【摘要】 花烟草(Nicotiala alata)的花中存在的植物防御素Nicotiala alata defensin 1(NaD1),对多种致病性真菌具有防御活性，在天然防御免疫系统中发挥着重要作用。本研究分别对花烟草根、茎、叶、花组织部分以及伸根期、旺长期、成熟期发育阶段叶片中NaD1基因的表达量进行qRT-PCR定量检测，结果显示，NaD1基因在花中表达量显著高于其他组织，在叶片中的表达量随着植物的生长发育先增加后降低。此外，通过热不对称交错PCR克隆得到NaD1基因5′端上游644 bp的片段，利用BDGP和Plant CARE在线软件分析启动子的转录起始位点及motif元件，发现其含有基本启动子元件和其他顺式作用元件，如光反应元件BOX-Ⅰ、乙烯反应元件ERE以及真菌诱导响应元件TGACG-motif等。通过对花烟草进行光胁迫处理，发现NaD1基因在花中的表达量随着暗处理时间的增长而显著增加，随着光处理时间的增长而显著降低，这与其启动子中存在的光反应元件BOX-Ⅰ相对应。通过启动子5′端缺失分析，构建了pCAMBIA1391z-pNaD1-644∶∶GUS与pCAMBIA1391z-pNaD1-310∶∶GUS两个植物表达载体，利用遗传转化获得包含-644～-1 bp和-310～-1 bp启动子片段的转基因烟草，GUS染色结果表明，NaD1基因启动子的核心区域在-310～-1 bp中，为进一步研究NaD1基因的功能和转录调控机制奠定了基础。更多还原

【Abstract】 Nicotiala alata defensin 1(NaD1) is a plant defensin in the flowers of Nicotiana alata, which has defense activity against many pathogenic fungi and plays an important role in the natural defense immune system. In this study, the expression levels of NaD1 gene in roots, stems, leaves, flowers and leaves at root extension stage, vigorous growth stage and mature stage were detected by qRT-PCR. The results showed that the expression level of NaD1 gene in flowers was significantly higher than that in other tissues, and the expression of NaD1 gene in leaves firstly increased and then decreased during the growth and development of plants. In addition, 644 bp upstream of NaD1 gene 5′ end was cloned by thermal asymmetric cross PCR. The transcription initiation site and motif element of NaD1 gene were analyzed by BDGP and Plant CARE online software. It was found that the promoter contained basic promoter elements and other cis acting elements, such as light response element box-Ⅰ, ethylene response element ERE and fungal induction response element TGACG-motif. It was found that NaD1 gene expression increased significantly with the increase of dark treatment time, and decreased significantly with the increase of light treatment time, which was corresponded to the light response element box-Ⅰ in the promoter. Two plant expression vectors pCAMBIA1391 z-pNaD1-644∶∶GUS and pCAMBIA1391 z-pNaD1-310∶∶GUS were constructed by 5′ promoter deletion analysis. Transgenic tobacco containing-644-1 bp and-310-1 bp promoter fragments were obtained by genetic transformation. GUS staining showed that the core region of NaD1 gene promoter was-310-1 bp, it laid a foundation for further study on the function and transcriptional regulation mechanism of NaD1 gene.更多还原