目录
摘要Ⅰ
关键词Ⅰ
AbstractⅡ
引言
引言1
1材料与方法3
1.1技术路线 3
1.2材料 4
1.2.1菌种来源4
1.2.2培养基的配置4
1.2.3实验药剂与仪器4
1.2.4实验所需引物4
1.3方法 5
1.3.1 RNA提取（试剂盒法）5
1.3.2反转录cDNA提取6
1.3.3 PCR扩增6
1.3.4 1%琼脂糖凝胶制备及电泳7
1.3.5 割胶回收7
1.3.6 加A到克隆基因上8
1.3.7 连接8
1.3.8 转化8
1.3.9 筛板（菌落PCR、跑胶、摇菌）9
1.3.10 提质粒10
1.3.11 双酶切连接10
1.3.12 转化筛板11
2结果与分析12
2.1病毒基因组结构12
2.2 RNA2编码的运动蛋白聚类分析13
2.3 RNA3编码的外壳蛋白聚类分析14
2.4 病毒基因组的小RNA分布图15
3讨论17
致谢18
参考文献19
图11克隆ARWV的技术路线3
图12鉴定ARWV编码的基因沉默抑制子的技术路线3
图13 ARWV的 PCR 扩增产物的琼脂糖凝胶电泳7
图14 ARWV的菌落PCR产物的琼脂糖凝胶电泳9
图21病毒基因组结构示意图12
图22 M链编码的MP的进化树13
图23 S链编码的CP的进化树14
图24 小RNA在L, M和S的分布图15
表11 病毒检测和基因组测定过程中涉及的引物 4
苹果胶质木病毒沉默抑制子鉴定
摘 要
苹果胶质木病害是在世界范围内发生的一种苹果病害，会严重影响苹果及梨的产量和品质，最高能够造成苹果30%的减产及梨50%的减产。 *51今日免费论文网|www.51jrft.com +Q: ^351916072^ 
最初它被归类为一种粗糙的树皮病害，其症状类似于茎部凹陷。但是关于这种病害的病原一直不是很清楚，直到近些年来在欧洲和北美洲首次确定了该病害是由植物病毒造成的。我们的前期研究首次发现苹果胶质木病的病原苹果胶质木病毒在我国果树中也广泛存在；但是关于该病毒在我国的分离物的基因组序列，是否具有地区特异性、在我国的发生流行情况、对我国苹果产区的苹果产量和品质是否会有影响、是否具有潜在的大流行趋势等都知之甚少。因此，在本研究中我们将对苹果胶质木病毒中国分离物进行全基因组克隆和测序，了解病毒的基因组结构及该病害的传播方式以及是否存在潜在的爆发风险。了解病毒的基因组结构、编码蛋白数量、传播方式、热稳定性、沉默抑制子等，均可用于病毒的检测预报，为防治该病害提供信息。本实验将利用MEGAX等软件比对我国苹果胶质木病毒序列和其他地区鉴定的病毒的同源性以及和其他相近种属的进化树分析，得出我国苹果胶质木病毒的进化聚类情况；通过研究病毒产生的小RNA的分布情况，了解基因沉默在苹果抗苹果胶质木病毒中发挥的作用；旨在为下一步鉴定病毒沉默抑制子打下良好的基础。
ABSTRACT
Apple rubbery wood disease（ARWD）causes severe damage on apple yield and the quality of fruit worldwide. It has been reported that ARWD can reduce fruit yield by up to 30% in apple and 50% in pear. It was initially classified as a rough bark disease with symptoms similar to stem depression, resulting in reduced apple yield and quality. The casual pathogen of this disease has not been identified until it was first determined in Europe and North America recently. The disease is caused by a plant virus named apple rubbery wood virus (ARWV). Our preliminary data identified ARWV occurrence in China for the first time by using small RNA deep sequencing technology. However, very few information is available about the genome and sequence similarity between China isolate and other viral lineages; the effect of this virus on the yield and quality of apples, the prevalence and distribution of this virus, and the potential epidemic trend are still unknown. In the present study, we will investigate the genome structure of ARWV and how the virus spread and whether there is a potential risk of outbreak in China. Understanding of viral genome structure will provide the preventive measure for the disease control. The genome structure, the number of viralencoded proteins, the mode of transmission, the thermal stability and the silencing suppressor of viruses can be utilized for the detection and prediction of viruses and provide information for prevention and treatment. In this experiment, software such as MEGAX is used to compare the homology of ARWV sequences in China with viruses identified in other regions, as well as the phylogenetic analysis of other species in same genus and family. The ARWVderived small RNAs are investigated and classified that will help us understand the role of RNA interference in apples against ARWV, providing foundations for the identification of virus genesilencing suppressors in the future.

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