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

以苹果褪绿叶斑病毒(ACLSV)和苹果茎沟病毒(ASGV)为研究对象，建立了利用苹果顶叶病病原病毒的互补DNA(CDNA)快速诊断苹果树病害的方法。结果表明：1.ACLSV和ASGV均含有单一的RNA物种，其相对分子质量分别为2.48×10~(-6)和2.30×10~(^6)。这两种RNA的3‘末端都是多聚腺苷化的。2.克隆病毒RNA，将插入第一链和第二链基因的载体转化到大肠杆菌中，用限制性内切酶从载体中去除其中的基因。获得的克隆与ACLSV-RNA的全长和ASGV-RNA的96%的长度一致。3.建立了ACLSV(或ASGV)与其cDNA的杂交形成方法。用~(32)P和~(32)P标记的每个病毒的cDNA进行斑点杂交，结果表明，用cDNA探针法对ACLSV和ASGV的检测下限为1.6 ng/点，对ACLSV-RNA的检测限为5.12 pg/点。4.探讨了基因探针技术在苹果病毒常规检索中的应用。当检测5月份采集的幼叶和花瓣时，观察到这两种病毒的特异杂交形成。然而，在8月和11月收集的老叶、树皮或果皮中，到目前为止还没有检测到任何病毒。该方法可用于苹果茎尖分生组织培养后繁殖的脱毒苹果幼苗的鉴定，该方法只需3-4天的时间即可完成，基本可应用于苹果花叶病等其他病毒的诊断。

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.