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

利用重组DNA技术建立抗病毒病苹果砧木转化植株的研究

基本信息

批准号：
04556006
负责人：
TAKAHASHI Tsuyoshi
金额：
$ 10.56万
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 (B)
财政年份：
1992
资助国家：
日本
起止时间：
1992 至 1994
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-04556006/
关键词：
apple top-working apple virus virus resintant rootstock transformed plant

项目摘要

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.

本研究利用重组DNA技术建立了抗病毒病的苹果砧木转基因植株。以苹果褪绿叶斑病病毒（ACLSV）和对ACLSV敏感的砧木海棠（Malus prunifolia）为材料，研究了苹果褪绿叶斑病病毒的防治效果. ACLSV在藜中繁殖并成功纯化。从纯化的病毒中提取ACLSV-RNA制备cDNA并用于产生ACLSV-cDNA克隆.用双脱氧核苷酸链终止法测定了ACLSV全基因组的核苷酸序列。基因组全长7552个核苷酸，不包括poly（A）尾，包含三个开放阅读框（ORF 1、2和3）.通过农杆菌Ti介导法将ACLSV外壳蛋白基因（ORF 3）导入烟草和海棠中。4. Western blot分析表明，ACLSV外壳蛋白在转基因烟草和海棠愈伤组织中均得到表达。将编码50 K运动蛋白的ORF 2反义基因导入烟草和海棠植株中。PCR分析表明，反义基因已成功导入烟草植株。以上结果表明，Ti介导的转化可以将ACLSV基因导入到砧木植株中。该方法将用于生产抗其它果树病毒病的转基因植物。