Method for obtaining glyphosate-resistant rice by site-directed nucleotide substitution

A method for obtaining a glyphosate-resistant plant, the method comprising:

• substituting threonine (T) at position 8 of the amino acid sequence of a conserved region of endogenous EPSPS protein of a target plant with isoleucine (I), and substituting proline (P) at position 12 with serine (S) to obtain a glyphosate-resistant plant,
• wherein the conserved region of the endogenous EPSPS protein of the target plant comprises the amino acid sequence of SEQ ID NO:
• 2,
• wherein the substituting step comprises introducing the following into a cell or tissue of the target plant via gene gun transformation:
• a first genetic material, a second genetic material and a donor vector:
• the first genetic material is a circular DNA plasmid, a linear DNA fragment or an RNA transcribed in vitro capable of expressing a first CRISPR/Cas9 nuclease;
• the second genetic material is a circular DNA plasmid, a linear DNA fragment or an RNA transcribed in vitro capable of expressing a second CRISPR/Cas9 nuclease;
• wherein the first CRISPR/Cas9 nuclease is able to specifically cleave a first target fragment in the genome of the target plant and the donor vector;
• and wherein the second CRISPR/Cas9 nuclease is able to specifically cleave a second target fragment in the genome of the target plant and the donor vector,
• wherein the donor vector additionally comprises a mutation target sequence released by the cleavage of the donor vector by the first and second CRISPR/Cas9 nucleases;
• the mutation target sequence contains a DNA fragment sequence corresponding to a sequence in the genome of the target plant from the 5′ end of a first target fragment to the 3′ end of a second target fragment, which contains the desired nucleotide substitution;
• wherein the first target fragment is positioned in an intron region or a promoter region in the genome of the target plant upstream of the nucleotide sequence encoding the amino acid sequence of the conserved region of the endogenous EPSPS protein,
• wherein the second target fragment is positioned in an intron region or a 3′-UTR region in the genome of the target plant downstream of a nucleotide sequence encoding the amino acid sequence of the conserved region of the endogenous EPSPS protein,
• wherein the nucleotide substitution is a mutation by which threonine (T) at position 8 of the amino acid sequence of the conserved region of endogenous EPSPS protein of a target plant is substituted with isoleucine (I), and proline (P) at position 12 is substituted with serine (S), and
• wherein the first genetic material, the second genetic material, and the donor vector are delivered simultaneously in a molar ratio of 1:1:2.