中国水稻科学 ›› 2022, Vol. 36 ›› Issue (3): 237-247.DOI: 10.16819/j.1001-7216.2022.210117
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,*()
收稿日期:
2021-01-17
修回日期:
2021-04-22
出版日期:
2022-05-10
发布日期:
2022-05-11
通讯作者:
林文雄
基金资助:
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
摘要:
【目的】探究OsARF7基因在水稻生长发育中的生物学功能及其对水稻农艺性状的影响。【方法】利用CRISPR/Cas9技术,在粳稻中花11背景下对OsARF7进行定向编辑,获得OsARF7基因突变植株,并考查其农艺性状。【结果】获得22株T0代转基因株系,经PCR扩增潮霉素基因鉴定出20株阳性植株。提取T2代转基因植株的基因组DNA,通过对OsARF7的2个编辑位点区域DNA片段进行PCR扩增及测序分析,筛选到15种突变类型的纯合基因型植株。基因表达分析显示,osarf7突变体的OsARF7和生长素转运关键基因OsPIN1b、OsPIN2和OsLAX2的表达量均显著低于野生型。田间调查发现,与野生型相比,osarf7突变体的分蘖成穗率显著降低,其中,分蘖数比野生型增多的osarf7突变体,有效穗数基本保持不变,而分蘖数与野生型相近的osarf7突变体,有效穗数少于野生型,最终表现为无效分蘖明显增多,分蘖成穗率降低。【结论】OsARF7基因与生长素调控水稻分蘖发生相关,并影响分蘖芽的生长发育及成穗;OsARF7基因突变会导致迟发分蘖芽不能正常发育成穗;粳稻OsARF7突变体的创建对揭示OsARF7基因生理功能和探索生长素促控分蘖发生与成穗机理等具有积极意义。
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.
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.
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 |
表1 本研究采用的gRNA靶点序列及相应的引物序列
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 |
图2 pYLCRISPR/Cas9-ARF7-T12表达载体组装 2个靶位点gRNA表达盒与pYLCRISPR/Cas9-MT载体组装成重组表达载体。LB―左边界;RB―右边界;参照文献[24]略作修改。
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.
图3 pYLCRISPR/Cas9-ARF7-T12重组载体中2个靶点序列的测序结果
Fig. 3. Sequencing results for the two target sequences inserted in pYLCRISPR/Cas9-ARF7-T12 recombinant vector.
图4 PCR扩增鉴定阳性转基因植株 “—”代表阴性对照,“+”代表阳性对照。
Fig. 4. Identification of the positive transgenic plants by PCR amplification. –, Negative control; +, Positive control.
图5 osarf7突变体株系的PCR检测及其与野生型序列比对分析 A–部分T2代(1~22)转化水稻株系OsARF7编辑位点附近DNA片段的PCR检测,靶点1的扩增长度为657 bp,靶点2的扩增长度为575 bp,野生型(WT)为中花11;B–转化植株编辑位点的PCR产物测序序列与野生型(WT)序列的比对结果,蓝色字母为靶点序列,黄色高亮显示PAM序列,删除线表示缺失碱基,红色小写字母为插入碱基,–表示缺失,+表示插入,WT为野生型。
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 |
表2 CRISPR/Cas9-OsARF7-T12潜在脱靶位点检测
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 |
图6 osarf7突变体及其野生型水稻OsARF7、OsPIN1b、OsPIN2、OsLAX2相对表达量
Fig. 6. Relative expression levels of OsARF7, OsPIN1b, OsPIN2 and OsLAX2 in osarf7 mutant and its wild type.
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 |
表3 不同类型osarf7突变体株系及其野生型水稻(WT)的农艺性状
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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