Isolation and analysis of CPD photolyase gene of spinach and fundamental study for the production pf transgenic plant resistant to UV

我们克隆了菠菜环丁烷嘧啶二聚体(CPD)光解酶基因，并测定了该基因在雄株和雌株中的功能和表达程度。以NiRNA为模板，以简并引物为模板，用RT-PCR方法克隆了该基因的全长c DNA。该基因由9个外显子组成，氨基酸序列与第二类CPD光解酶非常相似。该基因恢复了光修复缺陷突变体的光复活能力，表明该基因为光解酶基因。通过酶联免疫吸附试验确认CPD的清除。该基因是单拷贝的。在雄性和雌性菠菜中，根的表达水平低于叶、茎和花。紫外线会产生活性氧(ROS)。对大肠杆菌中ROS突变的特异性进行了研究。碱基改变突变在突变中占主导地位。

We isolated a gene for cyclobutane pyrimidine dimer (CPD) photolyase in spinach and determined the function and the extent of the expression in male and female plants. cDNA of the gene was cloned by RTPCR with the use of niRNA as a tremplate and degenerate primers. The gene consists of nine exsons and amino acid sequence was quite similar to class II CPD photolyase. The gene obtined here restored the photoreactivating ability of mutant E. coil deficient in photorepair indicating that the gene was photolyase gene. Removal of CPD was confirmed by ELISA procedure. The gene was a single copy. The level of expression in root was lower than in leaves, stems and flowers in male and female spinach. UV generates reactive oxygen species (ROS). Specificity of ROS mutation was investigated in E. coil. Base change mutations were predominant among mutations.