利用CRISPR/Cas9技术创制高效抗除草剂水稻

【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。

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

【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.