Functional characterization of homologues of repair enzyme genes for ultraviolet-induced DNA damages in higher plants.

Research Project

Project/Area Number	06660009
Research Category	Grant-in-Aid for General Scientific Research (C)
Allocation Type	Single-year Grants
Research Field	Breeding science
Research Institution	Kagawa University
Principal Investigator	IKEDA Shigeru Kagawa Univ., Fac., Agric., Research associate, 農学部, 助手 (90151290)
Project Period (FY)	1994 – 1995
Project Status	Completed (Fiscal Year 1995)
Budget Amount *help	¥1,900,000 (Direct Cost: ¥1,900,000) Fiscal Year 1995: ¥600,000 (Direct Cost: ¥600,000) Fiscal Year 1994: ¥1,300,000 (Direct Cost: ¥1,300,000)
Keywords	DNA Repair / Rice / Gene Tranfer
Research Abstract	Two sensitive detection methods, ELISA and immuno-PCR method, utilizing monoclonal antibodies were developed to estimate the ability of rice cells to repair thymine dimers induced by ultraviolet irradiation. After rice tissues were irradiated by ultraviolet, expression of PRHs (homologues of E.coli phr ; gene for photoreactivation enzyme) and ERHs (homologues of E.coli uvrB ; gene for excision repair enzyme) were estimated by Northern blotting, and repairability of thymine dimers was estimated by the immuno-PCR method. PRH1 and ERH1 showing UV irradiation-specific expression were selected to transformate rice cells. The rice protoplasts were co-transformed with plasmids harvoring E.coli phr or uvrB,PRH1, or ERH1 and plasmids harvoring kanamycin resistance gene by electroporation. Kanamycin resistant calli showed expression of introduced repair gene or its homologue and higher repairability of thymin dimers than non-transformants. The rice protoplasts were also electroporated with yeast phr gene by the method described above, but kanamycin resistant calli showed expression of phr and the same repairablity as of non-transformants. Delesion mutant genes, phrDELTA and PRH1DELTA, lost highly conservative amino acids sequence (PIVDAAMRQL) were construced and introduced into rice protoplasts by co-transformation method described above. Kanamycin resistant calli showed expression of mutant gene and the same repairability as of non-transformants. Therefore the conclusion that PRH1 is a rice gene for photoreactivation enzyme was drawn. Unfortunately any plantlets were not regenerated from kanamycin resistant calli.