利用CRISPR/Cas9技术改良南粳46抽穗期

doi:10.16819/j.1001-7216.2025.240707

中国水稻科学 ›› 2025, Vol. 39 ›› Issue (6): 760-770.DOI: 10.16819/j.1001-7216.2025.240707

利用CRISPR/Cas9技术改良南粳46抽穗期

陈伟¹^,³, 叶元妹¹, 赵剑华¹, 冯志明¹^,², 陈宗祥¹^,², 胡珂鸣¹^,², 左示敏¹^,²^,^*()

¹扬州大学农学院, 江苏省作物基因组学和分子育种重点实验室/生物育种钟山实验室/植物功能基因组学教育部重点实验室, 江苏扬州 225009
²扬州大学江苏省粮食作物现代产业技术协同创新中心/江苏省作物遗传生理重点实验室, 江苏扬州 225009
³江苏瑞华农业科技有限公司，江苏宿迁 223800

收稿日期:2024-07-09 修回日期:2024-08-12 出版日期:2025-11-10 发布日期:2025-11-19
通讯作者: * email:smzuo@yzu.edu.cn
基金资助:
生物育种钟山实验室项目(ZSBBL-KY2023-06-3);宿迁英才群英计划青年资助项目([2022]QNXM-0035);江苏省种业振兴揭榜挂帅项目(JBGS[2021]001)

Modifying Heading Date of Nanjing 46 via CRISPR/Cas9-mediated Genome Editing

CHEN Wei¹^,³, YE Yuanmei¹, ZHAO Jianhua¹, FENG Zhiming¹^,², CHEN Zongxiang¹^,², HU Keming¹^,², ZUO Shimin¹^,²^,^*()

¹Jiangsu Key Laboratory of Crop Genomics and Molecular Breeding/Zhongshan Biological Breeding Laboratory/Key Laboratory of Plant Functional Genomics of the Ministry of Education, Agricultural College of Yangzhou University, Yangzhou 225009, China
²Jiangsu Co-Innovation Center for Modern Production Technology of Grain Crops/Jiangsu Key Laboratory of Crop Genetics and Physiology, Yangzhou University, Yangzhou 225009, China
³Jiangsu Ruihua Agricultural Technology Co. Ltd, Suqian 223800, China

Received:2024-07-09 Revised:2024-08-12 Online:2025-11-10 Published:2025-11-19
Contact: * email:smzuo@yzu.edu.cn

摘要/Abstract

摘要：

【目的】抽穗期是决定水稻品种种植区域和生态适应性的关键农艺性状。本研究通过对优良食味中熟晚粳品种南粳46(简称NJ46)的抽穗期基因进行编辑，以缩短NJ46的生育期，扩大其种植区域。【方法】利用CRISPR/Cas9技术，以DTH8和Ghd2为靶基因，构建pCAMBIA1305-Actin:Cas9-sgRNA^DTH8与pCAMBIA1305-Actin:Cas9-sgRNA^Ghd2编辑载体，用农杆菌介导的植物转化方法转化NJ46，筛选不含外源成分的纯合编辑系，并考察其抽穗期、农艺性状及品质相关性状。【结果】对DTH8的编辑获得了3个比NJ46抽穗期提早22~36 d的无外源成分纯合突变系(NJ46-dth8-1~-3)，对Ghd2的编辑获得了3个比NJ46抽穗期提早14~15 d的无外源成分纯合突变系(NJ46-ghd2-1~-3)。分别在苏中和苏北测定了各基因纯合突变系的抽穗期、主要农艺性状及品质性状，与相应生态区推广品种南粳9108、南粳518进行比较，发现NJ46-ghd2-1单株产量显著高于南粳9108，但抽穗期和品质与之相当，好于另外2个敲除系；NJ46-dth8-1与南粳518在单株产量、主要品质性状上均无显著差异，好于另外2个敲除系。【结论】定向编辑南粳46中的DTH8和Ghd2基因，获得适宜苏北和苏中生态区种植的优良食味粳稻新种质，有助于扩大优良食味水稻品种的推广种植区域。

关键词: 水稻, CRISPR/Cas9, 基因编辑, 抽穗期, DTH8, Ghd2

Abstract:

【Objective】 Heading date is a key agronomic trait determining the regional and ecological adaptation of rice cultivars. We edited the heading date genes of the medium-maturing late japonica rice variety Nanjing 46 (NJ46) with excellent tasting to shorten its heading date thereby potentially expanding its planting area.【Method】 Using CRISPR/Cas9 technology, the editing constructs, pCAMBIA1305-Actin:Cas9-sgRNA_DTH8 and pCAMBIA1305-Actin:Cas9-sgRNA_Ghd2 targeting DTH8 and Ghd2, respectively, were developed and transformed into NJ46 via Agrobacterium-mediated transformation. Homozygous editing lines without exogenous components were screened and evaluated on heading date, agronomic traits and quality-related traits. 【Result】 Three homozygous mutant lines were obtained. The NJ46-dth8 lines (NJ46-dth8-1 to -3) presented 22-36 days earlier than NJ46 on heading date, while the NJ46-ghd2 lines (NJ46-ghd2-1 to -3) showed 14-15 days earlier. The heading date, major agronomic traits, and quality related components of the homozygous mutant lines were evaluated in different areas of central and northern regions of Jiangsu Province, and compared with the widely cultivated varieties Nanjing 9108 and Nanjing 518 in their respectively ecological zones. NJ46-ghd2-1 exhibited a significantly higher yield per plant than Nanjing 9108, while showing comparable heading date and quality traits, and performed better than the other two knockout lines. NJ46-dth8-1 showed no significant differences from Nanjing 518 in yield per plant and major quality traits, and performed better than the other two knockout lines.【Conclusion】 Knockout of DTH8 and Ghd2 genes in NJ46 yielded new germplasms suitable for planting in the northern and central region of Jiangsu Province, respectively, which will help expand the planting area of this excellent-tasting rice variety.

Key words: rice, CRISPR/Cas9, genetic editing, heading date, DTH8, Ghd2

陈伟, 叶元妹, 赵剑华, 冯志明, 陈宗祥, 胡珂鸣, 左示敏. 利用CRISPR/Cas9技术改良南粳46抽穗期[J]. 中国水稻科学, 2025, 39(6): 760-770.

CHEN Wei, YE Yuanmei, ZHAO Jianhua, FENG Zhiming, CHEN Zongxiang, HU Keming, ZUO Shimin. Modifying Heading Date of Nanjing 46 via CRISPR/Cas9-mediated Genome Editing[J]. Chinese Journal OF Rice Science, 2025, 39(6): 760-770.

图/表 13

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引物名称 Primer name	引物序列（5'-3'） Primer sequence（5'-3'）	用途 Purpose
DTH8-gF-1	GGCACGTGAGCCGCATCATGAAGCGG	构建 DTH8靶点1敲除载体 Construction of DTH8 target 1 knockout vector
DTH8-gR-1	AAACCCGCTTCATGATGCGGCTCACG
DTH8-gF-2	GGCAAAGATCTCCAAGGAGTCGAAGG	构建 DTH8靶点2敲除载体 Construction of DTH8 target 2 knockout vector
DTH8-gR-2	AAACCCTTCGACTCCTTGGAGATCTT
Ghd2-gF	GGCAGCGTCGGGCAGCTGCTCTCGAGG	构建 Ghd2敲除载体 Construction of Ghd2 knockout vector
Ghd2-gR	AAACCCTCGAGAGCAGCTGCCCGACGC	构建 Ghd2敲除载体 Construction of Ghd2 knockout vector
Hpt-F	CGGAAGTGCTTGACATTG	转基因检测 Transgenic detection
Hpt-R	GACCTCGTATTGGGAATCC	转基因检测 Transgenic detection
sgRNA-F	AAGGAATCTTTAAACATACGAACAGATC	单克隆测序鉴定 Monoclonal sequencing identification Sequencing for Monoclonal
Cas9-F	ACTCGAACGCGACCAACTTA	载体检测 Vector assay
Cas9-R	ATCTGCAGAATTGCCCTTCG	载体检测 Vector assay
DTH8-F	CGAAGGAGCAGGACAGGTTC	DTH8 敲除靶点测序鉴定 Sequencing for DTH8 knockout target
DTH8-R	TTGATGGTCTTCCGCTTCTCG	DTH8 敲除靶点测序鉴定 Sequencing for DTH8 knockout target
Ghd2-F	TGACGCCTTCCTGTGCCAGGGGTG	Ghd2 敲除靶点测序鉴定 Sequencing for Ghd2 knockout target
Ghd2-R	GTCCGACGACTCCACCGCCATCTC	Ghd2 敲除靶点测序鉴定 Sequencing for Ghd2 knockout target

引物名称 Primer name	引物序列（5'-3'） Primer sequence（5'-3'）	用途 Purpose
DTH8-gF-1	GGCACGTGAGCCGCATCATGAAGCGG	构建 DTH8靶点1敲除载体 Construction of DTH8 target 1 knockout vector
DTH8-gR-1	AAACCCGCTTCATGATGCGGCTCACG
DTH8-gF-2	GGCAAAGATCTCCAAGGAGTCGAAGG	构建 DTH8靶点2敲除载体 Construction of DTH8 target 2 knockout vector
DTH8-gR-2	AAACCCTTCGACTCCTTGGAGATCTT
Ghd2-gF	GGCAGCGTCGGGCAGCTGCTCTCGAGG	构建 Ghd2敲除载体 Construction of Ghd2 knockout vector
Ghd2-gR	AAACCCTCGAGAGCAGCTGCCCGACGC	构建 Ghd2敲除载体 Construction of Ghd2 knockout vector
Hpt-F	CGGAAGTGCTTGACATTG	转基因检测 Transgenic detection
Hpt-R	GACCTCGTATTGGGAATCC	转基因检测 Transgenic detection
sgRNA-F	AAGGAATCTTTAAACATACGAACAGATC	单克隆测序鉴定 Monoclonal sequencing identification Sequencing for Monoclonal
Cas9-F	ACTCGAACGCGACCAACTTA	载体检测 Vector assay
Cas9-R	ATCTGCAGAATTGCCCTTCG	载体检测 Vector assay
DTH8-F	CGAAGGAGCAGGACAGGTTC	DTH8 敲除靶点测序鉴定 Sequencing for DTH8 knockout target
DTH8-R	TTGATGGTCTTCCGCTTCTCG	DTH8 敲除靶点测序鉴定 Sequencing for DTH8 knockout target
Ghd2-F	TGACGCCTTCCTGTGCCAGGGGTG	Ghd2 敲除靶点测序鉴定 Sequencing for Ghd2 knockout target
Ghd2-R	GTCCGACGACTCCACCGCCATCTC	Ghd2 敲除靶点测序鉴定 Sequencing for Ghd2 knockout target

靶点 Target	潜在脱靶序列 Off-target sequence	脱靶指数 Off-score	潜在基因 Gene	区域 Region
DTH8-Target1	ACATCAGCCGCATCATGAAGAAG	0.153	LOC_Os01g61810	CDS
	ACATCAGCCGCATCATGAAGAAG	0.153	LOC_Os05g38820	CDS
	ACATGAGCAGCATCAAGAAGGAG	0.152	LOC_Os02g16909	Utr
	TCGTGAGTCGCTTCATGGAGCGG	0.117	LOC_Os08g36774	Intron
DTH8-Target2	CAAGAGCTCCAGGGAGACGGAGG	0.168	LOC_Os08g13020	Intron
	CAAAATCCCCAAGGAGTAGACAG	0.126	LOC_Os07g46910	CDS
	CAAGATCTCCAAAGTATTGATGG	0.119	Null	Intergenic
	GGAGCTCTCCAATGAGTCGATGG	0.110	LOC_Os01g32660	Intron
Ghd2-Target	CCTAGCCGAGCATCAAAACCGGG	0.270	Null	Intergenic
	GCGTCGTGCAGCTGGTCTCGCGG	0.197	LOC_Os01g03420	CDS
	GCGTCAGGGAGCTGCTATCGAGG	0.157	LOC_Os04g43350	CDS
	GCCTCCGGCTGCTGATCTCGTGG	0.143	LOC_Os05g45430	CDS

靶点 Target	潜在脱靶序列 Off-target sequence	脱靶指数 Off-score	潜在基因 Gene	区域 Region
DTH8-Target1	ACATCAGCCGCATCATGAAGAAG	0.153	LOC_Os01g61810	CDS
	ACATCAGCCGCATCATGAAGAAG	0.153	LOC_Os05g38820	CDS
	ACATGAGCAGCATCAAGAAGGAG	0.152	LOC_Os02g16909	Utr
	TCGTGAGTCGCTTCATGGAGCGG	0.117	LOC_Os08g36774	Intron
DTH8-Target2	CAAGAGCTCCAGGGAGACGGAGG	0.168	LOC_Os08g13020	Intron
	CAAAATCCCCAAGGAGTAGACAG	0.126	LOC_Os07g46910	CDS
	CAAGATCTCCAAAGTATTGATGG	0.119	Null	Intergenic
	GGAGCTCTCCAATGAGTCGATGG	0.110	LOC_Os01g32660	Intron
Ghd2-Target	CCTAGCCGAGCATCAAAACCGGG	0.270	Null	Intergenic
	GCGTCGTGCAGCTGGTCTCGCGG	0.197	LOC_Os01g03420	CDS
	GCGTCAGGGAGCTGCTATCGAGG	0.157	LOC_Os04g43350	CDS
	GCCTCCGGCTGCTGATCTCGTGG	0.143	LOC_Os05g45430	CDS

靶位点 Target	阳性株数Positive plants	突变率 Mutation ratio(%)	突变基因型比率 Mutant genotype ratio(%)			突变类型比率 Ratio of mutation types(%)
靶位点 Target	阳性株数Positive plants	突变率 Mutation ratio(%)	纯合突变率Homozygous	杂合突变率Heterozygous	双等位突变率 Bi-allele	碱基插入 Insert	碱基缺失Deletion
DTH8 Target 1	36	36.1	30.8	15.4	53.8	0.0	100.0
DTH8 Target 2	36	44.4	31.3	18.8	50.0	100.0	0.0
Ghd2 Target	25	84.0	28.6	23.8	47.6	9.5	90.5

利用CRISPR/Cas9技术改良南粳46抽穗期

Modifying Heading Date of Nanjing 46 via CRISPR/Cas9-mediated Genome Editing

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T₁代基因型 Genotype of T₁generation	T₂代分离群体抽穗期 Heading date of T₂segregating population plants(d)			1:3分离比的渐进显著性 Progressive significance of the separation ratio of 1:3
T₁代基因型 Genotype of T₁generation	早 Earlier	无变化 No change	晚 Later
Aa₁	44	142	0	0.672
Aa₂	62	171	0	0.570
Aa₃	56	162	0	0.815
Bb₁	53	170	0	0.671
Bb₂	47	152	0	0.653
Bb₃	62	200	0	0.618

地点 Site	株系 Line	株高 Plant height (cm)	穗长 Panicle length (cm)	单株有效穗 Effective panicle per plant	每穗实粒数 Number of grains per panicle	千粒重 1000-grain weigh(g)	单株产量 Yield per plant(g)
高邮Gaoyou	NJ9108	96.31±0.74 b	16.82±0.41 a	15.52±0.87 a	142.10±5.81 a	27.50±0.01 a	60.63±2.18 a
	NJ46-ghd2-1	90.23±2.23 a	16.54±0.62 a	16.25±1.01 b	148.33±3.21 b	28.51±0.05 b	68.51±3.32 b
	NJ46-ghd2-2	91.64±1.14 a	16.13±0.45 a	16.72±1.51 b	146.22±7.08 ab	28.32±0.03 b	69.10±4.23 b
	NJ46-ghd2-3	91.12±1.43 a	16.64±0.32 a	15.61±1.11 ab	152.24±3.21 b	28.13±0.02 ab	66.74±3.61 b
宿迁 Suqian	NJ518	100.20±1.14 b	17.80±0.36 b	15.00±1.51 b	155.31±5.81 b	27.80±0.03 a	64.82±4.21 b
	NJ46-dth8-1	98.55±2.32 b	18.31±0.28 b	14.23±0.87 a	156.24±5.77 b	28.31±0.02 a	62.82±3.32 b
	NJ46-dth8-2	98.12±0.74 b	18.62±0.28 b	14.94±0.87 b	151.35±5.77 b	28.12±0.03 a	63.34±2.18 b
	NJ46-dth8-3	89.30±0.74 a	15.53±0.30 a	14.41±0.87 a	122.11±5.77 a	30.52±0.01 b	53.61±2.18 a

地点 Site	株系 Line	整精米率 Rate of milled rice(%)	垩白粒率 Chalky grain percentage(%)	垩白度 Chalkiness(%)	食味值 Edibility value	直链淀粉含量 Amylose content(%)
高邮 Gaoyou	NJ9108	70.56±0.26 b	32.91±0.18 c	7.07±0.03 b	71.00±1.52 b	11.45±0.02 b
	NJ46-ghd2-1	72.21±0.29 b	12.87±0.20 a	2.60±0.02 a	68.70±1.46 ab	10.14±0.03 a
	NJ46-ghd2-2	64.52±0.33 a	17.26±0.14 a	6.95±0.02 b	68.30±1.38 a	10.49±0.03 a
	NJ46-ghd2-3	71.93±0.26 b	23.82±0.15 ab	5.40±0.01 ab	72.00±1.21 c	10.32±0.04 a
宿迁 Suqian	NJ518	72.22±0.26 a	22.26±0.15 a	5.58±0.02 a	76.70±1.44 b	10.00±0.02 a
	NJ46-dth8-1	73.04±0.26 b	26.14±0.14 a	6.45±0.01 a	74.30±1.26 b	10.31±0.02 a
	NJ46-dth8-2	72.06±0.26 a	35.72±0.13 b	9.00±0.02 b	53.70±2.20 a	9.81±0.04 a
	NJ46-dth8-3	72.32±0.26 a	28.26±0.23 a	6.60±0.02 a	60.30±1.65 a	10.72±0.03 a

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