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

高等植物紫外线诱导 DNA 损伤修复酶基因同源物的功能表征。

基本信息

批准号：
06660009
负责人：
IKEDA Shigeru
金额：
$ 1.22万
依托单位：
Kagawa University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1994
资助国家：
日本
起止时间：
1994 至 1995
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-06660009/
关键词：
DNA Repair Rice Gene Tranfer

项目摘要

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.

开发了两种敏感的检测方法，即ELISA和免疫PCR方法，利用单克隆抗体来估计水稻细胞修复紫外线辐射诱导的胸腺嘧啶二聚体的能力。在紫外线辐照水稻组织后，PRH的表达（E.Coli Phr的同源物；用于光电反应酶的基因）和ERH（E.Coli UVRB的同源物；切除酶的基因的同源物）通过北粒斑点估算了胸骨二聚体的修复方法，估算了北虫的修复能力。选择了显示紫外线特异性表达的PRH1和ERH1以转化水稻细胞。将水稻原生质体与收获大肠杆菌PHR或UVRB，PRH1或ERH1的质粒一起转化，并通过电穿孔收获卡纳米霉素耐药基因。卡纳霉素耐药的Calli表达了引入的修复基因或其同源物的表达，比非转化剂比非转化剂更高。通过上述方法，还用酵母PHR基因对水稻原生质体进行电穿孔，但是抗卡纳米霉素的Calli表现出PHR的表达和与非转化剂相同的修复性。 Delesion突变基因，Phrdelta和Prh1delta，高度保守的氨基酸序列（PIVDAAMRQL）丢失，并通过上述共转化方法被约束并引入水稻原生质体中。卡纳霉素耐药的Calli表现出突变基因的表达和与非转化剂相同的可修复性。因此，得出了PRH1是用于光电反应酶的水稻基因的结论。不幸的是，任何植被都没有从抗卡纳米霉素的calli中再生。