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

利用重组DNA技术构建烟草培养细胞中有用化合物的生产体系

基本信息

批准号：
05555221
负责人：
SHINMYO Atsuhiko
金额：
$ 3.39万
依托单位：
NARA INSTITUTE OF SCIENCE AND TECHNOLOGY (1994)Osaka University (1993)
依托单位国家：
日本
项目类别：
Grant-in-Aid for Developmental Scientific Research (B)
财政年份：
1993
资助国家：
日本
起止时间：
1993 至 1994
项目状态：
已结题

项目摘要

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.

本文研究了外源基因在烟草培养细胞中高表达体系的构建。BY2，在植物细胞间生长速度较快（倍增时间为10-11h）。将一组靶基因导入烟草细胞，必须将其置于强启动子、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，在翻译起始点289 bp处鉴定出一个诱导损伤的顺式元件，其核心序列为CACGTG。还克隆了一个与顺式元件相关的转录因子cDNA。在烟草培养细胞中，伤口诱导系统似乎是有效的基因表达。黄瓜抗坏血酸氧化酶基因的一个启动子也表现出较高的转录活性。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 mRNA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启动子。