Homologous recombination-mediated knock-in targeting of the MET1a gene for a maintenance DNA methyltransferase reproducibly reveals dosage-dependent spatiotemporal gene expression in rice

Authors

  • Takaki Yamauchi,

    1. National Institute for Basic Biology, Okazaki 444-8585, Japan
    2. Graduate School of Science and Technology, Chiba University, Matsudo 271-8510, Japan
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    • Present address: Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo 113-8657, Japan.

  • Yasuyo Johzuka-Hisatomi,

    1. National Institute for Basic Biology, Okazaki 444-8585, Japan
    2. Graduate School of Nutritional and Environmental Sciences, University of Shizuoka, Shizuoka 422-8526, Japan
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  • Sachiko Fukada-Tanaka,

    1. National Institute for Basic Biology, Okazaki 444-8585, Japan
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  • Rie Terada,

    1. National Institute for Basic Biology, Okazaki 444-8585, Japan
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  • Ikuo Nakamura,

    1. Graduate School of Horticulture, Chiba University, Matsudo 271-8510, Japan
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  • Shigeru Iida

    Corresponding author
    1. National Institute for Basic Biology, Okazaki 444-8585, Japan
    2. School of Pharmaceutical Sciences, University of Shizuoka, Shizuoka 422-8526, Japan
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*(fax +81 54 264 5503; e-mail shigiid@u-shizuoka-ken.ac.jp).

Summary

Although homologous recombination-promoted knock-in targeting to monitor the expression of a gene by fusing a reporter gene with its promoter is routine practice in mice, gene targeting to modify endogenous genes in flowering plants remains in its infancy. In the knock-in targeting, the junction sequence between a reporter gene and an endogenous target promoter can be designed properly, and transgenic plants carrying an identical and desired knock-in allele can be repeatedly obtained. By employing a reproducible gene-targeting procedure with positive–negative selection in rice, we were able to obtain fertile transgenic knock-in plants with the promoterless GUS reporter gene encoding β-glucuronidase fused with the endogenous promoter of MET1a, one of two rice MET1 genes encoding a maintenance DNA methyltransferase. All of the primary (T0) transgenic knock-in plants obtained were found to carry only one copy of GUS, with the anticipated structure in the heterozygous condition, and no ectopic events associated with gene targeting could be detected. We showed the reproducible, dosage-dependent and spatiotemporal expression of GUS in the selfed progenies of independently isolated knock-in targeted plants. The results in knock-in targeted plants contrast sharply with the results in transgenic plants with the MET1a promoter-fused GUS reporter gene integrated randomly in the genome: clear interindividual variation of GUS expression was observed among independently obtained plants bearing the randomly integrated transgenes. As our homologous recombination-mediated gene-targeting strategy with positive–negative selection is, in principle, applicable to modify any endogenous gene, knock-in targeting would facilitate basic and applied plant research.

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