Chinese Journal OF Rice Science ›› 2023, Vol. 37 ›› Issue (5): 478-485.DOI: 10.16819/j.1001-7216.2023.220907
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LI Jingfang1, WEN Shuyue2, ZHAO Lijun2, CHEN Tingmu1, ZHOU Zhenling1, SUN Zhiguang1, LIU Yan1, CHEN Haiyuan3, ZHANG Yunhui3, CHI Ming1, XING Yungao1, XU Bo1, XU Dayong1, WANG Baoxiang1,*()
Received:
2022-09-28
Revised:
2022-12-19
Online:
2023-09-10
Published:
2023-09-13
Contact:
*email:李景芳1, 温舒越2, 赵利君2, 陈庭木1, 周振玲1, 孙志广1, 刘艳1, 陈海元3, 张云辉3, 迟铭1, 邢运高1, 徐波1, 徐大勇1, 王宝祥1,*()
通讯作者:
*email:基金资助:
LI Jingfang, WEN Shuyue, ZHAO Lijun, CHEN Tingmu, ZHOU Zhenling, SUN Zhiguang, LIU Yan, CHEN Haiyuan, ZHANG Yunhui, CHI Ming, XING Yungao, XU Bo, XU Dayong, WANG Baoxiang. Development of Aromatic Salt-tolerant Rice Based on CRISPR/Cas9 Technology[J]. Chinese Journal OF Rice Science, 2023, 37(5): 478-485.
李景芳, 温舒越, 赵利君, 陈庭木, 周振玲, 孙志广, 刘艳, 陈海元, 张云辉, 迟铭, 邢运高, 徐波, 徐大勇, 王宝祥. 基于CRISPR/Cas9技术创制耐盐香稻[J]. 中国水稻科学, 2023, 37(5): 478-485.
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URL: http://www.ricesci.cn/EN/10.16819/j.1001-7216.2023.220907
引物名称 Primer name | 引物序列 Primer sequence(5’-3’) | 用途 Usage | |
---|---|---|---|
Badh2-S1-F | GGCATGGTGGAAAAAGTCCTATAG | 构建Badh2敲除载体 Construction of Badh2 knockout vector | |
Badh2-S1-R | AAACCTATAGGACTTTTTCCACCA | ||
Badh2-F | CCTTTGTCATCACACCCTGG | Badh2敲除靶点测序鉴定 | |
Badh2-R | TAACTGCCTTCCTTGCCACG | Sequencing for Badh2 knockout target | |
OsRR22-S1-F | GGCACTCAACTGACCATACAACTC | 构建OsRR22敲除载体 | |
OsRR22-S1-R | AAACGAGTTGTATGGTCAGTTGAG | Construction of OsRR22 knockout vector | |
OsRR22-F | TAGGAGGAAGTTCGGTAATCGT | OsRR22敲除靶点测序鉴定 | |
OsRR22-R | ACATTTTCCCTGGTGAGTTTCT | Sequencing for OsRR22 knockout target | |
Cas9-F | CCTTCTGTGTGGTCTGGTTC | 载体检测 | |
Cas9-R | AGACAATCACCCCCTGGAAC | Vector assay | |
Hyg-F | CTATTTCTTTGCCCTCGGAC | 转基因检测 | |
Hyg-R | ATGCCTGAACTCACCGCGAC | Transgenic detection | |
qPCR SOS1-F | ATTGCAGCTGAGCATGTACG | 实时荧光定量PCR | |
qPCR SOS1-R | AGAGCTTGCTTTCGTGTGAC | Quantitative real-time PCR | |
qPCR OsMYB2-F | GGGCTGAAACGCACAGGCAAGA | 实时荧光定量PCR | |
qPCR OsMYB2-R | CTGCTTGGCGTGCTTCTGC | Quantitative real-time PCR | |
qPCR OsNHX1-F | GTGACAGACCTGGCAAATCC | 实时荧光定量PCR | |
qPCR OsNHX1-R | TCGACACAGCTCCTCTCATC | Quantitative real-time PCR | |
qPCR OsHKT1;1-F | TGCCAGAAGTTGTTGAAGCC | 实时荧光定量PCR | |
qPCR OsHKT1;1-R | CCCAGGAACATCACCAGGAT | Quantitative real-time PCR | |
qPCR OsP5CS1-F | GTCAGAGTGGACTGATGGCT | 实时荧光定量PCR | |
qPCR OsP5CS1-R | GCCTTTCTAGTGCTGATGGC | Quantitative real-time PCR | |
UBIQUITIN-F | GCTCCGTGGCGGTATCAT | 实时荧光定量PCR | |
UBIQUITIN-R | CGGCAGTTGACAGCCCTAG | Quantitative real-time PCR |
Table 1. Primers used in this study.
引物名称 Primer name | 引物序列 Primer sequence(5’-3’) | 用途 Usage | |
---|---|---|---|
Badh2-S1-F | GGCATGGTGGAAAAAGTCCTATAG | 构建Badh2敲除载体 Construction of Badh2 knockout vector | |
Badh2-S1-R | AAACCTATAGGACTTTTTCCACCA | ||
Badh2-F | CCTTTGTCATCACACCCTGG | Badh2敲除靶点测序鉴定 | |
Badh2-R | TAACTGCCTTCCTTGCCACG | Sequencing for Badh2 knockout target | |
OsRR22-S1-F | GGCACTCAACTGACCATACAACTC | 构建OsRR22敲除载体 | |
OsRR22-S1-R | AAACGAGTTGTATGGTCAGTTGAG | Construction of OsRR22 knockout vector | |
OsRR22-F | TAGGAGGAAGTTCGGTAATCGT | OsRR22敲除靶点测序鉴定 | |
OsRR22-R | ACATTTTCCCTGGTGAGTTTCT | Sequencing for OsRR22 knockout target | |
Cas9-F | CCTTCTGTGTGGTCTGGTTC | 载体检测 | |
Cas9-R | AGACAATCACCCCCTGGAAC | Vector assay | |
Hyg-F | CTATTTCTTTGCCCTCGGAC | 转基因检测 | |
Hyg-R | ATGCCTGAACTCACCGCGAC | Transgenic detection | |
qPCR SOS1-F | ATTGCAGCTGAGCATGTACG | 实时荧光定量PCR | |
qPCR SOS1-R | AGAGCTTGCTTTCGTGTGAC | Quantitative real-time PCR | |
qPCR OsMYB2-F | GGGCTGAAACGCACAGGCAAGA | 实时荧光定量PCR | |
qPCR OsMYB2-R | CTGCTTGGCGTGCTTCTGC | Quantitative real-time PCR | |
qPCR OsNHX1-F | GTGACAGACCTGGCAAATCC | 实时荧光定量PCR | |
qPCR OsNHX1-R | TCGACACAGCTCCTCTCATC | Quantitative real-time PCR | |
qPCR OsHKT1;1-F | TGCCAGAAGTTGTTGAAGCC | 实时荧光定量PCR | |
qPCR OsHKT1;1-R | CCCAGGAACATCACCAGGAT | Quantitative real-time PCR | |
qPCR OsP5CS1-F | GTCAGAGTGGACTGATGGCT | 实时荧光定量PCR | |
qPCR OsP5CS1-R | GCCTTTCTAGTGCTGATGGC | Quantitative real-time PCR | |
UBIQUITIN-F | GCTCCGTGGCGGTATCAT | 实时荧光定量PCR | |
UBIQUITIN-R | CGGCAGTTGACAGCCCTAG | Quantitative real-time PCR |
Fig. 1. Badh2 and OsRR22 target sites and CRISPR/Cas9 vector construction. A, Badh2 and OsRR22 structure and target site. The PAM motif is shown in green. B, Construction of CRISPR/Cas9 vector for Badh2 and OsRR22 gene target; C, Target site sequencing results in the expression vector.
Fig. 2. Identification of Cas9 markers in some T1 plants. M, DNA Marker; “+”, Positive control; “−”, Negative control; Lanes 1 to 9, Some plants of T1. The size of the Cas9 marker target fragment is 906 bp.
Fig. 3. Mutation types at the Badh2 and OsRR22 loci of the homozygous lines in T1. Red arrows indicate mutation positions. “+” means insertion; “-” means deletion. Black arrows indicate amino acid positions.
Fig. 4. Salt tolerance identification of T2 homozygous lines 21-30, 21-31 at the seedling stage. Values are shown as mean ± SD. *, ** Significantly different at 0.05 and 0.01 levels by t-test, respectively.
Fig. 5. Relative expression levels of salt resistance related genes in the wild-type and its T2 line 21-30. Values are shown as mean ± SD.*Significantly different at 0.05 level by t-test.
Fig. 6. Performance of rice yield and fragrance related traits in the wild-type and its positive transgenic lines. Values are shown as mean ± SD.** Significantly different at 0.01 level by t-test.
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