Application of the Cre/loxP Site-Specific Recombination System for Gene Transformation in Aurantiochytrium limacinum

作者：Sun, Hengyi; Chen, Hao; Zang, Xiaonan^*; Hou, Pan; Zhou, Bingbing; Liu, Yuantao; Wu, Fei; Cao, Xiaofei; Zhang, Xuecheng

来源：Molecules, 2015, 20(6): 10110-10121.

DOI：10.3390/molecules200610110

• antibiotic resistance marker gene
• Aurantiochytrium limacinum
• Cre
• loxP site-specific recombination system
• homologous recombination

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.

• 出版日期2015-6
• 单位中国海洋大学