Isolation of a pea (Pisum sativum) seed lipoxygenase promoter by inverse polymerase chain reaction and characterization of its expression in transgenic tobacco

Abstract

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.

Module‐specific regulation of the β‐phaseolin promoter during embryogenesis

“…Mutations in the RY elements led to a decrease in the seed‐specific expression and an increase in expression in leaves, suggesting that the RY elements repress expression in leaves. Forster et al . (1994) have shown that deletion of a region of the pea lipoxygenase ( lox‐2 ) gene promoter that includes an RY element led to an increase in leaf expression, and hence ascribed a downregulatory role for the RY element in non‐seed tissues.…”

“…Mutations in the RY elements led to a decrease in the seed-specific expression and an increase in expression in leaves, suggesting that the RY elements repress expression in leaves. Forster et al (1994) have shown that deletion of a region of the pea lipoxygenase (lox-2) gene promoter that includes an RY element led to an increase in leaf expression, and hence ascribed a downregulatory role for the RY element in non-seed tissues. Investigation of various regions of the usp (unknown seed protein) promoter showed that elimination of an RY element led to threefold increase in seed-specific activity of the promoter, further implicating a negative role to the RY element (Fiedler et al, 1993).…”

Isolation of a family of resistance gene analogue sequences of the nucleotide binding site (NBS) type fromLensspecies

“…Inverse PCR is a convenient and versatile method of cloning unknown sequences upstream or downstream of known sequences (Triglia et al 1988). Inverse PCR circumvents the laborious procedures of producing and screening genomic libraries (Forster et al 1994). Nucleotide sequences flanking the NBS sequence have been previously isolated using IPCR in lettuce (Shen et al 1998).…”

Isolation by PCR-based methods of a plant antifungal polygalacturonase-inhibiting protein gene

“…Inverse PCR circumvents the laborious procedures of producing and screening genomic libraries (Forster et al, 1994). It has successfully been used to isolate the seed lipoxygenase promoter from pea as well as a wound-inducible promoter from asparagus (Forster et al, 1994).…”

“…An additional problem with Inverse PCR is the inefficient PCR amplification of closed circular double-stranded DNA. Forster et al (1994) found that digestion of the self-ligated DNA improved the yields of PCR amplification product. The presence of introns in the target gene must also be considered when carrying out Inverse PCR from genomic DNA.…”