Functional Study of CjRAV1 from Camellia japonica in Regulating Flowering Delay

doi:10.13560/j.cnki.biotech.bull.1985.2025-0019

Abstract

Abstract:

Objective The study aims to investigate the functional roles and molecular mechanisms of the Camellia japonica related to ABI3 and VP1 (CjRAV1) gene in flowering time regulation, providing a theoretical foundation for molecular breeding of everblooming C. japonica. Method A comprehensive approach combining bioinformatics analysis, gene expression profiling, transgenic technology, and DAP-seq was employed to systematically examine the function and regulatory mechanisms of CjRAV1. Bioinformatics analysis was conducted to identify the gene structure, conserved domains, and phylogenetic relationships of CjRAV1. The expression patterns of CjRAV1 under exogenous hormone induction, in different tissues, and during various developmental stages of flower buds were analyzed using RT-qPCR technology. Transgenic Arabidopsis plants of overexpressing CjRAV1 were generated to observe phenotypic changes, particularly alterations in flowering time. Finally, DAP-seq technology was utilized to identify potential DNA binding sites and downstream regulatory genes of CjRAV1, elucidating its molecular regulatory network. Result Bioinformatics analysis showed that the CDS of the CjRAV1 was 1 101 bp, encoding 366 amino acids and possessing conserved AP2 and B3 domains. Systematic evolutionary analysis revealed that the CjRAV1 protein in C. japonica was most closely related to the CsRAV protein in Camellia sinensis, suggesting potential functional similarities between the two proteins. Subcellular localization analysis confirmed that the CjRAV1 transcription factor was localized in the nucleus, suggesting its potential direct involvement in transcriptional regulation. The expression pattern analysis showed that CjRAV1 had the highest expression in the leaves; during the maturation process of flower buds, the expression of CjRAV1 showed a gradually decreasing trend overall. The overexpression of CjRAV1 in transgenic Arabidopsis thaliana demonstrated the delayed flowering phenotype. A potential downstream regulatory gene CjERF of CjRAV1 was obtained by DAP-seq screening. Conclusion Overexpression of CjRAV1 leads to delayed flowering phenotype in transgenic Arabidopsis plants, and this function might be performed in collaboration with the potential regulatory gene CjERF.

Key words: Camellia japonica, RAV transcription factor, gene function, regulation of flowering period, transgenosis, DAP-seq, late flowering phenotype

GUO Tao, AI Li-jiao, ZOU Shi-hui, ZHOU Ling, LI Xue-mei. Functional Study of CjRAV1 from Camellia japonica in Regulating Flowering Delay[J]. Biotechnology Bulletin, 2025, 41(6): 208-217.

Figures/Tables 10

Table 1 Sequences of primers

名称 Primer name	序列 Primer sequence （5′‒3′）
RAV1-f	ATGGATGGTAGTTACATAGATGAGA
RAV1-r	TTACAGAGCCCCAATTATCCTTTGT
3302-R1-1	GAACACGGGGGACTCTTGACATGGATGGTAGTTACATAGA
3302-R1-2	TTTACCCTCAGATCTACCATCAGAGCCCCAATTATCCTTT
RAV1-RT-1	TAAAGGTTGGAGCCGATTTGTGA
RAV1-RT-2	ACCCGTTTCTCGTATTCCAGTCA
CjGAPDH-1	GGGAATCCTTGGTTACACTGAG
CjGAPDH-2	ACCCCATTCGTTGTCATACC
Atactin-1	ACCACTGTCCACTCTATCACTGC
Atactin-2	TGAGGGATGGCAACACTTTCCC
AtFT-1	ACCCTCACCTCCGAGAATATCTCCAT
AtFT-2	CTAAAGTCTTCTTCCTCCGCAGCCAC
AtSOC-1	AGGCATACTAAGGATCGAGTCAGCAC
AtSOC-2	GAAGAACAAGGTAACCCAATGAACAA
p3302-f	TGACGCACAATCCCACTATCCTT
p3302-r	CCGTCCAGCTCGACCAGGAT

Fig. 1 Phylogenetic tree and homology analysis of RAVs proteins from different speciesA: CDS sequence and encoded protein of CjRAV1. B: A phylogenetic tree of 15 RAVs proteins from different species (Cs: Camellia sinensis. Cl: Camellia lanceoleosa. Cj: Camellia japonica. At: Arabidopsis thaliana; Os: Oryza sativa). C: The sequence homology matrix of RAVs proteins in the indicative species

Fig. 2 Expression profiles analysis of CjRAV1*: indicates significant differences at P<0.05. The same below

Fig. 3 Schematic diagram of overexpression vector construction

Fig. 4 PCR identification of expression vector bacterial solutionM: Marker. -: Negative control. +: Positive control. 1: p3302-RAV1

Fig. 5 Positive identification of CjRAV1 transgenic Arabidopsis linesA: PCR identification of DNA for positive strains of CjRAV-overexpressed Arabidopsis. B: Relative expressions of CjRAV1 intransgenic Arabidopsis detected by RT-qPCR. M: Marker. -: Negative control. +: Positive control. 1-8: From p3302-RAV1-1 to p3302-RAV1-8 transgenic Arabidopsis lines

Fig. 6 Comparative analysis of flowering time between wild type and CjRAV1 transgenic Arabidopsis plantsA: Phenotypic comparison between wild-type and CjRAV1 transgenic Arabidopsis plants. B: Flowering time statistics of wild-type and CjRAV1 transgenic Arabidopsis plants. C: Expressions of flowering regulatory genes in overexpressing CjRAV1 Arabidopsis. D: Total GAs content of wild type and CjRAV1 transgenic Arabidopsis plants. WT: Wild-type Arabidopsis; 1: p3302-RAV1-1 transgenic Arabidopsis line; 3: p3302-RAV1-3 transgenic Arabidopsis line

Fig. 7 Subcellular localization of the CjRAV1A: The localization prediction results of CjRAV1 in UniProt. B: Subcellular localization analysis of 35S::CjRAV1-GFP

Fig. 8 DAP-seq analysis of CjRAV1in C. japonicaA: Common peak Venn plot between repeated samples within the group. B: Distribution map of peak distance relative to gene TSS position. C: The largest DNA-motif bound by CjRAV1 in C. japonica. D: Distribution ratio diagram of peak on gene functional elements. E: KEGG enrichment pathway of the genes corresponding to all sites

Table 2 Potential downstream regulatory gene CjERF binding site of CjRAV1

结合序列 Motif	结合峰 Peak name	配对序列 Matched sequence	得分 Score	富集倍数 Fold enrichment
RMAGCAACAGM	CjRAV1_peak_6710	GCAGCAACAAC	14.956 5	31.405 34
RMAGCAACAGM	CjRAV1_peak_6710	CCAGCAGCAGA	14.318 8	31.405 34
RMAGCAACAGM	CjRAV1_peak_6710	AAAGCAGCAGC	14.217 4	31.405 34
RMAGCAACAGM	CjRAV1_peak_6710	CCATCAACAGA	11.826 1	31.405 34
RMAGCAACAGM	CjRAV1_peak_6710	AAAGCAGCAAC	11.391 3	31.405 34

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