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

doi:10.16819/j.1001-7216.2022.211004

Abstract

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

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

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.

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.

Figures/Tables 9

Fig. 1. Gene structure and target site of ALS. The black sequence is the target sequence and the underlined sequence is the PAM sequence.

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

Fig. 2. Transgenic detection of T0 generation plants. M, DM2000 DNA Marker; 1, Negative control; 2, Positive control; 3−23, T0 generation plants.

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

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

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

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

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

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

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