利用CRISPR/Cas9系统定向改良水稻粒长和穗粒数性状

doi:10.16819/j.1001-7216.2017.7029 223

中国水稻科学 ›› 2017, Vol. 31 ›› Issue (3): 223-231.DOI: 10.16819/j.1001-7216.2017.7029 223

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利用CRISPR/Cas9系统定向改良水稻粒长和穗粒数性状

沈兰^1,^2,#, 李健^3,#, 付亚萍², 王俊杰², 华宇峰², 焦晓真², 严长杰^1,^*(), 王克剑^2,^*()

¹扬州大学农学院/江苏省作物遗传生理国家重点实验室培育点/江苏省现代粮食作物生产协同创新中心/教育部植物功能基因组学重点实验室,江苏扬州225009
²中国水稻研究所水稻生物学国家重点实验室,杭州310006
³连云港市农业科学院,江苏连云港222000

收稿日期:2017-03-09 修回日期:2017-03-20 出版日期:2017-05-10 发布日期:2017-05-10
通讯作者: 严长杰,王克剑
基金资助:
国家自然科学基金资助项目(31371233);高校自然科学基金重大项目(14KJA210002);中国农业科学院科技创新工程资助项目;江苏省农业科技自主创新资金资助项目[CX(13)5075]

Orientation Improvement of Grain Length and Grain Number in Rice by Using CRISPR/Cas9 System

Lan SHEN^1,^2,#, Jian LI^3,#, Yaping FU², Junjie WANG², Yufeng HUA², Xiaozhen JIAO², Changjie YAN^1,^*(), Kejian WANG^2,^*()

¹Jiangsu Key Laboratory of Crop Genetics and Physiology/Co-Innovation Center for Modern Production Technology of Grain Crops/Key Laboratory of Plant Functional Genomics of the Ministry of Education/College of Agronomy, Yangzhou University, Yangzhou 225009, China
²State Key Laboratory of Rice Biology, China National Rice Research Institute, Hangzhou 310006, China
³Lianyungang Academy of Agricultural Sciences; Lianyungang 222000, China

Received:2017-03-09 Revised:2017-03-20 Online:2017-05-10 Published:2017-05-10
Contact: Changjie YAN, Kejian WANG

摘要/Abstract

摘要：

【目的】基因组定点编辑技术已成为分子育种的重要手段。本研究拟对GS3和Gn1a功能缺失突变对目标性状的改良效应进行分析,以期为培育高产水稻提供理论基础。【方法】利用CRISPR/Cas9系统,以控制粒型基因GS3和控制每穗粒数基因Gn1a为编辑对象,构建了共敲除载体pC1300-2×35S::Cas9-g^GS3-g^Gn1a,用农杆菌介导法转化4个优质水稻品种,分析了基因突变的特征和相应农艺性状。【结果】构建的敲除载体成功地实现了对GS3和Gn1a基因的定点编辑。在4个转化受体的T₀代均分别获得了gs3和gs3gn1a的移码突变体。对T₁代中无选择标记突变体的农艺性状分析表明,突变体 gs3和gs3gn1a与野生型相比粒长变长,千粒重增加;突变体gs3gn1a与突变体 gs3相比,每穗粒数显著增加。【结论】利用CRISPR/Cas9系统进行水稻基因编辑可以快速改良品种的目标性状,在水稻品种的定向改良方面具有巨大的潜力。

关键词: CRISPR/Cas9, 基因编辑, 水稻, GS3, Gn1a

Abstract:

【Objective】Gene orientation editing has become an important way for molecular breeding. We evaluated the improvement effects on the target traits following the construction of GS3 and Gn1a loss-of-function mutants so as to lay a solid foundation for high-yielding rice breeding. 【Method】GS3 and Gn1a were selected as targets for gene editing, which control the grain size and grain number in rice, respectively. The pC1300-2×35S:: Cas9-g^GS3-g^Gn1a expression vector was constructed for knocking out both GS3 and Gn1a by using CRISPR/Cas9 system, and transformed into four good quality rice varieties by the Agrobacterium-mediated method. And the properties of mutations and the target traits were analyzed in the transgenic lines.【Result】The sequencing results showed the GS3 and Gn1a in four rice varieties were successfully edited. In T₀ generation, we obtained mutants with frame shift mutations in GS3 and Gn1a in four rice varieties. In T₁generation, the marker-free plants were screened for analyzing the agronomic traits in four genetic backgrounds. For the agronomic characters, the gs3 and gs3gn1a mutants had the longer grain length and the increased 1000-grain weight compared to the wild type, and the gs3gn1a mutants had more grains per panicle compared to the gs3 mutants.【Conclusion】CRISPR/Cas9-mediated gene editing can improve rice target traits, which has great potential in orientation improvement of rice varieties.

Key words: CRISPR/Cas9, gene editing, rice, GS3, Gn1a

中图分类号:

沈兰, 李健, 付亚萍, 王俊杰, 华宇峰, 焦晓真, 严长杰, 王克剑. 利用CRISPR/Cas9系统定向改良水稻粒长和穗粒数性状[J]. 中国水稻科学, 2017, 31(3): 223-231.

Lan SHEN, Jian LI, Yaping FU, Junjie WANG, Yufeng HUA, Xiaozhen JIAO, Changjie YAN, Kejian WANG. Orientation Improvement of Grain Length and Grain Number in Rice by Using CRISPR/Cas9 System[J]. Chinese Journal OF Rice Science, 2017, 31(3): 223-231.

图/表 9

图1 GS3和Gn1a靶点位置带下划线的字母为起始密码子,灰色字母为PAM序列。

Fig. 1. Schematic diagram of the targeted sites in GS3 and Gn1a. The underlined letters are the initiation codons. The gray letters are the protospacer adjacent motif (PAM) sequences.

图2 CRISPR/Cas9载体构建流程中间载体SK-gRNA包含U3启动子和sgRNA骨架结构,双元载体pC1300-Cas9包含2×35S启动子和Cas9蛋白。GS3, Gn1a 的gRNA 分别用KpnⅠ/ SalⅠ和 XhoⅠ/ BglⅡ酶切,并通过一步法连入用KpnⅠ和 BamHⅠ酶切的pC1300-Cas9载体中。

Fig. 2. Flow diagram of CRISPR/Cas9 system for construction. The intermediate vector SK-gRNA contains the U3 promotor and sgRNA scaffold. Binary vector pC1300-Cas9 contains the 2×35S promotor and a Cas9 protein. Two gRNA scaffolds with GS3, Gn1a gene targets are respectively digested with KpnⅠ/ SalⅠ, and XhoⅠ/ BglⅡ, and cloned into pC1300-Cas9 between the KpnⅠand BamHⅠsites in a one-step ligation.

表1 本研究所用的引物

Table 1 Primers used in this research.

引物名称 Primer name	引物序列 Sequence(5′-3′)
GS3-g++		GGCAGTGACATGGCAATGGCGG
GS3-g––		AAACCCGCCATTGCCATGTCAC
Gn1a-g++		GGCAATGAAGCAAGAGCAGGTC
Gn1a-g––		AAACGACCTGCTCTTGCTTCAT
GS3-JC-F		CTATACATAGCTGCTGCAC
GS3-JC-R		GACAGATAGCAAGCCGTAC
Gn1a-JC-F		TTCCATCGTCAGCACACAAA
Gn1a-JC-R		ACGGAGAGGTTGCCAAAGTC
Hyg-F1		GCTGTTATGCGGCCATTGTC
Hyg-R1		GACGTCTGTCGAGAAGTTTC
Cas9-F2		ACCAGACACGAGACGACTAA
pC1300-R2		ATCGGTGCGGGCCTCTTC
Actin-F		TGCTATGTACGTCGCCATCCA
Actin-R		AATGAGTAACCACGCTCCGTC
Gn1a-F		CCATGGTATGCATGCAACACCATG
Gn1a-R		CGTTGTCACGTACTCCCTCCGTA
GS3-F		CTATACATAGCTGCTGCACCGTCT
GS3-R		CAATCACGTACTCATCATGGCAGCA
T3		ATTAACCCTCACTAAAGGGA

图3 4个水稻品种T0代GS3和Gn1a 突变类型分析靶序列用蓝色标注,PAM序列用红色标注,碱基插入的用红色小写字母表示,缺失碱基用黑色连字符表示。

Fig. 3. Mutation types at the GS3 and Gn1a loci of the four varieties in T0 generation. The targeted sequence is highlighted in blue and the PAM sequence in red. Mutations with 1 bp insertion are represented by red lowercase letters. The deleted sequences are shown by black hyphens.

图4 PCR鉴定无选择标记基因的突变株 M–标记D5000;1~9–T1代转基因株系;+,转基因阳性对照;NIP–无选择标记基因的对照(日本晴)。

Fig. 4. PCR identification of the marker-free transgenic plants. M, Marker D5000; Lanes 1 to 9, T1 transgenic lines; +, Positive control of transgenic line, NIP, Negative control(Nipponbare).

图5 4个品种T1代株型 J102–吉粳102; Ch 25–长白25; Ken 6–垦鉴稻6号; Ko 131–空育131。

Fig. 5. Grass morphology of the plants of the four varieties in T1 generation. J102, Jijing 102; Ch 25, Changbai 25; Ken 6, Kenjiandao 6; Ko 131, Kongyu 131.

图6 T1代4个水稻品种及其突变体粒型 A, D, G, J分别为吉粳102、长白25、垦鉴稻6号和空育131水稻品种及其突变体粒型;B, E, H, K 和C, F, I, L分别为其对应的粒长和粒宽。*, **分别表示在0.05和0.01水平上显著差异。图7~8同。

Fig. 6. Grain size of the four varieties and their mutants in T1 generation. A, D, G, J, Grain shapes of Jijing 102, Changbai 25, Kenjiandao 6 and Kongyu 131 and their mutants, respectively. B, E, H, K and C, F, I, L show the grain length and grain width, respectively. *, **, Significant difference at 0.05 and 0.01 levels, respectively. The same as in Fig 7 and 8.

图7 T1代4个水稻品种及其突变体的千粒重 A, B, C, D分别为吉粳102、长白25、垦鉴稻6号和空育131水稻品种及其突变体的千粒重。

Fig. 7. 1000-grain weight among the four varieties and their mutants in T1 generation. A, B, C, D shows the 1000-grain weight of Jijing 102, Changbai 25, Kenjiandao 6 and Kongyu 131 and their mutants, respectively.

图8 T1代4个水稻品种及其突变体的每穗粒数和结实率 A, D, G, J分别为吉粳102、长白25、垦鉴稻6号和空育131水稻品种及其突变体的穗型,B, E, H, K和C, F, I, L分别为其对应的每穗粒数和结实率。

Fig. 8. Grain number per panicle and the seed-setting rate among the four varieties in T1 generation and their mutants. A, D, G, J indicate the panicle phenotype of Jijing 102, Changbai 25, Kenjiandao 6 and Kongyu 131 and their mutants, respectively. B, E, H, K and C, F, I, L show the grain number per panicle and the seed-setting rate, respectively.

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利用CRISPR/Cas9系统定向改良水稻粒长和穗粒数性状

Orientation Improvement of Grain Length and Grain Number in Rice by Using CRISPR/Cas9 System

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