中国水稻科学 ›› 2021, Vol. 35 ›› Issue (1): 11-18.DOI: 10.16819/j.1001-7216.2021.0503
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,*()
收稿日期:
2020-05-03
修回日期:
2020-05-27
出版日期:
2021-01-10
发布日期:
2021-01-10
通讯作者:
段美娟,袁定阳
基金资助:
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
摘要: 目的 为鉴定水稻AFP1在非生物胁迫响应中的作用,创制非生物胁迫抗性的水稻新材料。方法 以优异籼稻恢复系华占为转化受体,利用CRISPR/Cas9技术创制afp1突变体,并对afp1突变体的耐逆性进行初步鉴定。结果 AFP1靶点1和靶点2的编辑效率分别为66.67%和75.00%。所有突变株系中,突变类型仅有插入和缺失突变,90%突变株系的突变长度为小片段突变(<5bp)。获得了6种无转基因成分的afp1纯合突变体。正常条件下,afp1突变体株高和结实率降低,有效分蘖增加,穗长显著升高,单株产量在-4.06%和11.75%之间变化。和野生型相比,afp1突变体的ABA敏感性和叶片水分散失率降低,耐干旱、热和渗透胁迫能力提高。结论 编辑AFP1基因可提高水稻多种非生物胁迫抗性。
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.
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.
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 |
表1 本研究所用引物
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 |
表2 T0代突变体突变基因型比率
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 |
图1 利用CRISPR/Cas9技术定向突变OsAFP1基因 A-敲除靶点设计; B-CRISPR/Cas9敲除载体,箭头代表扩增引物。
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.
图3 T2代纯合敲除突变体的突变类型 长方形代表推测蛋白;红色区域为突变蛋白序列,下方为相应蛋白序列;倒三角形和蓝色区域均表示靶位点。WT-野生型;KO1~KO6代表6个不含外源基因的纯合突变株系。
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.
图 4 afp1敲除突变体和野生型的表型比较 A-整体株型;B-穗部性状;C-株高;D-穗长;E-有效分蘖数;F-结实率;G-单株产量;均值±标准误,n=3;*和**分别表示突变体与野生型的差异达0.05和0.01显著水平(t检验)。HZ代表野生型华占,KO代表敲除突变体。
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.
图5 野生型和突变体ABA敏感性分析 A-3μmol/L ABA处理后的幼苗; B-ABA处理后幼苗的株高和根长; C-ABA处理后幼苗根和地上部分的鲜质量; D-3μmol/L ABA处理后种子的萌发率;均值±标准误,n=3;*和**表示在0.05和0.01水平上的差异(t检验)。
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.
图6 耐旱性和耐渗透性鉴定 A-干旱处理后幼苗表型;B-干旱处理后存活率;C-渗透胁迫处理后幼苗表型;D-水稻离体叶片水分散失率;E-渗透胁迫处理后幼苗鲜重;F-渗透胁迫处理后幼苗株高和根长;均值±标准误,n=3;*和**分别表示在0.05和0.01水平上的差异(t检验)。
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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