Abstract:

UV-induced DNA damage in plants has harmful effect on agriculture production. The UV-damaged DNA binding protein-2 （DDB2）has been identified to participate in DNA damage repair process caused by UV radiation in human, rice and Arabidopsis. DDB2 could interact with DDB1 and CUL4 to form the typical E3 ubiquitin ligase complex, which was involved in various developmental processes in plant life style, including root and shoot development, flowering control and photomorphogenesis. DDB2 gene was cloned from tomato and constructed to the DDB2-YFP fusion expression vector. Through semi-quantitive PCR analysis, DDB2 was found to be up-regulated upon UV irradiation in wild type tomato. Bioinformatic analysis showed DDB2 had two WD-40 repeats and one DWD box conserved domain. DDB2-YFP fusion protein was introduced into tomato and revealed that DDB2 was targeted to tomato nuclei exclusively. Yeast two-hybrid assays were performed and confirmed DDB1 could interact with DDB2 and CUL4, supporting the formation that DDB1 acted as a bridge to form the typical CUL4-DDB1-DDB2 complex.

Key words: tomato, DDB2, subcellular localization, yeast two-hybrid analysis

摘要：

紫外(UV)辐射诱导的DNA损伤可导致农作物减产。DDB2蛋白参与由紫外辐射造成的DNA损伤的修复过程已经在人类、水稻和拟南芥中得到证实。DDB2与DDB1、CUL4相互作用形成E3泛素连接酶复合体,该复合体对植物根茎生长、花期调控和光形态建成等多种植物发育过程起调控作用。成功克隆了番茄DDB2基因,构建了DDB2-黄色荧光蛋白融合表达载体。通过对野生型番茄UV辐射后DDB2基因表达水平的半定量分析,证明番茄DDB2基因受UV诱导表达;运用生物信息学分析对DDB2蛋白序列与模式生物进行同源比对,发现2个WD-40重复和一个DWD盒保守结构域;通过对DDB2融合黄色荧光蛋白转基因番茄根尖的荧光显微观察,证实番茄DDB2定位于细胞核。利用酵母双杂交实验证明了番茄DDB2与DDB1和CUL4之间的相互作用关系,推测DDB1蛋白作为DDB2与CUL4蛋白的桥梁形成CUL4-DDB1-DDB2复合体。

关键词: 番茄;DDB2;亚细胞定位;酵母双杂交分析