CRISPR/Cas9-mediated Editing Rice EPSPS Gene

• Main Authors: Yi-Wun Huang, 黃怡文
• Other Authors: Yuh-Chyang Charng
• Format: Others
• Language: zh-TW
• Published: 2019
• Online Access: http://ndltd.ncl.edu.tw/handle/8v6ubs

碩士 === 國立臺灣大學 === 農藝學研究所 === 107 === Clustered regularly interspaced short palindromic repeats-associated protein 9 (CRISPR/Cas9) has emerged as the most powerful technology for efficient genomic modification in many organisms. To obtain glyphosate resistant rice, VQR variant of Cas9 requires the sites containing NGAN PAM (protospacer adjacent motif) was expressed and expected to mutate one-point of rice EPSPS (5-enolpyruvylshikimate-3-phosphate synthase), of which the codon CCA for the glycine residue at position 177 of the protein was changed to encode a serine or threonine residue (DNA sequences are TCA or ACA, respectively). The position of the target base was 4 bases upstream PAM. Rice calli were transformed using Agrobacterium. Single-nucleotide substitution was detected by PCR technique between 20 bases within the protospacer. Analyzing 12 positions of each transgenic plants of 25 independent lines showed that there was no substitution; 1.6% of the tested results were pseudo. The concentration of glyphosate selection criteria was 5 mM in this study. Transgenic seedlings were regenerated until T1 seeds which are collected for further research.