【作者】 黄海群；

【导师】 林拥军；

【作者基本信息】 华中农业大学 ， 生化与分子生物学， 2006， 硕士

【摘要】 生物技术的发展推动了转基因产业化的进程，而与此同时，转基因植物及其食品也成了环境和健康的中心议题。实现外源基因在转基因植株中的定向、安全、高效表达是解决转基因植物安全性问题的关键之一。解决这个问题的一个重要途径是克隆使外源基因在特定器官或特定发育时期表达的启动子，构建高效表达遗传转化载体，实现可控的遗传转化。 本研究内容主要涉及水稻及拟南芥启动子的克隆、植物表达载体的构建、水稻rbcs基因启动子功能分析、Osrbcs-cry1C融合基因表达载体的构建及水稻转化、对获得的转基因植株及其分子检测等。主要结果如下： 1、通过PCR扩增分别获得水稻及拟南芥来源的rbcs基因启动子，分别命名为Posrbcs、Pats1A。对启动子序列进行比较分析，发现两者存在的光调控元件有同有异。 2、将Posrbcs、Pats1A分别与gus基因构成融合基因表达载体，转化水稻，得到相应的转化植株30株、28株，分别命名为1301r、1301a。GUS定性定量检测结果显示1301r中gus基因在绿色组织中特异表达，1301a中gus基因仅在叶片和叶鞘中表达且表达量低于1301r。 3、对Posrbcs序列进行5’端缺失，构建5个缺失表达载体，转化水稻。所得转化植株分别命名为OS62、OS344、OS550、OS941、OS1320。GUS定量检测显示启动子片段越短，GUS活性越低。 4、将1301r、OS62、OS344、OS550、OS941、OS1320转化植株的T₁代幼苗进行光诱导试验，结果表明1301r、OS1320、OS941、OS550、0S344随着光照时间的延长，GUS活性增强，而光照对OS62幼苗gus基因的表达无诱导作用；随着启动子片段缩短，Posrbcs中I box、T box、GT-1结合位点、GATA box等光调控元件的缺失造成各时间点gus基因受光诱导后的表达降低。 5、对Posrbcs序列进行凝胶阻滞分析结果显示I box、T box、GT-1结合位点、GATA box等光调控元件与核蛋白有特异结合条带。 6、将Osrbcs-cry1C融合基因表达载体转化水稻，得到的转化植株命名为3300rc，通过PCR阳性检测共有79株，Southern检测得到19株单拷贝植株。选择其更多还原

【Abstract】 The development of biotechnology promotes the transgenic commercialization process. In the meantime, transgenic plants and their products become the central issue on the environment and health. The realization of the directional, safe and efficient expression of foreign gene in plants is one of the key problems to be solved. The cloning of specific promoters expressed in specific organ or developmental stage, the construction of efficient expression vectors, and the controllable genetic transformation are the important ways to solve this problem.The research mainly focus on sequences cloned of ribulose-1,5-bisphosphate carboxylase small Subunit genes ( rbcS ) gene promoter of rice and Arabidopsis thaliana; construction of plant expression vectors; functional analysis of rice rbcS promoter; construction Osrbcs-crylC gene expression vector; identification of transgenic plants. The main results were as follows:1. The promoter sequences of ribulose-1,5-bisphosphate carboxylase small Subunit genes (rbcS ) of rice and Arabidopsis thaliana were cloned by PCR. Compared the tow promoter sequences, we found there were some same light responsive elements in both promoters; also there were some differences between them.2. The cloned Osrbcs and ats1A promoters were fused with gus gene and plant expression vectors were constructed respectively, then introduced into rice by Agrobacterium-mediated transformation. The transgenic plants designated as 1301r and 1301a. The result of GUS histochemical staining showed that gus gene driven by Osrbcs promoter expressed in green tissues, while in 1301a, gus gene expressed in leaf and sheath, but not in culm. Quantitative analysis of GUS activity showed that the gus gene expression level of 1301r was stronger than 1301a.3. 5’ end deletions of Osrbcs promoter with a length of 62, 344, 550, 941, 1320bp were fused with gus gene and plant expression vectors were constructed respectively. Quantitative analysis of GUS activity showed that the expression level of gus gene was reduced with the deletion of promoter.4. In our experiment we also found that light induction could enhance GUS activity, but light could not enchance the expression of the 62bp promoter fragment.5. Electrophoretic mobility shift assays showed that I box, GT1 factor, T box and GATA box更多还原