Creation of Germplasm Resources of Two-line Sterile Rice Resistant to ACCase-inhibiting Herbicides

doi:10.16819/j.1001-7216.2026.241109

Abstract

Abstract:

【Objective】The creation of herbicide-resistant rice germplasm is essential for breeding new herbicide-resistant rice varieties. By using CRISPR/Cas9 gene editing technology, the acetyl-CoA carboxylase OsACC1 gene was edited with the sterile line An 9S as the material, and a two-line sterile rice germplasm with herbicide resistance was created. 【Method】By using CRISPR/Cas9 technology, a single-base mutation vector, ePE2-ACC-I1879V, was constructed with acetyl-CoA carboxylase OsACC1 as the target gene. With An 9S as the transformation material, transgenic plants were obtained by Agrobacterium-mediated transformation. The genotypes and phenotypes were identified by sequencing the mutation sites of transgenic plants, conducting herbicide spraying tests and agronomic traits analysis. 【Result】Twenty-four ACC1 ^I1879V mutant plants were obtained as confirmed by molecular detection in T₀ generation; A total of 96 homozygous mutant plants were obtained from 24 lines in the T₁ generation through segregation and screening, all of which had herbicide resistance. High-throughput sequencing showed that the three tested lines (NS512, NS513, NS514) contained no exogenous transgenic elements. Furthermore, the survival rate after spraying with 4% haloxyfop-P-methyl was 100%. Compared with the control An 9S, the NS512 line had increased plant height and wider flag leaf; the NS513 line had increased plant height and longer and wider flag leaf; and the NS514 line had a narrower flag leaf; the fertility of the three tested lines remained unchanged, and there was no significant difference in the number of effective panicles per plant and the panicle length. The transgenic safety evaluation indicated that the OsACC1 protein shares no sequence similarity with any known toxic proteins, antinutritional factors or allergens. Therefore, it is considered safe for human consumption. 【Conclusion】Using CRISPR/Cas9 gene editing technology, we obtained homozygous mutant lines that are resistant to ACCase inhibitor herbicides. These lines are genetically stable, free of transgenic components, exhibit unaltered fertility, and are safe, providing fundamental materials for herbicide-resistant breeding in rice.

Key words: CRISPR/Cas9, gene editing, acetyl coenzyme a carboxylase, sterile line

摘要：

【目的】创制抗除草剂水稻新种质是培育抗除草剂水稻新品种的前提。利用 CRISPR/Cas9 基因编辑技术，以安9S不育系为材料，编辑乙酰辅酶A羧化酶OsACC1基因，创制具有抗除草剂特性的两系不育系水稻新种质。【方法】利用CRISPR/Cas9技术，以乙酰辅酶A羧化酶OsACC1为靶基因，构建单碱基突变载体ePE2-ACC-I1879V，以安9S为转化材料，利用农杆菌介导法转化获得转基因植株，通过对转基因植株的突变位点进行测序结合除草剂喷施试验、农艺性状分析，鉴定基因型及表型。【结果】经分子水平检测验证，T₀代获得 ACC1^I1879V突变植株24株；经分离筛选，T₁代24个株系共获得96株纯合突变单株，均具有除草剂抗性。高通量测序结果显示，三个试验组株系(NS512、NS513、NS514)均不含外源转基因成分，且喷施4%甲禾灵存活率仍达到100%。与对照安9S相比，NS512株系株高增加，剑叶变宽；NS513株系株高增高，剑叶变长变宽；NS514株系剑叶变窄；三个试验组株系的育性未发生改变，单株有效穗数、穗长、均没有显著差异。转基因安全性评价分析表明，OsACC1基因编码蛋白与已知毒蛋白、抗营养因子及过敏原均无相似序列，不存在毒性、致敏性问题。【结论】利用CRISPR/Cas9 基因编辑技术，能够获得具有抗ACCase抑制剂类除草剂特性、稳定遗传、不含转基因成分、育性不发生改变、安全无害的纯合株系，可为水稻抗除草剂育种提供材料基础。

关键词: CRISPR/Cas9, 基因编辑, 乙酰辅酶A羧化酶, 不育系

GUO Jinmeng, TAO Kailiang, ZHU Jun, ZHAO Xueyu, LI Kang, GAN Quan, LIN Cuixiang, WANG Nansheng, YU Hao, SONG Fengshun, LI Juan, SHI Yingyao, NI Dahu. Creation of Germplasm Resources of Two-line Sterile Rice Resistant to ACCase-inhibiting Herbicides[J]. Chinese Journal OF Rice Science, 2026, 40(2): 223-234.

郭金梦, 陶开亮, 朱俊, 赵学宇, 李康, 甘泉, 林翠香, 汪楠胜, 于豪, 宋丰顺, 李娟, 石英尧, 倪大虎. 水稻抗ACCase抑制剂类除草剂两系不育系种质资源的创制[J]. 中国水稻科学, 2026, 40(2): 223-234.

Figures/Tables 12

References 40

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引物名称 Primer name	正向引物 Forward primer (5’-3’)	反向引物 Reverse primer (5’-3’)
ACC-1	GGACCTTGAAGATGTCCACTCC	GCAGGAACATAACTGAGCCACC
T-1	GATATACTTGGATGATGGCA	GAGCCTGTGGAAGAAGCTG
T-2	TAGACATGCAATGCTCATTATC	ACAGGTAAGAGCTAAGCATC
T-3	GAATTCTGCAGGTCACTGGATT	TGACCCACTACCAGGCCCTG
T-4	CGCCACCCTCATCCACCAGTCT	CTCCTGAGACATTGGGTACTGC
HITOM	ggagtgagtacggtgtgcCTGTCATAGCACATAAGATGCA	gagttggatgctggatggTCCAACAGTTCTTCCAGTCACA

引物名称 Primer name	正向引物 Forward primer (5’-3’)	反向引物 Reverse primer (5’-3’)
ACC-1	GGACCTTGAAGATGTCCACTCC	GCAGGAACATAACTGAGCCACC
T-1	GATATACTTGGATGATGGCA	GAGCCTGTGGAAGAAGCTG
T-2	TAGACATGCAATGCTCATTATC	ACAGGTAAGAGCTAAGCATC
T-3	GAATTCTGCAGGTCACTGGATT	TGACCCACTACCAGGCCCTG
T-4	CGCCACCCTCATCCACCAGTCT	CTCCTGAGACATTGGGTACTGC
HITOM	ggagtgagtacggtgtgcCTGTCATAGCACATAAGATGCA	gagttggatgctggatggTCCAACAGTTCTTCCAGTCACA

甲禾灵浓度^1） Concentrations of haloxyfop-P-methyl^1）	对照组存活率 Survival rate of control group	试验组存活率 Survival rate of experimental group
甲禾灵浓度^1） Concentrations of haloxyfop-P-methyl^1）	安9S An 9S	NS512	NS513	NS514
0	100.0	100.0	100.0	100.0
2	0	100.0	100.0	100.0
4	0	100.0	100.0	100.0
6	0	91.6	75.0	79.2
8	0	66.7	87.5	91.6
10	0	33.3	25.0	79.2

甲禾灵浓度^1） Concentrations of haloxyfop-P-methyl^1）	对照组存活率 Survival rate of control group	试验组存活率 Survival rate of experimental group
甲禾灵浓度^1） Concentrations of haloxyfop-P-methyl^1）	安9S An 9S	NS512	NS513	NS514
0	100.0	100.0	100.0	100.0
2	0	100.0	100.0	100.0
4	0	100.0	100.0	100.0
6	0	91.6	75.0	79.2
8	0	66.7	87.5	91.6
10	0	33.3	25.0	79.2

农艺性状 Agronomic trait	对照组 Control group	试验组 Experimental group
农艺性状 Agronomic trait	安9S An 9S	NS512	NS513	NS514
株高 Plant height(cm)	69.65±5.10	84.05±2.47^**	82.63±1.59^**	73.30±2.35
抽穗期Heading date(d)	70	70	70	70
单株有效穗数Effective panicle number per plant	9.37±1.85	9.75±1.67	10.88±1.13	9.33±0.52
穗长Panicle length(cm)	25.00±0.51	23.12±2.28	24.40±0.90	24.62±0.43
剑叶长度Flag leaf length(cm)	33.52±1.52	34.60±3.74	39.59±1.53^**	32.81±3.02
剑叶宽度Flag leaf width(cm)	1.49±0.06	1.68±0.08^**	1.70±0.10^**	1.37±0.07^*