title: Function and regulation domains of a newly isolated putative β-actin promoter from pacific white shrimp.
abstract: Current development of transgenic shrimp research has been hampered due to the lack of the suitable promoters and efficient transfection methods for crustaceans. A 1642 bp sequence, containing 5'-upstream sequence, exon 1, intron 1 and partial exon 2, which is responsible for transcriptional initiation of the newly reported shrimp β-actin (actinT1), has been isolated from the Pacific white shrimp (Litopenaeus vannamei) and named as SbaP. To determine its function and potential application in marine biotechnology, the sequence and functional domains were examined by constitutive expression of the luciferase reporter gene. We have identified 5' regions that play a central role in the expression of the β-actin gene. The proximal promoter (-1642/-1325) contains two highly conserved transcriptional sites, CCAAT box and CArG motif. Two negative (-1140/-924, -222/-21) and one positive (-810/-425) regulatory elements have been identified in intron1. Transient transfection assay with a construct containing proximal promoter and enhancer (SbaPΔ-222/+1Δ-1325/-924) regions of the shrimp β-actin coupled with luciferase and EGFP (enhanced green fluorescent protein) showed that the promoter was not only functional in sf21 cells, but promoter activity was more than 8-fold higher than a viral-origin promoter (ie1, white spot syndrome virus immediate early gene promoter). Furthermore, SbaPΔ-222/+1Δ-1325/-924 drove a successful expression of luciferase injection assay in vivo injection and also showed higher promoter activity than the ie1 promoter, suggesting that the expression vectors constructed with SbaPΔ-222/+1Δ-1325/-924 have important potential in gene transfer studies for shrimp and other crustacean species.
authors.affiliation: Department of Public Health Sciences, University of Hawaii at Manoa, Honolulu, Hawaii, United States of America; Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Qingdao, Shandong, China; University of Chinese Academy of Sciences, Beijing, China.
authors.forename: Yingli
authors.initials: Y
authors.lastname: Shi
authors.affiliation: Department of Public Health Sciences, University of Hawaii at Manoa, Honolulu, Hawaii, United States of America.
authors.forename: Marcus
authors.initials: M
authors.lastname: Soderlund
authors.affiliation: Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Qingdao, Shandong, China.
authors.forename: Jianhai
authors.initials: J
authors.lastname: Xiang
authors.affiliation: Department of Public Health Sciences, University of Hawaii at Manoa, Honolulu, Hawaii, United States of America.
authors.forename: Yuanan
authors.initials: Y
authors.lastname: Lu
mesh_terms: D000199:Actins / Q000235:genetics* / Q000378:metabolism; D000818:Animals; D060829:Arthropod Proteins / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D003001:Cloning, Molecular; D004742:Enhancer Elements, Genetic; D005091:Exons; D015870:Gene Expression; D017930:Genes, Reporter; D007438:Introns; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D033561:Penaeidae / Q000235:genetics* / Q000378:metabolism; D011401:Promoter Regions, Genetic; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D061987:Sf9 Cells; D018411:Spodoptera / Q000166:cytology / Q000235:genetics; D015533:Transcriptional Activation
chemical_list: D000199:Actins; D060829:Arthropod Proteins; D011994:Recombinant Proteins; D008156:Luciferases
references.citation: Cell. 1983 Sep;34(2):343-58
references.pmid: 6616614
references.citation: Mol Cell Biol. 1990 Jul;10(7):3432-40
references.pmid: 2355913
references.citation: J Biol Chem. 1989 Jun 5;264(16):9539-46
references.pmid: 2722849
references.citation: Cell Transplant. 2000 Sep-Oct;9(5):675-80
references.pmid: 11144964
references.citation: Theriogenology. 2000 Dec 1;54(9):1421-32
references.pmid: 11191866
references.citation: Mar Biotechnol (NY). 2003 May-Jun;5(3):244-52
references.pmid: 14502396
references.citation: Annu Rev Biochem. 1981;50:349-83
references.pmid: 6791577
references.citation: Proc Natl Acad Sci U S A. 1987 Oct;84(19):6702-6
references.pmid: 3477800
references.citation: Mol Cell Biol. 1988 Jan;8(1):267-72
references.pmid: 3422098
references.citation: Nucleic Acids Res. 1989 Jan 25;17(2):601-15
references.pmid: 2915924
references.citation: Gene. 1991 Dec 15;108(2):211-7
references.pmid: 1748307
references.citation: Mol Reprod Dev. 1991 Nov;30(3):201-6
references.pmid: 1793597
references.citation: Mol Reprod Dev. 1993 Feb;34(2):117-26
references.pmid: 8442950
references.citation: Nucleic Acids Res. 1995 Apr 11;23(7):1223-30
references.pmid: 7739901
references.citation: Mol Biotechnol. 1996 Aug;6(1):17-30
references.pmid: 8887358
references.citation: Mol Reprod Dev. 1996 Oct;45(2):117-22
references.pmid: 8914067
references.citation: Mol Mar Biol Biotechnol. 1998 Sep;7(3):165-72
references.pmid: 9701610
references.citation: Virology. 2005 Apr 10;334(2):327-41
references.pmid: 15780883
references.citation: J Exp Zool A Comp Exp Biol. 2005 Dec 1;303(12):1104-9
references.pmid: 16254920
references.citation: J Biotechnol. 2006 Sep 1;125(2):173-84
references.pmid: 16621097
references.citation: Exp Parasitol. 2007 Nov;117(3):292-303
references.pmid: 17531975
references.citation: Virology. 2009 Sep 1;391(2):315-24
references.pmid: 19604531
references.citation: Virology. 2010 Oct 25;406(2):176-80
references.pmid: 20684968
references.citation: Transgenic Res. 2011 Dec;20(6):1333-55
references.pmid: 21437716
references.citation: Fish Shellfish Immunol. 2012 Jan;32(1):26-34
references.pmid: 22030024
references.citation: Fish Shellfish Immunol. 2013 Jan;34(1):397-401
references.pmid: 23032439
references.citation: Mol Cell Biol. 1985 Oct;5(10):2720-32
references.pmid: 3837182