METABOLIC ENGINEERING OF CULTURED TOBACCO CELLS

研究了烟草培养细胞（BY2）基因表达体系的构建。拟南芥（Arabidopsis thaliana）烟叶热休克蛋白基因（HSP18.2） 925 bp启动子片段在BY2细胞中表达β -葡糖苷酸酶（GUS）报告基因，显示出明显的热休克反应。在500ml烧瓶和3l罐发酵罐中也观察到类似的结果。尝试在BY2细胞中分离强启动子。分离出对数相细胞mRNA水平高、基因组拷贝数低的cDNA克隆。这些克隆分别与拟南芥的f1 - atp酶（线粒体型）、延伸因子1- α和一个功能未知的基因（克隆27）具有高度同源性。克隆27的A5′侧区启动子活性是BY2细胞中CaMV35S启动子活性的6.2倍。通过差异筛选，分离出3个在BY2细胞稳定生长期表达的cDNA克隆。这些克隆与乙醇脱氢酶、果胶酯酶和伸展酶具有较高的序列同源性。这些基因的启动子将在高密度培养中用于基因表达。

Construction of a gene expression system in tobacco cultured cells (BY2) was studied. A 925 bp promoter fragment of a heat-shock protein Nicotiana tabacum gene (HSP18.2) of Arabidopsis thaliana showed clear heat-shock response of expression of the beta-glucuronidase (GUS) reporter gene in BY2 cells. Similar results were observed in a 500ml flask and 3-l jar fermentor.Isolation of strong promoters in BY2 cells was tried. cDNA clones of which mRNA level is high in log phase cells and copy number in genome is low were isolated. These clones showed high homology with F1-ATPase (mitochondria type), elongation factor 1-alpha, and a gene with unknown function of A.thaliana (clone 27), respectively. A5'-flanking region of clone 27 showed 6.2 times of promoter activity of the CaMV35S promoter in BY2 cells.Three cDNA clones which are expressed in stationary growth phase of BY2 cells were isolated by a differential screening. There clones showed high sequence homologies to alcohol dehydrogenase, pectin esterase and extensin. Promoters of these genes will be useful in gene expression in high cell density culture.