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Development of simple method for diagnosis of apple virus diseases by using gene-manupulation technique

利用基因操作技术开发简单的苹果病毒病诊断方法

基本信息

批准号：
62860005
负责人：
TAKAHASHI T.
金额：
$ 4.86万
依托单位：
Iwate University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Developmental Scientific Research
财政年份：
1987
资助国家：
日本
起止时间：
1987 至 1989
项目状态：
已结题

项目摘要

The present study was undertaken to develop the method for rapid diagnosis of diseased apple trees by using complementary DNA (cDNA) of causal viruses of apple topworking disease, particularly apple chlorotic leaf spot virus (ACLSV) and apple stem grooving virus (ASGV). The results obtained are as follows: 1. ACLSV and ASGV contained a single RNA species with molecular weight of 2.48 x 10^6 and 2.30 x 10^6, respectively. The 3'termini of both RNA species were polyadenylated. 2. Cloning of virus RNA was made; Plasmid vector inserting first- and second-strand cDNA were transformed into Escherichia coli, then cDNA of them was removed from vector by restriction enzyme digestion. cDNA clones thus obtained were corresponded to full length for ACLSV-RNA and 96% length for ASGV-RNA. 3. Method for hybrid formation between ACLSV (or ASGV) and its cDNA was established. From dot hybridization using ^<32>P-labeled cDNA of each virus, it was evident that limitation for detection by cDNA probe method was 1.6ng per spot for ACLSV or ASGV and 5.12 pg per spot for ACLSV-RNA. 4. Application of cDNA probe method to routine apple virus-indexing was investigated. When young leaves and petals collected in May was tested, specific hybrid formation for both viruses was observed. However, in old leaves, barks or fruit skins collected in August and November, any virus could not be detected so far. This method allowed to apply the certification of virus-free apple seedlings which have been propagated after meristem-tip culture.The cDNA probe method takes 3-4 days to perform in vitro, and principally can apply to other virus diagnosis, such as apple mosaic disease.

本研究利用苹果顶梢病病原病毒，特别是苹果褪绿叶斑病毒（ACLSV）和苹果茎沟病毒（ASGV）的互补DNA，建立了快速诊断苹果病树的方法。主要研究结果如下：1. ACLSV和ASGV含有单一RNA种类，分子量分别为2.48 x 10^6和2.30 x 10^6。两种RNA的3 '末端都是聚腺苷酸化的。2.将插入第一和第二链cDNA的质粒载体转化到大肠杆菌中，然后通过限制性内切酶消化从载体中去除它们的cDNA。由此获得的cDNA克隆对应于ACLSV-RNA的全长和ASGV-RNA的96%长度。3.建立了ACLSV（或ASGV）与其cDNA杂交的方法。用13 <32>P标记各病毒的cDNA进行斑点杂交，结果表明，用cDNA探针法检测ACLSV和ASGV的最低检测限为1.6ng/斑点，ACLSV-RNA的最低检测限为5.12pg/斑点。4.研究了cDNA探针法在苹果病毒常规指标化中的应用。当5月收集的幼叶和花瓣进行测试时，观察到两种病毒的特异性杂交体形成。然而，在8月和11月收集的老叶、树皮或果皮中，迄今未能检测到任何病毒。该方法可用于苹果茎尖培养后脱毒苗的鉴定，cDNA探针法在离体条件下进行3-4天，主要可用于苹果花叶病毒等其他病毒的诊断。