中国水稻科学 ›› 2022, Vol. 36 ›› Issue (5): 459-466.DOI: 10.16819/j.1001-7216.2022.211004
尹丽颖1, 张元野1, 李荣田1(), 何明良2, 王芳权3, 许扬3, 刘欣欣4, 潘婷婷5, 田晓杰2, 卜庆云2, 李秀峰2(
)
收稿日期:
2021-10-13
修回日期:
2021-12-07
出版日期:
2022-09-10
发布日期:
2022-09-09
通讯作者:
李荣田,李秀峰
基金资助:
YIN Liying1, ZHANG Yuanye1, LI Rongtian1(), HE Mingliang2, WANG Fangquan3, XU Yang3, LIU Xinxin4, PAN Tingting5, TIAN Xiaojie2, BU Qingyun2, LI Xiufeng2(
)
Received:
2021-10-13
Revised:
2021-12-07
Online:
2022-09-10
Published:
2022-09-09
Contact:
LI Rongtian, LI Xiufeng
摘要:
【目的】培育抗除草剂品种在水稻育种中具有重要意义。利用CRISPR/Cas9基因编辑技术,以黑龙江优质粳稻品种为材料,编辑乙酰乳酸合酶ALS基因,创制具有抗除草剂特性的水稻材料。【方法】利用CRISPR/Cas9技术,以乙酰乳酸合酶ALS为靶基因,构建单碱基突变载体pH-nCas9-PBE-ALS,以松粳22、龙粳46和绥粳18为转化材料,利用农杆菌介导转化获得转基因植株,通过对转基因植株的突变位点进行测序结合除草剂喷施试验,鉴定基因型及表型。【结果】经分子水平检测验证,获得ALSS627N突变植株10株,ALSS627N且1884G-A但第628位氨基酸未改变突变植株1株,ALSS627N/G628E突变植株1株。相较于野生型,以上三类突变植株均具有较强抗除草剂特性。【结论】利用CRISPR/Cas9基因编辑技术获得具有抗除草剂特性,能够稳定遗传,不含转基因标记的纯合株系,可为抗除草剂水稻育种提供基础材料。
尹丽颖, 张元野, 李荣田, 何明良, 王芳权, 许扬, 刘欣欣, 潘婷婷, 田晓杰, 卜庆云, 李秀峰. 利用CRISPR/Cas9技术创制高效抗除草剂水稻[J]. 中国水稻科学, 2022, 36(5): 459-466.
YIN Liying, ZHANG Yuanye, LI Rongtian, HE Mingliang, WANG Fangquan, XU Yang, LIU Xinxin, PAN Tingting, TIAN Xiaojie, BU Qingyun, LI Xiufeng. Improvement of Herbicide Resistance in Rice by Using CRISPR/Cas9 System[J]. Chinese Journal OF Rice Science, 2022, 36(5): 459-466.
图1 ALS基因结构和靶点位置 黑色序列为靶点序列,下划线序列为PAM序列。
Fig. 1. Gene structure and target site of ALS. The black sequence is the target sequence and the underlined sequence is the PAM sequence.
引物名称 Primer name | 引物序列(5'-3') Primer sequence(5'-3') |
---|---|
CAS9-ALS-LP | ATGATCCCAAGTGGGGGCGC |
CAS9-ALS-RP | GCGCCCCCACTTGGGATCAT |
M13-F | GTAAAACGACGGCCAGT |
ALS-F | GCATTGAGAACCTCCCTGTG |
ALS-R | TGTGATGCATATGCCTACAG |
HPT-F | TGCGCCCAAGCTGCATCAT |
HPT-R | TGAACTCACCGCGACGTCTGT |
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') |
---|---|
CAS9-ALS-LP | ATGATCCCAAGTGGGGGCGC |
CAS9-ALS-RP | GCGCCCCCACTTGGGATCAT |
M13-F | GTAAAACGACGGCCAGT |
ALS-F | GCATTGAGAACCTCCCTGTG |
ALS-R | TGTGATGCATATGCCTACAG |
HPT-F | TGCGCCCAAGCTGCATCAT |
HPT-R | TGAACTCACCGCGACGTCTGT |
图2 T0代植株转基因检测 M―DM2000 DNA 标记; 1―阴性对照; 2―阳性对照; 3~23―T0 代植株。
Fig. 2. Transgenic detection of T0 generation plants. M, DM2000 DNA Marker; 1, Negative control; 2, Positive control; 3−23, T0 generation plants.
株系 Line | 对照 CK | 喷施除草剂 Exposure to herbicide | ||
---|---|---|---|---|
3 d | 15 d | 存活率 Survival rate/% | ||
松粳22 Songjing 22 | 10 | 10 | 0 | 0 |
SJ22-1 | 10 | 0 | 10 | 100 |
SJ22-2 | 10 | 0 | 10 | 100 |
SJ22-3 | 10 | 0 | 10 | 100 |
SJ22-4 | 10 | 0 | 10 | 100 |
龙粳46 Longjing 46 | 10 | 8 | 0 | 0 |
LJ46-1 | 10 | 0 | 10 | 100 |
LJ46-2 | 10 | 0 | 10 | 100 |
LJ46-3 | 9 | 0 | 9 | 100 |
LJ46-4 | 10 | 0 | 10 | 100 |
绥粳18 Suijing 18 | 10 | 9 | 0 | 0 |
SJ18-1 | 10 | 0 | 10 | 100 |
SJ18-2 | 10 | 0 | 10 | 100 |
SJ18-3 | 10 | 0 | 10 | 100 |
SJ18-4 | 10 | 0 | 10 | 100 |
表2 喷施除草剂咪唑乙烟酸后T2代植株存活率调查
Table 2. Survey of Survival rate of T2 plants sprayed with imidazole ethylnicotinic acid.
株系 Line | 对照 CK | 喷施除草剂 Exposure to herbicide | ||
---|---|---|---|---|
3 d | 15 d | 存活率 Survival rate/% | ||
松粳22 Songjing 22 | 10 | 10 | 0 | 0 |
SJ22-1 | 10 | 0 | 10 | 100 |
SJ22-2 | 10 | 0 | 10 | 100 |
SJ22-3 | 10 | 0 | 10 | 100 |
SJ22-4 | 10 | 0 | 10 | 100 |
龙粳46 Longjing 46 | 10 | 8 | 0 | 0 |
LJ46-1 | 10 | 0 | 10 | 100 |
LJ46-2 | 10 | 0 | 10 | 100 |
LJ46-3 | 9 | 0 | 9 | 100 |
LJ46-4 | 10 | 0 | 10 | 100 |
绥粳18 Suijing 18 | 10 | 9 | 0 | 0 |
SJ18-1 | 10 | 0 | 10 | 100 |
SJ18-2 | 10 | 0 | 10 | 100 |
SJ18-3 | 10 | 0 | 10 | 100 |
SJ18-4 | 10 | 0 | 10 | 100 |
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