Lee Kung-Ta, Chen Shih-Cheng, Chiang Bor-Luen, Yamakawa Takashi
Graduate Institute of Microbiology and Biochemistry, National Taiwan University, 1, Sec. 4, Roosevelt Road, Taipei, Taiwan, Republic of China.
Appl Microbiol Biotechnol. 2007 Jan;73(5):1047-53. doi: 10.1007/s00253-006-0576-2. Epub 2006 Sep 7.
The production of beta-glucuronidase (GUS) driven by the Arabidopsis small heat shock protein 18.2 promoter in liquid cultures of transgenic tobacco (Nicotiana tabacum) hairy roots is reported. Clone GD-3, showing high GUS heat induction and a moderate growth rate, was selected from 436 clones for study. Treatment of GD-3 with heat shock at 36-42 degrees C for 2 h then recovery at 27 degrees C resulted in an increase in GUS specific activity, while higher heat-shock temperatures led to a decline. These results were in accordance with the change in esterase activity, a measure of tissue viability. Using 2 h of 42 degrees C heat shock and a recovery phase at 27 degrees C, GUS specific activity increased rapidly and reached a maximum of 267.6 nmol 4-methylumbelliferyl beta-D-glucuronic acid (MU) min-1 mg-1 protein at 24 h of recovery. When tissues were continuously heated at 42 degrees C and tested without a recovery period, GUS mRNA was detectable at 2 h and peaked at 5 h, but GUS activity was not seen until 10 h and did not peak until 28 h; in addition, the maximum activity was lower than that seen after heat shock for only 30 min or 2 h, followed by recovery. This shows that recovery at normal temperature is crucial for the heat-inducible heterogeneous expression system of transgenic hairy roots. Multiple heat-shock treatments showed that this system was heat reinducible, although a gradual decline in GUS specific activity was seen in the second and third cycles.
报道了在转基因烟草（Nicotiana tabacum）毛状根液体培养物中，由拟南芥小热激蛋白18.2启动子驱动的β-葡萄糖醛酸酶（GUS）的产生。从436个克隆中选择了克隆GD-3进行研究，该克隆显示出高GUS热诱导和中等生长速率。将GD-3在36-42℃热激处理2小时，然后在27℃恢复，导致GUS比活性增加，而较高的热激温度导致活性下降。这些结果与酯酶活性的变化一致，酯酶活性是组织活力的一种度量。使用42℃热激2小时并在27℃进行恢复阶段，GUS比活性迅速增加，并在恢复24小时时达到最大值，为267.6 nmol 4-甲基伞形酮基β-D-葡萄糖醛酸（MU）min-1 mg-1蛋白质。当组织在42℃持续加热且不经过恢复期进行测试时，在2小时可检测到GUS mRNA，并在5小时达到峰值，但直到10小时才观察到GUS活性，直到28小时才达到峰值；此外，最大活性低于仅热激30分钟或2小时后再恢复的情况。这表明常温恢复对于转基因毛状根的热诱导异源表达系统至关重要。多次热激处理表明该系统可再次被热诱导，尽管在第二和第三个循环中GUS比活性逐渐下降。
