论文标题:糖皮质激素诱导绿色荧光蛋白（GFP）在烟草中的表达
The Glucocorticoid Inducible Expression of the Green Fluorescent Protein (GFP) in the Tobacco
论文作者 王俊杰
论文导师 王兴智,论文学位 硕士,论文专业 细胞生物学
论文单位 东北师范大学,点击次数 114,论文页数 40页File Size4674k
2005-05-01论文网 http://www.lw23.com/lunwen_704612232/ 化学诱导系统;诱导剂;绿色荧光蛋白;非翻译区
chemical-inducible system; inducer; green fluorescent protein; non-translated region
糖皮质激素化学诱导表达体系以嵌合型转录因子 GVG 为基础,由酵母的 Gal4 DNA结合域,单纯疱疹病毒蛋白 16 (VP16) 的酸性活化域和哺乳动物糖皮质受体调节域( GR)融合而成。在没有诱导剂糖皮质激素存在的情况下,GVG 蛋白与其他蛋白形成蛋白复合体保留在细胞质中;加入诱导剂后,诱导剂与 GVG 蛋白结合使蛋白发生变构,与其他蛋白脱离后进入细胞核内与诱导启动子结合,启动目的基因的表达。 本实验利用农杆菌介导的方法,将携带双元诱导表达系统的载体 pIndex3 转入到野生型烟草当中,经过鉴定,初步建立了糖皮质激素诱导表达系统。 在这个系统当中,转录因子 GVG 的基因由组成性启动子 35S 控制表达,绿色荧光蛋白基因 mgfp5 作为诱导表达的目的基因在诱导启动子的控制之下。诱导启动子由 4 个串联的 GAL4 上游激活序列(UAS)和一个微小 35S 启动子组成。绿色荧光蛋白的转录受到诱导剂的严格调控。未加诱导剂地塞米松(dexamethasone, Dex,人工合成糖皮质激素)时,检测不到绿色荧光蛋白的表达,加入诱导剂后首先在 RNA 水平检测到了mgfp5 的转录,绿色荧光蛋白随着诱导剂地塞米松的扩散沿叶脉开始表达,当诱导剂去除后绿色荧光蛋白停止表达。诱导剂对基因表达的调控首先表现在新生叶上,老叶对诱导剂的敏感程度相对滞后一些。在质粒构建过程中 mgfp5 两端分别加上马铃薯 X 病毒(PVX)的非翻译区,结果表明非翻译区并未抑制绿色荧光蛋白的表达,为下一步试验奠定了基础。GVG 系统对诱导剂具有非常高的专一性,并且对基因表达的控制非常严格,几乎没有基础表达,可以从时间空间及数量上对目的基因进行精确调控,这一系统的建立为今后利用化学诱导的方法进行理论和应用研究奠定了基础。
The glucocorticoid inducible expression system is based on the chimerical transcription factor, designated as GVG, which consists of the DNA-binding domain of the yeast transcription factor GAL4, the transactivating domain of the herpes viral protein VP16, and the receptor domain of the rat glucocorticoid receptor (GR). In the absence of its ligand, glucocorticoid hormone, GVG is produced and resides in the cytoplasm, where it forms complexes with multiple proteins. After binding to glucocorticoid, the modified GVG protein is released from the complexes, transferred into the nucleus and regulates specific gene expression. The binary vector pIndex3, carrying the glucocorticoid inducible expression system, is transferred into Agrobacterium in order to transform wild-type tobacco. After the detection, it is sure that the glucocorticoid inducible expression system has been established. In this system, the transcription factor GVG gene under the control of the 35S promoter and the mgfp5 transcribed from an inducible promoter containing four tandem copies of the GAL4 upstream activating sequence and a truncated 35S promoter. As the target gene of the chemically inducible system, the GFP expression is stringently controlled. In the presence of the inducer, the mRNA of mgfp5 was transcribed and the GFP began to express around the veins where the inducer (dexamethasone, Dex, a kind of man-made glucocorticoid) reached early. When the inducer was discarded, the GFP expression was stopped. According to the experiment, the young leaves were more sensitive to the inducer than the old ones. To the next experimental step, the non-translated region of the PVX was inserted flanking the mgfp5 gene and it did not repress the GFP expression. The GVG system has the merits, high stringency and specificity, and controls the transgene expression temporally, spatially and quantitatively with the help of exogenous chemicals. The establishment of this system offers the possibility for both theoretical and practical applications by the chemically inducible system.
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