Development of gene expression system by artificial chromosome

Research Project

Project/Area Number	60480007
Research Category	Grant-in-Aid for General Scientific Research (B)
Allocation Type	Single-year Grants
Research Field	植物生理学
Research Institution	Institute of Biological Sciences, Univ. of Tsukuba
Principal Investigator	UCHIMIYA H. Institute of Biological Sciences, Univ. of Tsukuba, 生物科学系, 講師 (50142229)
Project Period (FY)	1985 – 1986
Project Status	Completed (Fiscal Year 1986)
Budget Amount *help	¥5,000,000 (Direct Cost: ¥5,000,000) Fiscal Year 1986: ¥1,000,000 (Direct Cost: ¥1,000,000) Fiscal Year 1985: ¥4,000,000 (Direct Cost: ¥4,000,000)
Keywords	PROTOPLASTS / TRANSFORMATION / TOBACCO / イネ
Research Abstract	Protoplasts isolated from suspension cultures of rice cells were treated with bacterial plasmid DNA carrying a chimeric gene consisting of the nopaline synthase promoter, the aminoglycoside phosphotransferase <II> (APH(3') <II> ) struchural gene from bacterial transposon Tn5 and the terminator region from cauliflower mosaic virus DNA. Colonies capable of proliferating in medium containing kanamycin were selected. A transformation frequency of approximately 2% to 3% was recorded in several expriments. The enzyme (APH(3') <II> ) was also detected in kanamycin-resistant callus, which had survived after repeated selection. There was some variation in the APH(3') <II> activity in the transformation which paralleled to the copy number of the inserted genes. In the case of Nicotiana transformation, the plasmid containing an intact nopaline synthase gene and a chimeric gene consisting of promoter and terminater region from cauliflower mosaic virus gene VI and a structural gene, APH(3') <II> from bacterial transposon Tn5 was used. After direct DNA transformation of mesophyll protoplasts with this plasmid, several kanamycin resistant transformation were obtained. Intensive studies on drug tolerance of transformts in relation to growth and differentiation implicate the stable expression of a chimeric gene.