【摘要】 采用实时荧光定量聚合酶链式反应（RT-qPCR）技术对重组毕赤酵母基因组中左聚糖蔗糖酶基因（SacB）的拷贝数及信使核糖核酸（mRNA）转录水平进行检测分析，并用3,5-二硝基水杨酸（DNS）法测定菌株不同诱导时间下左聚糖蔗糖酶水解活力。结果表明，在BMMY液体培养基中经甲醇诱导24 h时，样品转录水平皆达到最大值，此时多拷贝菌株转录水平（2.13）为单拷贝菌株（0.42）的5.1倍；左聚糖蔗糖酶活力在甲醇诱导24 h后均随诱导时间不断上升，多拷贝菌株酶活（13.96 U/mL）较单拷贝菌株（5.48 U/mL）提高1.5倍。重组毕赤酵母整合的左聚糖蔗糖酶基因在1～3个拷贝范围内，随着拷贝数增加，多拷贝菌株较单拷贝菌株的mRNA转录水平及相应的蛋白表达量均显著增加（P<0.05），说明毕赤酵母是表达基因SacB的良好宿主。更多还原

【Abstract】 The copy numbers and messenger ribonucleic acid(m RNA) transcription of levansucrase(SacB) gene in recombinant Pichia pastoris genome were determined by real-time fluorescence quantitative polymerase chain reaction(RT-qPCR), and the levansucrase hydrolytic activity of the strains at different induction times was determined by 3,5-dinitrosalicylic acid(DNS) method. The results showed that the transcription of the samples reached the maximum after methanol induction in BMMY liquid medium for 24 h, the transcription of the multi-copy strain(2.13) was 5.1 times that of single-copy strain(0.42). The enzyme activity of levansucrase increased with induction time after methanol induction for 24 h, and the enzyme activity of multicopy strain(13.96 U/ml) was 1.5 times higher than that of single-copy strain(5.48 U/ml). The recombinant P. pastoris integrated levansucrase gene in the range of 1 to 3 copies, with increase of copy numbers, the m RNA transcription level and corresponding protein expression level of multi-copy strain were significantly increased compared to the single-copy strain(P<0.05), indicating that P. pastoris was a good host for expressing gene SacB.更多还原