Method of Obtaining Multileaflet Medicago Sativa Materials by Means of MsPALM1 Artificial Site-Directed Mutants

A method of obtaining multileaflet Medicago sativa materials by means of MsPALM1 artificial site-directed mutants, wherein, the method comprises the following steps:

• Step (1), Selecting a target site in an exon region of a compound leaf developmental regulatory gene MsPALM1 of Medicago sativa;
• wherein, one strand in the double-stranded structure of the target site has a structure of NGG, wherein N represents any one of bases A, T, C, G;
• Step (2), According to the nucleotide sequence of the target site, constructing a binary expression vector MsCRISPR/Cas9::PALM1 which is used for transforming Medicago sativa by Agrobacterium tumefaciens and further editing the MsPALM1 gene of Medicago sativa, the binary expression vector MsCRISPR/Cas9::PALM1 comprises an sgRNA expression frame and a Cas9 nuclease expression frame, the sgRNA expression frame comprises the guide sequence of the MsPALM1 target site;
• Step (3), Introducing the binary expression vector MsCRISPR/Cas9::PALM1 after being transformed by Agrobacterium tumefaciens into Medicago sativa cells, so that the sgRNA expression frame and the Cas9 gene expression frame are co-expressed in Medicago sativa cells;
• cutting the target site in the double-strand of MsPALM1 gene, inducing the DNA repairing function of Medicago sativa cells themselves;
• randomly inserting or deleting bases at target sites, to cause a loss-of-function mutation in the MsPALM1 gene of cells;
• Step (4), Regenerating plants from the Medicago sativa cells in step 3;
• Step (5), Performing PCR amplification on the DNA segment comprising the target site of MsPALM1 gene in the regenerated plants obtained from step 4, and then detecting by restriction endonuclease digestion and/or targeted deep sequencing;
• Step (6), Selecting the regenerated plants carrying four alleles with simultaneous loss-of-function mutation for phenotypic identification, observing the compound leaf phenotype of the regenerated plants, and selecting plants with more than 3 leaflets in all the compound leaves as the generated multileaflet Medicago sativa materials.