Ami Takeuchi, Mariko Ohnuma, Hiroshi Teramura, Kenji Asano, Takahiro Noda, Hiroaki Kusano, Koji Tamura, Hiroaki Shimada

Plant Biotechnology; Vol. 38 (2021), No. 3 pp. 345-353

Abstract

The potato tuber starch trait is changed depending on the composition of amylose and amylopectin. The amount of amylopectin is determined by the activity of the starch branching enzymes SBE1, SBE2, and SBE3 in potato. SBE3, a homolog of rice BEI, is a major gene that is abundant in tubers. In this study, we created mutants of the potato SBE3 gene using CRISPR/Cas9 attached to the translation enhancer dMac3. Potato has a tetraploid genome, and a four-allele mutant of the SBE3 gene is desired. Mutations in the SBE3 gene were found in 89 of 126 transformants of potato plants. Among these mutants, 10 lines contained four mutant SBE3 genes, indicating that 8% efficiency of target mutagenesis was achieved. These mutants grew normally, similar to the wild-type plant, and yielded sufficient amounts of tubers. The potato starch in these tubers was similar to that of the rice BEI mutant. Western blot analysis revealed the defective production of SBE3 in the mutant tubers, suggesting that these transformants were loss-of-function mutants of SBE3.

See:  https://doi.org/10.5511/plantbiotechnology.21.0727a

or https://www.jstage.jst.go.jp/article/plantbiotechnology/advpub/0/advpub_21.0727a/_article/-char/ja/

Figure 1. Structure of the potato SBE3 gene and location of the gRNAs used for targeted mutagenesis. (A) Schematic representation of the SBE3 gene. Exons are indicated by boxes. Arrows in the upper panel indicate the position of the PCR primers that were used for amplification of the fragment for the CAPS analysis. The positions of the gRNAs are shown in the lower panel. (B) Nucleotide sequences of the regions corresponding to the gRNAs. The upper panel shows the nucleotide sequences of gRNA-1, gRNA-2, and gRNA-3 used for this experiment. The BamHI site is shown in red letters. The lower panel shows the corresponding nucleotide sequences in WT-C. Polymorphic nucleotides found in the WT-C genome are highlighted. PAM sequences are underlined.