title: Application of the Cre/loxP Site-Specific Recombination System for Gene Transformation in Aurantiochytrium limacinum.
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.
authors.affiliation: Key Laboratory of Marine Genetics and Breeding, Ministry of Education, Ocean University of China, Qingdao 266003, China. sunhengyi1987@163.com.
authors.forename: Hengyi
authors.initials: H
authors.lastname: Sun
authors.affiliation: Key Laboratory of Marine Genetics and Breeding, Ministry of Education, Ocean University of China, Qingdao 266003, China. chenhao4680088@126.com.
authors.forename: Hao
authors.initials: H
authors.lastname: Chen
authors.affiliation: Key Laboratory of Marine Genetics and Breeding, Ministry of Education, Ocean University of China, Qingdao 266003, China. xnzang@ouc.edu.cn.
authors.forename: Xiaonan
authors.initials: X
authors.lastname: Zang
authors.affiliation: Key Laboratory of Marine Genetics and Breeding, Ministry of Education, Ocean University of China, Qingdao 266003, China. xiaohou_aishengwu@163.com.
authors.forename: Pan
authors.initials: P
authors.lastname: Hou
authors.affiliation: Key Laboratory of Marine Genetics and Breeding, Ministry of Education, Ocean University of China, Qingdao 266003, China. qzwnzn@yeah.net.
authors.forename: Bingbing
authors.initials: B
authors.lastname: Zhou
authors.affiliation: Key Laboratory of Marine Genetics and Breeding, Ministry of Education, Ocean University of China, Qingdao 266003, China. liuyuantao2418@126.com.
authors.forename: Yuantao
authors.initials: Y
authors.lastname: Liu
authors.affiliation: Key Laboratory of Marine Genetics and Breeding, Ministry of Education, Ocean University of China, Qingdao 266003, China. wufei0917@sina.cn.
authors.forename: Fei
authors.initials: F
authors.lastname: Wu
authors.affiliation: Key Laboratory of Marine Genetics and Breeding, Ministry of Education, Ocean University of China, Qingdao 266003, China. caoxiao1@126.com.
authors.forename: Xiaofei
authors.initials: X
authors.lastname: Cao
authors.affiliation: Key Laboratory of Marine Genetics and Breeding, Ministry of Education, Ocean University of China, Qingdao 266003, China. xczhang8@163.com.
authors.forename: Xuecheng
authors.initials: X
authors.lastname: Zhang
mesh_terms: D001761:Bleomycin / Q000494:pharmacology; D002702:Chloramphenicol Resistance / Q000235:genetics; D005690:Galactose / Q000494:pharmacology; D017353:Gene Deletion; D015870:Gene Expression; D005798:Genes, Bacterial; D005818:Genetic Engineering; D016678:Genome; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D019426:Integrases / Q000235:genetics*; D030781:Organisms, Genetically Modified; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism; D012337:RNA, Ribosomal, 18S / Q000235:genetics / Q000378:metabolism; D011995:Recombination, Genetic; D058009:Stramenopiles / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D014170:Transformation, Genetic
chemical_list: D012337:RNA, Ribosomal, 18S; C485184:enhanced green fluorescent protein; D001761:Bleomycin; D049452:Green Fluorescent Proteins; C105427:Zeocin; C045073:Cre recombinase; D019426:Integrases; D005690:Galactose
keywords: Aurantiochytrium limacinum; Cre/loxP  site-specific recombination system; antibiotic resistance marker gene; homologous recombination