Method for acquiring aromatic rice strain by targeting Badh2 gene via CRISPR/Cas9 gene editing technology

Abstract

The invention discloses a method for acquiring an aromatic rice strain by targeting a Badh2 gene via CRISPR/Cas9 gene editing technology. The method comprises the following steps: separately targeting sequences, recognizable by Cas9, of every exon and intron of an aromatic gene by using the CRISPR/Cas9 gene editing technology and then cutting a genomic DNA sequence to initiate DNA restoration so as to obtain an afunctional Badh2 gene; with the callus of Indica rice, japonica rice or glutinous rice as a receptor material of genetic transformation and an mature embryo, young ear, ovary or the like as explant, carrying out induction so as to obtain a diploid callus, introducing a targeting vector into cells of the callus by using an Agrobacterium mediated transformation method, screening and identifying positive plants and separating the plants from a T1 colony so as to obtain an aromatic rice strain; and with anther, pollen, unfertilized ovary or the like as explant, carrying out induction so as to obtain a haploid callus, introducing the targeting vector into cells of the callus by using the Agrobacterium mediated transformation method, screening a positive callus, reduplicating the positive callus by using colchicine, carrying out differentiation to form seedlings and identifying genetic transformation positive plants so as to eventually obtain the aromatic rice strain.

Description

technical field [0001] The invention relates to the technical field of gene editing, in particular to a method for obtaining a fragrant rice strain by targeting the Badh2 gene with the CRISPR / Cas9 gene editing technology. Background technique [0002] Transcription activator-like effector nuclease (TALEN), zinc-finger nuclease (ZFN) and clustered regularly interspaced short palindromic repeat (CRISPR) technology are currently three major technologies in the field of genome editing. technology. At present, TALEN and ZFN are two well-developed site-directed mutagenesis technologies. However, these two technologies are relatively cumbersome in the process of constructing vectors that can identify nucleases at specific sites, and each site requires the construction of two corresponding nucleases. Contents of the invention [0003] Based on the technical problems existing in the background technology, the present invention proposes a method of targeting the Badh2 gene with CRI...

AI Technical Summary

Problems solved by technology

At present, TALEN and ZFN are two well-developed site-directed mutagenesis technologies. However, these two technologies are relatively cumbersome in the process of constructing vectors to identify nucleases at specific sites, and each site needs to construct two corresponding nucleases.