Improvemed methods for cloning and mapping plant genes by DNA-tagging

在农杆菌介导的植物转化中，最终将T-DNA插入到功能基因中，导致突变产生隐性表型，并在初级转化子的后代中检测到，然后以插入的T-DNA为探针分离T-DNA标记的基因。由于检测T-DNA插入突变需要筛选大量的转化子，因此植物材料必须采用高效的转化体系。本研究以番茄和拟南芥为材料，建立了适合这两种植物的转化体系。以携带番茄部分基因组的番茄植株为材料，证实了在接种农杆菌的培养基中添加乙酰丁香酮对转化有促进作用。在拟南芥中，生态型Wassilewski ja的根段被发现是很好的转化材料，在拟南芥的转化体中发现了茎顶花异常的突变特征。遗传分析和Southern杂交的结果表明，该突变是由T-DNA插入一个被描述为“末端花”基因的等位基因引起的。克隆了插入T-DNA的染色体DNA片段，并以此为探针，从野生型植物基因组DNA文库中筛选出具有同源序列的DNA克隆，构建了筛选植物基因启动子的双元载体，获得了用于转化实验的结果。利用荧光原位杂交技术，在拟南芥中成功地定位了180bp的重复序列家族和10kb的单拷贝序列。

In Agrobacterium-mediated trans formation of a plant, eventual insertion of the T-DNA into a functional gene caused a mutation giving a recessive phenotype and being detectable in the progeny of the primary tranformant, and then the T-DNA-tagged gene can be isolated by using the inserted T-DNA as a probe. Since a large number of transformants are necessary to be screened for detecting T-DNA insertional mutations, an efficient transformation system must be employed for plant material.In this study, tomato and Arabidopsis thaliana were chosen as materials, and suitable transformation systems were established for the both species. Using a tomato plant that carries a part of the genome of Lycopersicon peruvianum as a material, it was confirmed that acetosyringon, when added to the medium for inoculation of Agrobacterium, showed a promoting effect on transformation. In Arabidopsis, root segments of ecotype Wassilewskija were found to be and excellent material for transformation.A mutant characteristic of abnormal flowers on the top of the stems were found among transformants of Arabidopsis. Results from genetic analysis and Southern hybridization suggested that the mutation was caused by a T-DNA insertion into an allele that had been described as "TERMINAL FLOWER" gene. A chromosomal DNA fragment franking the inserted T-DNA was cloned, and using the fragment as a probe, DNA clones having the homologous sequence were selected from a genomic DNA library of the wild type plants.Besides of these studies, a binary vector for screening promoters of plant genes was constructed, and obtained a result supporting its usefulness in a transformation experiment.Using FISH (fluorescent in situ hybridization), in A.thaliana, the 180 bp family of repetitive sequences and a 10 kb single-copy sequence were successfully localized on specific chromosomes.