山茶CjRAV1调控开花延迟的功能研究

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

摘要/Abstract

摘要：

目的探究山茶Related to ABI3 and VP1（CjRAV1）基因在开花调控中的功能及其分子机制，为四季山茶的分子育种提供理论依据。方法采用生物信息学分析、基因表达模式分析、转基因技术和DAP-seq等多种实验手段，系统研究CjRAV1的功能及其调控机制。生物信息学分析鉴定CjRAV1的基因结构、保守结构域及其系统进化关系。利用RT-qPCR技术分析CjRAV1在外源激素诱导下、不同组织以及花苞不同发育时期的表达模式。构建CjRAV1过表达转基因拟南芥植株，观察其表型变化，尤其是开花时间的改变。最后，采用DAP-seq技术筛选CjRAV1下游潜在的DNA结合位点及其调控基因，揭示CjRAV1的分子调控网络。结果生物信息学分析表明，CjRAV1的开放阅读框长度为1 101 bp，共编码366个氨基酸，具有AP2和B3保守结构域。系统进化分析显示，山茶CjRAV1蛋白与茶树CsRAV蛋白的亲缘关系最近，表明两者可能具有相似的功能。亚细胞定位分析证实，CjRAV1转录因子定位于细胞核，提示其可能在转录调控中发挥直接作用。表达模式分析显示，CjRAV1在山茶叶中表达量最高；在花苞成熟过程中，CjRAV1的表达量总体呈现逐步下降的趋势。CjRAV1的过表达转基因拟南芥表现出晚花的表型。通过DAP-seq筛选出潜在的下游调控基因CjERF。结论 CjRAV1过表达导致转基因拟南芥植株表现出晚花的表型，且这一功能可能与潜在的调控基因CjERF协作完成。

关键词: 山茶, RAV转录因子, 基因功能, 花期调控, 转基因, DAP-seq, 晚花表型

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

郭涛, 艾丽皎, 邹世慧, 周玲, 李学梅. 山茶CjRAV1调控开花延迟的功能研究[J]. 生物技术通报, 2025, 41(6): 208-217.

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.

图/表 10

表1 引物序列

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

图1 不同物种RAVs蛋白的系统发育树及其同源关系分析A：CjRAV1的CDS区序列及编码蛋白；B：来自不同物种的15个RAVs蛋白的系统发育树（Cs：茶树；Cl：狭叶油茶；Cj：山茶；At：拟南芥；Os：水稻）；C：指示物种中RAVs蛋白的序列同源矩阵

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

图2 CjRAV1的表达模式分析*：表示显著性差异（P<0.05）。下同

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

图3 过表达载体的构建示意图

Fig. 3 Schematic diagram of overexpression vector construction

图4 表达载体菌液PCR鉴定M：Marker；-：阴性对照；+：阳性对照；1：p3302-RAV1

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

图5 拟南芥转基因株系的阳性鉴定A：过表达CjRAV1拟南芥阳性株系DNA水平的PCR鉴定；B：实时荧光定量检测T1代转基因拟南芥中CjRAV1的相对表达水平；M：marker；-：阴性对照；+：阳性对照；1-8：p3302-RAV1-1至p3302-RAV1-8转基因拟南芥株系

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

图6 野生型和CjRAV1转基因拟南芥花期比较A：野生型和CjRAV1转基因拟南芥植株的表型比较；B：野生型和CjRAV1转基因拟南芥植株的开花时间统计；C：过表达CjRAV1拟南芥中开花调控基因的表达水平；D：野生型和过表达CjRAV1拟南芥中总GA含量；WT：野生型拟南芥；1：p3302-RAV1-1转基因拟南芥株系；3：p3302-RAV1-3转基因拟南芥株系

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

图7 CjRAV1的亚细胞定位分析A：CjRAV1在UniProt的定位预测结果；B：35S：：CjRAV1-GFP的亚细胞定位分析

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

图8 CjRAV1在山茶中的DAP-seq分析A：组内重复样本间的共有peak维恩图；B：Peak与距离相对基因TSS的位置分布图；C：在山茶中被CjRAV1结合的概率最大的DNA-motif；D：peak在基因功能元件上的分布比例图；E：所有位点对应基因的KEGG富集通路图

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

表2 CjRAV1潜在下游调控基因CjERF的结合位点

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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