小麦细胞增殖核抗原基因启动子的克隆及活性分析

细胞增殖核抗原（proliferating cell nuclear antigen，PCNA）基因是DNA聚合酶δ的辅助因子,在真核细胞DNA复制及其损伤修复中发挥着重要的作用.采用高效热不对称交互PCR法（high-efficiency thermal asymmetric interlaced PCR，hiTAIL PCR）从小麦西农1 376基因组中扩增得到小麦PCNA基因启动子片段，并命名为TaPCNA启动子. PlantCARE启动子在线分析软件预测含有光应答调控元件（Box I）、脱落酸应答元件（ABRE）、花粉发育应答元件（GGTT motif，GTGA motif）及细胞周期转换结合位点（E2F-binding site）等.为了分析其启动子活性, 通过替换pBI121载体上的CaMV35S启动子,构建了TaPCNA启动子与β-葡糖醛酸酶（GUS）基因的融合表达载体，通过农杆菌介导法在烟草叶片中进行瞬时表达. GUS组织化学染色结果表明，TaPCNA基因启动子能够驱动GUS基因在烟草叶片中表达，证实了所获得的启动子序列具有启动活性.本研究通过hiTAIL-PCR法克隆得到TaPCNA基因的启动子,为深入研究该基因的功能奠定了基础.

Molecular Cloning and Activity Analysis of the Promoter of Proliferating Cell Nuclear Antigen Gene in Wheat

Proliferating cell nuclear antigen (PCNA) is an auxiliary protein of DNA polymerase δ，and play an important role in DNA replication and repair in eukaryotic cells. To further investigate the regulation of the gene, a 894 bp genomic fragment at upstream of the PCNA translated sequence has been isolated by high efficiency thermal asymmetric interlaced PCR (hiTAIL-PCR), and designated as the TaPCNA promoter. Sequence analysis revealed that the promoter contained several cis acting elements such as light, heat, abscisic acid, pollen development, E2F-binding site, and so on. In order to verify the activity of promoter, the TaPCNA promoter was fused to the β-glucuronidase (GUS) reporter gene and the resulting construct was transferred into tobacco. Histochemical analysis in transgenic tobacco showed that the promoter exhibited activity to drive GUS gene expression In conclusion, the promoter region of PCNA gene was cloned by hiTAIL-PCR, and the results laid the foundation for us to study the role of PCNA gene.