1999 Fiscal Year Final Research Report Summary
DEVELOPMENT OF METABOLIC ENGINEERING OF PLANT ; CHLOROPLAST TECHNOLOGY OF CHILAMYDOMONAS
| Project/Area Number |
09450305
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
生物・生体工学
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| Research Institution | Nara Institute of Science and Technology |
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Principal Investigator |
SHINMYO Atsuhiko Nara Institute of Science and Technology, Graduate School of Biological Science, Professor, バイオサイエンス研究科, 教授 (30029235)
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| Co-Investigator(Kenkyū-buntansha) |
KATO Ko Nara Institute of Science and Technology, Graduate School of Biological Science, Assistant Professor, 助手 (80283935)
SEKINE Masami Nara Institute of Science and Technology, Graduate School of Biological Science, Assistant Professor, 助手 (70226653)
YOSHIDA Kazuya Nara Institute of Science and Technology, Graduate School of Biological Science, Associate Professor, 助教授 (50252622)
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| Project Period (FY) |
1997 – 1999
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| Keywords | Chloroplast / Chloroplast Transformation / Regulation of Gene Expression / Chlamydomonas / Foreign Gene Expression / β-Glucuronidase / Promoter / Untranslated Region |
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| Research Abstract |
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.
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