Chinese Journal OF Rice Science ›› 2022, Vol. 36 ›› Issue (6): 572-578.DOI: 10.16819/j.1001-7216.2022.211205
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ZHANG Yuanye1, YIN Liying1, LI Rongtian1,*(), HE Mingliang2, LIU Xinxin3, PAN Tingting4, TIAN Xiaojie2, BU Qingyun2, LI Xiufeng2,*()
Received:
2021-12-07
Revised:
2022-03-10
Online:
2022-11-10
Published:
2022-11-10
Contact:
LI Rongtian, LI Xiufeng
张元野1, 尹丽颖1, 李荣田1,*(), 何明良2, 刘欣欣3, 潘婷婷4, 田晓杰2, 卜庆云2, 李秀峰2,*()
通讯作者:
李荣田,李秀峰
基金资助:
ZHANG Yuanye, YIN Liying, LI Rongtian, HE Mingliang, LIU Xinxin, PAN Tingting, TIAN Xiaojie, BU Qingyun, LI Xiufeng. Breeding of Rc Function Restoration Red Rice via CRISPR/Cas9 Mediated Genome Editing[J]. Chinese Journal OF Rice Science, 2022, 36(6): 572-578.
张元野, 尹丽颖, 李荣田, 何明良, 刘欣欣, 潘婷婷, 田晓杰, 卜庆云, 李秀峰. 利用CRISPR/Cas9技术创制Rc基因恢复红稻[J]. 中国水稻科学, 2022, 36(6): 572-578.
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URL: http://www.ricesci.cn/EN/10.16819/j.1001-7216.2022.211205
引物名称 Primer name | 引物序列(5'-3') Primer sequence(5'-3') |
---|---|
CAS-U6a-Rc1-LP | GCCGCGCAAGTGGATGCCATCCA |
CAS-U6a-Rc1-RP | AAACTGGATGGCATCCACTTGCG |
CAS-U6b-Rc2-LP | GTTGGCACTGAAATCACCTTGGA |
CAS-U6b-Rc2-RP | AAACTCCAAGGTGATTTCAGTGC |
M13-F | GTAAAACGACGGCCAGT |
Rc-F | CACAGAGAATGCTCAAGA |
Rc-R | CGGCTTTATAGAAATAGAGG |
HPT-F | TGCGCCCAAGCTGCATCAT |
HPT-R | TGAACTCACCGCGACGTCTGT |
qRT-bZIP71-F | AGGGATGATGAGGAACTTGG |
qRT-bZIP71-R | GGTATGCATGCGATCATTTC |
qRT-NHX1-F | ACATTGGAACGCTGGATGTA |
qRT-NHX1-R | TCACAACACCTTCACCGAAT |
qRT-CHX11-F | AGATCCTGGGTGGCATCTT |
qRT-CHX11-R | AGGAAGAGGAAGAGCAGCAG |
qRT-IRO2-F | TCGCCGTGGCTGGACCTAGAC |
qRT-IRO2-R | CCCCAACACTCCTGGTTTGCAG |
qRT-IDEF1-F | GAGGCTAGTCTTCCACCTTTG |
qRT-IDEF1-R | TGGCCAGTACCTGTACTTAAAC |
qRT-IRT1-F | TCACCGCATCTGGTCATACT |
qRT-IRT1-R | GAGATTGAGGAGAGGCTTGG |
qRT-Rc-F | ACACTAACAACACTGACACT |
qRT-Rc-R | CTCTTACCACTTCTGACATCT |
Table 1. Primers used in this research.
引物名称 Primer name | 引物序列(5'-3') Primer sequence(5'-3') |
---|---|
CAS-U6a-Rc1-LP | GCCGCGCAAGTGGATGCCATCCA |
CAS-U6a-Rc1-RP | AAACTGGATGGCATCCACTTGCG |
CAS-U6b-Rc2-LP | GTTGGCACTGAAATCACCTTGGA |
CAS-U6b-Rc2-RP | AAACTCCAAGGTGATTTCAGTGC |
M13-F | GTAAAACGACGGCCAGT |
Rc-F | CACAGAGAATGCTCAAGA |
Rc-R | CGGCTTTATAGAAATAGAGG |
HPT-F | TGCGCCCAAGCTGCATCAT |
HPT-R | TGAACTCACCGCGACGTCTGT |
qRT-bZIP71-F | AGGGATGATGAGGAACTTGG |
qRT-bZIP71-R | GGTATGCATGCGATCATTTC |
qRT-NHX1-F | ACATTGGAACGCTGGATGTA |
qRT-NHX1-R | TCACAACACCTTCACCGAAT |
qRT-CHX11-F | AGATCCTGGGTGGCATCTT |
qRT-CHX11-R | AGGAAGAGGAAGAGCAGCAG |
qRT-IRO2-F | TCGCCGTGGCTGGACCTAGAC |
qRT-IRO2-R | CCCCAACACTCCTGGTTTGCAG |
qRT-IDEF1-F | GAGGCTAGTCTTCCACCTTTG |
qRT-IDEF1-R | TGGCCAGTACCTGTACTTAAAC |
qRT-IRT1-F | TCACCGCATCTGGTCATACT |
qRT-IRT1-R | GAGATTGAGGAGAGGCTTGG |
qRT-Rc-F | ACACTAACAACACTGACACT |
qRT-Rc-R | CTCTTACCACTTCTGACATCT |
Fig. 3. Phenotype and sequencing identification of T1 transgenic plants. A, Phenotype comparison of red rice and wild type(bar=1 cm); B, Sequence comparison between red rice and wild type; C, Comparison of amino acid sequences between red rice and wild type; D, Comparison of bHLH domains between red rice and wild type(the bHLH domain is in the red box).
Fig. 4. Identification of salt and alkali tolerance of T2 generation transgenic plants. A, Bud length in each group under different concentrations of salt treatment; E-F, Bud length in each group under different concentrations of alkali treatment; G, Growth of seedlings treated with 200 mmol/L NaCl (scale=2 cm); H, Growth of seedlings treated with 20 mmol/L Na2CO3 (scale =2 cm).
Fig. 5. Identification of saline-alkali tolerance related gene expression levels in T2 transgenic plants. A, Salt-tolerant gene expression level under 200 mmol/L NaCl treatment; D-F, Identification of alkali-tolerant gene expression level under 20 mmol/L Na2CO3 treatment; G-I, Rc expression level of 200 mmol/L NaCl, 20 mmol/L Na2CO3 and blank control. ns means no significant difference; **,*** and **** mean significant difference at 0.005, 0.001 and 0.0001 levels, respectively (t test). KY180, Kongyu 180;SY453, Shangyu 453;KY-1, Gene editing line of Kongyu 180; SY-1, Gene editing line of Shangyu 453.
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