期刊
MOLECULES
卷 20, 期 6, 页码 10110-10121出版社
MDPI
DOI: 10.3390/molecules200610110
关键词
antibiotic resistance marker gene; Aurantiochytrium limacinum; Cre; loxP site-specific recombination system; homologous recombination
资金
- Research Award Fund for Young and Middle-aged Scientists of Shandong Province [BS2012HZ017]
- Research Fund for the National High Technology Research and Development Program of China [2008AA09Z410]
The Cre/loxP site-specific recombination system was applied to Aurantiochytrium limacinum to obtain a transformant without the antibiotic resistance marker gene. First, the enhanced green fluorescent protein gene (egfp) and chloramphenicol resistance gene (Cm-r), along with the two loxP loci, were integrated into the genome of A. limacinum OUC88 using 18S rDNA sequences as the homologous recombination sites. Then plasmid pSH65, containing a zeocin resistance gene (Ble(r)) was transferred into A. limacinum OUC_CG. After induction with galactose, repeated passage in culture and PCR-based assessment, the pSH65 plasmid was lost and A. limacinum OUC_EG host was shown to no longer have resistance to 100 mg chloramphenicol/L or 5 mg zeocin/L. Through southern blotting and fluorescence detection, egfp was found to be integrated into the genome of A. limacinum OUC_EG, and EGFP was successfully expressed in the cells. The successful application of the Cre/loxP system demonstrates an experimental basis for genetic modification of A. limacinum so as to obtain transformed strains with no antibiotic resistance marker genes.
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