Exploring the Agrobacterium-mediated transformation with CRISPR/Cas9 in cucumber (Cucumis sativus L.)

文献类型： 外文期刊

第一作者： Zhao, Ziyao

作者： Zhao, Ziyao;Qi, Yaguang;Yang, Zhimin;Cheng, Liyu;Sharif, Rahat;Li, Yuhong;Zhao, Ziyao;Sharif, Rahat;Raza, Ali;Chen, Peng;Hou, Dong

作者机构：

关键词： Cucumber; GUS assay; Genetic transformation; CRISPR; Cas9

期刊名称：MOLECULAR BIOLOGY REPORTS （ 影响因子：2.742； 五年影响因子：2.702 ）

ISSN： 0301-4851

年卷期： 2022 年 49 卷 12 期

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收录情况： SCI

摘要： Backgrounds The narrow genetic basis of cucumber makes breeding of this species difficult. CRISPR/Cas9 system is characteristic of simple design, low cost and high efficiency, which has opened a new path for cucumber functional genetics and the development of cucumber mocular breeding. However, the immature genetic transformation system is the main limiting factor for applying this technology in cucumber. Methods and Results In this study, a Histochemical beta-glucuronidase (GUS) assay was used to analyze the effect of various parameters, including slight scratch of explants, pre-culture time, acetosyringone (AS) concentration, infection time in Agrobacterium solution, and co-culture period on the transformation efficiency. The results showed that the explants slightly scratched after cutting, pre-cultured for 1 day, Agrobacterium bacterial solution containing AS, and 20 min length of infection could significantly increase the GUS staining rate of explants. On this basis, two sequences with high specificity (sgRNA-1 and sgRNA-2) targeted different loci of gene CsGCN5 were designed. The corresponding vectors Cas9-sgRNA-1 and Cas9-sgRNA-2 were constructed and transformed using the above-optimized cucumber genetic transformation system, and three and two PCR positive lines were obtained from 210 and 207 explants, respectively. No sequence mutation at target loci of CsGCN5 was detected in the Cas9-sgRNA-1 transformed three PCR positive lines. However, one mutant line with targeted homozygous change was recognized from the Cas9-sgRNA-2 transformed two PCR positive lines. Conclusion In this study, 2.4 parts per thousand of total explants had directed mutation in the CsGCN5 gene. The results in the present study would be beneficial to further optimize and improve the efficiency of the genetic transformation of cucumber.

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