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

• 研究报告 • 上一篇    下一篇

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. 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. 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:

【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,ALSS627N1884G-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。

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 ALSS627N mutants, one ALSS627N and 1884G-A(the 628th amino acid unchanged mutant), and one ALSS627N/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