摘要：双生病毒是一类世界范围内广泛发生的唯一的单链环状DNA的单分体或二分体基因组病毒，已在多数重要经济作物上引起严重危害。

通过实验建立了一个能够同时分析ACMV病毒DNA复制和与烟草互作的体系——pOri2转基因体系。pOri2转基因系统内部环化分子的产生可以用引物P1和P2通过PCR来确定。ACMV中的Rep蛋白的基因序列里发生单核苷酸的突变后，该突变位点对病毒复制无影响，不会干扰病毒生活循环，还减低了双生病毒侵染植物后，叶片上针对抗病毒产生的过敏坏死反应。在以上的已有实验基础上，我们将Rep*蛋白基因（单位点突变的Rep蛋白）转入到转基因本氏烟1074植株及野生型的本氏烟中，构建了研究ACMV病毒复制调控系统，在这一系统里可以研究病毒复制与植物内源基因互作调控的机制。本论文开展的实验是验证Rep*:pOri2实验体系的有效性，及尝试将外源的基因在此系统内进行特异表达来研究植物与病毒表观互作过程，有望用于双生病毒病害的防治。 46819

毕业论文关键字：ACMV；Rep蛋白；ORi；转基因；

 Abstract：Geminiviruses is only a single split single-stranded circular DNA or bipartite viral genome within a class of widely occur worldwide, has caused serious harm to the most economically important crops.

Through the establishment of a laboratory capable of simultaneous analysis of CMV viral DNA replication and interaction of the system with the tobacco transgenic system --pOri2.Internal pOri2 transgenic system cyclized molecules generated may be determined by PCR using primers P1 and P2.After a single nucleotide mutation in the gene sequences occur ACMV Rep protein in the post, the mutations had no effect on viral replication, do not interfere with the viral life cycle, but also reduce the plant geminivirus infection, the leaves for antiviral necrosis resulting allergic reactions.More than the existing experimental basis, we will Rep* protein gene (single point mutation in the Rep proteins) into the transgenic Nicotiana benthamiana plants and wild-type 1074 N. benthamiana to construct a research ACMV viral replication and control system, in this system, where you can study viral replication and plant endogenous gene regulation mechanism of interaction.Prevention pOri2 experimental system effectiveness, and try to exogenous genes in this system to study the apparent specific expression during the interaction of plant viruses, are expected for geminivirus diseases: experiments carried out in this paper is to verify Rep * .

Keyword：ACMV; Rep protein; ORi; 

目    录

摘   要 2

Abstract 3

第一章 材料与方法 8

1. 实验材料与试剂 8

1.1 实验材料 8

1.1.1 菌株与质粒 8

1.1.2 病毒 8

1.1.3 受体植物 8

1.1.4 .植物生长条件 8

1.2 酶及化学试剂 8

1.3 实验仪器 8

1.4 常用试剂与培养基 8

2．基本实验方法 9

2.1 普通PCR 9

2.2 琼脂糖凝胶电泳技术 9

2.3样品酶切 10

2.4 样品纯化 10

2.5 目的片段与载体的连接 10