DEVELOPMENT OF METABOLIC ENGINEERING OF PLANT ; CHLOROPLAST TECHNOLOGY OF CHILAMYDOMONAS

叶绿体是植物细胞中最重要的细胞器之一。叶绿体基因组的遗传操作可能是未来构建有用的转基因植物的重要技术。为了在叶绿体中高效表达外源基因，必须对叶绿体基因表达的调控有一个透彻的了解。这些嵌合基因由来自叶绿体基因的启动子、rbcL、PSBA、psbD和ATPA、5‘和3’非翻译区以及作为外源基因的大肠杆菌β-葡萄糖醛酸酶结构基因IDA组成。获得了四种类型的叶绿体转化子(RG、PG、PDG和AG)。AG转化子在莱茵梭菌中表现出最高的GUS活性，积累的GUS蛋白占总可溶性蛋白的1.4%。然而，基因的表达模式与叶绿体中内源基因的表达模式不同，为了在叶绿体中高效表达外源基因，需要融合叶绿体基因的编码区，分析了起始密码子上游的核苷酸对翻译效率的影响。在chmeric-uidA(ATPA)的情况下，AUG上游的两个核键的取代改变了翻译效率。另一方面，在嵌合-uidA(PsbD)的情况下，这并没有效果。对衣藻叶绿体基因组基因表达调控机制的详细研究将在不久的将来应用于高等农业和工业重要植物叶绿体基因组的转化。

The chloroplast is one of the most important organelles in plant cells. Genetic manipulation of the chloroplast genome might be an important technique for construction of useful transgenic plants in the future. In order to express a foreign gene efficiently in the chloroplast, we have to obtain a thorough understanding of the regulation of chloroplast gene expression.Chimeric genes for expression of a foreign gene in the Chlamydomonas reinhardtii chloroplast were constructed. There chimeric genes are composed of the promoter from chloroplast genes, rbcL, psbA, psbD, and atpA, 5'- and 3'-untranslated regions, and the Escherichia coli b-glucuronidase (GUS) structural gene idA) as a foreign gene. Four types of chloroplast transformants (RG, PG, PDG, and AG) were generated. The AG transformant showed the highest GUS activity so far reported in C.reinhardtii, and the accumulated GUS protein accounted for 1.4% of the total soluble proteins. However, the pattern of gene expression was not the same as that of the endogenous genes in the chloroplast, in order to express a foreign gene efficiently in the chloroplast, it was necessary to fuse a coding region from chloroplast gene.We analyzed the effect of nucleoties immediately upstream of the initiation codon (AUG) on the translation efficiency. In the case of chmeric-uidA (atpA), the substitution of two nucleoties immediately upstream of the AUG changed the translation efficiency. On the other hand, in the case of chimeric-uidA (psbD), that had no effect. We also tried to construct a translation system from polycistronic mRNA, and partially successed.Study on the control mechanisms of gene expression of the Chlamydomonas chloroplast genome in detail will be applied to transformation of the chloroplast genome in higher agriculturally and industrially important plants in the near future.