title: Synthetic introns help identify sequences in the 5' UTR intron of the Glycine max polyubiquitin (Gmubi) promoter that give increased promoter activity.
abstract: Specific sequences within the leader intron of a soybean polyubiquitin gene stimulated gene expression when placed either within a synthetic intron or upstream of a core promoter. The intron in the 5' untranslated region of the soybean polyubiquitin promoter, Gmubi, seems to contribute to the high activity of this promoter. To identify the stimulatory sequences within the intron, ten different sequential intronic sequences of 40 nt were isolated, cloned as tetrameric repeats and placed upstream of a minimal cauliflower mosaic virus 35S (35S) core promoter, which was used to control expression of the green fluorescent protein. Intron fragment tetramers were also cloned within a modified, native intron, creating a Synthetic INtron Cassette (SINC), which was then placed downstream of Gmubi and 35S core promoters. Intron fragment tetramers and SINC constructs were evaluated using transient expression in lima bean cotyledons and stable expression in soybean hairy roots. Intron fragments, used as tetramers upstream of the 35S core promoter, yielded up to 80 times higher expression than the core promoter in transient expression analyses and ten times higher expression in stably transformed hairy roots. Tetrameric intronic fragments, cloned downstream of the Gmubi and 35S core promoters and within the synthetic intron, also yielded increased transient and stable GFP expression that was up to 4 times higher than Gmubi alone and up to 40 times higher than the 35S core promoter alone. These intron fragments contain sequences that seem to act as promoter regulatory elements and may contribute to the increased expression observed with this native strong promoter. Intron regulatory elements and synthetic introns may provide additional tools for increasing transgene expression in plants.
authors.affiliation: Department of Horticulture and Crop Science, OARDC, The Ohio State University, 1680 Madison Ave., Wooster, OH, 44691, USA.|Mid-Florida Research and Education Center, Institute of Food and Agricultural Sciences, University of Florida, 2725 Binion Road, Apopka, FL, 32703-8504, USA.
authors.forename: Trudi N L
authors.initials: TN
authors.lastname: Grant
authors.affiliation: Department of Horticulture and Crop Science, OARDC, The Ohio State University, 1680 Madison Ave., Wooster, OH, 44691, USA.|Division of Plant Sciences, 315 Christopher S. Bond Life Sciences Center, University of Missouri-Columbia, Columbia, MO, 65211, USA.
authors.forename: Carola M
authors.initials: CM
authors.lastname: De La Torre
authors.affiliation: Department of Horticulture and Crop Science, OARDC, The Ohio State University, 1680 Madison Ave., Wooster, OH, 44691, USA.|Boyce Thompson Institute for Plant Research, Cornell University, 533 Tower Rd, Ithaca, NY, 14853, USA.
authors.forename: Ning
authors.initials: N
authors.lastname: Zhang
authors.affiliation: Department of Horticulture and Crop Science, OARDC, The Ohio State University, 1680 Madison Ave., Wooster, OH, 44691, USA. finer.1@osu.edu.
authors.forename: John J
authors.initials: JJ
authors.lastname: Finer
mesh_terms: D020121:5' Untranslated Regions / Q000235:genetics* / Q000502:physiology; D001483:Base Sequence; D018506:Gene Expression Regulation, Plant / Q000235:genetics* / Q000502:physiology; D007438:Introns / Q000235:genetics*; D027805:Phaseolus / Q000235:genetics / Q000378:metabolism; D018517:Plant Roots / Q000378:metabolism; D025821:Polyubiquitin / Q000235:genetics* / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics*; D013025:Glycine max / Q000235:genetics* / Q000378:metabolism
chemical_list: D020121:5' Untranslated Regions; D025821:Polyubiquitin
keywords: Enhancer; Soybean; Synthetic intron; Synthetic promoter