摘要： CRISPR/Cas9基因编辑技术已经成为水稻育种新型手段，香稻育种一直是水稻育种行业的研究热点，水稻香味主要由8号染色体上的甜菜碱脱氢酶基因Badh2控制。为了改良原有品种的香味性状，促进香稻育种的发展，以非香型粳稻品种东农425为试验材料，构建了2个CRISPR/Cas9敲除载体对Badh2基因进行定点编辑，第1个载体（pYLCRISPR/Cas9-B1-gRNA）的2个靶点分别位于第2和第3外显子上，第2个载体（pYLCRISPR/Cas9-B2-gRNA）的2个靶点均在第2外显子上。采用农杆菌介导法对东农425进行遗传转化，成功获得了T₀纯合植株，并对其衍生出的T₁植株进行T-DNA元件检测，共获得8个突变类型不同且不含有转基因成分的纯合突变株系。同时采用咀嚼法和氢氧化钾浸泡法对这8个纯合突变株系的水稻籽粒进行香味检测，结果表明，8个Badh2株系均具有不同程度的香味，总体上载体pYLCRISPR/Cas9-B1-gRNA编辑的3个株系的香味更为浓郁。对这8个纯合突变株系的主要农艺性状进行考察，发现突变株系的穗数、穗粒数、结实率及千粒质量与野生型相比均没有明显差异。综上，本研究对东农425的Badh2基因成功地进行了编辑，并获得了香味显著提高且无转基因成分的纯合突变体材料，为加快香型粳稻品种的培育提供了技术支持。

关键词: 粳稻, CRISPR/Cas9, Badh2, 香味

Abstract: Rice is the main food crop planted in the world, and the fragrant rice breeding has always been a hotspot in the rice breeding. The aroma of rice is mainly controlled by betaine dehydrogenase gene(Badh2), located on chromosome 8. In this study, a non-fragrant japonica variety Dongnong 425 was used as the test material, and two CRISPR/Cas9 knockout vectors were constructed to edit Badh2. The two targets of the first vector(pYLCRISPR/Cas9-B1 -gRNA)were located on the second exon and the third exon, respectively, while the two targets of the second vector(pYLCRISPR/Cas9-B2 -gRNA)were both on the second exon. The Agrobacterium-mediated transformation was used to transform Dongnong 425, and the homozygous T₀ generation plants were successfully obtained. The T₁ plants derived from the homozygous T₀ generation plants were tested for T-DNA elements, and a total of eight homozygous mutant lines were obtained, which had different mutation types and non-transgenic components. At the same time, the seeds of the eight homozygous mutant lines were tested for fragrance by chewing method and potassium hydroxide immersion method. The results showed that the eight Badh2 lines had different degrees of fragrance, of which the fragrance of three lines edited by the vector pYLCRISPR/Cas9-B1 -gRNA were more intensive. The main agronomic traits of the eight homozygous mutants were investigated, indicating that the number of panicles per plant, the number of grains per panicle, the seed setting rate and the 1000-grain weight were not significantly different from those of the wild-type. In summary, this study successfully edited the Badh2 gene of Dongnong 425, and obtained the homozygous mutant lines with significantly improved fragrance and non-transgenic components, which provided the technical support for accelerating the breeding of fragrant japonica rice varieties.

Key words: japonica rice, CRISPR/Cas9, Badh2, Fragrance

中图分类号: 

• Q78

• S511.03