Characterization of a PttRPS18 promoter active in the vascular cambium region of hybrid aspen

Characterization of a PttRPS18 promoter active in the vascular cambium region of hybrid aspen By screening 273 hybrid aspen plants transformed with a luciferase-based promoter trap T-DNA vector, one plant was found in which the reporter gene (luxF2) was activated only in cells of the cambial region, i.e., vascular cambium, phloem and differentiating xylem. Southern blot analysis revealed that this plant denoted #24 had a single T-DNA insert. The chromosomal regions flanking the T-DNA were cloned by plasmid rescue. A 757 bp DNA fragment, originating from the rescued plasmid and covering the genomic region immediately upstream from the right-border sequence of the T-DNA, was used as a probe to isolate the corresponding chromosomal region from a wild-type hybrid aspen genomic library. A hybrid aspen small ribosomal protein gene, PttRPS18, was then isolated. By screening a wt cambial region-specific cDNA library, two cDNA clones encoding a putative 152 amino acid PttRPS18 protein were isolated. Comparison of the DNA sequence immediately flanking the T-DNA insert in #24 with the corresponding wild-type sequence showed that only a minor deletion occurred during the T-DNA integration. Northern analysis revealed that the PttRPS18 gene was expressed mainly in the cambial region. By RT-PCR and DNA sequencing analysis, the exact structures of the PttRPS18 and luxF2 transcripts were determined. Finally, the hybrid aspen PttRPS18 promoter was fused to the uidA reporter gene and transformed into hybrid aspen plants. Histochemical analysis of GUS activity showed that the PttRPS18promoter was expressed in the cambial region in the same manner as the luciferase reporter gene in the initial screening. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Plant Molecular Biology Springer Journals

Characterization of a PttRPS18 promoter active in the vascular cambium region of hybrid aspen

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Publisher
Springer Journals
Copyright
Copyright © 2003 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:1023919331037
Publisher site
See Article on Publisher Site

Abstract

By screening 273 hybrid aspen plants transformed with a luciferase-based promoter trap T-DNA vector, one plant was found in which the reporter gene (luxF2) was activated only in cells of the cambial region, i.e., vascular cambium, phloem and differentiating xylem. Southern blot analysis revealed that this plant denoted #24 had a single T-DNA insert. The chromosomal regions flanking the T-DNA were cloned by plasmid rescue. A 757 bp DNA fragment, originating from the rescued plasmid and covering the genomic region immediately upstream from the right-border sequence of the T-DNA, was used as a probe to isolate the corresponding chromosomal region from a wild-type hybrid aspen genomic library. A hybrid aspen small ribosomal protein gene, PttRPS18, was then isolated. By screening a wt cambial region-specific cDNA library, two cDNA clones encoding a putative 152 amino acid PttRPS18 protein were isolated. Comparison of the DNA sequence immediately flanking the T-DNA insert in #24 with the corresponding wild-type sequence showed that only a minor deletion occurred during the T-DNA integration. Northern analysis revealed that the PttRPS18 gene was expressed mainly in the cambial region. By RT-PCR and DNA sequencing analysis, the exact structures of the PttRPS18 and luxF2 transcripts were determined. Finally, the hybrid aspen PttRPS18 promoter was fused to the uidA reporter gene and transformed into hybrid aspen plants. Histochemical analysis of GUS activity showed that the PttRPS18promoter was expressed in the cambial region in the same manner as the luciferase reporter gene in the initial screening.

Journal

Plant Molecular BiologySpringer Journals

Published: Oct 7, 2004

References

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