荷兰鸢尾‘玉妃’花色变异关键结构基因分析

doi:10.3864/j.issn.0578-1752.2021.12.014

摘要/Abstract

摘要：

【目的】花色变异对丰富观赏植物花色具有重要意义,但由于花色变异的不确定性,使其变异机理尚未弄清。荷兰鸢尾是重要球根观赏植物,通过探索荷兰鸢尾蓝紫色野生型‘展翅’及白色突变株‘玉妃’的显色分子机制及色素积累差异,为花色变异机理提供依据。【方法】本研究通过超高效液相色谱-四级杆飞行时间串联质谱联用（UHPLC-QTOF-MS）方法测定荷兰鸢尾2个品种花的花色素苷和黄酮醇的种类及含量。以荷兰鸢尾‘展翅’和‘玉妃’的旗瓣为材料,通过转录组测序,筛选花色素苷合成相关差异基因。以2个品种花发育不同时期为材料,对差异基因进行荧光定量PCR验证。【结果】代谢组分析结果表明,飞燕草素和矢车菊素及其衍生物在蓝紫色花中积累,在白色花中几乎没有积累,而黄酮醇类物质在白色‘玉妃’中含量上升。RNA-seq结果表明,共获得46 485个unigenes,其中27 073个unigenes被公共数据库功能注释,占全部unigenes的58.24%。获得701个差异表达基因,其中485个基因在‘玉妃’中上调表达,216个基因在‘玉妃’中下调表达。花色素苷途径中,2个二氢黄酮醇-4-还原酶（dihydroflavonol 4-reductase,DFR）基因和1个黄酮醇合成酶（flavonol synthase,FLS）基因有差异表达,分别命名为IhDFR1、IhDFR2和IhFLS1。白色‘玉妃’中,IhDFR1和IhDFR2下调表达,IhFLS1上调表达,导致花色素苷含量显著降低和黄酮醇积累,使代谢流由花色素苷转向黄酮醇。3个基因的qRT-PCR结果显示,IhDFR1和 IhDFR2在蓝紫色花中随着花的发育表达量升高,在白色花中均低表达,IhFLS1在白色花中随着花的发育表达量升高,在蓝紫色花中均低表达,与RNA-seq结果保持一致。【结论】白色‘玉妃’中,IhDFR1和IhDFR2的低表达,及IhFLS1的高表达,使花色素苷积累受阻,部分代谢流由花色素苷转向黄酮醇,导致花色由蓝紫变为白色。

关键词: 荷兰鸢尾, 花色素苷, 黄酮醇, 转录组分析, 二氢黄酮醇-4-还原酶基因, 黄酮醇合成酶基因

Abstract:

【Objective】 Flower color variation is of great significance for enriching color of ornamental plants, but it is difficult to clarify variation mechanism due to uncertainty of flower color variation. Dutch iris (Iris hollandica) is an important bulbous ornamental plant. In this study, the blue-purple wild type ‘Zhanchi’ and white mutant strain 'Yufei' of Dutch iris were investigated to explore molecular mechanism and difference of pigment accumulation, so as to provide a basis for mechanism of flower color variation.【Method】In this study, using inner tepals of ‘Zhanchi’ and ‘Yufei’ from Dutch Iris as materials, UHPLC-QTOF-MS method was used to determine types and contents of anthocyanins and flavonols from two varieties of Dutch Iris, and the different expression genes related to anthocyanin synthesis were screened by transcriptome sequencing. Using different flowers in developmental stages of two varieties as materials, the different expression genes were verified by qRT-PCR.【Result】Results of metabolomics analysis revealed that delphinidin, cyanidin and its derivatives were accumulated in blue-purple flowers, which were almost no accumulation in white flowers, while the flavonol contents were increased in white ‘Yufei’. Results of RNA-seq analysis revealed that a total of 46 485 unigenes were obtained, and 27 073 unigenes of them were functionally annotated by public databases, accounting for 41.85% of the total. And 701 differentially expressed genes were obtained, 485 genes of which were up-regulated and 216 genes were down-regulated in white ‘Yufei’. Two dihydroflavonol-4-reductase genes and one flavonol synthase gene involving in anthocyanin biosynthetic pathway had different expression, named IhDFR1, IhDFR2 and IhFLS1. Down-regulated expression of IhDFR1 and IhDFR2 as well as up-regulated expression of IhFLS1 in white ‘Yufei’ led to significant decrease of anthocyanins and accumulation of flavonols, which caused metabolic flow from anthocyanin to flavonol. The qRT-PCR results of three genes showed that expression levels of IhDFR1 and IhDFR2 increased in blue-purple flowers during flower developmental stage, but low expression in white flowers, and expression level of IhFLS1 increased in white flowers during flower developmental stage, but low expression in blue-purple flowers, which was consistent with RNA-seq results.【Conclusion】Low expression of IhDFR1 and IhDFR2 as well as high expression of IhFLS1 in white ‘Yufei’ blocked accumulation of anthocyanins, and some of metabolic flow changed from anthocyanins to flavonols, resulting in the change of flower color from blue violet to white.

Key words: Iris hollandica, anthocyanin, flavonol, transcriptome analyses, dihydroflavonol-4-reductase gene, flavonol synthase gene

林兵,陈艺荃,钟淮钦,叶秀仙,樊荣辉. 荷兰鸢尾‘玉妃’花色变异关键结构基因分析[J]. 中国农业科学, 2021, 54(12): 2644-2652.

LIN Bing,CHEN YiQuan,ZHONG HuaiQin,YE XiuXian,FAN RongHui. Analysis of Key Genes About Flower Color Variation in Iris hollandica[J]. Scientia Agricultura Sinica, 2021, 54(12): 2644-2652.

0
/ / 推荐

导出引用管理器 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2021.12.014

https://www.chinaagrisci.com/CN/Y2021/V54/I12/2644

图/表 6

表1

图1

图2

表2

荷兰鸢尾花中花色素苷和黄酮醇的鉴定"

化合物种类 Species of compounds	Compound 化合物名称	含量 Content (μg·g^-1)
化合物种类 Species of compounds	Compound 化合物名称	展翅 Zhanchi	玉妃 Yufei
花青素苷Anthocyanidin	矢车菊素-3-(2G-木糖基芸香糖苷) Cyanidin -3-(2G-xylosylrutinoside)	25.22±1.66	1.34±0.05
	飞燕草素-3-[6-(4-咖啡酰基木糖基)葡萄糖苷]-5-葡萄糖苷 Delphinidin-3-[6-(4-(caffeoylrhamnosyl) glucoside]-5-glucoside	61.26±1.33	1.36±0.08
	矢车菊素-3-芸香糖苷 Cyanidin -3-rutinoside	144.42±6.46	16.90±0.63
	6-羟基矢车菊素-3-葡萄糖苷 6-Hydroxycyanidin -3-glucoside	5.88±0.19	0.56±0.01
	飞燕草素-3-(6-p-酰基葡萄糖苷)-5-(6-丙二酰基-4-鼠李糖基葡萄糖苷) Delphinidin-3-(6-p-coumaroylglucoside)-5-[6-(malonyl)-4-(rhamnosyl) glucoside)]	209.85±3.12	1.71±0.05
	飞燕草素-3-葡萄糖苷 Delphinidin-3-glucoside	4.37±0.24	0.08±0.01
	飞燕草素-3-芸香糖苷 Delphinidin 3-rutinoside	30.64±0.38	16.98±0.48
	矮牵牛素-3-(6-鼠李糖基-2-木糖基葡萄糖苷) Petunidin-3-[6-(rhamnosyl)-2-(xylosyl) glucoside]	61.86±0.59	8.29±0.20
	总计 Total	543.5	47.22
黄酮醇 Flavonol	山奈酚-3-O-半乳糖苷 Kaempferol-3-O-galactoside	73.37±2.13	69.21±1.69
	山奈酚-7-O-葡萄糖苷 Kaempferol-7-O-glucoside	62.36±2.61	56.64±0.85
	槲皮素-3-O-葡萄糖苷 Quercetin-3-O-glucoside	21.45±0.32	12.81±0.24
	鼠李素-3-O-葡萄糖苷 Rhamnetin-3-O-Glucoside	40.01±0.51	22.95±0.32
	异鼠李素-3-O-葡萄糖苷 Isorhamnetin-3-O-Glucoside	37.02±1.20	24.14±0.67
	苜蓿素-4'-甲基醚-3'-O-葡萄糖苷 Tricin-4'-methylether-3'-O-glucoside	39.31±0.45	23.55±0.33
	杨梅素-3-O-葡萄糖苷 Myricetin-3-O-glucoside	3.29±0.08	145.22±3.74
	杨梅素-3-O-半乳糖苷 Myricetin-3-O-galactoside	4.04±0.12	58.65±2.52
	槲皮素-3-O-(6''-丙二酰)半乳糖苷 Quercetin-3-O-(6''-malonyl)galactoside	4.99±0.12	6.27±0.32
	鼠李素-3-O-芸香糖苷 Rhamnetin-3-O-Rutinoside	4.36±0.21	15.05±1.23
	3,5,7,4'-四羟基-8-甲氧基黄酮-3-O-葡萄糖苷-7-O-鼠李糖苷 Sexangularetin-3-O-glucoside-7-O-rhamnoside	4.48±0.09	14.39±0.41
	羟基山奈酚-3,6-O-二葡萄糖苷 6-Hydroxykaempferol-3,6-O-Diglucoside	4.89±0.25	1.21±0.06
	槲皮素-3-O-芸香糖苷-7-O-鼠李糖苷 Quercetin-3-O-rutinoside-7-O-rhamnoside	28.55±1.01	3.75±0.31
	槲皮素-7-O-芸香糖苷-4'-O-葡萄糖苷 Quercetin-7-O-rutinoside-4'-O-glucoside	5.04±0.06	0.02±0.01
	总计 Total	333.15	453.81

表2

表3

图3

参考文献 32

[1]	MOL J, CORNISH E, MASON J, KOES R. Novel coloured flowers. Current Opinion in Biotechnology, 1999,10(2):198-201. doi: 10.1016/S0958-1669(99)80035-4
[2]	林兵, 钟淮钦, 黄敏玲, 樊荣辉, 罗远华. ⁶⁰Co-γ射线辐射对荷兰鸢尾花色诱变效应的研究. 核农学报, 2019,33(4):633-639.
	LIN B, ZHONG H Q, HUANG M L, FAN R H, LUO Y H. The study of ⁶⁰Co-γ ray irradiation effects on flower color of Iris hollandica. Journal of Nuclear Agricultural Sciences, 2019,33(4):633-639. (in Chinese)
[3]	ZHOU C B, MEI X, RORHENBERG D O N, YANG Z B, ZHANG W T, WAN S H, YANG H J, ZHANG L Y. Metabolome and transcriptome analysis reveals putative genes involved in anthocyanin accumulation and coloration in white and pink tea (Camellia sinensis) flower. Molecules, 2020,25:190. doi: 10.3390/molecules25010190
[4]	LOU Q, LIU Y L, QI Y Y, JIAO S Z, TIAN F F, JIANG L, WANG Y J. Transcriptome sequencing and metabolite analysis reveals the role of delphinidin metabolism in flower colour in grape hyacinth. Journal of Experimental Botany, 2014,65(12):3157-3164. doi: 10.1093/jxb/eru168
[5]	WU Q, WU J, Li S S, ZHANG H J, FENG C Y, YIN D D, WU R Y, WANG L S. Transcriptome sequencing and metabolite analysis for revealing the blue flower formation in waterlily. BMC Genomics, 2016,17:897. doi: 10.1186/s12864-016-3226-9
[6]	TANAKA Y, BRUGLIERA F, KALC G, SENIOR M, DYSON B, NAKAMURA N, KATSUMOTO Y, CHANDLER S. Flower color modification by engineering of the flavonoid biosynthetic pathway: Practical perspectives. Bioscience Biotechnology and Biochemistry, 2010,74(9):1760-1769. doi: 10.1271/bbb.100358
[7]	TANAKA Y, SASAKI N, OHMIYA A. Biosynthesis of plant pigments: Anthocyanins, betalains and carotenoids. The Plant Journal, 2008,54(4):733-749. doi: 10.1111/j.1365-313X.2008.03447.x
[8]	NAKATSUKA T, MISHIBA K, KUBOTA A, ABE Y, YAMAMURA S, NAKAMURA N, TANAKA Y, NISHIHARA M. Genetic engineering of novel flower colour by suppression of anthocyanin modification genes in gentian. Journal of Plant Physiology, 2010,167(3):231-237. doi: 10.1016/j.jplph.2009.08.007
[9]	WU X X, GONG Q H, NI X P, ZHOU Y, GAO Z H. UFGT: the key enzyme associated with the petals variegation in Japanese Apricot. Frontiers in Plant Science, 2017,8:108.
[10]	MIZUNO T, UEHARA A, MIZUTA D, YABUYA T, IWASHINA T. Contribution of anthocyanin-flavone copigmentation to grayed violet flower color of Dutch iris cultivar ‘Tiger’s Eye’ under the presence of carotenoids. Scientia Horticulturae, 2015,186(21):201-206. doi: 10.1016/j.scienta.2015.01.037
[11]	YOSHIHARA N, IMAYAMA T, FUKUCHI-MIZUTANI M, OKUHARA H, TANAKA Y, INO I, YABUYO T. cDNA cloning and characterization of UDP-glucose: Anthocyanidin 3-O-glucosyltransferase in Iris hollandica. Plant Science, 2005,169(3):496-501. doi: 10.1016/j.plantsci.2005.04.007
[12]	IMAYAMA T, YOSHIHARA N, FUKUCHIMIZUTANI M, TANAKA Y, INO I, YABUYA T. Isolation and characterization of a cDNA clone of UDP-glucose: anthocyanin 5-O-glucosyltransferase in Iris hollandica. Plant Science, 2004,167(6):1243-1248. doi: 10.1016/j.plantsci.2004.06.020
[13]	YOSHIHARA N, FUKUCHI-MIZUTANI M, OKUHARA H, TANAKA Y, YABUYA T. Molecular cloning and characterization of O-methyltransferases from the flower buds of Iris hollandica. Journal of Plant Physiology, 2008,165(4):415-422. doi: 10.1016/j.jplph.2006.12.002
[14]	TRAPNELLl C, WILLIAMS B A, PERTEA G, MORTAZAVI A, KWAN G, BAREN M J, SALZBERG S L, WOLD B J, PACHTER L. Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation. Nature Biotechnology, 2010,28:511-515. doi: 10.1038/nbt.1621
[15]	ANDERS S, HUBER W. Differential expression analysis for sequence count data. Genome Biology, 2010,11:R106. doi: 10.1186/gb-2010-11-10-r106
[16]	樊荣辉, 黄敏玲. 花青素苷调控研究进展. 中国细胞生物学学报, 2013,35(5):741-746.
	FAN R H, HUANG M L. Progress in regulation of anthocyanins. Chinese Journal of Cell Biology, 2013,35(5):741-746. (in Chinese)
[17]	ZHENG C, MA J Q, CHEN J D, MA C L, CHEN W, YAO M Z, CHEN L. Gene coexpression networks reveal key drivers of flavonoid variation in eleven tea cultivars (Camellia sinensis). Journal of Agricultural and Food Chemistry, 2019,67(35):9967-9978. doi: 10.1021/acs.jafc.9b04422
[18]	CASTELLARIN S D, GASPERO G D. Transcriptional control of anthocyanin biosynthetic genes in extreme phenotypes for berry pigmentation of naturally occurring grapevines. BMC Plant Biology, 2007,7:46. doi: 10.1186/1471-2229-7-46
[19]	WANG K L, BOLITHO K, GRAFTON K, KORTSTEE A, KARUNAIRETNAM S, MCGHIE T K, ESPLEY R V, HELLENS R P, ALLAN A C. An R2R3 MYB transcription factor associated with regulation of the anthocyanin biosynthetic pathway in Rosaceae. BMC Plant Biology, 2010,10:50. doi: 10.1186/1471-2229-10-50
[20]	FENG C, CHEN M, XU C J, BAI L, YIN X R, LI X, ALLAN A C, FERGUSON I B, CHEN K S. Transcriptomic analysis of Chinese bayberry (Myrica rubra) fruit development and ripening using RNA-Seq. BMC Genomics, 2012,13:19. doi: 10.1186/1471-2164-13-19
[21]	YUAN Y, MA X H, SHI Y M, TANG D Q. Isolation and expression analysis of six putative structural genes involved in anthocyanin biosynthesis in Tulipa fosteriana. Scientia Horticulturae, 2013,153(4):93-102. doi: 10.1016/j.scienta.2013.02.008
[22]	HUANG Y, GOU J Q, JIA Z C, YANG L, SUN Y M, XIAO X Y, SONG F, LUO K M. Molecular cloning and characterization of two genes encoding dihydroflavonol-4-reductase from Populus trichocarpa. PLoS ONE, 2012,7(2):e30364. doi: 10.1371/journal.pone.0030364
[23]	LUO P, NING G G, WANG Z, SHEN Y X, JIN H N, LI P H, HUANG S S, ZHAO J, BAO M Z. Disequilibrium of flavonol synthase and dihydroflavonol-4-reductase expression associated tightly to white vs. red color flower formation in plants. Frontiers in Plant Science, 2015,6:1257
[24]	HAN Y P, VIMOLMANGKANG S, SORIA-GUERRA R E, KORBAN S S. Introduction of apple ANR genes into tobacco inhibits expression of both CHI and DFR genes in flowers, leading to loss of anthocyanin. Journal of Experimental Botany, 2012,63(7):2437-2447. doi: 10.1093/jxb/err415
[25]	SAITO R, FUKUTA N, OHMIYA A, ITOH Y, OZEKI Y, KUCHITSU K, NAKAYAMA M. Regulation of anthocyanin biosynthesis involved in the formation of marginal picotee petals in Petunia. Plant Science, 2006,170(4):828-834. doi: 10.1016/j.plantsci.2005.12.003
[26]	SPITZER B, ZVI M M B, OVADIS M, MARHEVKA E, BAEKAI O, EDELBAUM O, MARTON I, MASCI T, ALON M, MORIN S, ROGACHEV I, AHARONI A, VAINSTEIN A. Reverse genetics of floral scent: Application of tobacco rattle virus-based gene silencing in Petunia. Plant Physiology, 2007,145(4):1241-1250. doi: 10.1104/pp.107.105916
[27]	HEMLEBEN V, DRESSEL A, EPPING B, LUKACIN R, MARTENS S, AUSTIN M. Characterization and structural features of a chalcone synthase mutation in a white-flowering line of Matthiola incana R. Br. (Brassicaceae). Plant Molecular Biology, 2004,55(3):455-465. doi: 10.1007/s11103-004-1125-y
[28]	ZHANG Y Z, CHENG Y W, YA H Y, XU S Z, HAN J M. Transcriptome sequencing of purple petal spot region in tree peony reveals differentially expressed anthocyanin structural genes. Frontiers in Plant Science, 2015,6:964.
[29]	CLARK S T, VERWOERD W S. A systems approach to identifying correlated gene targets for the loss of colour pigmentation in plants. BMC Bioinformatics, 2011,12:343. doi: 10.1186/1471-2105-12-343
[30]	MA H W, ZHAO X M, YUAN Y J, ZENG A P. Decomposition of metabolic network into functional modules based on the global connectivity structure of reaction graph. Bioinformatics, 2004,20(12):1870-1876. doi: 10.1093/bioinformatics/bth167
[31]	BOGS J, JAFFE F W, TAKOS A M, WALKER A R, ROBINSON S P. The grapevine transcription factor VvMYBPA1 regulates proanthocyanidin synthesis during fruit development. Plant Physiology, 2007,143:1347-1361. doi: 10.1104/pp.106.093203
[32]	HAN Y P, VIMOLMANGKANG S, SORIA-GUERRA R E, KORBAN S S. Introduction of apple ANR genes into tobacco inhibits expression of both CHI and DFR genes in flowers, leading to loss of anthocyanin. Journal of Experimental Botany, 2012,63(7):2437-2447. doi: 10.1093/jxb/err415

基因 Gene	正向引物（5′-3′）Forward primer sequence	反向引物（5′-3′）Reverse primer sequence
IhDFR1	GAGGTGGTCGCAGGATGCACT	CTCCGTCTGCTGATGTTCTTT
IhDFR2	AAGGCGGTCGCAGGATGCACC	TGATGAAAGGACCGACGACT
IhFLS1	GTTGGAGTGATGGACGGGATG	GGGACAGGGAGGGTAGTAGTTGA
Ihactin	ACGGAAATTGTATGTGGGT	CAGATGCGAAAGATGTGAG

样品 Sample	Clean read 数量 Number of clean reads	GC含量 GC content (%)	%≥Q30
展翅 Zhanchi	45850456	50.29	87.76%
玉妃 Yufei	48903721	50.30	87.08%