青鱼β-actin基因克隆及其启动子功能的初步检测.pdfVIP

遗 传 学 报 Acta Genetica Sinica , February 2006, 33 (2)：133–140 ISSN 0379-4172 Cloning of Black Carp β-actin Gene and Primarily Detecting the Function of Its Promoter Region FENG Hao*, CHENG Jia*, LUO Jian, LIU Shao-Jun, LIU Yun① Key Lab of Protein Chemistry and Developmental Biology of the Educational Department of China, College of Life Science, Hunan Normal University, Changsha 410081, China Abstract: A 3 338 bp DNA fragment including the open reading frame and 5′-flanking region of β-actin gene for black carp ge- nome was obtained through PCR amplification. Analysis of the sequencing results indicated the ORF of black carp β-actin gene encoding a 375 amino acid protein that shares a high degree of conservation to other known actins. The black carp β-actin sequence showed 100% identity to common carp, grass carp, and zebrafish, 99.2% identity to human and Norway rat β-actin gene, 98.9% and 98.1% identity to chicken and Kenyan clawed frog β-actin gene, respectively. The promoter region of black carp β-actin gene was inserted into the promoterless pEGFP1 vector. The recombinant plasmid was microinjected into the fertilized eggs of mud loach before two-cell stage as well as transfected into HeLa cell line. GFP expression was found in 50% of mud loach embryos and 2/3 HeLa cells. The GFP expression could be observed in every part of the mud loach embryos, and in some embryos, the GFP was expressed in the whole body. Thus, the usefulness of black carp β-actin promoter as a ubiquitous expression promoter was con- firmed using the EGFP as a reporter gene. Key words: black carp; β-actin; promoter; microinjection Actins are highly conserved proteins found in all rat [6], chicken [7], sea urchin [8], and Drosophila [9] and eukaryotic cells. They play a key role in maintaining the evolution of this gene family has been extensively cytoskeletal structure, cell m