【作者】 王莹；

【导师】 朱苏文； 江海洋；

【作者基本信息】 安徽农业大学 ， 生物物理学， 2011， 硕士

【摘要】 启动子是基因表达调控重要的元件，启动子的活性间接反映了它所控制的基因表达。组织特异性启动子作为启动子的一种，可以启动外源基因在受体植物的特定组织器官中高效表达，减少不必要的浪费。WRKY基因家族是在植物中起特异作用的一类转录调控因子，它被证明了参与植物的抗病反应，还影响植物的衰老、抗胁迫以及生长和发育。本实验克隆了一个玉米WRKY基因的启动子，采用GUS报告基因对WRKY基因的启动子功能进行了分析，通过启动子的删减实验，水稻的遗传转化及组织的GUS染色，取得如下结果：1、根据网上预测的WRKY基因设计引物，从玉米(玉米品种B73)中扩增了该基因的启动子部分，命名为P2880，在启动子顺式作用元件预测网站上分析该启动子序列，预测到存在TATA盒、CAAT盒和GATA盒等多个作用元件。2、克隆出WRKY基因上游的启动子并且克隆了4个5’端缺失启动子，5个启动子的长度依次为2880bp、1812bp、1254bp、680bp和355bp。将5个启动子与含有GUS报告基因的质粒pCAMBIA1301连接并构建了pCAM2880GUS和4个5’端缺失启动子载体，将缺失载体分别命名为pCAM1812GUS、pCAM1254GUS、pCAM680GUS和pCAM355GUS。3、用农杆菌介导法将所有植物表达载体转化水稻，获得了37棵转基因植株。由实验结果可知，pCAM2880GUS载体对应的转基因植株愈伤组织染色结果为蓝色，而pCAM1812GUS、pCAM1254GUS、pCAM680GUS、pCAM355GUS载体对应的转基因植株的愈伤组织均未染上蓝色，由此可知P2880启动子的核心区域位于转录起始位点前2880bp至1812bp之间。综上所述，本研究克隆了一个玉米WRKY基因启动子，并发现该启动子是一个愈伤特异性启动子，而目前WRKY基因未有愈伤组织表达特异性的报道，该启动子的克隆对玉米相关基因功能研究具有重要的意义。更多还原

【Abstract】 Promoters are important factors that regulated gene expression. The activity of promoters indirectly reflect gene expression that they control. As one kind of promoter, tissue-specific promoters could drive stable and effective expression of target gene in specific tissues of plants. As a result, wastes are reduced. WRKY gene family is a type of transcriptional regulatory factors which has specific functions in plants. Plant WRKY gene-encoded trans criptional regulators appear to be involved in various physiological programs, including disease-resistance, senescence, stress responses of biotic and abiotic, growth and development processes.We clones a WRKY gene promoter in Zea Maize, and analyse the functions with GUS reporter gene. Through the promoter reduction experiment, rice genetic transformation and GUS staining, we get the acheievements as below:1. A2880bp promoter fragment of WRKY gene is amplified by polymerase chain reaction (PCR) technique from maize B73, we called it P2880. We forecast some cis-elements in P2880from PLACE website.2. Besides upstream promoter of WRKY gene, four5’missing promoters are cloned, whose length are2880bp,1812bp,1254bp,680bp and355bp respectively. These five promoters and plasmid pCAMBIA1301which contains GUS reporter gene are attached to construct pCAM2880GUS and four5’missing promoter carriers whose name are pCAM1812GUS, pCAM1254GUS, pCAM680GUS and pCAM355GUS respectively.3. Transferring all plant expressional vectors to rice by Agrobacterium-mediated transformation,37transgenic plants are obtained. According to experimental results, the GUS staining result of callus tissue related to pCAM2880GUS vector is blue, whereas, other vectors such as pCAM1812GUS, pCAM1254GUS, pCAM680GUS and pCAM355GUS are diffrent. In conclusion, the core area of P2880promoter is between2880bp and1812bp before transcription start site.To sum up, we clone a WRKY gene promoter in maize, and found that the promoter is a callus-specific promoter. There is no report of WRKY gene which is callus-specific. The cloning of this promoter has the vital significance.更多还原