title: Application of gene targeting to designed mutation breeding of high-tryptophan rice.
abstract: Site-directed mutagenesis via gene targeting (GT) based on homologous recombination is the ultimate mutation breeding technology because it enables useful information acquired from structural- and computational-based protein engineering to be applied directly to molecular breeding, including metabolic engineering, of crops. Here, we employed this rationale to introduce precise mutations in OASA2--an α-subunit of anthranilate synthase that is a key enzyme of tryptophan (Trp) biosynthesis in rice (Oryza sativa)--via GT, with subsequent selection of GT cells using a Trp analog. The expression level of OASA2 in plants homozygous and heterozygous for modified OASA2 was similar to that of nontransformants, suggesting that OASA2 transcription in GT plants was controlled in the same manner as endogenous OASA2, and that GT could lead to a lower risk of gene silencing than in conventional overexpression approaches. Moreover, we showed that enzymatic properties deduced from protein engineering or in vitro analysis could be reproduced in GT plants as evidenced by Trp accumulation levels. Interestingly, mature seeds of homozygous GT plants accumulated Trp levels 230-fold higher than in nontransformants without any apparent morphological or developmental changes. Thus, we have succeeded in producing a novel rice plant of great potential nutritional benefit for both man and livestock that could not have been selected using conventional mutagenesis approaches. Our results demonstrate the effectiveness of directed crop improvement by combining precision mutagenesis via GT with a knowledge of protein engineering.
authors.affiliation: Plant Genome Engineering Research Unit, Agrogenomics Research Center, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-8602, Japan.
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Saika
authors.forename: Akira
authors.initials: A
authors.lastname: Oikawa
authors.forename: Fumio
authors.initials: F
authors.lastname: Matsuda
authors.forename: Haruko
authors.initials: H
authors.lastname: Onodera
authors.forename: Kazuki
authors.initials: K
authors.lastname: Saito
authors.forename: Seiichi
authors.initials: S
authors.lastname: Toki
mesh_terms: D019943:Amino Acid Substitution / Q000235:genetics; D001483:Base Sequence; D053898:Biosynthetic Pathways / Q000235:genetics; D001947:Breeding; D018556:Crops, Agricultural / Q000235:genetics / Q000254:growth & development; D018506:Gene Expression Regulation, Plant; D018390:Gene Targeting / Q000379:methods*; D017343:Genes, Plant / Q000235:genetics; D008969:Molecular Sequence Data; D016296:Mutagenesis / Q000235:genetics*; D009154:Mutation / Q000235:genetics*; D012275:Oryza / Q000235:genetics* / Q000378:metabolism*; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism; D030821:Plants, Genetically Modified; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012639:Seeds / Q000378:metabolism; D014364:Tryptophan / Q000031:analogs & derivatives / Q000378:metabolism*
chemical_list: D010940:Plant Proteins; D014364:Tryptophan