Rice Science ›› 2019, Vol. 26 ›› Issue (2): 118-124.DOI: 10.1016/j.rsci.2018.04.007
收稿日期:
2018-02-26
接受日期:
2018-04-27
出版日期:
2019-03-04
发布日期:
2018-12-18
. [J]. Rice Science, 2019, 26(2): 118-124.
Primer | Sequence (5′-3′) | Purpose |
T1-F | GGCACACGTCCCAGCTCGCCACCT | Vector construction |
T1-R | AAACAGGTGGCGAGCTGGGACGTG | Vector construction |
T2-F | GCCGTCCGTCGTCGTGTACGCCAC | Vector construction |
T2-R | AAACGTGGCGTACACGACGACGGA | Vector construction |
Wx-F | TAGCCACCCAAGAAACT | Amplification of the fragments across sgRNAs |
Wx-R | CTACCTCAGCCACAACG | Amplification of the fragments across sgRNAs |
hyg283-F | TCCGGAAGTGCTTGACATT | Vector deletion (HPTII) |
hyg283-R | GTCGTCCATCACAGTTTGC | Vector deletion (HPTII) |
Cas9-F | TTCGACCAGTCCAAGAACGG | Vector deletion (Cas9) ( |
Cas9-R | CTTGACCTTGGTGAGCTCGT | Vector deletion (Cas9) ( |
Table 1 Primers used in this study
Primer | Sequence (5′-3′) | Purpose |
T1-F | GGCACACGTCCCAGCTCGCCACCT | Vector construction |
T1-R | AAACAGGTGGCGAGCTGGGACGTG | Vector construction |
T2-F | GCCGTCCGTCGTCGTGTACGCCAC | Vector construction |
T2-R | AAACGTGGCGTACACGACGACGGA | Vector construction |
Wx-F | TAGCCACCCAAGAAACT | Amplification of the fragments across sgRNAs |
Wx-R | CTACCTCAGCCACAACG | Amplification of the fragments across sgRNAs |
hyg283-F | TCCGGAAGTGCTTGACATT | Vector deletion (HPTII) |
hyg283-R | GTCGTCCATCACAGTTTGC | Vector deletion (HPTII) |
Cas9-F | TTCGACCAGTCCAAGAACGG | Vector deletion (Cas9) ( |
Cas9-R | CTTGACCTTGGTGAGCTCGT | Vector deletion (Cas9) ( |
Fig. 1 Schematic diagrams of the Wx and CRISPR/Cas9 vector.A, Overview of the Wx gene structure and target sites for sgRNAs (T1 and T2). Exons and introns in Wx gene are depicted by orange rectangles and white rectangles, respectively. The target sequences of sgRNAs are shown at the top of the gene structure in red. B, The structure of the Wx gene editing system. T1 and T2 are driven by U3 and U6a promoters, respectively. RB, Right border; LB, Left border; Cas9, CRISPR associated protein 9 gene; HPTII, Hygromycin-resistance gene.
Fig. 2 CRISPR/Cas9-induced mutations in the Wx gene.A, PCR analysis of HPTII in T0 lines (part of the picture). M, DNA marker; 1, wx-SK118-1; 2, wx-SK118-2; 3, wx-SK118-3; 4, wx-HD5-1; 5, wx-HD5-2; 6, wx-HD5-3; 7, Positive control of pYLCRISPR/Cas9-MH-Wx; 8, Negative control of SK118; 9, Negative control of HD5; 10, Negative control of ddH2O. B, Polymerase chain reaction assays of Wx in T0 lines. M, DNA marker; 1, wx-SK118-1; 2, wx-SK118-2; 3, wx-SK118-3; 4, wx-HD5-1; 5, wx-HD5-2; 6, wx-HD5-3; 7, Control of SK118; 8, Control of HD5. C, Sequencing results of T0 mutant lines. sgRNA is shown in red letter; Insertion sequence is shown in green letter; Deletion sequence is shown by dashed line.
Line | No. of examined plants | No. of transgene-free plants |
wx-SK118-1 | 10 | 3 |
wx-SK118-2 | 23 | 5 |
wx-SK118-3 | 85 | 10 |
wx-HD5-1 | 53 | 6 |
wx-HD5-2 | 135 | 17 |
wx-HD5-3 | 47 | 13 |
Table 2 Segregation of transgene-free rice in T1 generation.
Line | No. of examined plants | No. of transgene-free plants |
wx-SK118-1 | 10 | 3 |
wx-SK118-2 | 23 | 5 |
wx-SK118-3 | 85 | 10 |
wx-HD5-1 | 53 | 6 |
wx-HD5-2 | 135 | 17 |
wx-HD5-3 | 47 | 13 |
Fig. 3 Phenotype of the Wx mutants.A, Intact polished rice. B, Grains stained with iodine solution. C, Amylose content of Wx mutants. Data represent Mean ± SD (n = 3). **, Significant at the 0.01 level by the t-test.
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