Chinese Journal OF Rice Science ›› 2021, Vol. 35 ›› Issue (1): 11-18.DOI: 10.16819/j.1001-7216.2021.0503
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Tianshun ZHOU1,2, Dong YU1,3, Ling LIU1,2, Ning OUYANG1,2, Guilong YUAN1,3, Meijuan DUAN4,*(), Dingyang YUAN1,2,3,*()
Received:
2020-05-03
Revised:
2020-05-27
Online:
2021-01-10
Published:
2021-01-10
Contact:
Meijuan DUAN, Dingyang YUAN
周天顺1,2, 余东1,3, 刘玲1,2, 欧阳宁1,2, 袁贵龙1,3, 段美娟4,*(), 袁定阳1,2,3,*()
通讯作者:
段美娟,袁定阳
基金资助:
Tianshun ZHOU, Dong YU, Ling LIU, Ning OUYANG, Guilong YUAN, Meijuan DUAN, Dingyang YUAN. CRISPR/Cas9-mediatedEditing of AFP1Improves Rice Stress Tolerance[J]. Chinese Journal OF Rice Science, 2021, 35(1): 11-18.
周天顺, 余东, 刘玲, 欧阳宁, 袁贵龙, 段美娟, 袁定阳. 利用CRISPR/Cas9技术编辑AFP1基因提高水稻耐逆性[J]. 中国水稻科学, 2021, 35(1): 11-18.
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URL: http://www.ricesci.cn/EN/10.16819/j.1001-7216.2021.0503
名称 Name | 正向引物 Forward primer (5' - 3') | 反向引物 Reverse primer (5' - 3') |
---|---|---|
AFP1-T1 | ggcaCTGTGCGGCATCGGCAAGGG | aaacCCCTTGCCGATGCCGCACAG |
AFP1-T2 | gccgCTGTCGCTCGGCGGCCGGTT | aaacAACCGGCCGCCGAGCGACAG |
AFP1-J | GGCGGATTACTCGCTCGGTCAGTTCT | TGGAGAGAAGCGATGACGCACCTTCAAG |
Hpt | GACAGCGTCTCCGACCTGAT | CATCGCCTCGCTCCAGTCAAT |
Cas9 | CTCTTCCTTCCAAGTACGTG | GAAAGGTCGATACGAGTCTC |
OT1-1 | CAAACGCCAAGGCGGGAATGCAG | GTGCGCCGCGGCGCAGCTGTTG |
OT1-2 | GATCCATGTGGGCCCCACATGTC | CGCTGGTCCATCCTCTCGATC |
OT1-3 | GAAGTTCTTGCCGATGACCTCG | GACGAGTGAAGAGGAGGAGAC |
OT2-1 | CAATCAGTGTAAGCTTGCTCTAG | GGTGAAGTCACACAATTACTG |
OT2-2 | CTTCTTGCTTGGAGGAGCATG | CTCAGCTGCAACCTGTTAACG |
OT2-3 | CAAGGAGCGCCGTGTACCTCG | GTAGTCCAGCTTCAGCTTCAC |
Table 1 Primers used in this study.
名称 Name | 正向引物 Forward primer (5' - 3') | 反向引物 Reverse primer (5' - 3') |
---|---|---|
AFP1-T1 | ggcaCTGTGCGGCATCGGCAAGGG | aaacCCCTTGCCGATGCCGCACAG |
AFP1-T2 | gccgCTGTCGCTCGGCGGCCGGTT | aaacAACCGGCCGCCGAGCGACAG |
AFP1-J | GGCGGATTACTCGCTCGGTCAGTTCT | TGGAGAGAAGCGATGACGCACCTTCAAG |
Hpt | GACAGCGTCTCCGACCTGAT | CATCGCCTCGCTCCAGTCAAT |
Cas9 | CTCTTCCTTCCAAGTACGTG | GAAAGGTCGATACGAGTCTC |
OT1-1 | CAAACGCCAAGGCGGGAATGCAG | GTGCGCCGCGGCGCAGCTGTTG |
OT1-2 | GATCCATGTGGGCCCCACATGTC | CGCTGGTCCATCCTCTCGATC |
OT1-3 | GAAGTTCTTGCCGATGACCTCG | GACGAGTGAAGAGGAGGAGAC |
OT2-1 | CAATCAGTGTAAGCTTGCTCTAG | GGTGAAGTCACACAATTACTG |
OT2-2 | CTTCTTGCTTGGAGGAGCATG | CTCAGCTGCAACCTGTTAACG |
OT2-3 | CAAGGAGCGCCGTGTACCTCG | GTAGTCCAGCTTCAGCTTCAC |
靶位点 Target | 阳性植株Positive plants | 突变率 Mutant ratio/% | 突变基因型比率Mutant genotype ratio/% | ||||
---|---|---|---|---|---|---|---|
纯合突变率 Homozygote ratio | 杂合突变率Heterozygote ratio | 双等位突变率 Bi-allele ratio | 嵌合突变率 Chimera ratio | ||||
靶点1 Target 1 | 12 | 66.7 | 12.5 | 12.5 | 75.0 | 0.0 | |
靶点2 Target 2 | 12 | 75.0 | 22.2 | 22.2 | 44.4 | 11.1 |
Table 2 Mutant genotype ratios of T0 mutations.
靶位点 Target | 阳性植株Positive plants | 突变率 Mutant ratio/% | 突变基因型比率Mutant genotype ratio/% | ||||
---|---|---|---|---|---|---|---|
纯合突变率 Homozygote ratio | 杂合突变率Heterozygote ratio | 双等位突变率 Bi-allele ratio | 嵌合突变率 Chimera ratio | ||||
靶点1 Target 1 | 12 | 66.7 | 12.5 | 12.5 | 75.0 | 0.0 | |
靶点2 Target 2 | 12 | 75.0 | 22.2 | 22.2 | 44.4 | 11.1 |
Fig. 1. Directed mutation of OsAFP1 using CRISPR/Cas9 technology. A, Design of mutated sites; B, Schematic diagram of CRISPR/Cas9 construction; Arrows represent amplification primers.
Fig. 3. Mutated types of T2 homozygous mutants. Rectangles are putative mutated proteins; Red shades indicate mutated protein regions and letters below are putative protein sequences;Inverted triangle and blue area represent target sites.WT, Wild type; KO1-KO6 indicate six homozygous mutants without exogenous genes.
Fig. 4. Phenotypic comparisons of afp1 knockdown mutants and the wildtype. A, Plant architectures; B, Panicle traits; C, Plant height; D, Panicle length; E, Effecttivepanicle per plant; F, Seed setting rate; G, Yield per plant;Mean±SE, n=3; * and ** represent significant difference between the mutant and the wild type at the 0.05 and 0.01 levels by t-test, respectively.HZ, Wild type Huazhan; KO, afp1knockdown mutants.
Fig. 5. ABA sensitivity of WT and mutants. A, Phenotype of seedlingsafter 3μmol/L ABA treatment; B, Length of root and shootafter 3μmol/L ABA treatment; C, Fresh weight of seedlings after 3μmol/L ABA treatment; D, Germination rate of seeds after 3μmol/L ABA treatment;Mean±SE, n=3; * and ** represent significant difference at the 0.05 and 0.01 levels by t-test, respectively.
Fig. 6. Identification of drought and osmotic stress tolerances. A, Phenotype of seedlings after drought treatment; B, Survival rate of seedlings after drought treatment; C, Phenotype of seedlings after osmotic stress treatment;D, Water loss rate of rice detached leaves;E, Fresh weight of seedlings after osmotic stress treatment; F, Length of root and shoot after osmotic stress treatment;Mean±SE, n=3; * and ** represent significant difference at the 0.05 and 0.01 levels by t-test, respectively.
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