水稻AHL基因家族鉴定及基于CRISPR敲除材料的种子萌发表型分析

doi:10.3724/SP.J.1006.2026.62025

摘要/Abstract

摘要： AT-hook基序核定位蛋白(AHL)蛋白是一种古老的转录因子，参与植物多个重要的生命活动过程。本研究对水稻AHL基因家族进行了全基因组鉴定，并系统分析了其基因结构、保守基序、启动子顺式作用元件、蛋白质理化性质、时空表达模式及系统发育关系。在此基础上，基于同源性构建了该基因家族的CRISPR-Cas9多靶点基因敲除材料，通过种子萌发试验和水淹低氧萌发试验进行了萌发表型的初步筛选与分析。结果显示，在水稻全基因组中共鉴定出26个AHL基因；其启动子区域包含多种胁迫响应和激素响应等相关的顺式作用元件。表达分析表明，AHL基因在种子胚和胚乳中广泛表达，且部分家族成员在无氧萌发过程中呈差异表达。通过种子萌发试验，初步筛选出10个与种子萌发相关的候选基因对；进一步对高活力材料开展种子水淹试验，获得了2个耐受低氧胁迫的基因集合。本研究系统鉴定了OsAHL基因家族，创制了相应的CRISPR敲除材料，并初步筛选出调控种子萌发及低氧胁迫响应的候选基因，为进一步解析其分子机制提供了基因资源和材料基础。

关键词: 水稻, AHL基因家族, CRISPR/Cas9, 种子萌发, 低氧胁迫

Abstract: AT-hook motif nuclear-localized (AHL) proteins are an ancient family of transcription factors involved in various essential biological processes in plants. In this study, genome-wide identification of the AHL gene family was performed in rice (Oryza sativa), followed by systematic analysis of gene structures, conserved motifs, promoter cis-acting regulatory elements, physicochemical properties, spatiotemporal expression patterns, and phylogenetic relationships. Furthermore, based on sequence homology, CRISPR/Cas9-mediated multiplex knockout lines were generated for members of this gene family. Preliminary screening and phenotypic analysis were conducted through seed germination and submergence-induced hypoxia assays. A total of 26 OsAHL genes were identified in the rice genome. Promoter analysis revealed various cis-acting elements associated with stress and phytohormone responses. Expression profiling showed that OsAHL genes were broadly expressed in the seed embryo and endosperm, with several members showing differential expression during anaerobic germination. Subsequent seed germination assays enabled the initial screening of 10 candidate gene sets associated with seed germination. Further submergence assays of the candidate lines successfully identified two gene sets associated with tolerance to hypoxic stress. In summary, this study systematically characterized the OsAHL gene family, established corresponding CRISPR/Cas9 knockout mutant resources, and identified candidate genes involved in seed germination and hypoxic stress responses, thereby providing an important genetic and material foundation for future mechanistic studies.

Key words: rice (Oryza sativa), AHL gene family, CRISPR/Cas9, seed germination, hypoxic stress

陈效博, 梁迪云, 李海波, 赵正昊, 王波. 水稻AHL基因家族鉴定及基于CRISPR敲除材料的种子萌发表型分析[J]. 作物学报, 2026, 52(11): 3308-3317.

Chen Xiao-Bo, Liang Di-Yun, Li Hai-Bo, Zhao Zheng-Hao, Wang Bo. Genome-wide identification of the AHL gene family in rice and phenotypic analysis of seed germination using CRISPR mutants[J]. Acta Agronomica Sinica, 2026, 52(11): 3308-3317.

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链接本文: https://zwxb.chinacrops.org/CN/10.3724/SP.J.1006.2026.62025

https://zwxb.chinacrops.org/CN/Y2026/V52/I11/3308

参考文献

[1] FAO. World Food and Agriculture - Statistical Yearbook 2024. Rome: Food and Agriculture Organization of the United Nations, 2024.

[2] 罗锡文, 王在满, 曾山, 等. 水稻机械化直播技术研究进展. 华南农业大学学报, 2019, 40(5): 1–13.

Luo X W, Wang Z M, Zeng S, et al. Recent advances in mechanized direct seeding technology for rice. J South China Agric Univ, 2019, 40(5): 1–13 (in Chinese with English abstract).

[3] Negi P, Rane J, Wagh R S, et al. Direct-seeded rice: genetic improvement of game-changing traits for better adaption. Rice Sci, 2024, 31: 417–433.

[4] Zhang W M, Cheng X Z, Fang D, et al. AT-hook motif nuclear localized (AHL) proteins of ancient origin radiate new functions. Int J Biol Macromol, 2022, 214: 290–300.

[5] Fujimoto S, Matsunaga S, Yonemura M, et al. Identification of a novel plant MAR DNA binding protein localized on chromosomal surfaces. Plant Mol Biol, 2004, 56: 225–239.

[6] Derelle E, Ferraz C, Rombauts S, et al. Genome analysis of the smallest free-living eukaryote Ostreococcus tauri unveils many unique features. Proc Natl Acad Sci USA, 2006, 103: 11647–11652.

[7] Aravind L, Landsman D. AT-hook motifs identified in a wide variety of DNA-binding proteins. Nucleic Acids Res, 1998, 26: 4413–4421.

[8] Širl M, Šnajdrová T, Gutiérrez-Alanís D, et al. AT-hook motif nuclear localised protein 18 as a novel modulator of root system architecture. Int J Mol Sci, 2020, 21: 1886.

[9] Zeng Q K, Song L, Xia M Z, et al. Overexpression of AHL proteins enhances root hair production by altering the transcription of RHD6-downstream genes. Plant Direct, 2023, 7: e517.

[10] Seo M, Lee J Y. Dissection of functional modules of AT-hook motif nuclear localized protein 4 in the development of the root xylem. Front Plant Sci, 2021, 12: 632078.

[11] Cai G Q, Kim S C, Li J W, et al. Transcriptional regulation of lipid catabolism during seedling establishment. Mol Plant, 2020, 13: 984–1000.

[12] Lee K, Seo P J. Coordination of matrix attachment and ATP-dependent chromatin remodeling regulate auxin biosynthesis and Arabidopsis hypocotyl elongation. PLoS One, 2017, 12: e0181804.

[13] Xiao C W, Chen F L, Yu X H, et al. Over-expression of an AT-hook gene, AHL22 delays flowering and inhibits the elongation of the hypocotyl in Arabidopsis thaliana. Plant Mol Biol, 2009, 71: 39–50.

[14] Wong M M, Bhaskara G B, Wen T N, et al. Phosphoproteomics of Arabidopsis Highly ABA-Induced1 identifies AT-Hook-Like10 phosphorylation required for stress growth regulation. Proc Natl Acad Sci USA, 2019, 116: 2354–2363.

[15] Zhou J, Wang X, Lee J Y, et al. Cell-to-cell movement of two interacting AT-hook factors in Arabidopsis root vascular tissue patterning. Plant Cell, 2013, 25: 187–201.

[16] Karami O, Rahimi A, Khan M, et al. A suppressor of axillary meristem maturation promotes longevity in flowering plants. Nat Plants, 2020, 6: 368–376.

[17] Rahimi A, Karami O, Balazadeh S, et al. miR156-independent repression of the ageing pathway by longevity-promoting AHL proteins in Arabidopsis. New Phytol, 2022, 235: 2424–2438.

[18] Rahimi A, Karami O, Lestari A D, et al. Control of cambium initiation and activity in Arabidopsis by the transcriptional regulator AHL15. Curr Biol, 2022, 32: 1764–1775.e3.

[19] Liu H, Hu D X, Du P X, et al. Single-cell RNA-seq describes the transcriptome landscape and identifies critical transcription factors in the leaf blade of the allotetraploid peanut (Arachis hypogaea L.). Plant Biotechnol J, 2021, 19: 2261–2276.

[20] Rayapuram N, Jarad M, Alhoraibi H M, et al. Chromatin phosphoproteomics unravels a function for AT-hook motif nuclear localized protein AHL13 in PAMP-triggered immunity. Proc Natl Acad Sci USA, 2021, 118: e2004670118.

[21] Jin Y, Luo Q, Tong H N, et al. An AT-hook gene is required for Palea formation and floral organ number control in rice. Dev Biol, 2011, 359: 277–288.

[22] Uzair M, Xu D W, Schreiber L, et al. PERSISTENT TAPETAL CELL2 is required for normal tapetal programmed cell death and pollen wall patterning. Plant Physiol, 2020, 182: 962–976.

[23] Zhou L G, Liu Z C, Liu Y H, et al. A novel gene OsAHL1 improves both drought avoidance and drought tolerance in rice. Sci Rep, 2016, 6: 30264.

[24] Liu X Y, Gao Y X, Guo Z Q, et al. MoIug4 is a novel secreted effector promoting rice blast by counteracting host OsAHL1-regulated ethylene gene transcription. New Phytol, 2022, 235: 1163–1178.

[25] Chen C J, Wu Y, Li J W, et al. TBtools-II: a “one for all, all for one” bioinformatics platform for biological big-data mining. Mol Plant, 2023, 16: 1733–1742.

[26] 曾栋昌, 马兴亮, 谢先荣, 等. 植物CRISPR/Cas9多基因编辑载体构建和突变分析的操作方法. 中国科学(生命科学), 2018, 48: 783–794.

Zeng D C, Ma X L, Xie X R, et al. A protocol for CRISPR/Cas9-based multi-gene editing and sequence decoding of mutant sites in plants. Sci Sin Vitae, 2018, 48: 783–794 (in Chinese with English abstract).

[27] Liu Q, Wang C, Jiao X Z, et al. Hi-TOM: a platform for high-throughput tracking of mutations induced by CRISPR/Cas systems. Sci China Life Sci, 2019, 62: 1–7.

[28] Wang S, Liu W N, He Y, et al. bZIP72 promotes submerged rice seed germination and coleoptile elongation by activating ADH1. Plant Physiol Biochem, 2021, 169: 112–118.

[29] Fu J, Wu H, Ma S Q, et al. OsJAZ1 attenuates drought resistance by regulating JA and ABA signaling in rice. Front Plant Sci, 2017, 8: 2108.

[30] Lin C C, Chao Y T, Chen W C, et al. Regulatory cascade involving transcriptional and N-end rule pathways in rice under submergence. Proc Natl Acad Sci USA, 2019, 116: 3300–3309.

[31] Reed R C, Bradford K J, Khanday I. Seed germination and vigor: ensuring crop sustainability in a changing climate. Heredity, 2022, 128: 450–459.

[32] Wasternack C, Song S S. Jasmonates: biosynthesis, metabolism, and signaling by proteins activating and repressing transcription. J Exp Bot, 2017, 68: 1303–1321.

[33] Ruan J J, Zhou Y X, Zhou M L, et al. Jasmonic acid signaling pathway in plants. Int J Mol Sci, 2019, 20: 2479.

[34] Dave A, Hernández M L, He Z S, et al. 12-oxo-phytodienoic acid accumulation during seed development represses seed germination in Arabidopsis. Plant Cell, 2011, 23: 583–599.

[35] Linkies A, Leubner-Metzger G. Beyond gibberellins and abscisic acid: how ethylene and jasmonates control seed germination. Plant Cell Rep, 2012, 31: 253–270.

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