CONSTRUCTION OF PRODUCTION SYSTEMS OF USEFUL COMPOUNDS IN PLANT CELL CULTURE BY APPLICATION OF PLANT GENE EXPRESSION SYSTEM

利用植物基因表达机制，研究构建植物细胞培养中有用化合物的生产体系。烟草细胞，烟叶细胞。选择BY2作为寄主细胞，因为它在植物细胞中生长速度较快（倍增时间为10-11h）。将一组靶基因导入烟草细胞中，必须根据植物细胞培养的操作条件，将其置于强启动子、增强子和顺式元件的控制下。辣根过氧化物酶同工酶基因启动子prxC2在烟草细胞中表现出高活性。prxC2基因通过损伤诱导表达，在翻译点289 bp处鉴定出一个诱导损伤的顺式元件，其核心序列为CACGTG。还克隆了一个编码顺式元件相关转录因子的cDNA。伤口诱导系统似乎对烟草培养细胞的基因表达有影响。将拟南芥HSP18.2基因的一个热休克启动子与β -葡萄糖醛酸酶（GUS）结构基因连接，导入烟草培养细胞的染色体中。当孵育温度从25ºC转移到37ºC时，GUS基因的表达被强烈诱导。GUS mRNA在温度变化后15 min检测到，2 h时GUS mRNA表达量达到最大值，4 h时GUS活性达到最大值。这表明通过控制烟草培养细胞的孵育温度，HSP18.2启动子是外源基因表达的良好候选基因。真核细胞的生长速度是由细胞周期调节蛋白控制的，如蛋白激酶、细胞周期蛋白和蛋白磷酸酶。分析这些基因的表达对了解植物细胞的生长机制和提高植物细胞的生长速度具有重要意义。本研究从烟草细胞中克隆了3个细胞周期蛋白cdna，发现这些细胞周期蛋白基因属于G2细胞周期蛋白。

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.