Creation of Rice <i>Gn1a</i> Mutants via CRISPR/Cas9 System and Their Application in Breeding

ZHAO Xiaoyu; JIN Di; JIANG Yutong; YANG Chaoyue; PENG Boran; LIU Xiao; WANG Liujing; LI Yunfeng; ZHUANG Hui

doi:10.13718/j.cnki.xdzk.2025.07.006

2025 Volume 47 Issue 7

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ZHAO Xiaoyu, JIN Di, JIANG Yutong, et al. Creation of Rice Gn1a Mutants via CRISPR/Cas9 System and Their Application in Breeding[J]. Journal of Southwest University Natural Science Edition, 2025, 47(7): 70-81. doi: 10.13718/j.cnki.xdzk.2025.07.006

Citation:

ZHAO Xiaoyu, JIN Di, JIANG Yutong, et al. Creation of Rice Gn1a Mutants via CRISPR/Cas9 System and Their Application in Breeding[J]. Journal of Southwest University Natural Science Edition, 2025, 47(7): 70-81. doi: 10.13718/j.cnki.xdzk.2025.07.006

Creation of Rice Gn1a Mutants via CRISPR/Cas9 System and Their Application in Breeding

Rice Research Institute, Southwest University/Academy of Agricultural Sciences/Key Laboratory of Application and Safety Control of Genetically Modified Crops, Chongqing 400715, China

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Corresponding author: ZHUANG Hui
Received Date: 31/03/2025
Available Online: 20/07/2025
MSC: S511

Abstract

The number of grains per panicle is an important factor determining the yield of rice.Gn1a, as the main effective QTL, can affect the number of grains per panicle by regulating cytokinin metabolism. Double-target knockout was performed in the coding region of the FAD binding domain of exon 1 of the Gn1a gene in indica rice variety Yujinxiang (YJX^WT) using CRISPR/Cas9 technology. The homozygous editing mutant YJX^Gn1a-1 without exogenous genes was screened and identified. Domain prediction indicates that compared with the wild-type YJX^WT, the FAD binding domain of the YJX^Gn1a-1 mutant is missing 26 amino acids. The investigation of agronomic traits showed that the height of the mutant plants and the yield per plant were increased significantly, and the number of primary and secondary branches, the number of grains per panicle were increased. Gene expression analysis indicated that Gn1a was specifically expressed in culm and young panicles of different lengths, suggesting that it plays an important role in the plant height and inflorescence development of rice. Haplotype analysis further verified that the absence of the FAD binding domain could significantly increase the plant height and the number of grains per panicle of rice.
- rice(Oryza sativa L.),
- CRISPR/Cas9 technology,
- Gn1a,
- germplasm resources

References

[1]	文钦, 贾思思, 王加峰, 等. 水稻单倍体诱导基因OsMATL突变体的创制与分析[J]. 作物学报, 2021, 47(5): 816-825. Google Scholar
[2]	CHEN J J, MIAO Z N, KONG D Y, et al. Application of CRISPR/Cas9 Technology in Rice Germplasm Innovation and Genetic Improvement[J]. Genes, 2024, 15(11): 1492. Google Scholar
[3]	HU H F, ZHAO J L, THOMAS W J W, et al. The Role of Pangenomics in Orphan Crop Improvement[J]. Nature Communications, 2025, 16(1): 118. Google Scholar
[4]	MCGINTY E L, SMITH-KEUNE C, JERRY D R. A High Through-put Protocol for Quantifying Nucleic Acids in Individual Microcrustaceans Using New Generation RNA and DNA Specific Dyes[J]. Journal of Shellfish Research, 2008, 27(2): 449-455. Google Scholar
[5]	MA X L, ZHANG Q Y, ZHU Q L, et al. A Robust CRISPR/Cas9 System for Convenient, High-efficiency Multiplex Genome Editing in Monocot and Dicot Plants[J]. Molecular Plant, 2015, 8(8): 1274-1284. Google Scholar
[6]	温一博, 陈淑婷, 徐正进, 等. DEP1、Gn1a和qSW5组合应用调控水稻穗部性状[J]. 中国农业科学, 2023, 56(7): 1218-1227. Google Scholar
[7]	WANG S G, MA B T, GAO Q, et al. Dissecting the Genetic Basis of Heavy Panicle Hybrid Rice Uncovered Gn1a and GS3 as Key Genes[J]. Theoretical and Applied Genetics, 2018, 131(6): 1391-1403. Google Scholar
[8]	李兆伟, 莫祖意, 孙聪颖, 等. OsNAC2d基因编辑水稻突变体的创建及其对干旱胁迫的响应[J]. 作物学报, 2023, 49(2): 365-376. Google Scholar
[9]	ASHIKARI M, SAKAKIBARA H, LIN S Y, et al. Cytokinin Oxidase Regulates Rice Grain Production[J]. Science, 2005, 309(5735): 741-745. Google Scholar
[10]	LI M R, LI X X, ZHOU Z J, et al. Reassessment of the Four Yield-related Genes Gn1a, DEP1, GS3, and IPA1 in Rice Using a CRISPR/Cas9 System[J]. Frontiers in Plant Science, 2016(7): 377. Google Scholar
[11]	YEH S Y, CHEN H W, NG C Y, et al. Down-regulation of Cytokinin Oxidase 2 Expression Increases Tiller Number and Improves Rice Yield[J]. Rice, 2015, 8(1): 36. Google Scholar
[12]	GUO T, LU Z Q, SHAN J X, et al. ERECTA1 Acts Upstream of the OsMKKK10-OsMKK4-OsMPK6 Cascade to Control Spikelet Number by Regulating Cytokinin Metabolism in Rice[J]. The Plant Cell, 2020, 32(9): 2763-2779. Google Scholar
[13]	苌兴超, 黄永兰, 唐秀英, 等. Gn1a基因靶向敲除对粳稻产量构成因素的影响[J]. 江西农业大学学报, 2023, 45(1): 10-16. Google Scholar
[14]	HU X X, MENG X B, LIU Q, et al. Increasing the Efficiency of CRISPR-Cas9-VQR Precise Genome Editing in Rice[J]. Plant Biotechnology Journal, 2018, 16(1): 292-297. Google Scholar
[15]	TU B, TAO Z, WANG S G, et al. Loss of Gn1a/OsCKX2 Confers Heavy-panicle Rice with Excellent Lodging Resistance[J]. Journal of Integrative Plant Biology, 2022, 64(1): 23-38. Google Scholar
[16]	殷得所, 李进波, 万丙良, 等. 利用Gn1a基因进行粳稻穗粒数改良的研究[J]. 湖北农业科学, 2016, 55(21): 5455-5457, 5518. Google Scholar
[17]	XIE X R, MA X L, ZHU Q L, et al. CRISPR-GE: A Convenient Software Toolkit for CRISPR-based Genome Editing[J]. Molecular Plant, 2017, 10(9): 1246-1249. Google Scholar
[18]	刘建国, 陈冬东, 陈玉玉, 等. 水稻MKKs家族基因成员OsMKK4的不同等位基因型及自然变异对籽粒的影响[J]. 作物学报, 2025, 51 (3): 598-608. Google Scholar
[19]	WANG W S, MAULEON R, HU Z Q, et al. Genomic Variation in 3 010 Diverse Accessions of Asian Cultivated Rice[J]. Nature, 2018, 557(7703): 43-49. Google Scholar
[20]	ALEXANDROV N, TAI S S, WANG W S, et al. SNP-seek Database of SNPS Derived from 3000 Rice Genomes[J]. Nucleic Acids Research, 2015, 43: 1023-1027. Google Scholar
[21]	LU Z F, YU H, XIONG G S, et al. Genome-wide Binding Analysis of the Transcription Activator Ideal Plant Architecture1 Reveals a Complex Network Regulating Rice Plant Architecture[J]. The Plant Cell, 2013, 25(10): 3743-3759. Google Scholar
[22]	HUANG X H, YANG S H, GONG J Y, et al. Genomic Architecture of Heterosis for Yield Traits in Rice[J]. Nature, 2016, 537(7622): 629-633. Google Scholar
[23]	HUANG X H, KURATA N, WEI X H, et al. A Map of Rice Genome Variation Reveals the Origin of Cultivated Rice[J]. Nature, 2012, 490(7421): 497-501. Google Scholar
[24]	DESCALSOTA-EMPLEO G I, AMPARADO A, INABANGAN-ASILOMA. Genetic Mapping of QTL for Agronomic Traits and Grain Mineral Elements in Rice[J]. The Crop Journal, 2019, 7(4): 560-572. Google Scholar
[25]	ISHIMARU K, HIROTSU N, MADOKA Y, et al. Loss of Function of the IAA-Glucose Hydrolase Gene TGW6 Enhances Rice Grain Weight and Increases Yield[J]. Nature Genetics, 2013, 45(6): 707-711. Google Scholar
[26]	RUAN B P, SHANG L G, ZHANG B, et al. Natural Variation in the Promoter of TGW2 Determines Grain Width and Weight in Rice[J]. The New Phytologist, 2020, 227(2): 629-640. Google Scholar
[27]	GUO L B, YE G Y. Use of Major Quantitative Trait Loci to Improve Grain Yield of Rice[J]. Rice Science, 2014(2): 65-82. Google Scholar
[28]	GALUSZKA P, FREBORT I, SEBELA M, et al. Cytokinin Oxidase or Dehydrogenase? Mechanism of Cytokinin Degradation in Cereals[J]. European Journal of Biochemistry, 2001, 268(2): 450-461. Google Scholar
[29]	WERNER T, KÖLLMER I, BARTRINA I, et al. New Insights into the Biology of Cytokinin Degradation[J]. Plant Biology, 2006, 8(3): 371-381. Google Scholar
[30]	MALITO E, CODA A, BILYEU K D, et al. Structures of Michaelis and Product Complexes of Plant Cytokinin Dehydrogenase: Implications for Flavoenzyme Catalysis[J]. Journal of Molecular Biology, 2004, 341(5): 1237-1249. Google Scholar
[31]	何勇, 刘耀威, 熊翔, 等. 利用CRISPR/Cas9技术编辑OsOFP30基因创制水稻粒型突变体[J]. 中国水稻科学, 2024, 38(5): 507-515. Google Scholar
[32]	JING F, SHI S L, KANG W J, et al. The Physiological Basis of Alfalfa Plant Height Establishment[J]. Plants, 2024, 13(5): 679. Google Scholar
[33]	Lu B R, Cai X X, JIN X, et al. Efficient Indica and Japonica Rice Identification Based on the Indel Molecular Method: Its Implication in Rice Breeding and Evolutionary Research[J]. Prog Nat Sci, 2009, 19(10): 1241-1252. Google Scholar
[34]	ZHAO H, LI J C, YANG L, et al. An Inferred Functional Impact Map of Genetic Variants in Rice[J]. Molecular Plant, 2021, 14(9): 1584-1599. Google Scholar
[35]	ZHAO H, YAO W, OUYANG Y D, et al. Rice Var Map: A Comprehensive Database of Rice Genomic Variations[J]. Nucleic Acids Research, 2015, 43: 1018-1022. Google Scholar
[36]	HECHANOVA S L, BHATTARAI K, SIMON E V, et al. Development of a Genome-wide InDel Marker Set for Allele Discrimination between Rice (Oryza sativa) and the Other Seven AA-Genome Oryza Species[J]. Scientific Reports, 2021, 11(1): 8962. Google Scholar
[37]	MOONSAP P, LAKSANAVILAT N, SINUMPORN S, et al. Genetic Diversity of Indo-China Rice Varieties Using ISSR, SRAP and InDel Markers[J]. Journal of Genetics, 2019, 98: 80. Google Scholar
[38]	YONEMARU J I, CHOI S H, SAKAI H, et al. Genome-wide Indel Markers Shared by Diverse Asian Rice Cultivars Compared to Japanese Rice Cultivar 'Koshihikari'[J]. Breeding Science, 2015, 65(3): 249-256. Google Scholar
[39]	SANG S, SUN X, MA T, et al. Efficient Promoter Editing of the SBEIIb Gene Enables Fine-tuning of the Resistant Starch Content in Rice[J]. Int J Biol Macromol, 2025, 290: 138904. Google Scholar
[40]	李光正, 李岩, 李建容, 等. 利用CRISPR/Cas 9基因编辑技术提高贵州禾产量研究[J]. 种子, 2021, 40(6): 1-5, 13. Google Scholar

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水稻是我国最重要的粮食作物之一, 提高其产量和品质对于保障我国粮食安全、增进民生福祉具有极为重要的意义^[1]。随着粮食缺口的逐步扩大和生物技术的不断发展, 传统的杂交选育方法已经很难满足水稻生产的实际需要, 种质创新成为亟待解决的重要课题。近年来, CRISPR/Cas9等基因编辑技术的出现, 为水稻等作物的遗传改良提供了新的工具^[2]。利用基因编辑技术创新种质, 通过对选定的基因进行特定修饰, 可以有针对性地引入理想性状, 实现作物的改良^[3-8]。Gn1a(Grain number 1a)是水稻第1染色体短臂上的主效QTL, 通过调控细胞分裂素(Cytokininin, CK)代谢影响每穗粒数, 在高产育种中具有重要的应用价值^{[6-7, 9]}。Gn1a编码的OsCKX2蛋白由FAD结合结构域和CKX催化结构域组成, 其中FAD结合结构域负责结合FAD(黄素腺嘌呤二核苷酸), 作为电子受体介导细胞分裂素侧链的氧化反应, 而CKX催化结构域是酶的催化核心, 两者协同作用, 特异性降解细胞分裂素, 从而调控水稻的生长发育^{[4, 6]}。Gn1a表达量的降低可导致花序分生组织中细胞分裂素的积累, 促进一次、二次枝梗的分化, 最终提升每穗粒数和产量^[9-10]。Gn1a是水稻产量性状改良的重要基因编辑靶点, 重穗型恢复系蜀恢498Gn1a基因第3外显子上11 bp碱基缺失会引起CKX催化结构域编码序列的移码突变, 使蛋白失活并显著增加每穗粒数^[7]。此外, 在籼稻中花11的遗传背景下, 将Gn1a基因序列前端的第135~157位碱基敲除, 也能引起类似效应^{[6, 10]}。一方面, 在转基因水稻中利用RNA干扰(shRNA)抑制Gn1a表达, 可以显著增加分蘖数和穗粒数并提升水稻的产量^[11]; 另一方面, OsMPK6与锌指转录因子DST的相互作用可以激活Gn1a的转录, 使水稻的每穗粒数显著减少^[12]。这些研究表明, 通过Gn1a关键位点的精确编辑可用于改良水稻的产量性状。当前Gn1a研究主要集中在CKX催化结构域, 而FAD结合结构域的功能解析仍较少, 限制了该位点在种质创新中的应用^[9-16]。本研究利用CRISPR/Cas9技术在籼稻渝金香(YJX^WT)中Gn1a基因的第1外显子上FAD结合结构域的编码序列进行靶向敲除, 筛选、鉴定出不含有外源基因的纯合单株, 成功创制出穗粒数增加、具有实际应用价值的突变体, 并系统分析其农艺性状、基因表达模式及单倍型变异, 为进一步筛选适宜的基因编辑位点、优化水稻高产育种策略提供理论依据和种质资源。

3. 讨论与结论

3.1. 讨论

水稻产量由单位面积内有效穗数、每穗粒数和粒质量等因子决定, 而这些产量决定因子则是由数量性状位点(Quantitative Trait Locus, QTLs) 控制^{[17, 23-24]}。筛选与穗粒数相关的理想QTL位点有利于培育高产水稻品种^{[23, 25-27]}。Gn1a(OsCKX2)是控制水稻每穗粒数的重要QTL, 编码一种细胞分裂素氧化酶, 其表达下降会导致细胞分裂素在花序分生组织中积累, 从而增加每穗粒数, 提高产量^{[9-10, 16, 23-24]}。Gn1a蛋白由FAD结合结构域和CKX催化结构域组成。CKX是催化活性核心, 而FAD结合结构域则负责结合辅酶黄素腺嘌呤二核苷酸(FAD), 作为电子受体完成细胞分裂素异戊烯基侧链的氧化反应。两个结构域相互协同, 通过β-氧化途径特异性降解细胞分裂素从而调控水稻的生长^{[9, 11, 15, 28-30]}。研究发现, Gn1a基因不仅能够调控穗粒数, 也影响结实率和分蘖数^[31]。Wang等^[7]研究表明, CKX催化结构域部分缺失可显著增加穗粒数但降低了结实率; Yeh等^[11]利用CRISPR/Cas9敲除CKX催化结构域编码区特定位点, 发现其可提高穗粒数但减少了分蘖。本研究利用CRISPR/Cas9基因编辑技术在籼稻品种渝金香(YJX^WT) Gn1a基因的第1外显子上设计双靶点敲除, 筛选、鉴定到不含外源基因的YJX^Gn1a-1突变体。结构域分析表明, YJX^Gn1a-1突变体存在78 bp碱基缺失, 引起FAD结合结构域中26个氨基酸缺失(图 4)。相较于野生型, YJX^Gn1a-1突变体的一次枝梗数、二次枝梗数极显著增加, 而籽粒形态和千粒质量并未发生显著变化(图 5), 最终单株产量显著提高(图 6)。值得注意的是, YJX^Gn1a-1突变体植株高度显著增加。基因的表达分析显示, Gn1a基因在茎及不同长度幼穗中均有表达(图 7), 提示其不仅调控穗粒数, 还可能影响株高, 这一功能或由FAD结合结构域介导。在未来研究中, 可进一步解析Gn1a在株高调控中的作用机制, 并探索利用其编辑效应提高水稻地上生物量, 以创制高光效品种用于育种^[32]。

随着CRISPR/Cas9基因编辑技术的发展, 研究者可快速实现目标基因的定向编辑, 以加速水稻的遗传改良^{[12, 33]}。解析高产单倍型突变位点并结合CRISPR/Cas9技术, 可筛选出具有育种价值的突变体^[25-26]。已有研究表明, Gn1a基因的缺失是重穗型水稻Shuhui498(R498)每穗粒数增加的决定性因素, 且部分携带InDel突变位点的水稻单倍型表现出显著的穗粒数增加^[6-7]。此外, 部分InDel位点与粳籼亚种分化密切相关^[33], 可能在水稻高产性状的形成中发挥着关键作用。目前对于水稻InDel位点与表型的关联性分析研究仍停留在不同品种InDel位点的鉴定层面^[34-38], 缺乏系统的突变体表型数据收集及数据库构建, 导致育种过程中难以精准筛选关键的InDel位点, 限制了高效创制具有目标性状的水稻种质。本研究基于CRISPR/Cas9技术成功敲除Gn1a蛋白的FAD结合结构域, 并获得YJX^Gn1a-1突变体, 可用于分析FAD结合结构域缺失的表型效应^[39-40]。为了进一步评估编辑位点的育种潜力, 我们在自然种质资源中筛选了编辑区段(Chr1: 5274920-5274997)携带SNP和InDel的单倍型, 用以评估其表型效力。在敲除区间内, 仅检测到1个SNP位点Chr1: 5274962, 其编码氨基酸发生了改变(由丙氨酸变为苏氨酸), 对应两种单倍型。数据库分析显示, 这两种单倍型在株高和枝梗数方面差异无统计学意义(图 8), 表明单一SNP位点的变异不足以作为水稻穗粒数提升的决定性因素, 而FAD结合结构域的完整性对Gn1a功能行使具有重大影响。相较之下, 本研究创制出的YJX^Gn1a-1突变体表现出株高和每穗颖花数的显著提升, 因此InDel突变及大片段缺失可能比单点突变对Gn1a功能的影响更为深远。未来可针对InDel相关单倍型进行系统的表型收集, 构建InDel位点与水稻高产表型的关联性数据库, 以筛选Gn1a基因与表型强关联的关键InDel位点。结合自然种质资源的InDel突变体筛选及CRISPR/Cas9技术, 可创制具有优良特性的高产单倍型, 以加速水稻品种的改良。

3.2. 结论

表达模式分析表明, Gn1a在水稻茎和不同长度幼穗中特异表达, 提示其可能在水稻株高和花序发育中发挥重要作用。结构域预测表明, Gn1a蛋白由FAD结合结构域和CKX催化结构域组成。本研究以籼稻品种渝金香(YJX^WT)为受体材料, 利用CRISPR/Cas9技术靶向敲除Gn1a基因FAD结合结构域编码区段的78 bp碱基序列, 测序检测出无外源片段插入的纯合突变体YJX^Gn1a-1。农艺性状调查显示, 突变体植株株高显著增加, 一次、二次枝梗数显著增加, 每穗颖花数显著提升, 单株产量显著提高。单倍型分析进一步验证了FAD结合结构域的缺失可显著增加水稻株高和穗粒数。本研究初步解析了FAD结合结构域缺失对株高和穗粒数的影响, 为Gn1a基因的功能解析提供了新的视角, 创制了可用于高产育种的新种质资源, 为培育高产水稻品种提供了理论依据和实践支持。

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Creation of Rice Gn1a Mutants via CRISPR/Cas9 System and Their Application in Breeding