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Biotechnology and Applied Biochemistry (2009) 52, (233–243) (Printed in Great Britain)
Construction and evaluation of a maize (Zea mays) chimaeric promoter with activity in kernel endosperm and embryo
Colin T. Shepherd* and M. Paul Scott†1
*Interdepartmental Genetics, Iowa State University, Ames, IA 50011, U.S.A., and †USDA-ARS (U.S. Department of Agriculture Agricultural Research Service), Iowa State University, Ames, IA 50011, U.S.A.
Key words: chimaeric (27 kDa γ-zein+Globulin-1) promoter, kernel embryo, kernel endosperm, maize (Zea mays), recombinant protein expression, transgene. Abbreviations used: A27znGlb1 promoter, chimaeric [27zn (27 kDa γ-zein)+Glb1 (Globulin-1)] promoter; ABA, abscisic acid; ABRE, ABA response element; Ct, threshold-cycle value; DAP, days after pollination; EST, expressed sequence tag; GFP, green fluorescent protein; pb, prolamin box; QRT–PCR, quantitative reverse-transcriptase PCR; RFU, relative fluorescence units. 1To whom correspondence should be addressed (email pscott@iastate.edu). The nucleotide sequence of the chimaeric promoter A27znGlb1 will appear in the GenBank®, EMBL, DDBJ and GSDB Nucleotide Sequence Databases under the accession number EF064989. Chimaeric promoters contain DNA sequences from different promoters. Chimaeric promoters are developed to increase the level of recombinant protein expression, to precisely control transgene activity or to combat homology-based gene silencing. Sets of chimaeric promoters, each containing different lengths of DNA from maize (Zea mays) 27zn (27 kDa γ-zein) endosperm-specific promoter and the Glb1 (Globulin-1) embryo-specific promoter were created and tested in a transient expression assay of GFP (green fluorescent protein). Promoter fragments with the highest activity were combined to create the chimaeric promoter A27znGlb1. In the context of the chimaeric promoter, the selected Glb1 promoter fragment was necessary and sufficient to activate expression in embryo tissue and was functionally equivalent to the native Glb1 promoter. Similarly, the selected 27zn promoter fragment in the chimaeric promoter was necessary and sufficient to activate expression in endosperm tissue and was functionally equivalent to the native 27zn promoter. Maize transgenic plants containing the A27znGlb1 chimaeric promoter fused to GFP were produced to characterize this promoter in vivo. Quantitative reverse-transcriptase PCR was used to determine that the promoter was active in the embryo, endosperm, pericarp and immature leaf tissues. GFP activity in plants containing the chimaeric promoter was not significantly different in endosperm than the activity of GFP fused to the full-length 27zn promoter, nor was it different in embryo from the activity of GFP fused to the full-length Glb1 promoter. Transgene copy numbers were shown to be between 4 and 12 copies in different events. Received 13 December 2007/2 July 2008; accepted 16 July 2008 Published as Immediate Publication 16 July 2008, doi:10.1042/BA20070269 © 2009 Portland Press Ltd |
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