小麦细胞增殖核抗原基因启动子的克隆及活性分析  

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

作　　者：于永昂[1] 张改生[1] 张姣[1] 宋瑜龙[1] 朱启迪[1] 巨岚 牛娜[1] 王军卫[1] 马守才[1] 

机构地区：[1]西北农林科技大学国家杨凌农业生物技术育种中心/国家小麦改良中心杨凌分中心/小麦育种教育部工程研究中心/陕西省作物杂种优势研究与利用重点实验室,陕西杨凌712100

出　　处：《中国生物化学与分子生物学报》2015年第5期527-534,共8页Chinese Journal of Biochemistry and Molecular Biology

基　　金：国家高技术研究发展计划(863计划;No.2011AA10A106);国家自然科学基金(No.31171611;No.31371697);陕西省科技统筹创新工程计划课题(No.2014KTZB02-02)项目资助~~

摘　　要：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(highefficiency thermal asymmetric interlaced PCR,hi TAIL-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Ta PCNA启动子.Plant CARE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p BI121载体上的Ca MV35S启动子,构建了Ta PCNA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,Ta PCNA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hi TAILPCR法克隆得到Ta PCNA基因的启动子,为深入研究该基因的功能奠定了基础.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（ hi TAIL-PCR）,and designated as the Ta PCNA 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 Ta PCNA 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 promoterregion of PCNA gene was cloned by hi TAIL-PCR,and the results laid the foundation for us to study the role of PCNA gene.

关 键 词：小麦 细胞增殖核抗原基因 启动子 顺式作用元件 瞬时表达 

分 类 号：Q51[生物学—生物化学] Q946.1