月季AP2/ERF转录因子RcERF4和RcRAP2-12的克隆及功能分析

doi:10.13560/j.cnki.biotech.bull.1985.2025-0598

摘要/Abstract

摘要：

目的 AP2/ERF（APETALA2/ethylene-responsive factor）转录因子家族在调控植物生长发育、类黄酮合成和响应逆境胁迫等方面发挥重要作用。克隆RcERF4和RcRAP2-12基因并分析其功能，为揭示AP2/ERF转录因子调控月季花色中的作用奠定基础。方法首先，通过RT-PCR技术从月季‘夏洛特夫人’中克隆RcERF4和RcRAP2-12，并进行生物信息学、表达模式、亚细胞定位的分析及检测；进一步，采用农杆菌介导的叶盘转化法创制转RcERF4和RcRAP2-12基因烟草阳性植株，观察转基因烟草表型；最后，利用酵母双杂交和双分子荧光互补试验验证RcERF4和RcRAP2-12蛋白之间的相互作用关系。结果 RcERF4的ORF（open reading frame）区为795 bp，编码264个氨基酸，RcRAP2-12的ORF区为1 179 bp，编码392个氨基酸；RcERF4和RcRAP2-12蛋白均含有1个AP2结构域，属于ERF亚族，与玫瑰、野草莓进化关系较近，蛋白同源性较高；RcERF4和RcRAP2-12主要在月季花瓣、雄蕊及子房中表达，属于花特异性表达基因，RcERF4在‘夏洛特夫人’花发育初期表达量较高，RcRAP2-12在整个花发育时期呈现出上调表达趋势；RcERF4和RcRAP2-12蛋白均定位于细胞核；在烟草中过量表达RcRAP2-12会提高烟草花冠中花青素含量和NtANS、NtUFGT、NtAN2的表达；RcERF4和RcRAP2-12存在相互作用关系。结论 RcERF4和RcRAP2-12为细胞核定位的ERF类转录因子，RcRAP2-12可能在月季花色形成与调控中具有潜在功能。

关键词: RcERF4, RcRAP2-12, 生物信息学分析, 表达模式, 亚细胞定位, 转基因, 蛋白互作, 花色

Abstract:

Objective The AP2/ERF transcription factor family plays crucial roles in regulating plant growth and development, flavonoid biosynthesis, and stress responses. Cloning the RcERF4 and RcRAP2-12 genes and analyzing their expression will lay the foundation for revealing the role of AP2/ERF transcription factors in regulating flower coloration in roses. Method First, RcERF4 and RcRAP2-12 were cloned from rose ‘Lady of Shalott’ via RT-PCR, followed by bioinformatic analysis, expression pattern profiling, and subcellular localization assays. Subsequently, positive transgenic tobacco plants overexpressing RcERF4 and RcRAP2-12 were generated via Agrobacterium-mediated leaf disk transformation, with transgenic phenotypes being systematically observed. Finally, the protein-protein interaction between RcERF4 and RcRAP2-12 was investigated through yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays. Result The open reading frame (ORF) of RcERF4 was 795 bp, encoding a protein of 264 amino acids, while the ORF of RcRAP2-12 was 1 179 bp, encoding a protein of 392 amino acids. Both RcERF4 and RcRAP2-12 proteins contained a single AP2 domain and belonged to the ERF subfamily. Phylogenetic analysis revealed a closer evolutionary relationship and higher protein homology with species such as Rosa rugosa and Fragaria vesca subsp. vesca. RcERF4 and RcRAP2-12 were primarily expressed in rose petals, stamens, and ovaries. RcERF4 had higher expressions during the early stages of flower development, while RcRAP2-12 showed an up-regulated expression trend throughout the entire petal coloring period. Subcellular localization demonstrated that both RcERF4 and RcRAP2-12 proteins localized to the nucleus. The overexpression of RcRAP2-12 in tobacco enhanced anthocyanin content in the corolla and upregulates the expression of NtANS, NtUFGT, and NtAN2. Furthermore, yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays confirmed that RcERF4 and RcRAP2-12 owned an interaction relationship. Conclusion RcERF4 and RcRAP2-12 are nuclear-localized ERF transcription factors, with RcRAP2-12 potentially playing a functional role in the formation and regulation of rose flower coloration.

Key words: RcERF4, RcRAP2-12, bioinformatics analysis, expression pattern, subcellular localization, genetic transformation, protein-protein interaction, flower coloration

杨娟, 冯慧, 吉乃喆, 孙丽萍, 王赟, 张佳楠, 赵世伟. 月季AP2/ERF转录因子RcERF4和RcRAP2-12的克隆及功能分析[J]. 生物技术通报, 2026, 42(1): 150-160.

YANG Juan, FENG Hui, JI Nai-zhe, SUN Li-ping, WANG Yun, ZHANG Jia-nan, ZHAO Shi-wei. Cloning and Functional Analysis of AP2/ERF Transcription Factors RcERF4 and RcRAP2-12 in Rose[J]. Biotechnology Bulletin, 2026, 42(1): 150-160.

图/表 12

图1 ‘夏洛特夫人’的不同花发育时期S1：萼片包裹紧实，花瓣未着色；S2：萼片张开，花瓣开始着色；S3：萼片完全张开，花瓣完全着色；S4：花瓣完全盛开。下同

Fig. 1 Different flower development stages of 'Lady of Shalott'S1: Sepals tightly wrapped, petals not colored. S2: Sepals open, petals begin to color. S3: Sepals fully open, petals fully colored. S4: Petals fully bloomed. The same below

表1 本研究所用引物

Table 1 Primers used in this study

用途 Application	引物名称Primer name	引物序列Primer sequence （5′‒3′）
基因克隆 Gene cloning	RcERF4-F1	ATGGCACCGAGAACCGAGA
	RcERF4-R1	TCAGGCGAGTTCCGCCGGA
	RcRAP2-12-F1	ATGTGCGGAGGTGCCATTAT
	RcRAP2-12-R1	GAAAACTCCCCCAGACATTG
实时荧光定量PCR Quantitative real-time PCR	RcERF4-F2	ACCACGGCTAAACCTAACGG
	RcERF4-R2	ATTCACATTCTCCGACGGCA
	RcRAP2-12-F2	CGACTTGGTCTGGCCTGATT
	RcRAP2-12-R2	TGCACTCCACGGATTTCACA
亚细胞定位和遗传转化烟草 Subcellular localization and genetic transformation of tobacco	RcERF4-F3	aagtccggagctagctctagaATGGCACCGAGAACCGAGA
	RcERF4-R3	gcccttgctcaccatggatccGGCGAGTTCCGCCGGAGG
	RcRAP2-12-F3	aagtccggagctagctctagaATGTGCGGAGGTGCCATTAT
	RcRAP2-12-R3	gcccttgctcaccatggatccGAAAACTCCCCCAGACATTGAA
酵母双杂交 Yeast two-hybrid	RcERF4-F4	gccatggaggccagtgaattcATGGCACCGAGAACCGAGA
	RcERF4-R4	cagctcgagctcgatggatccGGCGAGTTCCGCCGGAGG
	RcRAP2-12-F4	tcagaggaggacctgcatatgATGTGCGGAGGTGCCATTAT
	RcRAP2-12-R4	ctagttatgcggccgctgcagGAAAACTCCCCCAGACATTGAA
双分子荧光互补 Bimolecular fluorescence complementation	RcERF4-F5	tggcgcgccactagtggatccATGGCACCGAGAACCGAGA
	RcERF4-R5	ctccatcccgggagcggtaccGGCGAGTTCCGCCGGAGG
	RcRAP2-12-F5	tggcgcgccactagtggatccATGTGCGGAGGTGCCATTAT
	RcRAP2-12-R5	gtacatcccgggagcggtaccGAAAACTCCCCCAGACATTGAA

表2 生物信息学分析网址及用途

Table 2 Bioinformatics analysis websites and their applications

名称 Name	网址 Website	用途 Application
ExPASy-ProtParam	https：//web.expasy.org/protparam/	理化性质 Physicochemical properties
CDS	https：//www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi	结构域 Domain
TMHMM-2.0	https：//services.healthtech.dtu.dk/services/TMHMM-2.0/	跨膜结构 Transmembrane structure
SignalP-4.1	https：//services.healthtech.dtu.dk/services/SignalP-4.1/	信号肽 Signal peptide
NetGlycate-1.0	https：//services.healthtech.dtu.dk/services/NetGlycate-1.0/	糖基化位点 Glycosylation site
NetPhos-3.1	https：//services.healthtech.dtu.dk/services/NetPhos-3.1/	磷酸化位点 Phosphorylation site

图2 RcERF4和RcRAP2-12基因的ORF扩增M： DL2000 DNA marker

Fig. 2 ORF amplification of RcERF4 and RcRAP2-12 genes

图3 RcERF4和RcRAP2-12蛋白保守结构域

Fig. 3 Conserved domains of RcERF4 and RcRAP2-12 proteins

图4 RcERF4（A）和RcRAP2-12（B）氨基酸序列比对Aa：蕨麻，XP_050371303.1；Ej：枇杷，AKN10304.1；Fa：草莓，AZL19463.1；Fv：野草莓，XP_004293846.1；Mp：苹果，NP_001385262.1；Ms：欧洲野苹果，XP_050108098.1；Pp：桃，ALO81027.1；Ra：尖齿黑莓，KAK9936415.1；Rc：月季，XP_024158160.1；Rr：玫瑰，XP_062013106.1；Pa：欧洲甜樱桃，XP_021819942.1； Pb：白梨，XP_009338318.1；Ps：山樱花，APY26916.1。红色圆点表示WLG元件；红色方块表示目标蛋白；红色框表示第14位和第19位氨基酸；蓝色框表示AP2结构域

Fig. 4 Amino acid sequence alignment of RcERF4 (A) and RcRAP2-12 (B)Aa: Argentina anserina, XP_050371303.1. Ej: Eriobotrya japonica, AKN10304.1. Fa: Fragaria × ananassa, AZL19463.1. Fv: Fragaria vesca subsp. vesca, XP_004293846.1. Mp: Malus pumila, NP_001385262.1. Ms: Malus sylvestris, XP_050108098.1. Pp: Prunus persica, ALO81027.1. Ra: Rubus argutus, KAK9936415.1. Rc: Rosa chinensis, XP_024158160.1. Rr: Rosa rugosa, XP_062013106.1. Pa: Prunus avium, XP_021819942.1. Pb: Pyrus × bretschneideri, XP_009338318.1. Ps: Prunus serrulata, APY26916.1. The red dot indicates the WLG component. The red square indicates the target protein. The red box indicates the 14th and 19th amino acids. The blue box indicates the AP2 domain

图5 RcERF4 （A）和RcRAP2-12 （B）系统进化树

Fig. 5 Phylogenetic tree of RcERF4 (A) and RcRAP2-12 (B)

图6 RcERF4和RcRAP2-12在‘夏洛特夫人’不同组织部位和不同花发育时期的表达模式使用3次生物学重复；误差棒表示标准偏差；采用单因素方差进行统计分析，不同小写字母表示差异显著（P<0.05），下同

Fig. 6 Expression patterns of RcERF4 and RcRAP2-12 in different tissues and various floral developmental stages of 'Lady of Shalott'Three biological replicates were used. The error bars indicate standard deviation. Statistical analysis was performed using one-way ANOVA, different lower case letters indicate significant differences (P<0.05), the same below

图7 RcERF4 和RcRAP2-12 蛋白的亚细胞定位

Fig. 7 Subcellular localization of RcERF4 and RcRAP2-12proteins

图8 转基因烟草的鉴定及分析A：转基因烟草的PCR鉴定；B：转基因烟草中目的基因的qPCR检测；C：转基因烟草的表型分析；D：转RcRAP2-12基因烟草中花青素合成相关基因的表达；E：转基因烟草的花青素含量检测；OE-RcERF4：过表达RcERF4基因；OE-RcRAP2-12：过表达RcRAP2-12基因；使用3次生物学重复，误差棒表示标准偏差，星号（*）表示转基因与野生型株系之间存在显著差异（t-检验，*P<0.05，**P<0.01）

Fig. 8 Identification and analysis of transgenic tobaccoA: PCR identification of transgenic tobacco. B: qPCR detection of target genes in transgenic tobacco. C: Phenotypic analysis of transgenic tobacco. D: Expressions of anthocyanin biosynthesis-related genes in RcRAP2-12 transgenic tobacco. E: Detection of anthocyanin content in genetically modified tobacco. OE-RcERF4: Overexpression of RcERF4 gene. OE-RcRAP2-12: Overexpression of RcRAP2-12 gene. Three biological replicates were used. The error bars indicate standard deviation. Asterisks (*) indicate significant differences between transgenic lines and wild type lines (t-test, *P<0.05, **P<0.01)

图9 酵母双杂交A：pGBKT7-RAP2-12的毒性检测；B：pGBKT7-RAP2-12的自激活验证；C：pGBKT7-RAP2-12和pGADT7-RcERF4的互作验证

Fig. 9 Yeast two-hybrid verificationA: Toxicity testing of pGBKT7-RAP2-12. B: Self-activation verification of pGBKT7-RAP2-12. C: Interaction validation between pGBKT7-RAP2-12 and pGADT7-RcERF4

图10 双分子荧光互补

Fig. 10 Bimolecular fluorescence complementation

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