Chinese Journal OF Rice Science ›› 2017, Vol. 31 ›› Issue (3): 223-231.DOI: 10.16819/j.1001-7216.2017.7029 223
• Orginal Article • Next Articles
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
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,*()
通讯作者:
严长杰,王克剑
基金资助:
CLC Number:
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.
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.
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URL: http://www.ricesci.cn/EN/10.16819/j.1001-7216.2017.7029 223
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.
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 |
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 |
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.
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).
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.
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.
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.
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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