ApGA20ox1通过赤霉素代谢途径正向调控早花百子莲开花

doi:10.13560/j.cnki.biotech.bull.1985.2026-0356

摘要/Abstract

摘要：

目的揭示赤霉素（gibberellin, GA）合成酶关键基因ApGA20ox1调控早花百子莲（Agapanthus praecox）开花的机制，为其开花调控及遗传改良提供依据。方法提取早花百子莲叶片RNA，利用RACE法克隆ApGA20ox1基因，并进行序列同源性比对、亚细胞定位及原核表达分析；将该基因在拟南芥中进行异源过表达，在早花百子莲中通过RNA干扰（RNA interference, RNAi）技术沉默内源ApGA20ox1基因的表达，研究其开花调控功能，并结合GA靶向代谢组学和转录组测序，分析转基因早花百子莲GAs含量变化及开花相关基因的表达模式。结果从早花百子莲中克隆获得ApGA20ox1基因，其ORF全长为1 086 bp，编码361个氨基酸，包含3个外显子和2个内含子。该基因序列高度保守，编码蛋白分子量约为41.1 kD，定位于细胞质中。遗传转化结果表明，异源过表达ApGA20ox1可使拟南芥抽薹时间提前；通过RNAi抑制ApGA20ox1基因表达显著影响早花百子莲叶片GAs合成酶基因的表达和代谢流，其中GA20ox6、GA3ox1表达量显著升高，而GA20ox1、GA20ox3、GA20ox4和GA2ox3表达量显著降低，代谢产物GA₄、GA₂₄、GA₉、GA₄₄、GA₁₉和GA₂₀含量显著降低，而GA₃、GA₅₁、GA₂₉和GA₃₄含量显著升高。开花相关基因中，FLC家族、SOC1家族表达量全部显著下调；CO家族成员主要表现为上调；同时，FT及LFY基因的表达显著上调。结论 ApGA20ox1是早花百子莲GA合成的关键基因，通过GA通路正向调控开花；RNAi沉默该基因表达可显著抑制开花基因SOC1表达，阻断开花通路。

关键词: 早花百子莲, ApGA20ox1, 赤霉素, 开花调控, SOC1, 异源过表达, 靶向代谢组, 转录组

Abstract:

Objective To elucidate the mechanism by which ApGA20ox1, a key gibberellin (GA) biosynthesis gene, regulates flowering in Agapanthus praecox, and to provide a theoretical basis for flowering regulation and genetic improvement in this species. Method Total RNA was extracted from leaves of A. praecox, and the full-length coding sequence of ApGA20ox1 was cloned using RACE (rapid amplification of cDNA ends). Sequence homology, subcellular localization, and prokaryotic expression were analyzed. The gene was heterologously overexpressed in Arabidopsis thaliana, whereas its endogenous expression was silenced in A. praecox via RNA interference (RNAi). Changes in GA contents and expression patterns of flowering-related genes were investigated through transcriptome sequencing and GA-targeted metabolomics. Result The ApGA20ox1 gene was successfully cloned from A. praecox. Its full-length ORF was 1 086 bp, encoding a protein of 361 amino acids, and the genomic sequence contained three exons and two introns. The gene was evolutionarily conserved, and its encoded protein was predicted to have a molecular weight of approximately 41.1 kD and to localize in the cytoplasm. Genetic transformation assays demonstrated that heterologous overexpression of ApGA20ox1 significantly advanced bolting time in A. thaliana. RNAi-mediated suppression of ApGA20ox1 expression significantly affected the expression of GA synthase genes and metabolic flux in A. praecox leaves. Among these genes, the expression levels of GA20ox6 and GA3ox1 were significantly upregulated, whereas those of GA20ox1, GA20ox3, GA20ox4, and GA2ox3 were markedly downregulated. The contents of GA₄, GA₂₄, GA₉, GA₄₄, GA₁₉, and GA₂₀ decreased significantly, whereas those of GA₃, GA₅₁, GA₂₉, and GA₃₄ increased remarkably. Among flowering-related genes, all members of the FLC and SOC1 families were significantly downregulated, whereas most members of the CO family were upregulated. Additionally, the expression of FT and LFY genes was significantly upregulated. Conclusion ApGA20ox1 was identified as a critical gene that regulates GA biosynthesis and positively regulates flowering through the GA pathway in A. praecox. RNAi-mediated silencing of ApGA20ox1 markedly repressed the expression of the flowering integrator gene SOC1 and blocked the flowering pathway.

Key words: Agapanthus praecox, ApGA20ox1, gibberellin, flowering regulation, SOC1, heterologous overexpression, targeted metabolomics, transcriptome

岳建华, 方婷婷, 陈冠群, 申晓辉. ApGA20ox1通过赤霉素代谢途径正向调控早花百子莲开花[J]. 生物技术通报, 2026, 42(11): 186-195.

YUE Jian-hua, FANG Ting-ting, CHEN Guan-qun, SHEN Xiao-hui. ApGA20ox1 Positively Regulates Flowering of Agapanthus praecox via the Gibberellin Metabolic Pathway[J]. Biotechnology Bulletin, 2026, 42(11): 186-195.

图/表 6

图1 早花百子莲ApGA20ox1基因外显子、内含子结构分析ATG和TGA分别为起始密码子和终止密码子

Fig. 1 Analysis of exon-intron structure of ApGA20ox1 from A. praecoxATG and TGA are the start codon and stop codon, respectively

图2 早花百子莲ApGA20ox1基因系统进化分析

Fig. 2 Phylogenetic analysis of ApGA20ox1 in A. praecox

图3 ApGA20ox1蛋白在本氏烟草中的亚细胞定位（标尺100 μm）

Fig. 3 Subcellular localization of ApGA20ox1 in N. benthamiana (bar 100 μm)

图4 过表达ApGA20ox1对拟南芥开花的影响A：转基因植株DNA的PCR检测（M：DL2 000 DNA标准分子量；Col-0：野生型植株；1-8：过表达植株）；B：ApGA20ox1在拟南芥野生型和4个株系中表达水平的检测；C：过表达ApGA20ox1对拟南芥开花的影响；D：过表达ApGA20ox1促进拟南芥抽薹的时间统计；*表示差异显著（P<0.05）；下同

Fig. 4 Effect of ApGA20ox1 overexpression on flowering in A. thalianaA: PCR detection of transgenic plant DNA (M: DL2 000 DNA marker; Col-0: wild-type plant; 1-8: overexpression lines). B: Detection of ApGA20ox1 expression in wild-type and four overexpression lines of A. thaliana. C: Effect of overexpression of ApGA20ox1 on flowering in A. thaliana. D: Statistical analysis of bolting time promoted by overexpression of ApGA20ox1 in A. thaliana. * indicates significant differences compared with the control (P<0.05). The same below

图5 沉默ApGA20ox1表达对早花百子莲开花的影响A：pTCK303-ApGA20ox1载体构建；B：转基因植株PCR检测（CK：野生型植株；1-14：RNAi植株）；C：沉默ApGA20ox1对早花百子莲开花的影响；D：ApGA20ox1相对表达量检测

Fig. 5 Effect of RNAi-mediated silencing of ApGA20ox1 on flowering in A. praecoxA: Construction of vector pTCK303-ApGA20ox1. B: PCR detection of transgenic RNAi plants (CK: wild-type plant; 1-14: RNAi lines). C: Effect of silencing of ApGA20ox1 on flowering in A. praecox. D: Detection of relative expression of ApGA20ox1

图6 沉默ApGA20ox1表达对早花百子莲GAs生物合成的影响A：内源GAs组分的定量分析，*表示差异显著（P<0.05），数据为RNAi vs. CK；B：GAs合成后期催化酶相关DEGs表达谱，数据为RNAi vs. CK；C：GAs合成后期模式图

Fig. 6 Effects of silencing ApGA20ox1 expression on GAs biosynthesis in A. praecoxA: Quantification of endogenous GAs contents. * indicates significant difference at P<0.05, data shown in the figure represent RNAi vs. CK. B: Expression profiles of DEGs encoding key catalytic enzymes in the late GA biosynthesis pathway, data shown in the figure represent RNAi vs. CK. C: Schematic diagram of the late-stage GA biosynthesis pathway

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