Cloning and Functional Analysis of AP2/ERF Transcription Factors RcERF4 and RcRAP2-12 in Rose

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

Abstract

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

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.

Figures/Tables 12

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

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

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

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

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

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

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

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

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

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)

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

Fig. 10 Bimolecular fluorescence complementation

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