【作者】 杨坤；

【导师】 李艳红；

【作者基本信息】 首都师范大学 ， 细胞生物学， 2006， 硕士

【摘要】 随着人类基因组测序工作的完成，基因组研究已经由以全基因组测序为目标的结构基因组学转向以基因功能鉴定为目标的功能基因组学研究。近几年发展起来的RNAi技术，能快速有效的关闭相应基因，使生物体产生相应的功能缺陷表型，从而确定未知基因的功能。以其特异性、高效性和广泛性的优势，已经逐渐成为一项极具潜力的基因功能研究技术。RNA干扰技术的发现已经为我们深入了解基因功能、疾病发生以及植物—病毒互作关系提供了一个简捷高效的特异性手段。目前利用RNA干扰技术进行植物体发育过程中基因功能的研究，植物体内致病基因、程序性死亡有关基因的研究，动物的工程化抗病毒研究等还很少，这也为RNA干扰技术更广泛的应用提供了广阔的空间。 γ-微管蛋白(γ-TUBULIN)是动物细胞微管组织中心的重要组成成分，它广泛存在于真核生物细胞中。已有的研究证明，它在微管成核的起始、装配和取向等方面具有重要作用，也与有丝分裂纺锤体的形成及细胞周期调控相关。本研究通过转基因技术在烟草(N．tabacum var．Samsun NN)的基因组中同时引γ-tubulin部分cDNA的反义和正义片段得到了低水平表达γ-tubulin的烟草植株，发现降低该基因的表达对于植物的发育有明显的影响。与对照相比，低表达γ-tubulin的无菌苗植株表现出植株生长非常缓慢，叶片颜色黄绿，有的植株甚至继代多次该表型基本稳定；根的发生非常困难，偶有发生也与普通的转基因植株有明显区别，表现为根毛不发达、根较粗，皮层内侧细胞较大，输导组织不发达，对养分的吸收困难。因此我们认为烟草细胞内低表达γ-tubulin对植株发育具有明显的抑制作用。更多还原

【Abstract】 As the accomplishment of human genome project, more and more researches were concentrated on the area of functional genomics. One of the best methods is RNA interference. It was developed recently and would be a very potential technology on gene functional research because of its discrepancy, efficiency and universality. It can make corresponding gene inactive quickly and efficiently to cause organism behave the corresponding phenotype, thereby we can identify the function of unknown gene.γ-Tubulin is the very important component in the microtubule organizingcenter in animal cells, it exists nearly all the eukaryotic cells. As reported, it not only played very important role in the microtubule nucleation, assemble and array, but also involved in the cell spindle formation and cell cycle regulation. But its function in higher plants was still uncertain. In this paper, we got the down-regulated γ-tubulin tobacco plant of N. tabacum L. cv. Sumsun NN. by genetic engineering. The dsRNA expression vector was constructed by inserting sense and antisense partial γ-tubulin cDNA fragment and used to transfer tobacco by leaf disc method. The result showed that low expression γ-tubulin inhibited the tobacco transformants growth and development obviously. The down-regulated γ-tubulin tobacco plants grew very slowly, its plant height and leaves number were less than one half of the control plants and this phenotype was still stable after subculturing several times; The root regenerated from the low expressed γ-tubulin tobacco plants after 30 days, but it appeared brown and the root hair and transport system were undeveloped, which maybe the key factor that maybe effected on absorbing the nutrient material in the medium. Therefore, we concluded that downregulating the γ-tubulin in tobacco cells could inhibit plant growth and development obviously, but the direct mechanism of inhibiting still need to be discovered from small RNA level in the future.更多还原