Hughes Erik H, Hong Seung-Beom, Shanks Jacqueline V, San Ka-Yiu, Gibson Susan I
Department of Chemical Engineering, Rice University, PO Box 1892, Houston, Texas 77251, USA.
Biotechnol Prog. 2002 Nov-Dec;18(6):1183-6. doi: 10.1021/bp025603o.
Transgenic hairy root cultures of Catharanthus roseus were established with a glucocorticoid-inducible promoter controlling the expression of green fluorescent protein (GFP), and GFP expression was characterized. The inducible system shows a tightly controlled, reversible, and dosage-dependent response to the glucocorticoid dexamethasone in C. roseus hairy roots. Full induction was noted after 12-18 h in the mature regions of the root tips and after 6 h in the meristem tissue. Upon removal of the inducing agent, GFP expression declined to undetectable levels in the mature tissues after 24 h and in the meristem after 48 h. Although no dosage-dependent response was noted in the meristem region, such a response was apparent in the mature region of the tip and verified by quantitative GFP analysis. The inducible promoter system allowed quantitative control of GFP expression between 0.01 and 10 microM dexamethasone with saturation occurring at higher levels. Using GFP as a model system allowed demonstration of the ability to control temporal and quantitative gene expression with the glucocorticoid-inducible promoter in transgenic C. roseus hairy roots.
利用控制绿色荧光蛋白(GFP)表达的糖皮质激素诱导型启动子建立了长春花转基因毛状根培养物,并对GFP表达进行了表征。该诱导系统在长春花毛状根中对糖皮质激素地塞米松表现出严格控制、可逆且剂量依赖性的反应。在根尖成熟区域12 - 18小时后以及分生组织6小时后观察到完全诱导。去除诱导剂后,24小时后成熟组织中GFP表达下降至检测不到的水平,48小时后分生组织中GFP表达下降至检测不到的水平。虽然在分生组织区域未观察到剂量依赖性反应,但在根尖成熟区域这种反应明显,并通过GFP定量分析得到验证。诱导型启动子系统允许在0.01至10微摩尔地塞米松之间对GFP表达进行定量控制,在较高水平时出现饱和。以GFP作为模型系统,证明了在转基因长春花毛状根中利用糖皮质激素诱导型启动子控制基因表达的时间和定量的能力。