大肠杆菌胞内L-丙氨酸响应型启动子的筛选与表征

Screening and characterization of intracellular L-alanine responsive promoter in Escherichia coli

该研究通过对L-丙氨酸压力下大肠杆菌的转录组进行分析,利用mKate荧光蛋白进行表征,筛选响应L-丙氨酸的启动子元件。转录组筛选了ArgK、FlgG、GlgP、CpxP、BetA、GlpA、KdpB、PulG、LivH和YhfX十个在L-丙氨酸压力下转录水平显著提高的基因,并将基因前500 bp潜在的启动子片段与荧光报告蛋白基因m Kate融合,筛选得到特异性响应L-丙氨酸浓度的启动子PAla-7。结果表明,PAla-7启动子在10 g/L、20 g/L、30 g/L、40 g/L、50 g/L和60 g/L L-丙氨酸诱导后的相对荧光强度分别增加了49%、122%、181%、275%、432%和705%,表明PAla-7启动子在L-丙氨酸质量浓度0~60 g/L范围内有着良好的灵敏性。选择11种常见氨基酸诱导PAla-7启动子表达,只有L-丙氨酸在诱导质量浓度从10 g/L增加到20 g/L时,相对荧光强度增加了52.4%,表明PAla-7启动子是特异性响应L-丙氨酸的启动子。

In this study,the transcriptome of Escherichia coli under L-alanine pressure was analyzed and characterized by mKate fluorescent protein to screen the promoter elements with responsive to L-alanine.The transcriptome screened ten genes,ArgK,FlgG,GlgP,CpxP,BetA,GlpA,KdpB,PulG,LivH and YhfX,which transcription levels were significantly increased under L-alanine pressure.Then,the possible promoter fragments of the first 500 bp of these genes were fused with the fluorescent reporter gene mKate to construct a promoter library to screen the promoter with responsive to L-alanine,indicating that PAla-7 specifically responds to the concentration of L-alanine.The results showed that the relative fluorescence intensity of PAla-7 promoter induced by 10 g/L,20 g/L,30 g/L,40 g/L,50 g/L and 60 g/L L-alanine increased by 49%,122%,181%,275%,432%and 705%,respectively,indicating that the PAla-7 promoter had good sensitivity in the concentration range of 0-60 g/L L-alanine.Eleven common amino acids were selected to induce the expression of PAla-7 promoter.Only L-alanine increased the relative fluorescence intensity by 52.4%when the induction concentration increased from 10 g/L to 20 g/L,indicating that PAla-7 promoter was a specific promoter responsive to L-alanine.