【作者】 陈泠；

【导师】 何光源；

【作者基本信息】 华中科技大学 ， 生物化工， 2005， 硕士

【摘要】 小麦是世界重要的粮食作物之一,分离麦类作物花粉、花药特异性基因不仅为了解小麦有性生殖中的基因调控提供重要基础,同时也为构建小麦雄性不育系和恢复系创造了条件。为了在作物中分离新的启动子,以小大麦tritordeum 为材料,采用无启动子转化策略,获得了一调控序列被uidA 基因标记的转基因材料,初步检测为花粉特异性启动子被标记材料。为更明确该材料后代中标记基因uidA 的花粉特异性表达遗传特性,以便为该上游的启动子分离工作打下基础,本文对筛选的该材料阳性单株进行了两代遗传表达分析。结果表明,后代51 株材料的基因组中都含有uidA 基因,uidA 基因没有发生分离。组织化学检测显示,各材料都只在花粉中检测到GUS 活性,而在其他组织没有检测到GUS 活性。进一步的RT-PCR 分析显示,uidA 基因在子代的根和叶中没有发生转录,因而在根和叶中没有uidA 基因的表达。通过以上分析说明,该株材料为花粉特异性阳性纯合体,并且该特异性能稳定遗传,从而可以肯定该材料被标记的启动子为花粉特异性启动子,仅启动基因在花粉中表达。为了得到调控uidA 基因表达的上游启动子序列,采用了TAIL-PCR 法进行克隆。结果显示,标记基因uidA 为多拷贝且发生了重排或者丢失,这为分离启动子增加了难度也提供了帮助。更多还原

【Abstract】 Wheat is one of the important crops in the world. Isolation of pollen/anther -specific expessing gene and promoter could not only understand gene regulation in the sexual reprodution of flowering plants,but also could generate male-sterility by gene engineering. As an approach to isolate novel cereal promoters, promoterless uidA constructs and particle bombardment were used to transform tritordeum. Then one transgenic material was got, primarily detection showed that it’s a pollen-specific regulater being tagged by uidA gene. In order to confirm the inheritance of uidA gene, the pollen-tissue specific expression of uidA gene in the next genetation,and to prepare the materials for the promoter isolation, two generations of one positive material were analysed. All fity-one F1 and F2 progeny plants had uidA gene, and they did not separate. Histochemical analysis of progeny plants showed that GUS activity was only detected in pollen. Further RT -PCR analyses showed that uidA gene was not transcripted in the root and leaf, therefore there is no GUS activity in the root and leaf. All above indicated the material was pollen-specific promoter tagged by uidA gene and they were homozygotes. Our experiments confirmed this specificity could inherit steadily. TAIL-PCR was adoptted to isolate the upsteam promoter, which regulates the expression of uidA gene. The results showed that uidA gene was multicopy and some copies had undergone rearrangment/deletion. So they made the isolation of the promoter harder, it also gave some help to the isolation.更多还原