Isolation of a pea (Pisum sativum) seed lipoxygenase promoter by inverse polymerase chain reaction and characterization of its expression in transgenic tobacco.
Part of the 5'-flanking sequence of a pea (Pisum sativum) lipoxygenase (LOX) gene was cloned, after amplification from genomic DNA by inverse polymerase chain reaction. Translational and transcriptional fusions of 818 bp of the 5'-flanking region and its deletion derivatives (-513 and -356) were made to a beta-glucuronidase (GUS)-coding sequence and introduced into tobacco. Analysis of T1 transformants showed that the 818 bp 5'-flanking sequence drove GUS expression in seeds that was temporally regulated in a fashion similar to the accumulation of LOX mRNA in developing pea seeds. Contrary to expectations, however, expression of the 818 bp promoter-GUS fusion was not seed-specific; GUS activity was highest in leaves and also present in stems and, to a lesser extent, roots. Deletion analyses identified the region between -818 and -513 as essential for high-level, temporally regulated expression in seeds and also indicated that the sequence between -513 and -356 plays a negative role in leaf/stem, but not seed, expression. Comparison of translational and transcriptional fusions indicated that the LOX initiation codon was used more efficiently than the GUS initiation codon by the tobacco leaf translational apparatus.
Forster
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John Innes Centre, Colney Lane, Norwich, UK.
Arthur
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Crespi
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Hobbs
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Mullineaux
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Casey
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D001483:Base Sequence; D003001:Cloning, Molecular; D016384:Consensus Sequence; D018628:Gene Dosage; D018507:Gene Expression Regulation, Developmental; D018506:Gene Expression Regulation, Plant; D005966:Glucuronidase / Q000096:biosynthesis / Q000235:genetics; D008084:Lipoxygenase / Q000235:genetics*; D008969:Molecular Sequence Data; D018532:Pisum sativum / Q000201:enzymology / Q000235:genetics*; D030821:Plants, Genetically Modified; D010947:Plants, Toxic; D016133:Polymerase Chain Reaction / Q000379:methods; D011401:Promoter Regions, Genetic / Q000235:genetics*; D012333:RNA, Messenger / Q000032:analysis; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012639:Seeds / Q000737:chemistry / Q000201:enzymology / Q000235:genetics; D017422:Sequence Analysis, DNA; D017384:Sequence Deletion / Q000502:physiology; D014026:Nicotiana / Q000235:genetics*
D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D008084:Lipoxygenase; D005966:Glucuronidase