A method for site-specific mutagenesis of a medicago sativa gene by using a CRISPR/Cas9 system. The method comprises: first constructing a universal binary expression vector MsCRISPR/Cas9 mediated and transformed by agrobacterium tumefaciens in medicago sativa; then designing a CRISPR/Cas9-based sgRNA sequence for a target gene in the medicago sativa, and connecting a DNA fragment for encoding the sgRNA sequence to MsCRISPR/Cas9 to construct a carrier MsCRISPR/Cas9::target; and then mediating and transforming the medicago sativa by using the agrobacterium tumefaciens, and obtaining, by screening, a mutant transformed plant with the target gene knocked out. According to the method, an MtU6 promoter is used for initiating sgRNA transcription in the medicago sativa.
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