检索
E-mail
RSS
高级检索 图表检索

当期目录 我要投稿 过刊浏览
高级检索 图表检索

作物杂志,2026, 第4期: 17–26 doi: 10.16035/j.issn.1001-7283.2026.04.003

• 遗传育种·种质资源·生物技术 • 上一篇    下一篇

利用CRISPR/Cas9技术编辑GS3、GS9和GW2基因改良水稻粒型

阙涛1(), 吴娴2, 王忠妮2, 吴钱蓉3, 龙武华2,4, 王倩2,4, 朱速松5()   

  1. 1 贵州师范大学生命科学学院， 550025， 贵州贵阳
    2 贵州省农业科学院水稻研究所， 550006， 贵州贵阳
    3 贵州大学生命科学学院， 550000， 贵州贵阳
    4 农业农村部喀斯特山区作物基因资源与种质创新重点实验室， 550006， 贵州贵阳
    5 贵州省农业科学院农作物品种资源研究所， 550006， 贵州贵阳
  • 收稿日期:2025-04-17 修回日期:2025-05-21 出版日期:2026-08-15 发布日期:2026-08-11
  • 通讯作者: 朱速松
  • 作者简介:阙涛，研究方向为水稻分子遗传育种，E-mail：3290376576@qq.com
  • 基金资助:
    贵州省优质特色水稻研发与转化及园区技术服务能力建设(黔科合平台人才（2017）5719号);贵州省农业农村厅种业资金项目“贵州优质资源优异基因挖掘及其分子改良”;喀斯特山区特色作物生物育种创新能力建设(黔科合服企[2024]003-1)

Improvement of Rice Grain Shape by Editing of GS3, GS9 and GW2 Genes Using CRISPR/Cas9 Technology

Que Tao1(), Wu Xian2, Wang Zhongni2, Wu Qianrong3, Long Wuhua2,4, Wang Qian2,4, Zhu Susong5()   

  1. 1 School of Life Sciences, Guizhou Normal University, Guiyang 550025, Guizhou, China
    2 Rice Research Institute, Guizhou Academy of Agricultural Sciences, Guiyang 550006, Guizhou, China
    3 College of Life Sciences, Guizhou University, Guiyang 550000, Guizhou, China
    4 Key Laboratory of Crop Genetic Resources and Germplasm Innovation in Karst Mountainous Areas, Ministry of Agriculture and Rural Affairs, Guiyang 550006, Guizhou, China
    5 Institute of Crop Variety Resources, Guizhou Academy of Agricultural Sciences, Guiyang 550006, Guizhou, China
  • Received:2025-04-17 Revised:2025-05-21 Online:2026-08-15 Published:2026-08-11
  • Contact: Zhu Susong

RichHTML

1

PDF (PC)

3

摘要：

利用CRISPR/Cas9基因编辑技术同时对水稻品种锡贡6号的粒型相关基因GS3、GS9和GW2进行编辑，通过多基因编辑策略改良水稻粒型，分别在这3个基因的第一外显子设计敲除靶点，并构建CRISPR/Cas9表达载体，通过农杆菌介导的遗传转化获得T0代转基因植株。结果表明，T0代植株中出现了多种基因编辑类型，包括双等位基因纯合突变和双杂合突变。通过自交获得T1代植株，并筛选出不含外源转基因成分且GS3、GS9和GW2基因均发生与T0代一致突变的突变体材料。对T1代突变体的农艺性状分析表明，突变体1188-42-1的粒长最高增加了21.62%，1188-4-12的粒宽最高增加了13.16%，1188-4-13的籽粒长宽比最高增加了26.65%。同时，各突变体的千粒重、单株粒重、单株产量、出糙率、精米率、整精米率和垩白度均显著提高，但各突变体的结实率均有轻微降低，降幅为4.12%~13.40%。另外，1188-18-8的垩白粒率最高，增加了175.00%，而1188-42-2的垩白粒率最低，降低了50.50%。

关键词: CRISPR/Cas9, 水稻, 粒型, 多基因编辑

Abstract:

The CRISPR/Cas9 gene-editing technique was used to simultaneously modify the grain shape-related genes GS3, GS9, and GW2 in the rice cultivar Xigong 6. The aim was to improve the rice grain shape through a multi-gene editing strategy. Knockout target sites were designed within the first exons of each gene, and CRISPR/Cas9 expression vectors were constructed to produce T0 generation transgenic plants via Agrobacterium- mediated genetic transformation. The results showed that various types of gene-editing mutations, including biallelic homozygous and double heterozygous mutations, were observed in the T0 generation. T1 generation plants were obtained through self-pollination, and mutant materials lacking exogenous transgenic components, while mutations in the GS3, GS9, and GW2 consistent with those in the T0 generation were screened. Agronomic trait analysis of T1 generation mutants revealed significant improvements in grain morphology: mutants 1188-42-1 showed a maximum grain length increase of 21.62%, 1188-4-12 exhibited a 13.16% increase in grain width, and 1188-4-13 displayed a 26.65% enhancement in length-width ratio. Concurrently, 1000-grain weight, grain weight per plant, yield per plant, brown rice rate, rice milling rate, head rice rate, and chalkiness degree were all significantly increased. However, a minor reduction in seed-setting rate 4.12%-13.40% was observed across all mutants. Notable variations in chalky rate were recorded: line 1188-18-8 showed a 175.00% increase, while 1188-42-2 demonstrated a 50.50% decrease.

Key words: CRISPR/Cas9, Rice, Grain shape, Multi-gene editing

表1

引物信息

引物名称Primer name 引物序列（5′-3′）Primer sequence (5′-3′)
Cas-9-gRT1 ACGCGCTCCACCGCGAGATgttttagagctagaaat
Cas-9-OsU6aT1-R ATCTCGCGGTGGAGCGCGTCggcagccaagccagca
Cas-9-gRT2 CGATTGCTTCCTGCTCGGTTgttttagagctagaaat
Cas-9-OsU6bT2-R AACCGAGCAGGAAGCAATCGCaacacaagcggcagc
Cas-9-gRT3 AAGCTCGCGCCGTGCTACATgttttagagctagaaat
Cas-9-OsU3T3-R ATGTAGCACGGCGCGAGCTTCtgagcctcagcgcag
U-F CTCCGTTTTACCTGTGGAATCG
gRNA-R CGGAGGAAAATTCCATCCAC
Pps-R TTCAGAggtctcT accg ACTAGTATGGAATCGGCAGCAAAGG
Pgs-L AGCGTGggtctcG ctcg ACGCGTATCCATCCACTCCAAGCTC
SP-L GCGGTGTCATCTATGTTACTAG
RB-R AAGTTGGGTAACGCCAGGGT
Cas9-F GAGACTATCACCCCTTGGAA
Cas9-R GATGAGCGTAAGTCTTGAGC
GS3-F CGGAGTGACATGGCAATGG
GS3-R TTCGACAGATAGCAAGCCGT
GS9-F AGCTGCAGGGAGTGTCCT
GS9-R AGCAGGGCACGTACAGAGT
GW2-F GAGTGGTGAGGGTTTCATCTG
GW2-R TACCAGGAAGCAGATGGGG

图1

GS3、GS9和GW2基因敲除靶点位置

图2

载体图谱 插入片段区域包含35S启动子激活的潮霉素磷酸转移酶基因，泛素启动子激活的Cas9基因，U6a、U6b和U3启动子激活的具有敲除靶点的GS3/GS9/GW2-sgRNA表达盒；LB：T-DNA左边界；RB：T-DNA右边界。

图3

筛选T0代阳性转化植株 M：2000 bp marker；“+”：阳性对照；“-”：阴性对照，下同。1~7依次为1188-2、1188-4、1188-11、1188-18、1188-31、1188-33和1188-42。

图4

T0代3个靶点的Sanger测序分析 “-”代表缺失；绿色代表插入位点；蓝色代表碱基替换位点；红色代表PAM序列。下同。

图5

PCR筛选无外源转基因成分的突变植株

图6

T1代突变类型 1188-4-12、1188-4-13、1188-18-8、1188-42-1和1188-42-2为T1代突变体。下同。

图7

T1代粒型农艺性状差异 WT为野生型。不同小写字母表示差异显著（P < 0.05），下同。

图8

T1代粒长、粒宽和糙米外观形态观察 (a) 粒长；(b) 粒宽；(c) 糙米外观形态。

图9

T1代单株产量农艺性状差异

图10

T1代稻米外观品质农艺性状差异

[15] Barrangou R, Fremaux C, Horvath P, et al. CRISPR provides acquired resistance against viruses in prokaryotes. Science, 2007, 315(5819):1709-1712.
doi: 10.1126/science.1138140 pmid: 17379808
[16] 梁楚炎, 巫明明, 黄凤明, 等. 基因编辑及全基因组选择技术在水稻育种中的应用展望. 中国水稻科学, 2024, 38(1):1-12.
[17] 李海燕. 玉米基因编辑技术在抗性育种中的应用. 种子科技, 2025, 43(5):111-113.
[18] 黎华, 李冬秀, 林秀芳, 等. 在籼稻中突变GS9同时影响粒型和株型. 分子植物育种. (2024-09-14) [2025-09-17]. https://link.cnki.net/urlid/46.1068.S.20240914.1422.002.
[19] Hua K, Zhang J, Zhu J K, et al. Perspectives on the application of genome-editing technologies in crop breeding. Molecular Plant, 2019, 12(8):1047-1059.
doi: S1674-2052(19)30230-8 pmid: 31260812
[20] Huang Y, Bai X F, Cheng N N, et al. Wide Grain 7 increases grain width by enhancing H3K4me3 enrichment in the OsMADS1 promoter in rice (Oryza sativa L.). The Plant Journal, 2020,102:517-528.
[21] Tan W C, Miao J, Xu B, et al. Rapid production of novel beneficial alleles for improving rice appearance quality by targeting a regulatory element of SLG7. Plant Biotechnology Journal, 2023, 21(7):1305-1307.
doi: 10.1111/pbi.v21.7
[22] Zhu X Y, Gou Y J, Heng Y Q, et al. Targeted manipulation of grain shape genes effectively improves outcrossing rate and hybrid seed production in rice. Plant Biotechnology Journal, 2023, 21(2):381-39.
doi: 10.1111/pbi.v21.2
[23] Tu B, Tao Z, Wang S G, et al. Loss of Gn1a/OsCKX2 confers heavy-panicle rice with excellent lodging resistance. Journal of Integrative Plant Biology, 2022, 64(1):23-38.
doi: 10.1111/jipb.v64.1
[24] Rong C Y, Liu Y X, Chang Z Y, et al. Cytokinin oxidase/ dehydrogenase family genes exhibit functional divergence and overlap in rice growth and development, especially in control of tillering. Journal of Experimental Botany, 2022,73:3552-3568.
[25] Wang C G, Wang G K, Gao Y, et al. A cytokinin-activation enzyme-like gene improves grain yield under various field conditions in rice. Plant Molecular Biology, 2020, 102(4/5):373-388.
doi: 10.1007/s11103-019-00952-5
[1] FAO. Cereal supply and demand brief database. Rome: Food and Agriculture Organization of the United Nations. (2025-03-06) [2025-04-17]. https://www.fao.org/worldfoodsituation/csdb/en/.
[2] 康云海, 方玉, 李潜龙, 等. 水稻粒型基因研究进展及其育种应用. 杂交水稻, 2022, 37(3):7-10.
[26] Huang J, Gao L J, Luo S M, et al. The genetic editing of GS3 via CRISPR/Cas 9 accelerates the breeding of three-line hybrid rice with superior yield and grain quality. Molecular Breeding, 2022, 42(4):22.
doi: 10.1007/s11032-022-01290-z
[27] 沈兰, 李健, 付亚萍, 等. 利用CRISPR/Cas9系统定向改良水稻粒长和穗粒数性状. 中国水稻科学, 2017, 31(3):223-231.
doi: 10.16819/j.1001-7216.2017.7029 223
[3] Fan C C, Xing Y Z, Mao H L, et al. GS3, a major QTL for grain length and weight and minor QTL for grain width and thickness in rice,encodes a putative transmembrane protein. Theoretical and Applied Genetics, 2006, 112(6):1164-1171.
doi: 10.1007/s00122-006-0218-1
[4] Qi P, Lin Y S, Lin H X, et al. The novel quantitative trait locus GL3.1 controls rice grain size and yield by regulating Cyclin-T1; 3. Cell Research, 2012, 22(12):1666-1680.
[28] 吴才军. 特色优质稻锡贡6号品种特征特性及高产栽培技术. 农技服务, 2011, 28(8):1107,1109.
[29] 国家水稻数据中心. 水稻品种信息. [2025-04-17]. https://www.ricedata.cn/variety/varis/606804.htm.
[5] Wu W G, Liu X Y, Wang M H, et al. A single-nucleotide polymorphism causes smaller grain size and loss of seed shattering during African rice domestication. Nature Plants, 2017, 3(6):17064.
doi: 10.1038/nplants.2017.64
[6] Ishimaru K, Hirotsu N, Katoh E, et al. Loss of function of the IAA-glucose hydrolase gene TGW6 enhances rice grain weight and increases yield. Nature Genetics, 2013, 45(6):707-711.
doi: 10.1038/ng.2612 pmid: 23583977
[7] Song X J, Huang W, Lin H X, et al. A QTL for rice grain width and weight encodes a previously unknown RING-type E3 ubiquitin ligase. Nature Genetics, 2007, 39(5):623-630.
doi: 10.1038/ng2014
[8] Ruan B P, Shang L G, Zhang B, et al. Natural variation in the promoter of TGW2 determines grain width and weight in rice. New Phytologist, 2020,227:629-640.
[9] Gao Q S, Li G, Sun H, et al. Targeted mutagenesis of the rice FW 2.2-like gene family using the CRISPR/Cas 9 system reveals OsFWL4 as a regulator of tiller number and plant yield in rice. International Journal of Molecular Sciences, 2020, 21(3):809.
doi: 10.3390/ijms21030809
[10] Liu J F, Chen J, Zheng X M, et al. GW5 acts in the brassinosteroid signalling pathway to regulate grain width and weight in rice. Nature Plants, 2017, 3(5):17043.
doi: 10.1038/nplants.2017.43
[11] Wang S K, Li S, Liu Q, et al. The OsSPL16-GW7 regulatory module determines grain shape and simultaneously improves rice yield and grain quality. Nature Genetics, 2015, 47(8):949-954.
doi: 10.1038/ng.3352
[12] Zhao D S, Li Q F, Liu Q Q, et al. GS9 acts as a transcriptional activator to regulate rice grain shape and appearance quality. Nature Communications, 2018, 9(1):1240.
doi: 10.1038/s41467-018-03616-y
[13] Jinek M, Chylinski K, Fonfara I, et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science, 2012,337:816-821.
[14] Gasiunas G, Barrangou R, Horvath P, et al. Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria. Proceedings of the National Academy of Sciences of the United States of America, 2012,109:2579-2586.
[30] Ma X, Zhang Q, Liu Y G, et al. A robust CRISPR/Cas 9 system for convenient, high-efficiency multiplex genome editing in monocot and dicot plants. Molecular Plant, 2015, 8(8):1274-1284.
doi: 10.1016/j.molp.2015.04.007
[31] 陈盈盈. 水稻RECQ4、FANCM、ZEP1和FIGNL1基因突变材料的创建及鉴定. 扬州:扬州大学, 2024.
[32] 王学良, 王忠妮, 李祖军, 等. 香稻苟当3号香味成分与土壤养分相关性分析. 江苏农业科学, 2023, 51(7):168-173.
[33] Liu W Z, Xie X R, Liu Y G, et al. Dsdecode:A web-based tool for decoding of sequencing chromatograms for genotyping of targeted mutations. Molecular Plant, 2015, 8(9):1431-1433.
doi: 10.1016/j.molp.2015.05.009
[34] Zeng D C, Liu T L, Ma X L, et al. Quantitative regulation of waxy expression by CRISPR/Cas9-based promoter and 5’UTR- intron editing improves grain quality in rice. Plant Biotechnology Journal, 2020, 18(12):2385-2387.
doi: 10.1111/pbi.v18.12
[35] Hao J Q, Wang D K, Wu Y B, et al. The GW2-WG1-OsbZIP47 pathway controls grain size and weight in rice. Molecular Plant, 2021, 14(8):1266-1280.
doi: 10.1016/j.molp.2021.04.011
[36] Mao H L, Sun S Y, Yao J L, et al. Linking differential domain functions of the GS3 protein to natural variation of grain size in rice. Proceedings of the National Academy of Sciences of the United States of America, 2010, 107(45):19579-19584.
[37] 韩政宏, 段宇轩, 徐善斌, 等. 利用CRISPR/Cas9技术敲除GS3和GS9基因改良水稻粒型性状. 华北农学报, 2022, 37 (2):9-17.
doi: 10.7668/hbnxb.20192762
[38] 孟帅, 徐鹏, 张迎信, 等. 利用CRISPR/Cas9技术编辑粒长基因GS3改善粳稻花时. 中国水稻科学, 2018, 32(2):119-127.
[39] 赵春芳, 梁文化, 赫磊, 等. CRISPR/Cas9编辑GS3和qGL3基因创制大粒水稻新种质. 植物遗传资源学报, 2022, 23(6):1709-1717.
[40] 颜静宛, 陈子强, 周淑芬, 等. 利用CRISPR/Cas9系统创制水稻品种GW2基因的突变体. 江苏农业科学, 2024, 52(3):73-78.
[41] 徐善斌, 郑洪亮, 刘利锋, 等. 利用CRISPR/Cas9技术高效创制长粒香型水稻. 中国水稻科学, 2020, 34(5):406-412.
doi: 10.16819/j.1001-7216.2020.0104
[1] 殷佳, 张男, 孙茹梦, 汝艳, 景文疆, 张耗. 根系分泌物在水稻氮素吸收利用中的作用机制及氮肥调控研究进展[J]. 作物杂志, 2026, (4): 1–7
[2] 吴子帅, 黄秋要, 陈传华, 罗芳媚, 张世冠, 刘广林, 路一平, 罗群昌, 李虎. 施氮量和栽插密度对桂香99产量和稻米品质的影响[J]. 作物杂志, 2026, (4): 140–145
[3] 阮新民, 从夕汉, 杜弘杨, 岳伟, 施伏芝, 罗彦长, 王元垒, 罗志祥. 江淮稻作区氮肥用量对高产优质中籼稻产量、品质及氮肥利用效率的影响[J]. 作物杂志, 2026, (4): 146–154
[4] 王鑫琦, 车欣洋, 张海洋, 王旭, 李玉涵, 赵硕, 刘思贝, 王雪贺缘, 贺琳, 徐晶宇. 玉米二酰甘油激酶基因耐低温分析[J]. 作物杂志, 2026, (4): 27–35
[5] 黄新元, 张英, 陶迎梅, 江青山, 魏丕伟. 水稻OsDOG1-like基因家族突变体种子的萌发势及其GA敏感性的研究[J]. 作物杂志, 2026, (4): 36–45
[6] 吴钱蓉, 吴娴, 姜雪, 王倩, 宫彦龙, 王忠妮, 朱速松, 宋莉. 利用CRISPR/Cas9技术编辑Badh2基因改良水稻优质恢复系QR79的香味[J]. 作物杂志, 2026, (4): 57–66
[7] 郭威, 孙冬, 闫治霖, 丁蓓, 杜跃辰, 吴慧扬, 徐强, 窦志, 高辉. 灌浆结实期半深水灌溉对水稻产量、干物质转运及籽粒灌浆的影响[J]. 作物杂志, 2026, (3): 21–29
[8] 刘冬, 李君霞, 马梦娟, 张骐飞, 李明, 付景, 王越涛, 王付华, 尹海庆, 王亚. 淹水深度对水稻胚芽鞘伸长的影响及其生理机制[J]. 作物杂志, 2026, (3): 216–224
[9] 张清芮, 凌凤楼, 高子婷, 王玉, 苏庆旺, 韩笑. 不同叶施时期和锌肥种类对水稻产量和品质的影响[J]. 作物杂志, 2026, (3): 38–47
[10] 潘群肖, 龙超, 卓乐, 殷超, 肖敏, 周文新, 易镇邪. 施氮量与氮磷钾配比对小粒型杂交稻源库特性的影响[J]. 作物杂志, 2026, (2): 160–171
[11] 王金玲, 潘越, 李思宇, 沈炘垭, 刘立军. 枯草芽孢杆菌和哈茨木霉菌对水稻稻米品质的影响[J]. 作物杂志, 2026, (2): 202–208
[12] 陆楚盛, 赖嘉俊, 唐偲, 梁开明, 覃元钰, 钟旭华, 潘俊峰, 刘彦卓, 胡香玉, 胡锐, 李妹娟, 王昕钰, 尹媛红, 叶群欢, 沈宏, 傅友强. 铵、硝营养条件下不同酸碱度对水稻苗期生长和养分吸收的影响[J]. 作物杂志, 2026, (2): 90–97
[13] 孙茹梦, 张男, 殷佳, 汝艳, 景文疆, 张耗. 水稻根系分泌物对干旱胁迫的响应研究进展[J]. 作物杂志, 2026, (1): 1–8
[14] 刘晴, 孙露宏, 高世伟, 刘宇强, 常汇琳, 马成, 王婧泽, 王翠玲, 聂守军. 水稻叶片的生理性状和形态特征受铬胁迫的影响研究[J]. 作物杂志, 2026, (1): 143–151
[15] 闸雯俊, 李行润, 周发松, 冯芳, 吴边, 陈俊孝, 石少阶, 周雷, 王静, 游艾青. 水稻光温敏不育系19XS的选育及其特性分析[J]. 作物杂志, 2026, (1): 9–14
Viewed
Full text


Abstract

Cited
  Shared   
  Discussed   
No Suggested Reading articles found!