[1]郭冬姝,杨郁文,李笑,等.基于基因编辑的水稻4-羟基苯丙酮酸双加氧酶（HPPD）基因突变体库构建及抗HPPD抑制剂类除草剂种质筛选[J].江苏农业学报,2026,42(05):865-875.[doi:doi:10.3969/j.issn.1000-4440.2026.05.001]
　GUO Dongshu,YANG Yuwen,LI Xiao,et al.Genome editing-mediated mutant library construction of rice 4-hydroxyphenylpyruvate dioxygenase (HPPD) gene and identification of germplasm with resistance to HPPD-inhibiting herbicides[J].,2026,42(05):865-875.[doi:doi:10.3969/j.issn.1000-4440.2026.05.001]
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基于基因编辑的水稻4-羟基苯丙酮酸双加氧酶（HPPD）基因突变体库构建及抗HPPD抑制剂类除草剂种质筛选()

江苏农业学报[ISSN:1006-6977/CN:61-1281/TN]

卷:
42
期数:
2026年05期
页码:
865-875
栏目:
遗传育种·生理生化
出版日期:
2026-05-31

文章信息/Info

Title:
Genome editing-mediated mutant library construction of rice 4-hydroxyphenylpyruvate dioxygenase (HPPD) gene and identification of germplasm with resistance to HPPD-inhibiting herbicides
作者:
郭冬姝; 杨郁文; 李笑; 余月
(江苏省农业科学院种质资源与生物技术研究所,江苏南京210014)
Author(s):
GUO Dongshu; YANG Yuwen; LI Xiao; YU Yue
(Institute of Germplasm Resources and Biotechnology, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China)
关键词:
水稻; HPPD基因; 抗HPPD抑制剂类除草剂种质
Keywords:
rice; HPPD gene; germplasm resistant to HPPD-inhibiting herbicides
分类号:
S511
DOI:
doi:10.3969/j.issn.1000-4440.2026.05.001
文献标志码:
A
摘要:
为鉴定更多的抗性新位点,创制更多的抗4-羟基苯丙酮酸双加氧酶(HPPD)抑制剂类除草剂水稻种质,实现HPPD抑制剂类除草剂在水稻生产中的应用,本研究采用基因编辑技术介导的体内定向进化策略,针对水稻HPPD基因的编码区、上游启动子区及5′非翻译区,设计300个基因编辑靶标位点,分别利用碱基编辑器和多基因敲除突变载体构建突变体库,并进行基因突变当代植株基因型鉴定和测序,及突变体对除草剂硝磺草酮的抗性鉴定。结果表明,在HPPD基因编码区的99个基因编辑靶标位点检测到碱基突变,在编码区上游区域检测到43种不同形式的纯合插入突变或缺失突变。鉴定到2个对0.3 μmol/L硝磺草酮抗性提高的突变体株系,包括1个编码区碱基突变的突变体株系和1个编码区上游碱基缺失的突变体株系。编码区碱基突变的突变体株系的突变类型为HPPD蛋白第419位甘氨酸(Glycine,G)突变为天冬氨酸(Aspartic acid,D)；编码区上游碱基缺失突变体株系存在168 bp的碱基缺失,且其HPPD基因表达水平是野生型的8.0倍。本研究为培育抗HPPD抑制剂类除草剂的水稻品种提供了新的遗传材料和方法。
Abstract:
To identify more new resistance sites, create more rice germplasm resistant to 4-hydroxyphenylpyruvate dioxygenase (HPPD)-inhibiting herbicides, and realize the application of HPPD-inhibiting herbicides in rice production, this study used genome editing technology-mediated in vivo directed evolution strategy to design 300 gene editing target sites for the coding region, upstream promoter region and 5′ untranslated region of rice HPPD gene. The base editor and multi-gene knockout mutation vectors were used to construct the mutant library, and the genotype identification and sequencing of transgenic contemporary plants were carried out, and the resistance of the mutants to mesotrione was identified. The results showed that base mutations were detected at 99 genome editing target sites in the coding region of HPPD gene, and 43 different forms of homozygous insertion mutations or deletion mutations were detected in the upstream region of the coding region. Two mutant lines with increased resistance to 0.3 μmol/L mesotrione were identified, including one mutant line carrying a base mutation in the coding region and one mutant line carrying a base deletion mutation in the upstream region of the coding region. The mutation type in the mutant line carrying a base mutation in the coding region was the mutation of glycine (G) to aspartic acid (D) at the 419th site of the HPPD protein. The mutant line carrying a base deletion in the upstream of the coding region had a base deletion of 168 bp, and its HPPD gene expression level was 8.0 times that of the wild type. This study provides new genetic materials and methods for the cultivation of rice varieties resistant to HPPD-inhibiting herbicides.

参考文献/References:

[1]FAROOQ M, SIDDIQUE K H M, REHMAN H, et al. Rice direct seeding:experiences,challenges and opportunities[J]. Soil and Tillage Research,2011,111(2):87-98.
[2]廖萍,翁文安,高辉,等. 我国直播稻栽培技术应用现状与发展建议[J]. 中国农业科学,2024,57(24):4854-4870.
[3]YANG Y W, ZHOU Z Z, LIU T L, et al. Multisite mutagenesis of 4-hydroxyphenylpyruvate dioxygenase (HPPD) enhances rice resistance to HPPD inhibitors and its carotenoid contents[J]. Journal of Agricultural and Food Chemistry,2024,72(40):22063-22072.
[4]SYNOWIEC A, CZEKAJ M, VERDEGUER M, et al. Herbicides in use:current status and perspectives in the different biogeographic regions of Europe[J]. Journal of Agricultural and Food Chemistry,2025,73(36):22089-22109.
[5]JIN M, CHEN L, DENG X W, et al. Development of herbicide resistance genes and their application in rice[J]. The Crop Journal,2022,10(1):26-35.
[6]DUKE S O. Why have no new herbicide modes of action appeared in recent years?[J]. Pest Management Science,2012,68(4):505-512.
[7]SIEHL D L, TAO Y M, ALBERT H, et al. Broad 4-hydroxyphenylpyruvate dioxygenase inhibitor herbicide tolerance in soybean with an optimized enzyme and expression cassette[J]. Plant Physiology,2014,166(3):1162-1176.
[8]NDIKURYAYO F, MOOSAVI B, YANG W C, et al. 4-hydroxyphenylpyruvate dioxygenase inhibitors:from chemical biology to agrochemicals[J]. Journal of Agricultural and Food Chemistry,2017,65(39):8523-8537.
[9]WU Y Y, XIAO N, CAI Y, et al. CRISPR-Cas9-mediated editing of the OsHPPD 3′ UTR confers enhanced resistance to HPPD-inhibiting herbicides in rice[J]. Plant Communications,2023,4(5):100605.
[10]CHEN L, LIU R, TAN Q, et al. Improving the herbicide resistance of rice 4-hydroxyphenylpyruvate dioxygenase by DNA shuffling basis-directed evolution[J]. Journal of Agricultural and Food Chemistry,2023,71(41):15186-15193.
[11]WANG M G, ZHONG Y L, HE Y X, et al. Identifying resistant mutations in the herbicide target site of the plant 4-hydroxyphenylpyruvate dioxygenase[J]. Plant Biotechnology Journal,2025,23(1):75-77.
[12]LU Y, WANG J Y, CHEN B, et al. A donor-DNA-free CRISPR/Cas-based approach to gene knock-up in rice[J]. Nature Plants,2021,7(11):1445-1452.
[13]DONG J, DONG J Q, WANG X L, et al. Structure-based directed evolution of rice 4-hydroxyphenylpyruvate dioxygenase confers enhanced herbicide tolerance[J]. Journal of Agricultural and Food Chemistry,2025,73(30):18923-18931.
[14]MAEDA H, MURATA K, SAKUMA N, et al. A rice gene that confers broad-spectrum resistance to β-triketone herbicides[J]. Science,2019,365(6451):393-396.
[15]WANG Y, PHELPS A, GODBEHERE A, et al. Revolutionizing agriculture with CRISPR technology:applications,challenges,and future perspectives[J]. Biotechnology Journal,2025,20(9):e70113.
[16]QIAN H J, SHI H Z. Herbicide-resistant 4-hydroxyphenylpyruvate dioxygenase variants identified via directed evolution[J]. Journal of Experimental Botany,2024,75(22):7096-7106.
[17]WANG X, PAN W B, SUN C, et al. Creating large-scale genetic diversity in Arabidopsis via base editing-mediated deep artificial evolution[J]. Genome Biology,2024,25(1):215.
[18]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.
[19]MIAO J, GUO D S, ZHANG J Z, et al. Targeted mutagenesis in rice using CRISPR-Cas system[J]. Cell Research,2013,23(10):1233-1236.
[20]WANG Y H, ZHAO Y, BOLLAS A, et al. Nanopore sequencing technology,bioinformatics and applications[J]. Nature Biotechnology,2021,39(11):1348-1365.
[21]HUA K, TAO X P, LIANG W Y, et al. Simplified adenine base editors improve adenine base editing efficiency in rice[J]. Plant Biotechnology Journal,2020,18(3):770-778.
[22]RICHTER M F, ZHAO K T, ETON E, et al. Phage-assisted evolution of an adenine base editor with improved Cas domain compatibility and activity[J]. Nature Biotechnology,2020,38(7):883-891.
[23]ZENG D C, LIU T L, TAN J T, et al. PhieCBEs:plant high-efficiency cytidine base editors with expanded target range[J]. Molecular Plant,2020,13(12):1666-1669.
[24]PARK T K, KANG I A, PARK C H, et al. Inhibition of 4-hydroxyphenylpyruvate dioxygenase expression by brassinosteroid reduces carotenoid accumulation in Arabidopsis[J]. Journal of Experimental Botany,2022,73(5):1415-1428.
[25]LI Y, LING X T, ZHANG W T, et al. OsDNR1 as a key regulator of rice resistance to 4-hydroxyphenylpyruvate dioxygenase-inhibiting herbicides[J]. Journal of Integrative Plant Biology,2025,67(9):2262-2264.
[26]SUN C, LI H C, LIU Y J, et al. Iterative recombinase technologies for efficient and precise genome engineering across kilobase to megabase scales[J]. Cell,2025,188(17):4693-4710.
[27]ZHOU Z Z, JIANG Q, QIU Z Y, et al. Differential resistance to acetyl-CoA carboxylase inhibitors in rice:insights from two distinct target-site mutations[J]. Journal of Agricultural and Food Chemistry,2024,72(21):12029-12044.
[28]FALK J O N, KRAUB N, DHNHARDT D, et al. The senescence associated gene of barley encoding 4-hydroxyphenylpyruvate dioxygenase is expressed during oxidative stress[J]. Journal of Plant Physiology,2002,159(11):1245-1253.
[29]REN W W, ZHAO L X, ZHANG L D, et al. Molecular cloning and characterization of 4-hydroxyphenylpyruvate dioxygenase gene from Lactuca sativa[J]. Journal of Plant Physiology,2011,168(10):1076-1083.
[30]YU X H, WEN X, DONG J Q, et al. Hydroxylation of HPPD facilitates its PUB11-mediated ubiquitination and degradation in response to oxidative stress in Arabidopsis[J]. Plant Communications,2025,6(11):101521.

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备注/Memo

备注/Memo:
收稿日期：2025-12-31基金项目：江苏省重点研发计划项目——现代农业(BE2022365)作者简介：郭冬姝(1988-),女,辽宁锦州人,博士,副研究员,主要从事水稻基因编辑技术和抗除草剂育种研究。(Tel)13811136765;(E-mail)guodongshuguodong@163.com
更新日期/Last Update: 2026-06-17