文献类型： 外文期刊

作者： Liu, Han ¹ ; Lin, Baogang ¹ ; Ren, Yun ³ ; Hao, Pengfei ¹ ; Huang, Lan ¹ ; Xue, Bowen ¹ ; Jiang, Lixi ⁴ ; Zhu, Yang ⁴ ; Hua, Shuijin ¹ ;

作者机构： 1.Zhejiang Acad Agr Sci, Inst Crops & Nucl Technol Utilizat, Hangzhou, Peoples R China

2.Yongding Agr & Rural Bur Longyan, Dept Seed Management, Longyan, Peoples R China

3.Huzhou Agr Sci & Technol Dev Ctr, Inst Crop Sci, Huzhou, Peoples R China

4.Zhejiang Univ, Coll Agr & Biotechnol, Hangzhou, Peoples R China

关键词： CRISPR/Cas9; genome editing; mutation; oleic acid; rapeseed; sequencing; yield

期刊名称：FRONTIERS IN PLANT SCIENCE （ 影响因子：6.627； 五年影响因子：7.255 ）

ISSN： 1664-462X

年卷期： 2022 年 13 卷

页码：

收录情况： SCI

摘要： Seed oleic acid is an important quality trait sought in rapeseed breeding programs. Many methods exist to increase seed oleic acid content, such as the CRISPR/Cas9-mediated genome editing system, yet there is no report on seed oleic acid content improvement via this system's precise editing of the double loci of BnFAD2. Here, a precise CRISPR/Cas9-mediated genome editing of the encoded double loci (A5 and C5) of BnFAD2 was established. The results demonstrated high efficiency of regeneration and transformation, with the rapeseed genotype screened in ratios of 20.18% and 85.46%, respectively. The total editing efficiency was 64.35%, whereas the single locus- and double locus-edited ratios were 21.58% and 78.42%, respectively. The relative proportion of oleic acid with other fatty acids in seed oil of mutants was significantly higher for those that underwent the editing on A5 copy than that on C5 copy, but it was still less than 80%. For double locus-edited mutants, their relative proportion of oleic acid was more than 85% in the T-1 and T-4 generations. A comparison of the sequences between the double locus-edited mutants and reference showed that no transgenic border sequences were detected from the transformed vector. Analysis of the BnFAD2 sequence on A5 and C5 at the mutated locus of double loci mutants uncovered evidence for base deletion and insertion, and combination. Further, no editing issue of FAD2 on the copy of A1 was detected on the three targeted editing regions. Seed yield, yield component, oil content, and relative proportion of oleic acid between one selected double loci-edited mutant and wild type were also compared. These results showed that although the number of siliques per plant of the wild type was significantly higher than those of the mutant, the differences in seed yield and oil content were not significant between them, albeit with the mutant having a markedly higher relative proportion of oleic add. Altogether, our results confirmed that the established CRISPR/Cas9-mediated genome editing of double loci (A5 and C5) of the Bn FAD2 can precisely edit the targeted genes, thereby enhancing the seed oleic acid content to a far greater extent than can a single locus-editing system.