AP2转录因子OsERF34正向调控水稻种子休眠

doi:10.16819/j.1001-7216.2026.250411

摘要/Abstract

摘要：

【目的】水稻种子休眠性变弱会导致收获前穗发芽(PHS)，严重影响水稻的产量与品质，因而研究水稻种子休眠性的遗传调控机制至关重要。【方法】以中花11为背景通过CRISPR/Cas9技术创制水稻AP2/ERF转录因子OsERF34功能缺失突变体，结合转录组、激素代谢组及生理生化分析，解析其调控种子休眠的分子途径。【结果】发现oserf34突变体种子穗发芽严重，休眠性极显著减弱。转录组分析显示，oserf34与野生型之间差异表达基因(DEGs)显著富集于植物激素信号转导、ROS代谢及糖酵解通路，表明OsERF34可能通过调控激素合成与代谢、氧化还原平衡及糖代谢，维持种子休眠状态。进一步研究发现，oserf34突变体种子中生长素(IAA)、赤霉素(GA19/GA20)及水杨酸(SA)含量显著升高，而ROS清除剂脯氨酸含量降低，过氧化氢(H₂O₂)和超氧阴离子积累加剧，同时抗氧化酶(SOD、POD、CAT)活性上调。此外，突变体胚乳中可溶性糖含量和α-淀粉酶活性显著升高，为打破休眠提供能量基础。【结论】OsERF34通过参与激素合成与代谢、氧化还原平衡及糖代谢正向调控水稻种子休眠，为解析水稻种子休眠与萌发分子机制及创制抗穗发芽水稻新品种提供了新的理论依据与遗传资源。

关键词: 穗发芽, 休眠, 激素代谢组, 活性氧, 可溶性糖, α-淀粉酶

Abstract:

【Objective】 Weakened seed dormancy in rice leads to pre-harvest sprouting (PHS), which severely impacts grain yield and quality, making it crucial to investigate the genetic regulatory mechanisms underlying rice seed dormancy. 【Method】We generated loss-of-function mutants of the AP2/ERF transcription factor OsERF34 in the Zhonghua 11 (ZH11) background using CRISPR/Cas9 technology. 【Result】 The oserf34 mutants exhibited severe pre-harvest sprouting and significantly reduced seed dormancy. Transcriptome analysis revealed that differentially expressed genes (DEGs) between oserf34 and the wild type were significantly enriched in plant hormone signal transduction, ROS metabolism, and glycolytic pathways, suggesting that OsERF34 may maintain seed dormancy by regulating hormone biosynthesis/metabolism, redox homeostasis, and carbohydrate metabolism. Further investigation demonstrated that the oserf34 mutant seeds showed substantially increased levels of auxin (IAA), gibberellins (GA19/GA20), and salicylic acid (SA), along with decreased proline content and elevated hydrogen peroxide (H₂O₂) and superoxide anion level. Meanwhile, the activities of antioxidant enzymes (SOD, POD, CAT) were upregulated. Additionally, the mutant endosperm exhibited significantly elevated soluble sugar content and α-amylase activity, providing an energy foundation for dormancy release. This study demonstrates that OsERF34 positively regulates rice seed dormancy through its involvement in hormone biosynthesis/metabolism, redox homeostasis regulation, and carbohydrate metabolism. 【Conclusion】 These findings provide novel theoretical insights and genetic resources for deciphering the molecular mechanisms of seed dormancy and germination, as well as for developing PHS-resistant rice varieties.

Key words: pre-harvest sprouting, dormancy, hormone metabolome, reactive oxygen species, soluble sugars, α-amylase

杜志敏, 贾一楠, 段影青, 曹妮, 马刘洋, 董欣丽, 徐海, 焦桂爱, 唐绍清, 胡培松. AP2转录因子OsERF34正向调控水稻种子休眠[J]. 中国水稻科学, 2026, 40(2): 196-209.

DU Zhimin, JIA Yinan, DUAN Yingqing, CAO Ni, MA Liuyang, DONG Xinli, XU Hai, JIAO Guiai, TANG Shaoqing, HU Peisong. AP2 Transcription Factor OsERF34 Positively Regulates Seed Dormancy in Rice[J]. Chinese Journal OF Rice Science, 2026, 40(2): 196-209.

图/表 8

图1 oserf34突变体种子休眠性减弱 A：表示OsERF34 的基因结构、突变位点及突变方式，箭头表示CRISPR/Cas 9 编辑的靶位点，灰色矩形表示外显子，白色部分表示内含子。B：中花11靶位点序列和2种突变类型的突变信息及测序峰图。红色矩形位置是碱基插入和缺失的位置。C：收获授粉后30 d（DAP）的中花11和oserf34穗子，立即泡在ddH2O中，每24 h换一次水，C图为第4天的萌发表型。比例尺：2 cm。D：新鲜种子的萌发百分比随时间的变化。误差条表示标准差（n=3）。

Fig. 1. The oserf34 mutant exhibits a weaker dormancy phenotype A, Schematic diagram illustrating the gene structure of OsERF34, mutation sites, and mutation types. Arrows indicate CRISPR/Cas9 editing target sites. Gray rectangles represent exons, and white regions denote introns. B, Target site sequence in ZH11, mutation details of two mutation types, and sequencing chromatograms. Red rectangles highlight positions of nucleotide insertions and deletions (InDels). C, Panicles of ZH11 and oserf34 harvested at 30 days after pollination (DAP) were immediately soaked in ddH2O, with the water changed every 24 hours. The image shows the sprouting phenotype on the fourth day. Scale bar: 2 cm. D, Germination percentage of fresh seeds over time. Error bars represent standard deviation (n=3).

图2 OsERF34 的蛋白保守结构域及三维结构 A：NCBI网站分析OsERF34的蛋白保守结构域。B：SWISS-MODEL分析OsERF34突变前后的蛋白三级结构。红色圆圈表示变化的氨基酸位置。C：OsERF34突变后氨基酸序列的改变，红色字体表示变化的氨基酸。

Fig. 2. Analysis of conserved protein domains and tertiary structure in OsERF34 A, Conserved protein domains of OsERF34 identified using the NCBI database. B, Tertiary protein structures of OsERF34 before and after mutation analyzed using SWISS-MODEL. Red circles indicate positions of altered amino acids. C, Changes in the amino acid sequence of OsERF34 post-mutation. Amino acids modified by the mutation are highlighted in red.

图3 OsERF34在不同发育时期种子中的表达模式及亚细胞定位 A：使用qRT-PCR 检测OsERF34在中花11不同授粉天数种子中的相对表达量表达量，以Ubi基因的表达量为内参进行标准化，将DAP5的表达量设为1，数值代表平均值±标准差，生物学重复3次。B：OsERF34-GFP 在水稻原生质体中的亚细胞定位。将OsERF34-GFP 和细胞核定位的Nuclear localization signal (NLS)-mCherry 共转进水稻原生质体。GFP，绿色荧光蛋白，标尺=10 μm。

Fig. 3. Expression patterns in seeds at different developmental stages of OsERF34 and its subcellular localization A, Relative expression levels of OsERF34 in Zhonghua11 seeds at different days after pollination (DAP) analyzed by qRT-PCR. Expression levels were normalized to the Ubi gene as an internal control, with the DAP5 value set to 1. Data represent mean ± standard deviation (n = 3). B, Subcellular localization of OsERF34-GFP in rice protoplasts. OsERF34-GFP and nuclear-localized NLS-mCherry were co-transfected into rice protoplasts. GFP, Green fluorescent protein. Scale bar: 10 μm.

图4 Oserf34 和中花11 的转录组测序分析 A: Oserf34与中花11差异表达基因的数目。B: oserf34与中花11授粉后20 d种子的总基因表达谱火山图。C: DEGs的GO富集中显著性排名前20的功能分析(Padj<0.05）。D: DEGs的KEGG富集中显著性排名前20的通路分析。

Fig. 4. Transcriptome sequencing analysis of oserf34 and Zhonghua11 A, Number of differentially expressed genes (DEGs) between oserf34 and Zhonghua11. B, Volcano plot of global gene expression profiles in seeds of oserf34 and Zhonghua11 at 20 days after pollination (DAP). C, Gene Ontology (GO) enrichment analysis of DEGs, showing the top 20 significantly enriched functional terms(Padj<0.05). D, KEGG pathway enrichment analysis of DEGs, displaying the top 20 significantly enriched pathways.

图5 中花11和oserf34激素含量的分析 A：授粉后25 d 中花11和oserf34中生长素类激素的含量。MEIAA：吲哚-3-乙酸甲酯；IAA：生长素；OxIAA：氧化吲哚乙酸；IAA-Phe：N-(3-吲哚乙酰基)-L-苯丙氨酸；ILA：吲哚-3-乳酸。B，授粉后25 d 中花11和oserf34赤霉素类激素的含量。GA19：赤霉素19；GA20：赤霉素20。C，授粉后25 d 中花11和oserf34细胞分裂素类激素的含量。DHZ7G：双氢玉米素-7-糖苷；iP7G：异戊烯腺嘌呤-7-葡糖苷；cZRMP：顺式玉米素-糖苷磷酸盐；tZOG：反式-玉米素-9-Β-葡萄糖苷。D：授粉后25 d 中花11和oserf34水杨酸类激素的含量。SA：水杨酸；Phe：L-苯丙氨酸；SAG：水杨酸-2-O-β-葡萄糖苷。数值代表平均值±标准差（n=3）。E：授粉后25 d 中花11和oserf34中脱落酸（ABA）含量。野生型中花11和突变体oserf34之间进行差异比较分析，**代表在P <0.01水平上存在显著差异, *代表在P <0.05水平上存在显著差异(学生氏t检验)。

Fig. 5. Analysis of hormone contents between Zhonghua11 and oserf34 A, Auxin content in Zhonghua11 and oserf34 at 25 days after pollination. MEIAA, Methyl indole-3-acetate; IAA, Indole-3-acetic acid; OxIAA, 2-oxindole-3-acetic acid; IAA-Phe, N-(3-Indolylacetyl)-L-phenylalanine; ILA, Indole-3-lactic acid. B, Gibberellin content in Zhonghua11 and oserf34 at 25 days after pollination. GA19, Gibberellin A19; GA20, Gibberellin A20. C, Cytokinin content in Zhonghua11 and oserf34 at 25 days after pollination. DHZ7G, Dihydrozeatin-7-glucoside; iP7G, N6-Isopentenyl-adenine-7-glucoside; cZRMP, cis-Zeatin riboside monophosphate; tZOG, trans-Zeatin-O-glucoside. D, Salicylic acid content in Zhonghua 11 and oserf34 at 25 days after pollination. SA, Salicylic acid; Phe, L-Phenylalanine; SAG, Salicylic acid 2-O-β-glucoside. E, ABA content in Zhonghua 11 and oserf34. Differential comparative analysis between Zhonghua 11 and oserf34, error bars represent SD (n = 3). **, P < 0.01; *, P < 0.05(Student’s t-test).

图6 ZH11和oserf34活性氧途径分析 A：ZH11和oserf34 授粉后20 d的种子中脯氨酸含量。B：ZH11和oserf34授粉后20 d的种子中过氧化氢含量。C：ZH11和oserf34授粉后20 d的种子中超氧阴离子含量。D：ZH11和oserf34授粉后20 d的种子中ROS清除酶含量。数值代表平均值±标准差（n=3）。野生型ZH11和突变体oserf34之间进行差异比较分析，**代表在P <0.01水平上存在显著差异, *代表在P <0.05水平上存在显著差异，t检验。

Fig. 6. Analysis of reactive oxygen species pathways between ZH11 and oserf34 A, Determination of proline content in seeds of ZH11 and oserf34 at 20 days after pollination (DAP). B, Determination of hydrogen peroxide (H2O2) content in seeds of ZH11 and oserf34 at 20 DAP. C, Determination of superoxide anion content in seeds of ZH11 and oserf34 at 20 DAP. D, Determination of ROS-scavenging enzyme activities in seeds of ZH11 and oserf34 at 20 DAP. Differential comparative analysis between wild-type ZH11 and mutant oserf34, error bars represent SD (n = 3). **, P < 0.01; *, P < 0.05, Student’s t-test.

图7 ZH11和oserf34糖代谢相关指标分析 A：授粉后20 d的种子中可溶性糖含量的测定。B：授粉后20 d的种子中α-淀粉酶活性的测定。数值代表平均值±标准差（n=3）。**代表野生型ZH11和突变体oserf34在P <0.01水平上存在显著差异, *代表在P <0.05水平上存在显著差异(t检验)。

Fig. 7. Analysis of sugar metabolism-related parameters in ZH11 and oserf34 A, Measurement of soluble sugar content in seeds at 20 days after pollination (DAP). B, Measurement of α-amylase activity in seeds at 20 days after pollination (DAP). Differential comparative analysis between ZH11 and oserf34, error bars represent SD (n = 3). **, P < 0.01; *, P < 0.05 (Student’s t-test).

图8 田间农艺性状统计 A-D：ZH11和oserf34分蘖数（A），株高（B），每穗粒数（C）和穗长（D）。柱状图代表平均值±标准差（n=10）。E-F：千粒重（E）和结实率（F）。柱状图代表平均值±标准差（n=3）。**代表野生型ZH11和突变体oserf34在P<0.01水平上存在显著差异, ns代表无显著差异(t检验)。

Fig. 8. Statistical analysis of agronomic traits in field conditions A-D, Tiller number(A), plant height(B), grains per panicle(C), and panicle length(D) of ZH11 and oserf34. Bar graphs represent mean±SD(n=10). E-F, 1000-grain weight (E) and seed setting rate(F). Values are means±SD(n=3). ** and "ns" indicate extremely significant differences (Student’s t-test, P<0.01) and no significant difference between ZH11 and oserf34, respectively.

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