解淀粉芽孢杆菌内源启动子的筛选及表达碱性果胶酶的应用研究

Screening of endogenous promoters of Bacillus amyloliquefaciens and application of them in the expression of alkaline pectinase

【目的】通过检测目的基因的转录水平和表达强度,筛选来源于解淀粉芽孢杆菌(Bacillus amyloliquefaciens)的内源启动子,确定适合碱性果胶酶基因表达的强启动子,并进一步对选用的强启动子进行分析。【方法】通过生物信息学手段对启动子片段进行预测与筛选,采用相对荧光强度、酶活力等表征手段进行分析,同时采用实时荧光定量聚合酶链式反应(real-time fluorescence quantitative polymerase chain reaction,RT-qPCR)技术检测不同启动子的转录水平。【结果】启动子PrapA、PmetE-1、Phin-1表达碱性果胶酶的活力分别是P43启动子的9.8倍、4.8倍、3.0倍,筛选出的这3个强启动子为其他异源基因在解淀粉芽孢杆菌中的表达奠定了基础。【结论】通过生物信息学手段预测及筛选启动子,筛选得到比较强的启动子PrapA、PmetE-1和Phin-1,有效提高了碱性果胶酶的表达量。

[Objective]To screen out the strong promoters suitable for the expression of alkaline pectinase in Bacillus amyloliquefaciens based on the transcription level and expression of the target gene and further analyze the selected strong promoters.[Methods]The promoter fragments were predicted and screened out by bioinformatics tools on the basis of the relative fluorescence intensity and enzyme activity.Further,real-time fluorescence quantitative polymerase chain reaction(RT-qPCR)was carried out to determine the transcription levels of different promoters.[Results]The activities of PrapA,PmetE-1,and Phin-1 expressing alkaline pectinase were 9.8,4.8,and 3.0 times that of P43 promoter,respectively.The three strong promoters screened out laid a foundation for the expression of other heterologous genes in B.amyloliquefaciens.[Conclusion]The strong promoters PrapA,PmetE-1,and Phin-1 screened out in this study can effectively improve the expression of alkaline pectinase.