利用CRISPR/Cas9技术创制高效抗除草剂水稻

doi:10.16819/j.1001-7216.2022.211004

中国水稻科学 ›› 2022, Vol. 36 ›› Issue (5): 459-466.DOI: 10.16819/j.1001-7216.2022.211004

利用CRISPR/Cas9技术创制高效抗除草剂水稻

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()

1.黑龙江大学生命科学学院，哈尔滨 150080
2.中国科学院东北地理与农业生态研究所，哈尔滨 150081
3.江苏省农业科学院粮食作物研究所，南京 210014
4.东北林业大学生命科学学院，哈尔滨 150040
5.黑龙江八一农垦大学农学院，大庆 163319

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:
黑龙江省杰出青年基金资助项目(JQ2020C003)

Improvement of Herbicide Resistance in Rice by Using CRISPR/Cas9 System

YIN Liying¹, ZHANG Yuanye¹, LI Rongtian¹(), HE Mingliang², WANG Fangquan³, XU Yang³, LIU Xinxin⁴, PAN Tingting⁵, TIAN Xiaojie², BU Qingyun², LI Xiufeng²()

1. College of Life Science, Heilongjiang University, Harbin 150080, China
2. Northeast Institute of Geography and Agroecology, Key Laboratory of Soybean Molecular Design Breeding, Chinese Academy of Sciences, Harbin 150081, China
3. Institute of Food Crops, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China
4. College of Life Science, Northeast Forestry University, Harbin 150040, China
5. College of Agriculture, Heilongjiang Bayi Agricultural University, Daqing 163319, China

Received:2021-10-13 Revised:2021-12-07 Online:2022-09-10 Published:2022-09-09
Contact: LI Rongtian, LI Xiufeng

摘要/Abstract

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ALS^S627N突变植株10株，ALS^S627N且¹⁸⁸⁴G-A但第628位氨基酸未改变突变植株1株，ALS^S627N/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。

关键词: 水稻, 乙酰乳酸合酶, 抗除草剂, 咪唑乙烟酸, CRISPR/Cas9, 基因编辑

Abstract:

【Objective】 It is of great significance to create herbicide-resistant rice varieties. The acetyllactic acid synthase (ALS) gene was edited to create rice varieties with excellent herbicide resistance using non-herbicide-resistant japonica rice varieties with good quality as materials. 【Methods】Using CRISPR/Cas9 technology, the single-base mutant vector pH-NCas9-PBA-ALS was constructed with acyllactic acid synthase (ALS) as the target gene. Songjing 22, Longjing 46 and Suijing 18 were used as transformation materials to obtain transgenic plants through Agrobacterium-mediated genetic transformation. Genotypes and phenotypes were identified by sequencing the mutation sites of transgenic plants and herbicide spraying test. 【Results】 Ten ALS^S627N mutants, one ALS^S627N and ¹⁸⁸⁴G-A(the 628^th amino acid unchanged mutant), and one ALS^S627N/G628E mutant were obtained by molecular level detection. Compared with the wild type, the three types of mutants had stronger herbicide resistance. 【Conclusion】 Using CRISPR/Cas9 gene editing technology, we obtained homozygous mutant lines which are genetically stable and herbicide-resistant, providing basic materials for herbicide-resistant rice breeding.

Key words: rice, ALS, herbicide resistance, imazethapyr, CRISPR/Cas9, gene editing

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/表 9

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.

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.

图3 T0代转基因植株测序结果

Fig. 3. Sequencing results of T0 generation transgenic plants.

图4 T1代转基因植株测序结果

Fig. 4. Sequencing results of T1 generation transgenic plants.

图5 T1代转基因植株氨基酸突变类型

Fig. 5. Amino acid mutation of the target gene of T1 generation transgenic plants.

图6 T2代除草剂抗性鉴定（标尺=10 cm）

Fig. 6. Herbicide resistance identification of T2 generation (Bar=10 cm).

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
株系 Line	对照 CK	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

图7 T2代农用除草剂抗性鉴定（标尺=10 cm）

Fig. 7. Resistance identification of T2 generation to herbicides for agricultural use (Bar=10 cm).

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利用CRISPR/Cas9技术创制高效抗除草剂水稻

Improvement of Herbicide Resistance in Rice by Using CRISPR/Cas9 System

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