Basic studies on the chloroplast transformation using photoautotrophic cultured cells

叶绿体在光合作用、氮代谢和氨基酸生物合成中起着重要作用，叶绿体基因组编码许多叶绿体蛋白。然而，这些特性使得高等植物叶绿体突变体的分离变得困难；突变通常是致命的，叶绿体基因组的高拷贝数使得突变体的选择效率低下。最近，我们已经证明了烟草（Nicotiand tabacum cv Samsun）的光嗜营养细胞系。NN)对筛选抗除草剂阿特拉津细胞系具有重要意义，可作为分离叶绿体突变体的模型系统。在此基础上，开展了建立叶绿体基因组转化方法的基础研究。构建了将卡那霉素抗性基因（NPTII）克隆在psbA基因启动子下游的载体，转化叶绿体基因组。2. 将psbA基因置于小麦Rubisco小亚基启动子和转运肽下游，构建了将叶绿体psbA基因转化为核基因的载体。3. 建立了光嗜营养细胞原生质体的分离培养条件，为光嗜营养细胞的直接基因转移奠定了基础。转化光营养细胞的进一步实验正在进行中。4. 抗阿特拉津原生质体和叶肉原生质体不对称融合，以突变叶绿体再生植株。通过这种方法，获得了可移植的抗阿特拉津苗。5. 为了了解突变体细胞的耐药机制和D-1蛋白的功能，我们分析了突变体D1蛋白的投影二级结构。结果表明，苏氨酸264 D1蛋白对三嗪类和尿素类除草剂的交叉抗性主要是由于其结合位点的构象改变所致。

Chloroplasts have an important role in photosynthesis, nitrogen metabolism and amino acid biosynthesis and many chloroplast proteins are encoded by the chloroplast genome. However, these properties make the isolation of chloroplast mutants of higher plants difficult ; the mutations are often lethal and the high copy number of the chloroplast genome makes the selection of mutants inefficient. Recently, we have shown that a photomixotrophic cell line of tobacco (Nicotiand tabacum cv Samsun. NN) was very useful to select a cell line resistant to the herbicide atrazine, as a model system for the isolation of chloroplast mutants. Based on this success, the basic studies were carried out to establish the method to transform chloroplast genome.1. A vector, in which kanamycin resistant gene (NPTII) was cloned downstream the promoter of psbA gene, was constructed to transform chloroplast genome. 2. Another vector, in which psbA gene was placed downstream the promoter and transit peptide of wheat Rubisco small subunit, was constructed to convert chloroplast psbA gene into a nuclear gene. 3. The conditions for the isolation and culture of protoplasts from photomixotrophic cells were established for the direct gene transfer of photomixotrophic cells. Further experiments to transform photomixotrophic cells is in progress. 4. Atrazine-resistant protoplasts and mesophyll protoplasts were fused asymmetrically to regenerate plants with mutant chloroplasts. By this method, putable atrazine-resistant shoots were obtained. 5. To understand the resistance mechanism of mutant cells and the function of D-1 protein, the projected secondary structures of the mutant D1 proteins were analyzed. This result indicates that the cross-resistantce of threonine264 D1 protein to triazine and urea herbicides is mainly due to a conformational change of the binding site for the herbicides.