茶树咖啡碱合成酶基因cDNA在烟草中的表达

为了在烟草中合成咖啡碱,将咖啡碱合成酶(Tea caffeine synthase,TCS)基因cDNA全序列克隆到双元载体pBI121中,通过农杆菌叶盘转化法将TCS基因成功转入烟草,结果得到72株转基因烟草植株。对其中3株转基因植株进行PCR检测和PCR-Southern检测,证实TCS基因已整合到基因组中;RT-PCR和Northern分析显示,TCS在这些植株中均能正常转录。用从转基因植株中提取的粗酶液进行体外酶促反应,能催化7-甲基黄嘌呤和可可碱向咖啡碱的转变,由此表明,转基因烟草植株中能产生具有正常生物学活性的TCS,但从3株转基因烟草中均未检测到咖啡碱。

Expression of tea caffeine synthase cDNA in tobacco

In order to synthesze caffeine,the full length of the tea caffeine synthase(TCS) cDNA was cloned into the binary vector pBI121.TCS cDNA was introduced into tobacco by employing the leaf disk method of Agrobacterium mediated transformation,and 72 transgenic tobacco plants were obtained,among which,three tobacce plants were tested by PCR and PCR-Southern blot,and the result confirmed that the TCS cDNA had been integrated into tobacco genome.RT-PCR and Northern blot indicated that the TCS gene could be transcribed normally.The results of in vitro enzymatic reaction indicated that the crude enzyme extracted from transgenic tobacco leaves could catalyze the methylation of 7-methylxanthine and theobromine.Therefore,transformed TCS gene could be translated and formed normal secondary structure in tobacco plant and the expressed protein had normal TCS biological activities.However,caffeine was not detected in three transgenic genetic tobacco plants.