| Co-Investigator(Kenkyū-buntansha) |
YOSHIDA Kazuya Osaka University, Department of Biotechnology, Assistant Professor, 工学部, 助手 (50252622)
SEKINE Masami Osaka University, Department of Biotechnology, Assistant Professor, 工学部, 助手 (70226653)
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| Research Abstract |
We studied construction of a production system of useful compounds in plant cell culture by application of plant gene expression mechanism. Tobacco cells, Nicotiana tabacum L.cv. BY2, was selecteed as a hosat cell, since it grows quite faster among plant cells (doubling time is 10-11h). A set of target genes introduced into tobacco cells must be placed under the control of strong promoters, enhancers, and cis-elements respondiog to operation conditions of plant cell culture. A promoter of horseradish peroxidase isozyme gene, prxC2, showed high activity in tobacco cells. Expression of the prxC2 gene was induced by wounding, and a cis-element, of which core sequence was CACGTG,for wound-induction was identified at-289 bp from the translation point. A cDNA encodig a transcription factor associated with the cis-element was also cloned. The wound-induction system seems to be effective on gene expression in the tobacco cultured cells.
A heat shock promoter of HSP18.2 gene of Arabidopsis thaliana was ligated to the structural gene for beta-glucuronidase (GUS), and introduced into chromosome of the tobacco cultured cells. Expression of the GUS gene was strongly induced when incubation temperature was shifted from 25ﾟC to37ﾟC.GUS mRNA was detected at 15 min after temperature ahift and was maximum at 2 h. Maximun GUS sctivity was found at 4 h. This result indicated that the HSP18.2 promoter is a good candidate for expression of foreign genes by the control of incubation temperature of tobacco cultured cells.
Growth rate of eukaryotic cells is controlled by cell cycle regulating proteins, such as protein kinases, cyclins, and protein phosphatases. Analysis of these gene expression will be important to understand mechanism of cell growth and enhancement of growth rate of plant cells. In the present study, three cyclin cDNAs were cloned grom tobacco cells, and it was found that these cyclin genes belonged to G2 cyclins.
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