论文部分内容阅读
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.
The proliferating cell nuclear antigen (PCNA) gene is a cofactor of DNA polymeraseδ and plays an important role in DNA replication and damage repair in eukaryotic cells.Using high efficiency thermal asymmetric reciprocal PCR (PCR-RFLP) was used to amplify the promoter region of wheat PCNA gene from the wheat XiNong 1 376 genome and named it as Ta PCNA promoter.The on-line analysis software of Plant CARE promoter predicted that the promoter region containing the light response regulatory element (ABRE), GGTT motif (GTG motif), E2F-binding site, etc. In order to analyze its promoter activity, Of CaMV35S promoter, a fusion expression vector of Ta PCNA promoter and β-glucuronidase (GUS) gene was constructed and transiently expressed in tobacco leaves by Agrobacterium tumefaciens method.GUS histochemical staining results showed that, The promoter of Ta PCNA gene can drive the expression of GUS gene in tobacco leaves, which confirmed that the obtained promoter sequence has the priming activity.This study was cloned by hi TAILPCR To Ta PCNA gene promoter, which lay the foundation for further study of the function of this gene.