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

菠菜CPD光解酶基因的分离分析及抗紫外线转基因植株生产基础研究

• 批准号: 13680628
• 负责人: TAKIMOTO Koichi
• 金额: $ 2.3万
• 依托单位: Yamaguchi University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13680628/
• 关键词: ultraviolet light; DNA damage; DNA repair; pvrimidine dimers; photoreactivation; photoreactivating enzyme; spinach; plant; ピリミジン二量体

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.

我们在菠菜中分离了一个环丁烷嘧啶二聚体（CPD）光裂合酶基因，并确定了该基因在雄性和雌性植株中表达的功能和程度。以mRNA为模板，采用简并引物，通过RTPCR方法克隆了该基因的cDNA。该基因由9个外显子组成，氨基酸序列与Ⅱ类CPD光裂合酶十分相似。该基因恢复了突变体E. coil缺乏光修复功能，表明该基因为光裂合酶基因。通过ELISA程序确认CPD的去除。该基因是单拷贝的。在雌雄菠菜中，根中的表达水平低于叶、茎和花。UV产生活性氧物质（ROS）。在大肠杆菌中研究了ROS突变的特异性。线圈突变中以碱基改变突变为主。

项目成果

Tano, K., Iwamatsu, Y., Utsumi, H., Takimoto, K.: "Increased base change mutations at G:C pairs in Escherichia coli deficient in endonuclease III and VIII"J. Radiat. Res.. 42・4. 409-413 (2001)

Tano, K.、Iwamatsu, Y.、Utsumi, H.、Takimoto, K.：“内切核酸酶 III 和 VIII 缺陷的大肠杆菌中 G:C 对碱基变化突变增加”J Radiat。 .409-413 (2001)

Keizo Tano: "Increased base change mutations at G: C pairs in Escherichia coil deficient in endonuclease III and VIII"J. Radiat. Res.. 42.4. 409-413 (2001)

Keizo Tano：“内切核酸酶 III 和 VIII 缺陷的大肠杆菌中 G: C 对的碱基变化突变增加”J.

K.Tano, Y.Iwamatsu, S.Yasuhira, Utsumi, H., K.Takimoto: "Increased base change mutations at G : C pairs in Escherichia coli deficient in Endonuclease III and VIII"J. Radiat Res.. 42(4). 409-413 (2001)

K.Tano、Y.Iwamatsu、S.Yasuhira、Utsumi、H.、K.Takimoto：“缺乏核酸内切酶 III 和 VIII 的大肠杆菌中 G 处碱基变化突变增加：C 对”J.

Mano, J., Torii, Y., Hayashi, S., Takimoto, K., Matsui, K., Nakamura, K., Inze, D., Babiychuk, E., Kushnir, S., Asada, K.: "The NADPH : Quinone oxidoreductase P1-ζ-crystallin in Arabidopsis catalyzes the a,b-hydrogenation of 2-alkenals : Detoxication of t

Mano, J.、Torii, Y.、Hayashi, S.、Takimoto, K.、Matsui, K.、Nakamura, K.、Inze, D.、Babiychuk, E.、Kushnir, S.、Asada, K.： “NADPH：拟南芥中的醌氧化还原酶 P1-ζ-晶状体蛋白催化 2-烯醛的 a,b-氢化：t 的解毒

Junichi Mano: "The NADPH: Quinone oxidoreductase P1-ζ-crystallin in Arabidopsis catalyzes the a, b-hydrogenation of 2-alkenals : Detoxication of the lipid peroxide-derived reactive aldehydes"Plant Cell Physiol.. 43.12. 1445-1455 (2002)

Junichi Mano：“NADPH：拟南芥中的醌氧化还原酶 P1-δ-晶状体蛋白催化 2-烯醛的 a、b-氢化：脂质过氧化物衍生的反应性醛的解毒”植物细胞生理学.. 43.12。 ）