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

利用单抗建立了两种敏感的检测方法--酶联免疫吸附试验和免疫-聚合酶链式反应，以评价水稻细胞修复紫外线辐射诱导的胸腺嘧啶二聚体的能力。经紫外线照射后，用Northern印迹法检测光复活酶基因PRHs和切除修复酶基因ERHs的表达，并用免疫-聚合酶链式反应方法检测胸腺嘧啶二聚体的修复能力。筛选出紫外光特异表达的PRH1和ERH1转化水稻细胞。利用电穿孔技术，将获得的pHR、uvrB、PRH1、ERH1表达载体与获得卡那霉素抗性基因的表达载体共转化水稻原生质体。卡那霉素抗性愈伤组织表达了导入的修复基因或其同系物，胸腺素二聚体的修复能力高于未转化的愈伤组织。用上述方法也将酵母phr基因电击转化水稻原生质体，但卡那霉素抗性愈伤组织表现出phr基因的表达和与非转化体相同的修复能力。构建了缺失高度保守氨基酸序列(PIVDAAMRQL)的缺失突变基因PhrDELTA和PRH1DELTA，并通过共转化的方法将其导入水稻原生质体。卡那霉素抗性愈伤组织表达突变基因，修复能力与非转化子相同。由此推断，prh1是一个水稻光复活酶基因。不幸的是，没有任何植株是从卡那霉素抗性愈伤组织中再生出来的。

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.