Cloning of a wheat puroindoline gene promoter by IPCR and analysis of promoter regions required for tissue-specific expression in transgenic riceseeds

Cloning of a wheat puroindoline gene promoter by IPCR and analysis of promoter regions required... A genomic DNA fragment containing the 5′-upstream sequence and part of the open reading frame corresponding to Triticum aestivum puroindoline-b cDNA, was isolated by inverse PCR. Promoter fragments extending to −1068, −388, −210 or −124 upstream of the translation initiation ATG codon and the sequence coding for the first 13 amino acids of the puroindoline-b, were translationally fused to the uidA reporter gene encoding β-glucuronidase and transferred to rice calli via particle bombardment-mediated transformation. The 1068 bp and 124 bp promoters were also transcriptionally fused to the uidA reporter gene. Out of the 196 plants regenerated from transformed rice calli, 118 plants set seeds. No GUS activity was detectable in the stems, roots, leaves or pollen of the transgenic rice which had integrated the puroindoline-b promoter or its deletions; GUS activity was detected only in seeds, except in those having integrated the 124 bp promoter. Within seeds, histological localisation showed GUS activity as being restricted to the endosperm, aleurone cells and pericarp cell layers; no GUS activity was detected in the embryonic axis. Analysis of 5′ promoter deletions identified the region between −388 and −210 as essential for endosperm expression, and the region between −210 and −124 as essential for expression in the epithelium of the scutellum. No difference of expression was observed between the translational and transcriptional fusion genes. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Plant Molecular Biology Springer Journals

Cloning of a wheat puroindoline gene promoter by IPCR and analysis of promoter regions required for tissue-specific expression in transgenic riceseeds

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Publisher
Kluwer Academic Publishers
Copyright
Copyright © 1999 by Kluwer Academic Publishers
Subject
Life Sciences; Biochemistry, general; Plant Sciences; Plant Pathology
ISSN
0167-4412
eISSN
1573-5028
D.O.I.
10.1023/A:1006194326804
Publisher site
See Article on Publisher Site

Abstract

A genomic DNA fragment containing the 5′-upstream sequence and part of the open reading frame corresponding to Triticum aestivum puroindoline-b cDNA, was isolated by inverse PCR. Promoter fragments extending to −1068, −388, −210 or −124 upstream of the translation initiation ATG codon and the sequence coding for the first 13 amino acids of the puroindoline-b, were translationally fused to the uidA reporter gene encoding β-glucuronidase and transferred to rice calli via particle bombardment-mediated transformation. The 1068 bp and 124 bp promoters were also transcriptionally fused to the uidA reporter gene. Out of the 196 plants regenerated from transformed rice calli, 118 plants set seeds. No GUS activity was detectable in the stems, roots, leaves or pollen of the transgenic rice which had integrated the puroindoline-b promoter or its deletions; GUS activity was detected only in seeds, except in those having integrated the 124 bp promoter. Within seeds, histological localisation showed GUS activity as being restricted to the endosperm, aleurone cells and pericarp cell layers; no GUS activity was detected in the embryonic axis. Analysis of 5′ promoter deletions identified the region between −388 and −210 as essential for endosperm expression, and the region between −210 and −124 as essential for expression in the epithelium of the scutellum. No difference of expression was observed between the translational and transcriptional fusion genes.

Journal

Plant Molecular BiologySpringer Journals

Published: Sep 29, 2004

References

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