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

作　　者：张莹[1] 韩晓静 蔡逸安 李登科 李庆刚 路福平[1] 李玉[1] ZHANG Ying;HAN Xiaojing;CAI Yi’an;LI Dengke;LI Qinggang;LU Fuping;LI Yu(Key Laboratory of Industrial Fermentation Microbiology,Ministry of Education,School of Bioengineering,Tianjin University of Science and Technology,Tianjin 300457,China)

机构地区：[1]天津科技大学生物工程学院、工业发酵微生物教育部重点实验室,天津300457

出　　处：《微生物学报》2023年第4期1575-1586,共12页Acta Microbiologica Sinica

基　　金：国家重点研发计划(2021YFC2104002)。

摘　　要：【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.

关 键 词：解淀粉芽孢杆菌 启动子 转录水平 生物信息学 碱性果胶酶 

分 类 号：Q78[生物学—分子生物学]