METABOLIC ENGINEERING OF CULTURED TOBACCO CELLS

培养烟草细胞的代谢工程

• 批准号: 07555258
• 负责人: SHINMYO Atsuhiko
• 金额: $ 10.82万
• 依托单位: NARA INSTITUTE OF SCIENCE AND TECHNOLOGY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (A)
• 财政年份: 1995
• 资助国家: 日本
• 起止时间: 1995 至 1997
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07555258/
• 关键词: TABACCO CULTURED CELLS; PROMOTER; ARABIDOPSIS THALIANA; HEAT-SHOCK PROTEIN; LOG PHASE CELLS; STATIONARY GROWTH PHASE; DIFFERENTIAL SCREENING; PECTIN ESTERASE; 制御シス配列; 増殖停止期; シロイヌナズナ; 転写因子; タバコ細胞; 熱ショックプロモーター; テアニン

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.

研究了烟草细胞（BY2）基因表达系统的构建。拟南芥热休克蛋白基因（HSP 18.2）的一个925 bp的启动子片段在BY2细胞中表达的β-葡萄糖醛酸酶（GUS）报告基因显示出明显的热休克反应。在500 ml摇瓶和3升发酵罐中也观察到类似的结果。分离在对数期细胞中mRNA水平高且在基因组中拷贝数低的cDNA克隆。这些克隆分别与F1-ATP酶（线粒体型）、延伸因子1-α和拟南芥的一个功能未知的基因（克隆27）具有高度同源性。克隆27的A5 '侧翼区在BY2细胞中的启动活性是CaMV 35 S启动子的6.2倍。这些克隆与乙醇脱氢酶、果胶酯酶和伸展蛋白的同源性较高。这些基因的启动子将可用于高细胞密度培养中的基因表达。

项目成果

K.Yoshida A.Shinmyo 他: "Heat-inducible expression system for a foreign gene in cultured Tobacco cells using the HSP18.2 promoter of Arabidopsis thaliana" Appl Microbiol Biotechnol. 44. 466-472 (1995)

K. Yoshida A. Shinmyo 等人：“使用拟南芥 HSP18.2 启动子在培养的烟草细胞中热诱导表达系统”Appl Microbiol Biotechnol. 44. 466-472 (1995)。

Shinmyo A, et al.: "Metabolic Engineering of Cultured Tobacco Cells." Biotechnol.Bioeng.(1997)

Shinmyo A 等人：“培养烟草细胞的代谢工程”。

Shinmyo A,Shoji A,Bando E,Nagaya S,Nakai Y,Kato K,Sekine M,Yoshida K: "Metabolic Engineering of Cultured Tabacco Cells." Biotechnol.Bioeng. (in press). (1997)

Shinmyo A、Shoji A、Bando E、Nagaya S、Nakai Y、Kato K、Sekine M、Yoshida K：“培养烟草细胞的代谢工程”。

Yoshida K,Kasai T,Garcia M R C,Sawada S,Shoji T,Shimizu S,Yamazaki K,Komeda Y,Shinmyo A: "Heat-inducible expression system for a foreign gene in cultured tobacco cells using the HSP18.2 promoter of Arabidopsis thaliana." Appl.Microbiol.Biotechnol.43. 466-

Yoshida K、Kasai T、Garcia M R C、Sawada S、Shoji T、Shimizu S、Yamazaki K、Komeda Y、Shinmyo A：“使用拟南芥 HSP18.2 启动子在培养烟草细胞中热诱导表达外源基因

吉田和哉,新名惇彦 他: "植物における外来遺伝子発現システムの構築を考えたプロモーター解析" 植物組織培養. 12. (1995)

Kazuya Yoshida、Atsuhiko Niina 等：“在植物中构建外源基因表达系统的启动子分析”植物组织培养 12。（1995）