Cloning of black carp beta-actin gene and primarily detecting the function of its promoter region.
A 3 338 bp DNA fragment including the open reading frame and 5'-flanking region of beta-actin gene for black carp genome was obtained through PCR amplification. Analysis of the sequencing results indicated the ORF of black carp beta-actin gene encoding a 375 amino acid protein that shares a high degree of conservation to other known actins. The black carp beta-actin sequence showed 100% identity to common carp, grass carp, and zebrafish, 99.2% identity to human and Norway rat beta-actin gene, 98.9% and 98.1% identity to chicken and Kenyan clawed frog beta-actin gene, respectively. The promoter region of black carp beta-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 beta-actin promoter as a ubiquitous expression promoter was confirmed using the EGFP as a reporter gene.
Feng
Hao
H
Key Lab of Protein Chemistry and Developmental Biology of the Educational Department of China, College of Life Science, Hunan Normal University, Changsha 410081, China.
Cheng
Jia
J
Luo
Jian
J
Liu
Shao-Jun
SJ
Liu
Yun
Y
D024506:5' Flanking Region / Q000235:genetics; D000199:Actins / Q000145:classification / Q000235:genetics*; D000595:Amino Acid Sequence; D000818:Animals; D002347:Carps / Q000235:genetics*; D003001:Cloning, Molecular; D003531:Cypriniformes / Q000196:embryology / Q000235:genetics; D004625:Embryo, Nonmammalian / Q000378:metabolism; D029941:Fish Proteins / Q000235:genetics*; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D010802:Phylogeny; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014162:Transfection
D000199:Actins; D029941:Fish Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins