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摘要：植物内源RBR基因（retinoblastoma-related）对细胞周期有负调控作用，决定植物细胞周期G1到S期的转变，控制着细胞的增殖分化及核内复制。而双生病毒可以通过劫持寄主植物的DNA复制系统，通过直接作用于植物内源RBR基因，调节细胞周期来进入复制周期从而起始自身的复制。幼嫩本氏烟植株的叶片细胞处于的S期，细胞中NbRBR1具有低水平的表达，ACMV（African cassava mosaic virus）的Rep蛋白可和NbRBR1直接互作，从而调控病毒DNA的复制起始，进入转录状态。然而在老的叶片中存在高水平表达的NbRBR1，维持叶肉细胞处于最小复制活性的G相位，从而使得植物系统DNA复制活性降低。通过已有实验发现通过病毒诱导的基因沉默手段使得 NbRBR1的表达降低即可使得老叶中G1期细胞转向S期, 在这种状态下，ACMV的Rep可以重新激活植物本身的DNA的复制功能，从而使得ACMV可以在成熟老叶中进行复制转录。本研究拟通过运用分子生物学手段构建NbRBR1 基因的干涉与过表达的转基因本氏烟，然后接种ACMV病毒的侵染性克隆以及可瞬时表达的ACMV 复制蛋白Rep，通过分析接种实验的植株表型，以及相应的植物内源基因的表达水平变化来分析NbRBR1基因表达量水平对于双生病毒持续复制表达转录的影响，并寻找其它未知的可以和NbRBR1以及ACMV Rep 互作的病毒或植物内源因子。47665
毕业论文关键词：RBR；ACMV；基因过表达；基因干涉；转基因植株；
Abstract： The RBR endogenous (retinoblastoma-related) gene from the plant can regulate the cell cycle, and determine the cell cycle transition from G1 to S phase, RBR gene control the proliferation and differentiation of cells and replicate within the nucleus. Geminivirus can infect terminally differentiated tissues and cells in plants by interact with the plant RBR to modulate host DNA synthesis protein expression, reprogram host cellular machinery for its own DNA replication. This process is thought to be mediated by geminviral replication initiator protein (Rep). Rep can interactions with several host proteins through N. benthamiana NbRBR1 have been described in previous study, all these different proteins interacting with rep were identified by using the yeast two-hybrid system. However, very little is known about the interaction of actual plant RBR complexes during geminviral replication, require generation of transgenic plants. In this study, NbRBR1 interference and over-expression transgenic plant were constructed. In future experiment ACMV will inoculate with transgenic plants. The analysis of inoculation experiments will help to find other uncharacterized viral and plant factors.
Keyword: RBR; African cassava mosaic virus (ACMV); Over-expression; RNA-interference; transgenic plants
目录：
摘要： 1
Abstract： 2
引言： 6
第一章 材料与方法 7
1.1 材料 7
1.1.1 菌株与质粒 7
1.1.2 受体植物 7
1.1.3 酶及化学试剂 7
1.1.4 抗生素 7
1.1.5 常见试剂配制 7
1.1.6 实验仪器 7
1.2 实验方法 7
1.2.1 野生型本氏烟植物总RNA的提取 7
1.2.2 反转录mRNA 8
1.2.3 PCR技术 8
1.2.4琼脂糖凝胶电泳技术