中国水稻科学 ›› 2017, Vol. 31 ›› Issue (3): 223-231.DOI: 10.16819/j.1001-7216.2017.7029 223
• • 下一篇
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,*()
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:
Lan SHEN1,2,#, Jian LI3,#, Yaping FU2, Junjie WANG2, Yufeng HUA2, Xiaozhen JIAO2, Changjie YAN1,*(), Kejian WANG2,*()
Received:
2017-03-09
Revised:
2017-03-20
Online:
2017-05-10
Published:
2017-05-10
Contact:
Changjie YAN, Kejian WANG
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-gGS3-gGn1a,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。
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.
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 |
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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