苎麻花叶病病毒检测及序列分析

摘  要：苎麻花叶病是苎麻上比较严重的病害，其病毒是苎麻花叶病毒。但是国内对苎麻花叶病病原的分类地位和基因组结构方面的研究比较少。为了研究病毒的基因组结构，本实验采集具有典型症状的苎麻叶片，通过SDS法提取苎麻花叶叶片的DNA。用引物PA/PB，F/MR，MF/R 以提取的DNA为模板进行PCR。分别扩增出大约500bp，1200bp和1300bp的片段。割胶后回收试剂盒回收，将回收的1200bp和1300bp片段连接转化，筛选出重组质粒进行PCR检测，扩增出目的片段的菌液可送样测序。序列拼接后获得病毒的DNA全2738bp的序列，通过比对此病毒与番茄黄化曲叶病毒同源性达95%，初步确定苎麻花叶病毒病原为双生病毒。

关键词： 苎麻；苎麻花叶病毒；序列分析

 

The detection of Ramie mosaic virus and analysis of its sequence

Abstract：Ramie mosaic disease is one of the major diseases in ramie, and its virus is Ramie mosaic virus. But there are few studies about the classification and the genome structure of the Ramie mosaic’s etiology. For this reason, we collected some ramie leaves with typical symptoms, used SDS method to extract the DNA of ramie leaves, and analysed with PCR by using PA/PB, F/MR,MF/R as the primer and the extracted DNA as the template. Amplified by 500bp, 1200bp and 1300bp fragments. After purification kit tapping recycling, the recycling of 1200bp and 1300bp fragments connected transformation, PCR screening recombinant plasmid was identified, and finally sequenced. Mosaic virus sequences obtained after DNA sequence of full-2738bp. Contrasted the Ramie mosaic virus with  the Tomato Yellow Leaf Curl Virus, the homology reached 95%. Therefor, we can draw the conclusion preliminary that the virus is a twin Ramie mosaic virus pathogenic virus.

Key words：Ramie；Ramie mosaic virus；analysis of sequences；

目    录

摘  要 1

关键词 1

1. 前言 2

1.1  苎麻 2

1.2  苎麻花叶病 3

1.2.1  苎麻花叶病症状 3

1.2.2  苎麻花叶病的传播方式 3

1.2.3  苎麻花叶病的发病条件 3

1.2.4  苎麻花叶病的防治方法 3

1.3  苎麻花叶病病原 4

1.4  立题依据 4

1.4.1  目的意义 4

1.4.2  内容简要 4

2. 材料与方法 4

2.1  材料 4

2.1.1  植物材料 4

2.1.2  试剂 5

2.1.3  仪器 5

2.2  方法 6

2.2.1  SDS法提取DNA 6

2.2.2  PCR 6

2.2.3  割胶回收 7

2.2.4  T-vector连接 7

2.2.5  转化 8

2.2.6  菌落PCR 8

2.2.7  碱裂解法提取质粒DNA 9

2.2.8  测序 9

3. 结果与分析 9

3.1  症状图 10

3.2  DNA图 10

3.3  PCR图 11

3.4  菌落筛选PCR图 12

3.5  测序序列 13

3.6  苎麻花叶病病毒系统进化树图 14

4. 结论与讨论 15

参考文献 15

致  谢 16