中国水稻科学 ›› 2020, Vol. 34 ›› Issue (5): 406-412.DOI: 10.16819/j.1001-7216.2020.0104
徐善斌1, 郑洪亮1, 刘利锋3, 卜庆云2, 李秀峰2,*(), 邹德堂1,*()
收稿日期:
2020-01-10
修回日期:
2020-04-16
出版日期:
2020-09-10
发布日期:
2020-09-10
通讯作者:
李秀峰,邹德堂
基金资助:
Shanbin XU1, Hongliang ZHENG1, Lifeng LIU3, Qingyun BU2, Xiufeng Li2,*(), Detang ZOU1,*()
Received:
2020-01-10
Revised:
2020-04-16
Online:
2020-09-10
Published:
2020-09-10
Contact:
Xiufeng Li, Detang ZOU
摘要:
【目的】CRISPR/Cas9基因编辑技术已成为水稻分子育种的重要手段。为了促进水稻育种的发展,本研究以非香型粳稻品种龙粳11为试验材料,对GS3、GS9和Badh2基因进行编辑,以期获得能稳定遗传的长粒香水稻材料。【方法】利用CRISPR/Cas9技术,以GS3、GS9和Badh2为靶基因,构建敲除载体pYLCRISPR/Cas9-GS3/ GS9/Badh2-gRNA,通过农杆菌介导法,在龙粳11的GS3、GS9和Badh2基因中引入了特定的突变。【结果】T2代无转基因的gs3/gs9/badh2纯合突变体与野生型龙粳11相比,粒长增加26.43%~27.01%,单株产量增加10.82%~12.11%,千粒重增加18.34%~41.36%,稻米变香,高效地将圆粒水稻变成长粒香型水稻。【结论】利用CRISPR/Cas9技术获得能够稳定遗传并具有长粒香品质的纯合突变株系,为组合多个品质性状提供了一种方便有效的方法,从育种角度加快了新品系创制过程。
中图分类号:
徐善斌, 郑洪亮, 刘利锋, 卜庆云, 李秀峰, 邹德堂. 利用CRISPR/Cas9技术高效创制长粒香型水稻[J]. 中国水稻科学, 2020, 34(5): 406-412.
Shanbin XU, Hongliang ZHENG, Lifeng LIU, Qingyun BU, Xiufeng Li, Detang ZOU. Improvement of Grain Shape and Fragrance by Using CRISPR/Cas9 System[J]. Chinese Journal OF Rice Science, 2020, 34(5): 406-412.
引物名称 Primer name | 引物序列(5'-3') Primer sequence(5'-3') |
---|---|
B1-F | GCCGAGTGACATGGCAATGGCGG |
B1-R | AAACCCGCCATTGCCATGTCACT |
B2-F | GCCGCGATTGCTTCCTGCTCGGTT |
B2-R | AAACAACCGAGCAGGAAGCAATCG |
B3-F | GCCGCAAGTACCTCCGCGCAATCG |
B3-R | AAACCGATTGCGCGGAGGTACTTG |
U-F | CTCCGTTTTACCTGTGGAATCG |
gRNA-R | CGGAGGAAAATTCCATCCAC |
B1' | TTCAGAGGTCTCTCTCGCACTGGAATCGGCAGCAAAGG |
B2 | AGCGTGGGTCTCGTCAGGGTCCATCCACTCCAAGCTC |
B2' | TTCAGAGGTCTCTCTGACACTGGAATCGGCAGCAAAGG |
B3 | AGCGTGGGTCTCGTCTTGGTCCATCCACTCCAAGCTC |
B3' | TTCAGAGGTCTCTAAGACACTGGAATCGGCAGCAAAGG |
BL | AGCGTGGGTCTCGACCGGGTCCATCCACTCCAAGCTC |
Seq1-F | TTCAAAGCAAAGCACCAAGC |
Seq1-R | GCTGGGGAAAACTTACAATG |
Seq2-F | TCCACTCGACTCCTCACTCA |
Seq2-R | GATTAGCGTAGGCCCTCACG |
Seq3-F | ATCCATCTCCGTATCTCT |
Seq3-R | GGTAGTCACCACCCTA |
Hyg-F | ACGGTGTCGTCCATCACAGTTTGCC |
Hyg-R | TTCCGGAAGTGCTTGACATTGGGGA |
NOFS | GCGGTGTCATCTATGTTACTAG |
M13F-47 | CGCCAGGGTTTTCCCAGTCACGAC |
表1 本研究中所用的引物
Table 1 Primers used in this research.
引物名称 Primer name | 引物序列(5'-3') Primer sequence(5'-3') |
---|---|
B1-F | GCCGAGTGACATGGCAATGGCGG |
B1-R | AAACCCGCCATTGCCATGTCACT |
B2-F | GCCGCGATTGCTTCCTGCTCGGTT |
B2-R | AAACAACCGAGCAGGAAGCAATCG |
B3-F | GCCGCAAGTACCTCCGCGCAATCG |
B3-R | AAACCGATTGCGCGGAGGTACTTG |
U-F | CTCCGTTTTACCTGTGGAATCG |
gRNA-R | CGGAGGAAAATTCCATCCAC |
B1' | TTCAGAGGTCTCTCTCGCACTGGAATCGGCAGCAAAGG |
B2 | AGCGTGGGTCTCGTCAGGGTCCATCCACTCCAAGCTC |
B2' | TTCAGAGGTCTCTCTGACACTGGAATCGGCAGCAAAGG |
B3 | AGCGTGGGTCTCGTCTTGGTCCATCCACTCCAAGCTC |
B3' | TTCAGAGGTCTCTAAGACACTGGAATCGGCAGCAAAGG |
BL | AGCGTGGGTCTCGACCGGGTCCATCCACTCCAAGCTC |
Seq1-F | TTCAAAGCAAAGCACCAAGC |
Seq1-R | GCTGGGGAAAACTTACAATG |
Seq2-F | TCCACTCGACTCCTCACTCA |
Seq2-R | GATTAGCGTAGGCCCTCACG |
Seq3-F | ATCCATCTCCGTATCTCT |
Seq3-R | GGTAGTCACCACCCTA |
Hyg-F | ACGGTGTCGTCCATCACAGTTTGCC |
Hyg-R | TTCCGGAAGTGCTTGACATTGGGGA |
NOFS | GCGGTGTCATCTATGTTACTAG |
M13F-47 | CGCCAGGGTTTTCCCAGTCACGAC |
图1 GS3、GS9和Badh2基因结构和靶点位置黑色序列为靶点序列,红色下划线序列为PAM序列。
Fig. 1. Gene structure and target site of GS3, GS9 and Badh2. The black sequence is the target sequence and the red underlined sequence is the PAM sequence.
图3 T0代植株转基因检测 M-DM2000 DNA标记; N-阴性对照; P-阳性对照; 1~16代表T0植株。
Fig. 3. Transgenic detection of T0 generation plants. M, DM2000 DNA marker; N, Negative control; P, Positive control; Lanes 1-16, T0 generation plants.
图4 三基因纯合突变植株测序结果红色小写字母表示插入1 bp的突变,红色连字符表示缺失的序列。
Fig. 4. Sequencing result of homozygous plants with triple gene mutation. Red lowercase letters represent 1 bp insertions and the deleted sequences are shown by red hyphens.
图5 T2代植株农艺性状考查数值用平均数±标准差表示,*,**分别表示在0.05和0.01水平上显著差异(t检验)。
Fig. 5. Agronomic traits of T2 generation plants. Values are shown as mean ± SD.*,**Significantly different at 0.05 and 0.01 levels by t-test, respectively.
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