CONSTRUCTION OF PRODUCTION SYSTEMS OF USEFUL COMPOUNDS IN TOBACCO CULTURED CELLS BY RECOMBINANT DNA TECHNOLOGY

我们研究了烟草BY2细胞中外源基因的高效表达系统的构建。BY2细胞在植物细胞间生长较快（倍增时间为10- 11 h）。将一组目的基因导入烟草细胞必须置于强启动子、增强子和顺式元件的控制下，以响应植物细胞培养的操作条件。辣根过氧化物酶同工酶基因启动子prxC 2在烟草细胞中表现出较高的活性。prxC 2基因的表达被创伤诱导，并且在距翻译起始点的-289bp处鉴定了用于创伤诱导的顺式元件，其核心序列为CACGTG。还克隆了编码与顺式元件相关的转录因子的cDNA。在烟草培养细胞中，创伤诱导系统似乎是有效的基因表达。将黄瓜抗坏血酸氧化酶基因启动子与拟南芥热激蛋白18. 2基因启动子连接，并将其导入烟草培养细胞染色体。当温度从25 ℃升高到37 ℃时，GUS基因的表达被强烈诱导，温度变化后15 min检测到GUS mRNA，2 h时达到最大值。GUS活性在4 h时达到最高。这一结果表明，HSP 18.2启动子是一个很好的候选表达外源基因的烟草培养细胞的温育温度控制。

We studied construction of a high expression system of foreign genes in tobacco cultured cells, Nicotiana tabacum L.cv.BY2, which can grow 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 responding 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 intiation point. A cDNA encoding a transcription factor associated with the cis-element was also cloned. The wound-induction system seems to be effective gene expression in the tobacco cultured cells. A promoter of ascorbate oxidase gene in cucumber was also showed high transcription activity.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 shift and was maximum at 2 h. Maximun GUS activity 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.