Identification of a Short Putative 5' Regulatory Sequence from Transgenic Hairy Root of Tomato-regulating Specific Expression Pattern

The Solanum lycopersicum cultivar MT1 was transformed by the A4 strain of Agrobacterium rhizogenes harboring the promoter trapping binary vector pPRF120. Transformed hairy root lines generated from the transformation were verified for stable expression of the inserted gus (beta-glucuronidase) transg...

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Bibliographic Details
Published in:Plant omics 2009-09, Vol.2 (5), p.206-213
Main Authors: Chan Kok Fei, Ismanizan Ismail, Siti Izera Ismail, Dinesh Natorajan, Zamri Zainal
Format: Article
Language:English
Subjects:
DNA polymerases
Genetic engineering
Plant molecular biology
Polymerase chain reaction
Roots (Botany)
Tomatoes
Transgenic plants
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Summary:The Solanum lycopersicum cultivar MT1 was transformed by the A4 strain of Agrobacterium rhizogenes harboring the promoter trapping binary vector pPRF120. Transformed hairy root lines generated from the transformation were verified for stable expression of the inserted gus (beta-glucuronidase) transgene in the genomic DNA. The 68i line was found to be stably transformed by pPRF120 via PCR analysis. This positive line was used for further study to detect promoter sequences that control expression of the integrated T-DNA, which contains a promoterless gus reporter gene. By using a 'DNA walking' approach, a single unknown flanking sequence was successfully amplified by two rounds of adaptor ligation PCR (AL-PCR). The AL-PCR patterns obtained were specific and reproducible for a given genomic library. AL-PCR products from the genomic libraries digested with either DraI (clone 1B) or HpaI (clone 5B) contain roughly 350 bp and roughly 1400 bp of upstream region respectively. The alignment of the sequences from clones 1B and 5B showed a very high percentage of similarity (97%). Resulting BLASTn analysis of the 5B clone indicated that 183 bp was 97% similar to chromosome 11 of Solanum lycopersicum, whereas 1105 bp was 97% similar to integrated Ri plasmid agropine of A. rhizogenes strain A4 for complete TL-DNA. The bioinformatics-based characterization of the short 256 bp putative novel promoter region directly upstream of T-DNA insertion via PlantCARE revealed the presence of several motifs for plant transcription factors such as circadian, TGA-element and motifs involved in light responsive control including CATT, ATCT, Sp1 and G-box.
ISSN:1836-0661
1836-3644