Cloning and Expression Analysis of DFR Gene from Meconopsis with Different Colors

doi:10.7668/hbnxb.20194661

Abstract

Abstract:

Dihydroflavone 4-reductase(DFR)is a key enzyme in the anthocyanidin synthesis pathway.Its expression is closely related to the accumulation of anthocyanidin,which affects flower color.In order to explore the relationship between DFR gene and the formation of flower color in Meconopsis,red-flowered M.punicea and yellow-flowered M.integrifolia were selected as experimental materials.The DFR gene was successfully cloned from them using RT-PCR technology,and bioinformatics and RT-qPCR analysis were performed on them.The results showed that the cDNA full-length of the two DFR genes in Meconopsis was 1 131,1 125 bp,respectively,encoding 376 and 374 amino acids,named MpDFR and MiDFR. Bioinformatics analysis revealed that both proteins belonged to hydrophilic proteins and possessed the unique NADPH-binding domain and substrate-specific binding domain of DFR.The phylogenetic tree indicated that MpDFR and MiDFR proteins had the closest genetic relationships with Papaver somniferum,Cinnamomum micranthum,Epimedium sagittatum, Macadamia integrifolia and Telopea speciosissima. Motif analysis found that the DFR protein motif is relatively conserved in different plants.The RT-qPCR results showed that MpDFR and MiDFR were expressed in different tissues with tissue specificity.During the bud stage,the expression levels of MpDFR and MiDFR genes were the highest and significantly higher than the other two stages.In addition,the expression level of DFR in M.punicea is higher than that in M.integrifolia.Based on the research findings of Quyan's research team at Southwest Forestry University,it was found that the accumulation pattern of anthocyanin-like compounds in these two species of Meconopsis was consistent with the expression pattern of the DFR gene.Therefore,studying the function of DFR genes is crucial for gaining a deeper understanding of anthocyanin biosynthesis pathways and improving plant flower color through genetic engineering methods.

Key words: Meconopsis punicea, Meconopsis integrifolia, Dihydroflavone 4-reductase, Gene cloning, Expression analysis

WANG Haiju, CHEN Xiaojuan, LI Tuojian, LUO Jun, QU Yan. Cloning and Expression Analysis of DFR Gene from Meconopsis with Different Colors[J]. Acta Agriculturae Boreali-Sinica, 2024, 39(3): 88-95. doi: 10.7668/hbnxb.20194661.

/ / Recommend / Download Citations

URL: http://www.hbnxb.net/EN/10.7668/hbnxb.20194661

http://www.hbnxb.net/EN/Y2024/V39/I3/88

Figures/Tables 13

References 31

[1]	Ni J, Ruan R J, Wang L J, Jiang Z F, Gu X J, Chen L S, Xu M J. Functional and correlation analyses of dihydroflavonol-4-reductase genes indicate their roles in regulating anthocyanin changes in Ginkgo biloba[J]. Industrial Crops and Products, 2020, 152:112546.doi:10.1016/j.indcrop.2020.112546.
[2]	Tsuda T, Horio F, Uchida K, Aoki H, Osawa T. Dietary cyanidin 3-O-beta-D-glucoside-rich purple corn color prevents obesity and ameliorates hyperglycemia in mice[J]. The Journal of Nutrition, 2003, 133(7):2125-2130.doi:10.1093/jn/133.7.2125.
[3]	Xie S, Zhao T, Zhang Z W, Meng J F. Reduction of dihydrokaempferol by Vitis vinfera dihydroflavonol 4-reductase to produce orange pelargonidin-type anthocyanins[J]. Journal of Agricultural and Food Chemistry, 2018, 66(13):3524-3532.doi:10.1021/acs.jafc.7b05766.
[4]	Tanaka Y, Brugliera F. Flower colour and cytochromes P450[J]. Philosophical Transactions of the Royal Society of London Series B, Biological Sciences, 2013, 368(1612):20120432.doi:10.1098/rstb.2012.0432.
[5]	刘静. 四季秋海棠花色苷相关基因的克隆及功能验证[D]. 郑州: 河南农业大学, 2022.doi:10.27117/d.cnki.ghenu.2022.000314.
	Liu J. Cloning and functional verification of anthocyanin-related genes in Begonia semperflorens[D]. Zhengzhou: Henan Agricultural University, 2022.
[6]	O'Reilly C, Shepherd N S, Pereira A, Schwarz-Sommer Z, Bertram I, Robertson D S, Peterson P A, Saedler H. Molecular cloning of the a1 locus of Zea mays using the transposable elements En and Mu1[J]. The EMBO Journal, 1985, 4(4):877-882.doi:10.1002/j.1460-2075.1985.tb03713.x.
[7]	Liu H L, Lou Q, Ma J R, Su B B, Gao Z Z, Liu Y L. Cloning and functional characterization of dihydroflavonol 4-reductase gene involved in anthocyanidin biosynthesis of grape hyacinth[J]. International Journal of Molecular Sciences, 2019, 20(19):4743.doi:10.3390/ijms20194743.
[8]	蒋宝鑫, 汪庆昊, 杨国霞, 贾永红, 谢晓鸿, 吴月燕. 比利时杜鹃花RhDFR基因克隆及分析[J]. 西北植物学报, 2023, 43(1):10-20.doi:10.7606/j.issn.1000-4025.2023.01.0010.
	Jiang B X, Wang Q H, Yang G X, Jia Y H, Xie X H, Wu Y Y. Cloning and analysis of RhDFR gene in Rhododendron hybridum hort[J]. Acta Botanica Boreali-Occidentalia Sinica, 2023, 43(1):10-20.
[9]	Qian X Z, Zheng W Y, Hu J, Ma J X, Sun M Y, Li Y, Liu N, Chen T H, Wang M Q, Wang L, Hou X Z, Cai Q G, Ye Z S, Zhang F G, Zhu Z H. Identification and expression analysis of DFR gene family in Brassica napus L.[J]. Plants, 2023, 12(13):2583.doi:10.3390/plants12132583.
[10]	Qin S J, Liu Y T, Cui B Q, Cheng J L, Liu S Y, Liu H Z. Isolation and functional diversification of dihydroflavonol 4-Reductase gene HvDFR from Hosta ventricosa indicate its role in driving anthocyanin accumulation[J]. Plant Signaling & Behavior, 2022, 17(1):2010389.doi:10.1080/15592324.2021.2010389.
[11]	郑好, 张芬, 简越, 黄文莉, 梁莎, 江敏, 袁巧, 汪俏梅, 孙勃. 芥蓝二氢黄酮醇4-还原酶基因BoaDFR的克隆与功能鉴定[J]. 园艺学报, 2021, 48(1):73-82.doi:10.16420/j.issn.0513-353x.2020-0378.
	Zheng H, Zhang F, Jian Y, Huang W L, Liang S, Jiang M, Yuan Q, Wang Q M, Sun B. Cloning and function identification of dihydroflavonol 4-reductase gene BoaDFR in Chinese kale[J]. Acta Horticulturae Sinica, 2021, 48(1):73-82.
[12]	Helariutta Y, Elomaa P, Kotilainen M, Seppänen P, Teeri T H. Cloning of cDNA coding for dihydroflavonol-4-reductase(DFR)and characterization of dfr expression in the corollas of Gerbera hybrida var.Regina(Compositae)[J]. Plant Molecular Biology, 1993, 22(2):183-193.doi:10.1007/BF00014927. pmid: 8507822
[13]	吴征镒. 西藏植物志-第二卷[M]. 北京: 科学出版社,1985.
	Wu Z Y. Flora of Tibet[M]. Beijing: Science Press,1985.
[14]	Li X X, Tan W, Sun J Q, Du J H, Zheng C G, Tian X X, Zheng M, Xiang B B, Wang Y. Comparison of four complete chloroplast genomes of medicinal and ornamental Meconopsis species:genome organization and species discrimination[J]. Scientific Reports, 2019, 9(1):10567.doi:10.1038/s41598-019-47008-8.
[15]	吴征镒, 庄璇. 绿绒蒿属分类系统的研究[J]. 云南植物研究, 1980, 2(4):371-381.
	Wu Z Y, Zhuang X. Study on the taxonomic system of Meconopsis[J]. Acta Botanica Yunnan, 1980, 2(4):371-381.
[16]	王海菊, 李拓键, 罗军, 屈燕. 三种不同花色绿绒蒿FLS基因克隆与表达分析[J]. 植物生理学报, 2023, 59(11):2063-2073.doi:10.13592/j.cnki.ppj.100653.
	Wang H J, Li T J, Luo J, Qu Y. Cloning and expression analysis of FLS gene in three different color Meconopsis species[J]. Plant Physiology Journal, 2023, 59(11):2063-2073.
[17]	鲁丹丹, 谭政委, 李磊, 余永亮, 许兰杰, 杨红旗, 董薇, 梁慧珍. 红花花青素还原酶基因ANR的克隆及表达分析[J]. 核农学报, 2022, 36(3):517-526.doi:10.11869/j.issn.100-8551.2022.03.0517.
	Lu D D, Tan Z W, Li L, Yu Y L, Xu L J, Yang H Q, Dong W, Liang H Z. Cloning and expression analysis of anthocyanidin reductase gene ANR in Carthamus tinctorius L.[J]. Journal of Nuclear Agricultural Sciences, 2022, 36(3):517-526.
[18]	陈丽琦, 严朋飞, 贾维嘉, 区智, 屈燕. 威氏绿绒蒿(Meconopsis wilsonii)花色相关基因MwF3H的克隆及表达分析[J]. 基因组学与应用生物学, 2022, 41(4):854-861.doi:10.13417/j.gab.041.000854.
	Chen L Q, Yan P F, Jia W J, Ou Z, Qu Y. Cloning and expression analysis of flower color related gene MwF3H from Meconopsis wilsonii[J]. Genomics and Applied Biology, 2022, 41(4):854-861.
[19]	Tian J, Han Z Y, Zhang J, Hu Y J, Song T T, Yao Y C. The balance of expression of dihydroflavonol 4-reductase and flavonol synthase regulates flavonoid biosynthesis and red foliage coloration in crabapples[J]. Scientific Reports, 2015, 5:12228.doi:10.1038/srep12228. pmid: 26192267
[20]	Nakatsuka A, Izumi Y, Yamagishi M. Spatial and temporal expression of chalcone synthase and dihydroflavonol 4-reductase genes in the Asiatic hybrid lily[J]. Plant Science, 2003, 165(4):759-767.doi:10.1016/s0168-9452(03)00254-1.
[21]	Watanabe K, Kobayashi A, Endo M, Sage-Ono K, Toki S, Ono M. CRISPR/Cas9-mediated mutagenesis of the dihydroflavonol-4-reductase-B(DFR-B)locus in the Japanese morning glory Ipomoea(Pharbitis)nil[J]. Scientific Reports, 2017, 7(1):10028.doi:10.1038/s41598-017-10715-1. pmid: 28855641
[22]	严朋飞, 张莹欣, 贾维嘉, 刘建, 郭小雪, 江元祺, 屈燕. 威氏绿绒蒿二氢黄酮醇4-还原酶DFR基因克隆与表达分析[J]. 分子植物育种, 2021, 19(24):8064-8070.doi:10.13271/j.mpb.019.008064.
	Yan P F, Zhang Y X, Jia W J, Liu J, Guo X X, Jiang Y Q, Qu Y. Cloning and expression analysis of dihydroflavonol 4-reductase gene from Meconopsis wilsonii[J]. Molecular Plant Breeding, 2021, 19(24):8064-8070.
[23]	Luo X N, Sun D Y, Wang S, Luo S, Fu Y Q, Niu L X, Shi Q Q, Zhang Y L. Integrating full-length transcriptomics and metabolomics reveals the regulatory mechanisms underlying yellow pigmentation in tree peony(Paeonia suffruticosa Andr.)flowers[J]. Horticulture Research, 2021, 8(1):235.doi:10.1038/s41438-021-00666-0.
[24]	Davies K M, Schwinn K E, Deroles S C, Manson D G, Lewis D H, Bloor S J, Bradley J M. Enhancing anthocyanin production by altering competition for substrate between flavonol synthase and dihydroflavonol 4-reductase[J]. Euphytica, 2003, 131(3):259-268.doi:10.1023/A:1024018729349.
[25]	Tian J, Chen M C, Zhang J, Li K T, Song T T, Zhang X, Yao Y C. Characteristics of dihydroflavonol 4-reductase gene promoters from different leaf colored Malus crabapple cultivars[J]. Horticulture Research, 2017, 4:17070.doi:10.1038/hortres.2017.70. pmid: 29263792
[26]	刘红利. 蓝色花葡萄风信子关键基因DFR和FLS调控花青苷积累的功能研究[D]. 杨凌: 西北农林科技大学, 2019:85-86.doi:10.27409/d.cnki.gxbnu.2019.000032.
	Liu H L. Characterization of DFR and FLS genes regulating of the anthocyanin accumulation in blue grape hyacinth[D]. Yangling: Northwest A&F University, 2019:85-86.
[27]	Qu Y, Ou Z, Wang S. Coloration differences in three meconopsis species: M.punicea, M.integrifolia and M.wilsonii[J]. South African Journal of Botany, 2022, 150:171-177.doi:10.1016/j.sajb.2022.07.016.
[28]	于婷婷, 倪秀珍, 高立宏, 韩国军, 朱长甫, 盛彦敏. 高等植物二氢黄酮醇4-还原酶基因研究进展[J]. 植物研究, 2018, 38(4):632-640.doi:10.7525/j.issn.1673-5102.2018.04.020.
	Yu T T, Ni X Z, Gao L H, Han G J, Zhu C F, Sheng Y M. Advances in study of dihydroflavonol 4-reductase(DFR)genes of higher plants[J]. Bulletin of Botanical Research, 2018, 38(4):632-640.
[29]	Johnson E T, Ryu S, Yi H, Shin B, Cheong H, Choi G. Alteration of a single amino acid changes the substrate specificity of dihydroflavonol 4-reductase[J]. The Plant Journal, 2001, 25(3):325-333.doi:10.1046/j.1365-313x.2001.00962.x.
[30]	Meyer P, Heidmann I, Forkmann G, Saedler H. A new petunia flower colour generated by transformation of a mutant with a maize gene[J]. Nature, 1987, 330(6149):677-678.doi:10.1038/330677a0.
[31]	Katsumoto Y, Fukuchi-Mizutani M, Fukui Y, Brugliera F, Holton T A, Karan M, Nakamura N, Yonekura-Sakakibara K, Togami J, Pigeaire A, Tao G Q, Nehra N S, Lu C Y, Dyson B K, Tsuda S, Ashikari T, Kusumi T, Mason J G, Tanaka Y. Engineering of the rose flavonoid biosynthetic pathway successfully generated blue-hued flowers accumulating delphinidin[J]. Plant & Cell Physiology, 2007, 48(11):1589-1600.doi:10.1093/pcp/pcm131.

试验材料 Materials	比色卡卡号 RHSCC code	采集地 Locality
红花绿绒蒿 M.punicea	RED GROUP 48B 红色系	四川省阿坝州巴郎山 102°54'29.58″E,30°53'45.94″N
全缘叶绿绒蒿 M.integrifolia	GREEN-YELLOW GROUP 1C 黄绿色系	云南省丽江市玉龙雪山 100°10'56″E,27°0'10″N

试验材料 Materials	比色卡卡号 RHSCC code	采集地 Locality
红花绿绒蒿 M.punicea	RED GROUP 48B 红色系	四川省阿坝州巴郎山 102°54'29.58″E,30°53'45.94″N
全缘叶绿绒蒿 M.integrifolia	GREEN-YELLOW GROUP 1C 黄绿色系	云南省丽江市玉龙雪山 100°10'56″E,27°0'10″N

软件及网站 Software and website	用途 Application
Expasy (https://web.expasy.org/protparam/)	蛋白质理化特性进行
NetPhos (https://services.healthtech.dtu.dk/services/NetPhos-3.1/)	蛋白磷酸化位点预测
NCBI CD-Search (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi)	保守结构域预测
SOPMA (https://npsa-pbil.ibcp.fr/cgi-bin/npsaautomat.pl?page=npsasopma.html)	蛋白质二级结构预测
SWISS-MODEL (https://swissmodel.expasy.org/interactive/)	蛋白质三级结构预测
MEME (https://meme-suite.org/meme/tools/meme)	Motif分析
DNAMAN 6.0	氨基酸同源序列比对
MEGA 7.0	系统进化树的构建

软件及网站 Software and website	用途 Application
Expasy (https://web.expasy.org/protparam/)	蛋白质理化特性进行
NetPhos (https://services.healthtech.dtu.dk/services/NetPhos-3.1/)	蛋白磷酸化位点预测
NCBI CD-Search (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi)	保守结构域预测
SOPMA (https://npsa-pbil.ibcp.fr/cgi-bin/npsaautomat.pl?page=npsasopma.html)	蛋白质二级结构预测
SWISS-MODEL (https://swissmodel.expasy.org/interactive/)	蛋白质三级结构预测
MEME (https://meme-suite.org/meme/tools/meme)	Motif分析
DNAMAN 6.0	氨基酸同源序列比对
MEGA 7.0	系统进化树的构建

引物名称 Primer name	核苷酸序列(5'—3') Nucleotide sequence(5'—3')	功能 Function
MpDFR-F	ATGGCTTCTATTGGCGTTAGTG	序列扩增
MpDFR-R	TTATGCAAAAACAGTAGACTTTTCTTGCG	序列扩增
MiDFR-F	ATGGGTTCTATTGGCGTTAGTGATGAAG	序列扩增
MiDFR-R	TTATGCAGTAGACTTTTCTTGCGGTGG	序列扩增
GAPDH-F	TTCCGTGTTCCTACCGTTGA	RT-qPCR
GAPDH-R	TCCCTTCATCTTACCCTCGG	RT-qPCR
qMpDFR-F	AGGCGGTGTATGACGAATCC	RT-qPCR
qMpDFR-R	AGCTTTCTCGGCCAGTGTTT	RT-qPCR
qMiDFR-F	AGATGAAGGGCCCTGTTGTG	RT-qPCR
qMiDFR-R	CAGATTTAGTGGGGTCGCGT	RT-qPCR

Please choose a citation manager

Content to export