Chinese Journal OF Rice Science ›› 2022, Vol. 36 ›› Issue (3): 237-247.DOI: 10.16819/j.1001-7216.2022.210117
• Research Papers • Previous Articles Next Articles
LI Zhaowei1, SUN Congying1, LING Donglan1, ZENG Huiling1, ZHANG Xiaomei1, FAN Kai2, LIN Wenxiong1,*()
Received:
2021-01-17
Revised:
2021-04-22
Online:
2022-05-10
Published:
2022-05-11
Contact:
LIN Wenxiong
李兆伟1, 孙聪颖1, 零东兰1, 曾慧玲1, 张晓妹1, 范凯2, 林文雄1,*()
通讯作者:
林文雄
基金资助:
LI Zhaowei, SUN Congying, LING Donglan, ZENG Huiling, ZHANG Xiaomei, FAN Kai, LIN Wenxiong. Construction of osarf7 Mutants in Rice Based on CRISPR/Cas9 Technology and Investigation on Their Agronomic Traits[J]. Chinese Journal OF Rice Science, 2022, 36(3): 237-247.
李兆伟, 孙聪颖, 零东兰, 曾慧玲, 张晓妹, 范凯, 林文雄. 利用CRISPR/Cas9创建osarf7突变体及其农艺性状调查[J]. 中国水稻科学, 2022, 36(3): 237-247.
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URL: http://www.ricesci.cn/EN/10.16819/j.1001-7216.2022.210117
引物名称Primer name | 引物序列Primer sequence (5′-3′) | 用途Usage |
---|---|---|
OsARF7-T1-fwd | gccgACACGAACCTGTGCCACCGC | 靶序列1构建 |
OsARF7-T1-rev | aaacGCGGTGGCACAGGTTCGTGT | Construction of the target sequence 1 |
OsARF7-T2-fwd | gttGAGCTATGGCATGCTTGCGC | 靶序列2构建 |
OsARF7-T2-rev | aaacGCGCAAGCATGCCATAGCT | Construction of the target sequence 2 |
Hyp-F | ACGGTGTCGTCCATCACAGTTTGCC | 阳性转基因植株鉴定 |
Hyp-R | TTCCGGAAGTGCTTGACATTGGGGA | Identification of the positive transgenic plant |
OsARF7-T1-F | ACCACCACCTCCTCCTCAC | 靶点1测序检测 |
OsARF7-T1-R | TCGCACGAATCAGCACAAC | Sequencing for target 1 |
OsARF7-T2-F | TTACGGATCTGAGAACCTG | 靶点2测序检测 |
OsARF7-T2-R | TAACTACATTGCCGAAGAA | Sequencing for target 2 |
Table 1. Sequences of gRNA targets and other primers used in this study.
引物名称Primer name | 引物序列Primer sequence (5′-3′) | 用途Usage |
---|---|---|
OsARF7-T1-fwd | gccgACACGAACCTGTGCCACCGC | 靶序列1构建 |
OsARF7-T1-rev | aaacGCGGTGGCACAGGTTCGTGT | Construction of the target sequence 1 |
OsARF7-T2-fwd | gttGAGCTATGGCATGCTTGCGC | 靶序列2构建 |
OsARF7-T2-rev | aaacGCGCAAGCATGCCATAGCT | Construction of the target sequence 2 |
Hyp-F | ACGGTGTCGTCCATCACAGTTTGCC | 阳性转基因植株鉴定 |
Hyp-R | TTCCGGAAGTGCTTGACATTGGGGA | Identification of the positive transgenic plant |
OsARF7-T1-F | ACCACCACCTCCTCCTCAC | 靶点1测序检测 |
OsARF7-T1-R | TCGCACGAATCAGCACAAC | Sequencing for target 1 |
OsARF7-T2-F | TTACGGATCTGAGAACCTG | 靶点2测序检测 |
OsARF7-T2-R | TAACTACATTGCCGAAGAA | Sequencing for target 2 |
Fig. 2. Diagrammatic sketch of pYLCRISPR/Cas9-ARF7-T12 recombinant vector. Cloning of two gRNA cassettes into the pYLCRISPR/Cas9-MT vector. LB, Left border; RB, Right border.
Fig. 5. PCR identification and sequence alignment of osarf7 mutants compared to the WT line. A, PCR amplification results of the DNA fragments near the OsARF7 locus for partial osarf7 T2 mutants, the fragment length of target 1 and 2 is 657 bp and 575 bp, respectively, and WT is ZH11; B, Sequence alignment of the edited locus for the osarf7 mutants and WT line. The blue letters are the target genome sequence; The yellow highlighted letters denote PAM; Dashes strikethrough indicate the deleted bases; Insertion nucleotides are shown in red lowercase letters; –, Deletion; +, Insertion; WT, Wild type.
编号 Number | 潜在脱靶位置 Putative off-target locus | 潜在脱靶序列 Sequence of the putative off-target site (5′-3′) | 错配碱基数 No. of mismatched bases | 测试株数 No. of plants tested | 突变株数 No. of plants with mutations |
---|---|---|---|---|---|
Off-target 1-1 | Chr08: 12898391-12898413 | ACCCTAACCTCTCCCACCGC | 4 | 9 | 0 |
Off-target 1-2 | Chr10: 15856432-15856454 | AAACGGACGTGTGCCGCCGC | 4 | 9 | 0 |
Off-target 1-3 | Chr02: 27855035-27855057 | AGACGGGCCTGTGCCAACGC | 4 | 9 | 0 |
Off-target 1-4 | Chr12: 12787156-12787178 | CCAAGAACCTGCGCCGCCGC | 4 | 9 | 0 |
Off-target 1-5 | Chr07: 2126716-2126738 | ACAGCAGCCGGTGCCACCGC | 4 | 9 | 0 |
Off-target 2-1 | Chr12: 26007034-26007056 | GAGCTGTGGCATGCATGCGC | 2 | 10 | 0 |
Off-target 2-2 | Chr02: 3488312-3488334 | GAGTTGTGGCATGCATGCGC | 3 | 9 | 0 |
Off-target 2-3 | Chr04: 34285188-34285210 | GAGCTATGGCACGCCTGCGC | 2 | 8 | 0 |
Off-target 2-4 | Chr06: 4926812-4926834 | GAGCTATGGCATGCTTGTGC | 1 | 10 | 0 |
Off-target 2-5 | Chr04: 22007551-22007573 | GAGCTATGGCATGCTTGTGC | 1 | 10 | 0 |
Off-target 2-6 | Chr01: 40696054-40696076 | GAGCTGTGGCACGCCTGCGC | 3 | 10 | 0 |
Table 2. Mutation detections in the putative CRISPR/Cas9-OsARF7-T12 off-target sites.
编号 Number | 潜在脱靶位置 Putative off-target locus | 潜在脱靶序列 Sequence of the putative off-target site (5′-3′) | 错配碱基数 No. of mismatched bases | 测试株数 No. of plants tested | 突变株数 No. of plants with mutations |
---|---|---|---|---|---|
Off-target 1-1 | Chr08: 12898391-12898413 | ACCCTAACCTCTCCCACCGC | 4 | 9 | 0 |
Off-target 1-2 | Chr10: 15856432-15856454 | AAACGGACGTGTGCCGCCGC | 4 | 9 | 0 |
Off-target 1-3 | Chr02: 27855035-27855057 | AGACGGGCCTGTGCCAACGC | 4 | 9 | 0 |
Off-target 1-4 | Chr12: 12787156-12787178 | CCAAGAACCTGCGCCGCCGC | 4 | 9 | 0 |
Off-target 1-5 | Chr07: 2126716-2126738 | ACAGCAGCCGGTGCCACCGC | 4 | 9 | 0 |
Off-target 2-1 | Chr12: 26007034-26007056 | GAGCTGTGGCATGCATGCGC | 2 | 10 | 0 |
Off-target 2-2 | Chr02: 3488312-3488334 | GAGTTGTGGCATGCATGCGC | 3 | 9 | 0 |
Off-target 2-3 | Chr04: 34285188-34285210 | GAGCTATGGCACGCCTGCGC | 2 | 8 | 0 |
Off-target 2-4 | Chr06: 4926812-4926834 | GAGCTATGGCATGCTTGTGC | 1 | 10 | 0 |
Off-target 2-5 | Chr04: 22007551-22007573 | GAGCTATGGCATGCTTGTGC | 1 | 10 | 0 |
Off-target 2-6 | Chr01: 40696054-40696076 | GAGCTGTGGCACGCCTGCGC | 3 | 10 | 0 |
株系 Line | 株高 Plant height / cm | 总分蘖数 Tiller number per plant | 有效穗数 Effective panicle per plant | 穗长 Length of main panicle /cm | 穗粒数 Grain number per panicle | 结实率 Seed setting rate / % | 千粒重 1000-grain weight / g | 单株产量 Grain yield per plant / g |
---|---|---|---|---|---|---|---|---|
osarf7-1 | 73.5±1.6 ab | 11.2±1.3 a | 24.6±2.1 a | 119.3±30.8 ab | 63.4±19.0 bc | 23.3±2.9 e | 8.6±4.6 ab | |
osarf7-2-1 | 72.2±3.4 ab | 20.6±3.3 a | 7.4±1.5 abc | 22.8±1.5 ab | 135.9±34.9 ab | 74.5±14.7 abc | 25.5±0.2 bcde | 12.9±6.3 ab |
osarf7-2-2 | 67.3±4.9 b | 11.0±2.6 e | 5.0±1.0 c | 23.5±2.5 ab | 131.8±35.0 ab | 82.5±8.8 ab | 23.5±0.3 de | 9.3±4.2 ab |
osarf7-2-3 | 72.7±1.5 ab | 20.0±1.0 a | 7.3±3.5 abc | 23.7±1.2 ab | 167.5±13.8 a | 78.4±5.8 abc | 22.6±0.6 e | 9.0±3.3 ab |
osarf7-5-1 | 67.3±3.2 b | 12.7±1.2 de | 5.0±0.9 c | 22.6±1.2 ab | 119.4±12.1 ab | 81.8±5.2 ab | 25.5±0.6 bcde | 10.4±0.5 ab |
osarf7-5-2 | 70.8±3.2 ab | 12.8±1.5 de | 6.2±0.8 bc | 22.8±2.0 ab | 112.8±29.5 b | 75.9±9.5 abc | 25.2±1.3 cde | 9.7±1.2 ab |
osarf7-6 | 68.5±3.6 b | 14.5±1.1 bcd | 7.6±1.8 bc | 23.0±1.5 ab | 126.2±23.0 ab | 69.9±11.9 abc | 24.4±1.2 de | 11.3±3.0 ab |
osarf7-7 | 73.0±3.7 ab | 20.2±1.8 a | 10.2±1.9 ab | 23.2±1.4 ab | 135.9±21.2 ab | 73.7±14.6 abc | 25.0±1.5 cde | 14.5±4.2 ab |
osarf7-8-1 | 74.8±2.2 a | 13.8±0.5 bcde | 6.5±2.1 bc | 23.7±2.0 ab | 144.8±34.6 ab | 76.5±10.0 abc | 25.4±1.2 bcde | 14.1±3.0 ab |
osarf7-8-2 | 75.4±2.4 a | 16.0±0.7 bc | 7.0±2.6 bc | 23.1±1.8 ab | 140.5±32.2 ab | 83.21±9.3 a | 24.7±0.7 cde | 12.0±3.3 ab |
osarf7-9 | 72.3±0.8 ab | 15.5±1.0 bc | 6.8±1.9 bc | 22.3±1.6 ab | 119.9±23.1 ab | 86.5±8.3 a | 26.7±1.6 bcd | 12.5±2.4 ab |
osarf7-12 | 75.0±2.0 a | 17.3±0.6 ab | 6.3±1.5 bc | 24.0±1.0 ab | 139.8±36.0 ab | 90.8±3.3 a | 23.6±0.8 cde | 17.4±9.8 a |
osarf7-15-1 | 75.8±2.4 a | 13.7±1.2 de | 5.1±1.7 c | 21.9±1.3 b | 117.2±22.5 b | 83.0±5.9 a | 28.1±1.1 ab | 10.0±3.2 ab |
osarf7-15-2 | 74.7±1.4 a | 11.9±1.2 de | 4.9±2.3 c | 21.8±1.9 b | 122.0±24.5 ab | 79.1±15.0 ab | 27.4±1.3 abc | 9.4±3.1 ab |
osarf7-16 | 72.0±2.3ab | 15.4±1.6 bc | 5.6±0.9 c | 21.8±1.3 b | 103.0±26.7 b | 57.2±26.4 c | 30.1±2.2 a | 13.0±3.9 b |
WT | 71.2±1.9 ab | 11.3±0.6 de | 10.2±1.8 ab | 20.8±1.6 b | 111.8±15.0 b | 88.4±4.4 a | 24.3±0.7 de | 14.2±4.8 ab |
Table 3. Agronomic traits of different types of osarf7 mutants and its corresponding wild type grown in paddy field.
株系 Line | 株高 Plant height / cm | 总分蘖数 Tiller number per plant | 有效穗数 Effective panicle per plant | 穗长 Length of main panicle /cm | 穗粒数 Grain number per panicle | 结实率 Seed setting rate / % | 千粒重 1000-grain weight / g | 单株产量 Grain yield per plant / g |
---|---|---|---|---|---|---|---|---|
osarf7-1 | 73.5±1.6 ab | 11.2±1.3 a | 24.6±2.1 a | 119.3±30.8 ab | 63.4±19.0 bc | 23.3±2.9 e | 8.6±4.6 ab | |
osarf7-2-1 | 72.2±3.4 ab | 20.6±3.3 a | 7.4±1.5 abc | 22.8±1.5 ab | 135.9±34.9 ab | 74.5±14.7 abc | 25.5±0.2 bcde | 12.9±6.3 ab |
osarf7-2-2 | 67.3±4.9 b | 11.0±2.6 e | 5.0±1.0 c | 23.5±2.5 ab | 131.8±35.0 ab | 82.5±8.8 ab | 23.5±0.3 de | 9.3±4.2 ab |
osarf7-2-3 | 72.7±1.5 ab | 20.0±1.0 a | 7.3±3.5 abc | 23.7±1.2 ab | 167.5±13.8 a | 78.4±5.8 abc | 22.6±0.6 e | 9.0±3.3 ab |
osarf7-5-1 | 67.3±3.2 b | 12.7±1.2 de | 5.0±0.9 c | 22.6±1.2 ab | 119.4±12.1 ab | 81.8±5.2 ab | 25.5±0.6 bcde | 10.4±0.5 ab |
osarf7-5-2 | 70.8±3.2 ab | 12.8±1.5 de | 6.2±0.8 bc | 22.8±2.0 ab | 112.8±29.5 b | 75.9±9.5 abc | 25.2±1.3 cde | 9.7±1.2 ab |
osarf7-6 | 68.5±3.6 b | 14.5±1.1 bcd | 7.6±1.8 bc | 23.0±1.5 ab | 126.2±23.0 ab | 69.9±11.9 abc | 24.4±1.2 de | 11.3±3.0 ab |
osarf7-7 | 73.0±3.7 ab | 20.2±1.8 a | 10.2±1.9 ab | 23.2±1.4 ab | 135.9±21.2 ab | 73.7±14.6 abc | 25.0±1.5 cde | 14.5±4.2 ab |
osarf7-8-1 | 74.8±2.2 a | 13.8±0.5 bcde | 6.5±2.1 bc | 23.7±2.0 ab | 144.8±34.6 ab | 76.5±10.0 abc | 25.4±1.2 bcde | 14.1±3.0 ab |
osarf7-8-2 | 75.4±2.4 a | 16.0±0.7 bc | 7.0±2.6 bc | 23.1±1.8 ab | 140.5±32.2 ab | 83.21±9.3 a | 24.7±0.7 cde | 12.0±3.3 ab |
osarf7-9 | 72.3±0.8 ab | 15.5±1.0 bc | 6.8±1.9 bc | 22.3±1.6 ab | 119.9±23.1 ab | 86.5±8.3 a | 26.7±1.6 bcd | 12.5±2.4 ab |
osarf7-12 | 75.0±2.0 a | 17.3±0.6 ab | 6.3±1.5 bc | 24.0±1.0 ab | 139.8±36.0 ab | 90.8±3.3 a | 23.6±0.8 cde | 17.4±9.8 a |
osarf7-15-1 | 75.8±2.4 a | 13.7±1.2 de | 5.1±1.7 c | 21.9±1.3 b | 117.2±22.5 b | 83.0±5.9 a | 28.1±1.1 ab | 10.0±3.2 ab |
osarf7-15-2 | 74.7±1.4 a | 11.9±1.2 de | 4.9±2.3 c | 21.8±1.9 b | 122.0±24.5 ab | 79.1±15.0 ab | 27.4±1.3 abc | 9.4±3.1 ab |
osarf7-16 | 72.0±2.3ab | 15.4±1.6 bc | 5.6±0.9 c | 21.8±1.3 b | 103.0±26.7 b | 57.2±26.4 c | 30.1±2.2 a | 13.0±3.9 b |
WT | 71.2±1.9 ab | 11.3±0.6 de | 10.2±1.8 ab | 20.8±1.6 b | 111.8±15.0 b | 88.4±4.4 a | 24.3±0.7 de | 14.2±4.8 ab |
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