Construction and Expression Analysis of 18S rDNA-mediated Expression Vector of Nuclease P1 from Penicillium citrinum
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Abstract:
In this study, the Aspergillus niger Bdel4 strain was used as the host strain, and the four major extracellular secretion proteins, glaA, aamA, An05g02100 and An12g06930, were knocked out to provide a pathway for the expression and secretion of nuclease P1. The rDNA sequence of A. niger Bdel4 was amplified by PCR, and the recombinant plasmids, pRpT-nucP1-18S and pRpT-2A-nucP1-18S were constructed through using the rDNA sequence as integration sites. The constructed recombinant plasmids pRpT-nucP1-18S and pRpT-2A-nucP1-18S were then transferred into A. niger Bdel4. Prescreening of the transformants was carried out by semi-quantitative fluorescent PCR, and the activity of nuclease P1 was then measured to screen a strain with high activity for industrial production. The total genomic samples containing the exogenous target gene of nuc P1 were repeatedly detected by SYBR Green real-time fluorescent quantitative PCR, and the average value was taken as the copy number of the target gene nuc P1 to explore the relationship between the copy number and the expression level of the enzyme. The result showed that the enzymatic activity of the strain containing 9 nuc P1 gene copies reached 88.52 U/ml, which was 3.86 times higher than that of the nuclease P1 for the single copy strain, and 1.2-fold as high as that of the heterologous nuclease P1 reported in the previous studies. When the copy number of nuc P1 was increased to over 9, the enzyme activity decreased.
