水稻抗ACCase抑制剂类除草剂两系不育系种质资源的创制

doi:10.16819/j.1001-7216.2026.241109

摘要/Abstract

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

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

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.

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.

图/表 12

图1 OsACC1在第34外显子上的靶位点

Fig. 1. Target site of OsACC1 on exon 34

表1 本研究中所用的引物

Table 1. Primers used in this study

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

图2 T0代转基因植株（部分）测序结果

Fig. 2. Sequencing results of T0 transgenic plants (partial)

图3 T1代T-DNA分离（部分）检测结果 M: 标记；1~22: T1代单株；23: 阳性对照；24: 阴性对照。

Fig. 3. T-DNA isolation (partial) test results of T1 generation M, Marker；Lanes 1-22, T1 plants; Lane 23, Positive control; Lane 24, Negative control.

表2 喷施不同浓度甲禾灵后的存活率

Table 2. Survival rate after spraying different concentrations of haloxyfop-P-methyl %

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

图4 对照组和试验组NS514经不同浓度甲禾灵处理后对比图（田间）

Fig. 4. Comparison of control group and experimental group NS514 treated with different concentrations of haloxyfop-P-methyl (field)

图5 NS514在0%、4%、8%甲禾灵处理下的生长状况（标尺=10 cm）

Fig. 5. Growth of NS514 under 0%, 4% and 8% haloxyfop-P-methyl treatments (bar=10 cm)

图6 试验组在Ⅰ期、Ⅱ期、Ⅲ期及割茬后的花粉育性 Ⅰ、Ⅱ、Ⅲ期分别指播种于2024年6月7日、18日和25日。

Fig. 6. Pollen fertility of the experimental group in the first, second and third stages and after stubble cutting The sowing dates for stage Ⅰ, Ⅱ, and Ⅲ were June 7, 18 and 25, 2024, respectively.

图7 不同浓度甲禾灵处理后的株高和穗粒数不同小写字母表示同一株系在不同处理之间差异显著（P<0.05）。

Fig. 7. Plant height and grain number per panicle of mutants treated with different concentrations of haloxyfop-P-methyl Different lowercase letters indicate significant difference among different treatments (P < 0.05).

表3 T3代纯合株系农艺性状

Table 3. Agronomic traits of T3 homozygous lines

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^*

图8 OsACC1蛋白在 NCBI 中与已知毒蛋白序列同源性比对结果

Fig. 8. Results of homology comparison between OsACC1 protein and known toxic protein sequence in NCBI

图9 OsACC1在Allergen Online中的检索结果

Fig. 9. Search results of OsACC1 in Allergen Online

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