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

我们研究了外源基因在烟草培养细胞中高效表达系统的构建，该系统能在植物细胞中生长较快(倍增时间为10-11h)。一组导入烟草细胞的目的基因必须置于强烈的启动子、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prxC2在烟草细胞中表现出较高的活性。PrxC2基因在创伤诱导下表达，从翻译起始点的-289bp处鉴定到一个以CACGTG为核心序列的创伤诱导顺式元件。还克隆了编码与顺式元件相关的转录因子的cDNA.创伤诱导系统似乎是烟草培养细胞中有效的基因表达系统。将拟南芥HSP18.2基因的热休克启动子连接到β-葡萄糖醛酸酶结构基因(GUS)上，并导入烟草细胞的染色体中。当培养温度从25ﾟC改变到37ﾟC时，GUS基因的表达被强烈诱导，温度改变后15min检测到GUS基因，2 h达到高峰，4 h达到最大活性，这表明通过控制培养细胞的培养温度，HSP18.2启动子是表达外源基因的良好候选基因。

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.