Abstract
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 (Cmr), 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 (Bler) 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.
Highlights
In recent years, a genetic transformation method has been successfully established in Aurantiochytrium limacinum
18S rDNA sequences of A. limacinum were located at 1893–2507 (615 bp of 18S+) and 6605–7284 (680 bp of 18S−), respectively, which constituted the two ends of the transformation fragments
Electrotransformation and Agrobacterium tumefaciens-mediated transformation have been successfully applied to A. limacinum
Summary
A genetic transformation method has been successfully established in Aurantiochytrium limacinum. The advantage of the Cre/loxP site-specific recombination system is that it can eliminate the antibiotic resistance gene markers that are transformed into the host in the first step, and the pSH65 plasmid can be lost by repeated sub-culturing of the host after the second step. The Cre/loxP recombination system was used to remove the marker genes from genetically modified organisms This has been performed successfully in yeast and other organisms [9,10,11,12,13]. The Cre/loxP site-specific recombination system was, for the first time, applied to genetic transformation of A. limacinum, where it has provided the experimental basis for directional transformation of A. limacinum without leaving behind an antibiotic marker
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