摘要经调查，建兰花叶病毒（Cymbidium mosaic virus，CymMV）和齿兰环斑病毒（Odontoglossum ringspot virus，ORSV）在杭州地区花卉市场的蝴蝶兰等兰科植物中广泛存在，严重影响其观赏价值及经济价值。兰科植物是多年生重要经济作物，其培育周期较长，外植体生长缓慢，但病毒病危害严重防治困难。抗病品种培育周期长且抗病性能不稳定；多年生植物转基因抗病品种培育，其外植体离体培养周期长是重要瓶颈。一旦发生兰花病毒病，会造成严重的经济损失，所以对于兰花病毒的研究具有重要的科研及经济价值。61317

利用pCAMBIA2300植物双元表达载体构建了CymMV/ORSV杭州分离物病毒全基因组侵染性克隆：pCAMBIA2300-CymMV FL和pCAMBIA2300-ORSV FL。通过农杆菌介导的浸润注射法接种本氏烟，综合发病率的统计、病症表型的分析结果比较以及分子生物学技术手段RT-PCR法和Southern blot技术检测结果，本研究构建的农杆菌介导的CymMV/ORSV两种病毒的侵染性克隆具有生物学活性，对本氏烟的侵染效率分别为29%-43%以及43%-64%。

通过构建上述两种兰花病毒ORSV和CymMV的侵染性克隆可以在本氏烟叶片上产生病毒症状，通过对接种量的精确定量，所得到实验结果可以进行较精确的分析统计，从而为下一步的病毒学生物学功能及抗病毒兰花的研究打下基础。

Abstract  According to the investigation result, we found that Cymbidium mosaic virus (CymMV) and Odontoglossum ringspot virus (ORSV) are widespread in orchid plants such as Phalaenopsis in Hangzhou flower market shown. These virus diseases seriously reduced the ornamental value and economic value of Phalaenopsis. Orchidaceae is one of the most important economic crop of perennial, its development cycle long, slow the growth of explants, but disease virus harm serious and difficult to control. The breeding period of disease resistant varieties is long and the resistance to disease resistance is not stable. The cultivation of transgenic resistant varieties of perennial plants is the important bottleneck of the culture cycle length of the external explants in vitro. Once the occurrence of orchid virus disease, will cause serious economic losses, so for the study of the orchid virus has an important scientific research and economic value.

We also constructed infectious clones of CymMV/ORSV Hangzhou isolate by insert full-length sequences into plant binary expression vector pCAMBIA2300, and named as pCAMBIA2300-CymMV FL and pCAMBIA2300-ORSV FL, then 7-weeks of N. benthamiana were agro-inoculated with these constructs. Symptom observation and genetic analysis (RT-PCR, Southern blot detection) results show constructed CymMV/ORSV infectious clones both have biological activity and the infection efficiency is 29%-43% and 43% -64%, respectively.

The infectious clones of ORSV and CymMV can induce virus symptoms in the tobacco leaf, quantified analysis results can be achieved in these experiment. These constructions would help further more antiviral research on orchid.

毕业论文关键词：齿兰环斑病毒：建兰花叶病毒；本氏烟； 侵染性克隆

Keyword: Odontoglossum ringspot  virus,  ORSV ； Cymbidium  mosaic  virus，CymMV； Nicotiana  benthamiana，NB；Infectious clone

目录

摘要 2

Abstract 2

目录 3

引言 4

1.建兰花叶病毒CymMV侵染性克隆毒性检测 5

引言 5

1.1材料与方法 5

1.1.1 植物材料 5

1.1.2 质粒材料