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2019/11/18 ABRC Seminar

Speaker: Dr. Seiichi Toki (Research Unit Head, Plant Genome Engineering Research Unit, Institute of Agrobiological Sciences, National Agriculture and Food Research Organization (NARO), Japan)
Topic: Application of the precision genome editing system to develop novel plant traits and to understand basic plant biological processes

Dominant expression of splicing variants of the Orange (Or) gene causes β-carotene accumulation in cauliflower curd. We focused on manipulating, by CRISPR/Cas9 mutagenesis, the putative rice orthologue of cauliflower Or, OsOr, to accumulate β-carotene in rice. CRISPR/Cas9 vectors targeting OsOr were constructed and transformed into rice calli. Some transformed calli became orange due to β-carotene hyper-accumulation. Molecular analyses suggested that orange-colored calli resulted from the abundance of in-frame aberrant OsOr transcripts.
Rice bran oil (RBO) contains many valuable healthy constituents, including oleic acid. Improvement of the fatty acid composition in RBO by increasing the content of oleic acid, would increase health benefits. The enzyme fatty acid desaturase 2 (FAD2) catalyzes the conversion of oleic acid to linoleic acid in plants. Rice harbors three functional FAD2 genes; expression of OsFAD2-1 gene is highest in rice seeds. We produced homozygous OsFAD2-1 knockout rice plants. The content of oleic acid increased more than two-fold and, surprisingly, linoleic acid, a catabolite of oleic acid by FAD2, decreased dramatically to undetectable levels.
Tomato rin mutants fail to ripen: they do not produce red pigmentation, soften, or induce an ethylene burst. RIN has long been believed to function as a major regulator that is essential for ripening induction. We provide evidence contradicting this concept of RIN function by showing induction of fruit ripening in the absence of RIN. A CRISPR/Cas9-mediated RIN-knockout mutation did not repress initiation of ripening, and the mutant fruits showed moderate red coloration. Moreover, inactivation of the rin mutant allele partially restored induction of ripening. Therefore, RIN is not required for the initiation of ripening and rin is not a null mutation, but is rather a gain-of-function mutation that produces a protein that actively represses ripening.
Site-directed mutagenesis via gene targeting (GT) based on homologous recombination is the ultimate mutation breeding technology. We introduced precise mutations in OASA2, which encodes the α-subunit of anthranilate synthase, a key enzyme of tryptophan (Trp) biosynthesis, via GT, with subsequent selection of GT cells using a Trp analog. Enzymatic properties deduced from protein engineering or in vitro analysis were reproduced in GT plants as evidenced by Trp accumulation levels. Mature seeds of homozygous GT plants accumulated Trp levels 230-fold higher than did non-transformants without any apparent morphological or developmental changes.

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