不同花色绿绒蒿DFR基因克隆及表达分析

doi:10.7668/hbnxb.20194661

摘要/Abstract

摘要：

二氢黄酮4-还原酶(DFR)是花青素合成通路中的关键酶,其表达与花青素积累紧密相关,影响花朵呈色。为了探究DFR基因与绿绒蒿花色形成的关系,以红色的红花绿绒蒿和黄色的全缘叶绿绒蒿为试验材料,使用RT-PCR技术成功从中克隆出DFR基因,并对其进行生物信息学及RT-qPCR分析。结果显示,2种绿绒蒿DFR基因的cDNA全长为1 131,1 125 bp,分别编码376,374个氨基酸,命名为MpDFR、 MiDFR。生物信息学分析发现,2种蛋白均属于亲水性蛋白,且具有DFR特有的NADPH结合结构域和底物特异性结合结构域。系统进化树表明,MpDFR、MiDFR蛋白与鸦片罂粟、沉水樟、三枝九叶草、澳洲坚果、蒂罗花的亲缘关系最近。Motif分析发现,DFR蛋白基序在不同植物中均较为保守。RT-qPCR结果显示,MpDFR、MiDFR在不同组织中均有表达,具有组织特异性。花蕾期中MpDFR、MiDFR基因表达量最高,且显著高于其他2个时期。另外,红花绿绒蒿中DFR表达量均高于全缘叶绿绒蒿。结合西南林业大学屈燕课题组研究发现,这2种绿绒蒿中花青素类化合物的积累模式与DFR基因的表达模式一致,所以,研究花青素生物合成途径中的关键结构基因DFR对于了解该途径以及通过基因工程手段改良植物花色均十分重要。

关键词: 红花绿绒蒿, 全缘叶绿绒蒿, 二氢黄酮4-还原酶, 基因克隆, 表达分析

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

王海菊, 陈晓涓, 李拓键, 罗军, 屈燕. 不同花色绿绒蒿DFR基因克隆及表达分析[J]. 华北农学报, 2024, 39(3): 88-95. doi: 10.7668/hbnxb.20194661.

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.

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

图/表 13

图1 绿绒蒿图片 A.红花绿绒蒿;B.全缘叶绿绒蒿。

Fig.1 Meconopsis images A.M.punicea;B.M.integrifolia.

表1 绿绒蒿花瓣信息

Tab.1 Petal information of Meconopsis

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

表2 生物信息学分析软件及网站

Tab.2 Bioinformatics analysis software and website

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

表3 目的基因引物信息

Tab.3 Target gene primer information

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

图2 DFR基因PCR扩增电泳条带

Fig.2 PCR amplification electrophoresis band of DFR gene

表4 MpDFR、MiDFR蛋白理化性质

Tab.4 Physicochemical properties of MpDFR and MiDFR proteins

蛋白名称 Protein name	分子式 Molecular formula	总原子数 Total number of atoms	分子质量/ku Molecular weight	等电点 Isoelectric point	脂肪系数 Aliphatic index	负电荷残基数 Negative charge residue number	正电荷残基数 Positive charge residue number	亲水性平均值 GRAVY	不稳定指数 Instability index
MpDFR	C₁₈₈₂H₂₉₆₉N₄₈₃O₅₅₈S₂₂	5 914	42.00	6.04	81.04	48	44	-0.261	38.93
MiDFR	C₁₈₉₆H₂₉₇₅N₄₈₅O₅₆₅S₂₂	5 943	42.31	5.40	82.18	50	40	-0.225	43.39

图3 MpDFR、MiDFR蛋白的磷酸化位点分析 A.MpDFR蛋白的磷酸化位点预测;B.MiDFR蛋白的磷酸化位点预测。

Fig.3 Analysis of phosphorylation sites of MpDFR and MiDFR proteins A.Prediction of phosphorylation sites of MpDFR protein;B.Prediction of phosphorylation sites of MiDFR protein.

图4 2种花色绿绒蒿DFR蛋白保守结构的预测 A.MpDFR蛋白的保守结构域预测;B.MiDFR蛋白的保守结构域预测。

Fig.4 Prediction of the conservative structure of DFR protein in two flower colors of Meconopsis A.Prediction of conservative domain of MpDFR protein;B.Prediction of conservative domain of MiDFR protein.

图5 MpDFR、MiDFR蛋白质二级结构预测 A.MpDFR蛋白的二级结构预测;B.MiDFR蛋白的二级结构预测;蓝色表示α-螺旋;紫色表示无规则卷曲;红色表示延伸链;绿色表示β-折叠。

Fig.5 Prediction of MpDFR and MiDFR protein secondary structure A.Prediction of the secondary structure of MpDFR protein;B.Prediction of the secondary structure of MiDFR protein;Blue represents alpha helix;Purple represents random coil;Red represents extended strand;Green represents beta turn.

图6 MpDFR、MiDFR蛋白质三级结构预测 A.MpDFR蛋白三级结构预测;B.MiDFR蛋白三级结构预测。

Fig.6 Prediction of MpDFR and MiDFR protein tertiary structure A.Prediction of the tertiary structure of MpDFR protein;B.Prediction of the tertiary structure of MiDFR protein.

图7 MpDFR、MiDFR蛋白与其他植物DFR蛋白编码的氨基酸序列的同源性比对红色标识为NADPH结合位点;蓝色标识为底物特异性结合结构域。

Fig.7 Homology comparison of amino acid sequences encoded by MpDFR and MiDFR proteins with other plant DFR proteins The red mark indicates the NADPH binding site;the blue mark represents the substrate specific binding domain.

图8 MpDFR、MiDFR蛋白与其他植物DFR蛋白系统进化树的构建及模体分析

Fig.8 Construction of phylogenetic trees and motif analysis of MpDFR,MiDFR proteins and other plant DFRs proteins

图9 绿绒蒿DFR基因在不同组织的表达不同小写字母代表差异显著(P<0.05)。

Fig.9 Expression of DFR gene in Meconopsis at different tissue Different lowercase letters indicate significant differences(P<0.05).

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