia Ni. 2008. Flower color,pigment composition and their changes during flowering in Chimonanthus praecox Link. Acta Horticulturae Sinica, 35 (9):1331-1338.(in Chinese)
	葛雨萱, 王亮生, 徐彦军, 刘政安, 李崇晖, 贾妮. 2008. 蜡梅的花色和花色素组成及其在开花过程中的变化. 园艺学报, 35 (9):1331-1338.
[9]	Giovanni G, Arzu Y. 2013. Elevation of lutein content in tomato:a biochemical tug-of-war between lycopene cyclases. Metabolic Engineering, 20:167-176. doi: 10.1016/j.ymben.2013.10.007 pmid: 24141052
[10]	Hannoufa A, Hossain Z. 2012. Regulation of carotenoid accumulation in plants. Biocatalysis & Agricultural Biotechnology, 1 (3):198-202.
[11]	Hermanns A S, Zhou X S, Xu Q, Tadmor Y, Li L. 2020. Carotenoid Pigment Accumulation in Horticultural Plants. Horticultural Plant Journal, 6 (6):343-360. doi: 10.1016/j.hpj.2020.10.002 URL
[12]	Hirschberg J. 2001. Carotenoid biosynthesis in flowering plants. Current Opinion in Plant Biology, 4:210-280. pmid: 11312131
[13]	Lewinsohn E, Sitrit Y, Bar E, Azulay Y, Ibdah M, Meir A, Yosef E, Zamir D, Tadmor Y. 2005. Not just colors-carotenoid degradation as a link between pigmentation and aroma in tomato and watermelon fruit. Trends in Food Science & Technology, 16 (9):407-415.
[14]	Li C, Du H, Wang L, Shu Q, Zheng Y, Xu Y, Zhang J, Zhang J, Yang R, Ge Y. 2009. Flavonoid composition and antioxidant activity of tree peony(Paeonia Section Moutan)yellow flowers. Journal of Agricultural & Food Chemistry, 57 (18):8496-8503.
[15]	Li L, Yuan H. 2013. Chromoplast biogenesis and carotenoid accumulation. Archives of Biochemistry and Biophysics, 539 (2):102-109. doi: 10.1016/j.abb.2013.07.002 pmid: 23851381
[16]	Li Jia-jue, Zhang Xi-fang, Zhao Xiao-qing. 2011. Tree peony of China. Beijing: Encyclopedia of China Publishing House.(in Chinese)
	李嘉珏, 张西方, 赵孝庆. 2011. 中国牡丹. 北京: 中国大百科全书出版社.
[17]	Li X, Lu M, Tang D, Shi Y, Gómez-Gómez L. 2015. Composition of carotenoids and flavonoids in narcissus cultivars and their relationship with flower color. PLoS ONE, 10 (11):e0142074. doi: 10.1371/journal.pone.0142074 URL
[18]	Li Xinlei, Wang Jiatong, Sun Zhenyuan, Wang Jie, Yin Hengfu, Fan Zhengqi, Li Jiyuan. 2019. Flavonoid components and their effects on flower colors in Camellia nitidissima,white C. japonica and their three hybrid cultivars. Acta Horticulturae Sinica, 46 (6):1145-1154.(in Chinese)
	李辛雷, 王佳童, 孙振元, 王洁, 殷恒福, 范正琪, 李纪元. 2019. 金花茶和白色山茶及其3个杂交品种类黄酮成分与花色的关系. 园艺学报, 46 (6):1145-1154.
[19]	Martins T R, Berg J J, Blinka S, Rausher M D, Baum D A. 2013. Precise spatio-temporal regulation of the anthocyanin biosynthetic pathway leads to petal spot formation in Clarkia gracilis(Onagraceae). New Phytologist, 197 (3):958-969. doi: 10.1111/nph.2013.197.issue-3 URL
[20]	Nambara E, Marion-Poll A. 2005. Abscisic acid biosynthesis and catabolism. Annual Review of Plant Biology, 56 (56):165-185. doi: 10.1146/arplant.2005.56.issue-1 URL
[21]	Ng J, Smith S D. 2016. Widespread flower color convergence in Solanaceae via alternate biochemical pathways. New Phytologist, 209 (1):407-417. doi: 10.1111/nph.2016.209.issue-1 URL
[22]	Ohmiya A, Sumitomo K, Aida R. 2009. “Yellow Jimba”:suppression of carotenoid cleavage dioxygenase(CmCCD4a)expression turns white chrysanthemum petals yellow. Journal of the Japanese Society for Horticultural Science, 78 (4):450-455. doi: 10.2503/jjshs1.78.450 URL
[23]	Parks C R, Scogin R. 1988. The elusive yellow camellia:results from breeding and pigment analysis. American Camellia Yearbook:31-37.
[24]	Ruiz-Sola M Á, Rodríguez-Concepción M. 2012. Carotenoid biosynthesis in Arabidopsis:a colorful pathway. Arabidopsis Book, 10:e015.
[25]	Song L, Ma Q, Zou Z, Kang S, Tao J, Kaleri N A, Li X. 2017. Molecular link between leaf coloration and gene expression of flavonoid and carotenoid biosynthesis in Camellia sinensis cultivar‘Huangjinya’. Frontiers in Plant Science, 8:803. doi: 10.3389/fpls.2017.00803 URL
[26]	Tadmor Y, Burger J, Yaakov I, Feder A, Libhaber S E, Portnoy V, Meir A, Tzuri G, Sa’ar U, Rogachev I, Aharoni A. 2010. Genetics of flavonoid,carotenoid,and chlorophyll pigments in melon fruit rinds. Journal of Agricultural & Food Chemistry, 58 (19):10722-10728.
[27]	Tanaka Y, Sasaki N, Ohmiya A. 2008. Biosynthesis of plant pigments:anthocyanins,betalains and carotenoids. The Plant Journal, 54 (4):733-749. doi: 10.1111/j.1365-313X.2008.03447.x URL
[28]	Wang Lian-ying, Yuan Tao. 2005. Sequel of Chinese tree peony(II). Beijing: China Forestry Publishing House.(in Chinese)
	王莲英, 袁涛. 2015. 中国牡丹品种图志(续志). 北京: 中国林业出版社.
[29]	Yamamizo C, Kishimoto S, Ohmiya A. 2010. Carotenoid composition and carotenogenic gene expression during Ipomoea petal development. Journal of Experimental Botany, 61 (3):709. doi: 10.1093/jxb/erp335 pmid: 19933319
[30]	Yang Nian-yun, Duan Jin-ao, Qian Shi-hui, Tian Li-juan. 2003. Studies on the chemical constituents of Tagetes erecta L. Journal of Shenyang Pharmaceutical University,(4):258-259.(in Chinese)
	杨念云, 段金廒, 钱士辉, 田丽娟. 2003. 万寿菊花的化学成分研究. 沈阳药科大学学报,(4):258-259.
[31]	Yang Y, Li B, Feng C, Wu Q, Wang L. 2020a. Chemical mechanism of flower color microvariation in Paeonia with yellow flowers. Horticultural Plant Journal, 6 (3):179-190. doi: 10.1016/j.hpj.2020.04.002 URL
[32]	Yang Y, Sun M, Li S, Chen Q, Wang L. 2020b. Germplasm resources and genetic breeding of Paeonia:a systematic review. Horticulture Research, 7:107. doi: 10.1038/s41438-020-0332-2 URL
[33]	Yuan H, Zhang J, Nageswaran D, Li L. 2015. Carotenoid metabolism and regulation in horticultural crops. Horticulture Research, 2:15036. doi: 10.1038/hortres.2015.36 pmid: 26504578
[34]	Yuan Y W, Sagawa J M, Stilio V, Bradshaw H D. 2013. Bulk segregant analysis of an induced floral mutant identifies a MIXTA-Like R2R3 MYB controlling nectar guide formation in Mimulus lewisii. Genetics, 194:523-528. doi: 10.1534/genetics.113.151225 URL
[35]	Zhao Chang-ling, Guo Wei-ming, Chen Jun-yu. 2003. Biochemical and molecular biological mechanism of the expression of the flower color of higher plant and its ameliorating by gene engineering. Acta Botanica Boreali-Occidentalia Sinica, 23 (6):1024-1035.(in Chinese)
	赵昶灵, 郭维明, 陈俊愉. 2003. 植物花色呈现的生物化学、分子生物学机制及其基因工程改良. 西北植物学报, 23 (6):1024-1035.
[36]	Zhou Lin, Wang Yan,Lv Chun-yan,Peng Zhen-hua. 2011. Identification of components of flower pigments in petals of Paeonia lutea wild population in Yunnan. Journal of Northeast Forestry University,(8):52-54.(in Chinese)
	周琳, 王雁, 律春燕, 彭镇华. 2011. 云南野生黄牡丹花色素成分的鉴定. 东北林业大学学报,(8):52-54.
[37]	Zhu K J, Wu Q J, Huang Y, Ye J L, Xu Q, Deng X X. 2020. Genome-wide characterization of cis-acting elements in the promoters of key carotenoid pathway genes from the main species of genus citrus. Horticultural Plant Journal, 6 (6):385-395. doi: 10.1016/j.hpj.2020.10.003 URL

分类 Classification	标准品 Standard	线性方程 Equation	相关系数 Coefficient of correlation
胡萝卜素	α-胡萝卜素α-Carotene	y = 4.98584e5x-1108.06123	0.99999
Carotene	番茄红素Lycopene	y = 3.49839e5x-242.82230	0.99982
	γ-胡萝卜素γ-Carotene	y = 4.37283e5x-547.23525	0.99998
	β-胡萝卜素β-Carotene	y = 5.74793e5x-11445.03070	0.99999
	六氢番茄红素Phytofluene	y = 1.42297e5x + 23435.01369	0.99995
	八氢番茄红素(E/Z)-Phytoene	y = 7.11827e4x-567.43881	1.00000
	ε-胡萝卜素ε-Carotene	y = 1.16042e6x + 1186.48166	0.99980
叶黄素	花药黄质Antheraxanthin	y = 5.55136e5x-1297.82785	1.00000
Lutein	玉米黄质Zeaxanthin	y = 1.82585e5x + 9928.65201	0.99985
	紫黄质Violaxanthin	y = 1.47435e6x-3.30834e4	0.99994
	新黄质Neoxanthin	y = 1.22081e6x-21272.87715	0.99928
	叶黄素Lutein	y = 9.00512e5x + 1.17150e5	0.99998
	β-隐黄质β-Cryptoxanthin	y = 2.29006e5x + 5179.02939	0.99998
	虾青素Astaxanthin	y = 4.24452e6x + 1051.48536	0.99668
	β-阿朴胡萝卜素醛Apocarotenal	y = 8.55527e6x-1234.24238	1.00000
	辣椒红素Capsanthin	y = 1.11119e7x + 4273.93502	0.99901
	α-隐黄质α-Cryptoxanthin	y = 5.99746e5x + 388.44092	0.99988
	辣椒玉红素Capsorubin	y = 2.33996e6x-1575.19663	0.99991

分类 Classification	标准品 Standard	线性方程 Equation	相关系数 Coefficient of correlation
胡萝卜素	α-胡萝卜素α-Carotene	y = 4.98584e5x-1108.06123	0.99999
Carotene	番茄红素Lycopene	y = 3.49839e5x-242.82230	0.99982
	γ-胡萝卜素γ-Carotene	y = 4.37283e5x-547.23525	0.99998
	β-胡萝卜素β-Carotene	y = 5.74793e5x-11445.03070	0.99999
	六氢番茄红素Phytofluene	y = 1.42297e5x + 23435.01369	0.99995
	八氢番茄红素(E/Z)-Phytoene	y = 7.11827e4x-567.43881	1.00000
	ε-胡萝卜素ε-Carotene	y = 1.16042e6x + 1186.48166	0.99980
叶黄素	花药黄质Antheraxanthin	y = 5.55136e5x-1297.82785	1.00000
Lutein	玉米黄质Zeaxanthin	y = 1.82585e5x + 9928.65201	0.99985
	紫黄质Violaxanthin	y = 1.47435e6x-3.30834e4	0.99994
	新黄质Neoxanthin	y = 1.22081e6x-21272.87715	0.99928
	叶黄素Lutein	y = 9.00512e5x + 1.17150e5	0.99998
	β-隐黄质β-Cryptoxanthin	y = 2.29006e5x + 5179.02939	0.99998
	虾青素Astaxanthin	y = 4.24452e6x + 1051.48536	0.99668
	β-阿朴胡萝卜素醛Apocarotenal	y = 8.55527e6x-1234.24238	1.00000
	辣椒红素Capsanthin	y = 1.11119e7x + 4273.93502	0.99901
	α-隐黄质α-Cryptoxanthin	y = 5.99746e5x + 388.44092	0.99988
	辣椒玉红素Capsorubin	y = 2.33996e6x-1575.19663	0.99991

基因 Gene	正向引物（5′-3′） Forword primer	反向引物（5′-3′） Reverse primer
PdPSY	ACACCGGCAGCTTTGGATAGGT	AGCCAACAGTTCCAGCGACA
PdCRISTO	TGATGCCGAGGGAATACGCA	TCAAATGGCTCGGTTCCTGGTG
PdCHYB	TGCTCTTTCTGTGGGTGCTGCT	ACCAGCGCCAAAACAGAGTCCA
PdLCYB	TCGACCTGGACTCTGCATGGAA	GCCGTACCACCAATCCCAACAA
PdCHYE	TGGTGGTTATTGGCTGTGGTCCA	CCGCCAAACATGCTCAATACACCC
PdNXS	TATTGGTGGCACGGCTGGGTTA	ATAGGCCACAACCCGTTCCACA
PdZDS	AAGGAGCCAGATTTCGCGGGTT	TTGACATGCCTGCAAGCCCA
PdCCD	GGTTGGCCGGAAACTTTGCT	ACCATGCCATCGCCATCAAACC
PsPP2A	CGTGTTTGGATGTTCTCAAGGC	GGCGAGTGAGTTTTCAGTTGGA

基因 Gene	正向引物（5′-3′） Forword primer	反向引物（5′-3′） Reverse primer
PdPSY	ACACCGGCAGCTTTGGATAGGT	AGCCAACAGTTCCAGCGACA
PdCRISTO	TGATGCCGAGGGAATACGCA	TCAAATGGCTCGGTTCCTGGTG
PdCHYB	TGCTCTTTCTGTGGGTGCTGCT	ACCAGCGCCAAAACAGAGTCCA
PdLCYB	TCGACCTGGACTCTGCATGGAA	GCCGTACCACCAATCCCAACAA
PdCHYE	TGGTGGTTATTGGCTGTGGTCCA	CCGCCAAACATGCTCAATACACCC
PdNXS	TATTGGTGGCACGGCTGGGTTA	ATAGGCCACAACCCGTTCCACA
PdZDS	AAGGAGCCAGATTTCGCGGGTT	TTGACATGCCTGCAAGCCCA
PdCCD	GGTTGGCCGGAAACTTTGCT	ACCATGCCATCGCCATCAAACC
PsPP2A	CGTGTTTGGATGTTCTCAAGGC	GGCGAGTGAGTTTTCAGTTGGA