Studies on the establishment of transformed apple-rootstock plants resistant to virus diseases by means of recombinant DNA technology

本研究采用重组DNA技术，建立了苹果砧木抗病转化植株。利用苹果褪绿叶斑病病毒（ACLSV）和对ACLSV敏感的砧木植物苹果（Malus prunifolia），得到了以下结果：ACLSV在藜麦上进行了繁殖并成功纯化。从纯化的病毒中提取aclv - rna制备cdna，并用于制备aclv - cdna克隆。采用双脱氧核苷酸链终止法测定ACLSV基因组全核苷酸序列。基因组包含7552个核苷酸（不包括poly (A)尾巴），包含三个开放阅读框（ORFs1,2和3）。通过农杆菌ti介导的转化，将外皮蛋白基因（ORF3）引入烟草和李树中。Western blot分析表明，ACLSV外壳蛋白在转基因烟草和转基因苹果愈伤组织中均有表达。改造后的苹果正在生长。将一个与ORF2区互补的编码50K运动蛋白的反义基因转移到烟草和苹果植株上。PCR分析表明，该反义基因已成功导入烟草植株。改造后的海棠植物正在生长。待总结。上述结果表明，可以利用ti介导的转化信息将ACLSV基因引入砧木植物。该方法将用于生产抗其他果树病毒病害的转基因植物。

The present study was undertaken to establish the transformed apple-rootstock plants resistant to virus diseases by means of recombinant DNA technology. Using apple chlorotic leaf spot uirus (ACLSV) and a rootstock plant (Malus prunifolia) sensiive to ACLSV,following results are obtained.1. ACLSV was propagated in Chenopodium quinoa and succesfully purified. cDNAs were prepared from ACLSV-RNA extracted from purified virus and used to produce ACLSV-cDNA clones.2. The complete nucleotide sequence of ACLSV genome was determined by the dideoxynucleotide chain termination method. The genome is 7552 nucleotides excluding the poly (A) tail and contains three open reading frames (ORFs1,2, and3) .3. Coat protein gene (ORF3) was introduced to tobacco and Malus prunifolia plants by Agrobacterium Ti-mediated transformation.4.Western blot analysis indicated that ACLSV coat protein was expressed in transgenic tobacco plants and transgenic Malus prunifolia caluses. Tansformed Malus prunifolia plants are now growing.5.An antisense gene complementary to ORF2 region encoding 50K movement protein was transffered to tobacco and Malus prunifolia plants. PCR analysis indicated that the antisense gene was successfully introduced to tobacco plants. Transformed Malus prinofolia plants are now growing.6.To be summarized.The results mentioned above indicated that Ti-mediated trasnformation can be ued to introduce ACLSV genes to a rootstock plant. The method will be applied to produce transgenic plants resistant to other fruit tree virus diseases.