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CLONING AND CHARACTERIZATION OF A WOUND-RESPONSIVE REGULATORY ELEMENT FROM ARABIDOPSIS THALIANA

KrishiKosh

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Title CLONING AND CHARACTERIZATION OF A WOUND-RESPONSIVE REGULATORY ELEMENT FROM ARABIDOPSIS THALIANA
 
Creator NAVIN CHANDRA GUPTA
 
Contributor Srinivasan
 
Subject biological phenomena, sampling, social sciences, economic systems, research methods, manpower, statistical methods, participation, environment, wells
 
Description T-8564
From a population of Arabidopsis thaliana (L.) tagged with a promoterless β-glucuronidase (GUS) in the T-DNA, a line T90, exhibiting a stem-specific and wound-responsive GUS expression was identified. Southern blot analysis showed that a single copy of T-DNA was present. The plant sequence flanking the T-DNA from right border end was amplified and cloned by TAIL-PCR. The insertion was located in the third chromosome, 57 bp upstream of the ATG start codon in 5′ untranslated region (UTR) of fatty acyl-CoA reductase 6 (FAR6) gene. The transcription initiation site (TSS) was identified by 5′ RACE PCR. Histochemical and fluorometric analyses of GUS activity revealed that the promoter of this gene is induced by wounding in internodal region of stem epidermis. However, some GUS expression was also detected in pollen grains of anther tissue. No expression of GUS was detected in the leaves and root tissues. Molecular characterization by RT-PCR analysis of the GUS and FAR6 genes in mutant and wild type plants, respectively, revealed that the gene is expressed predominantly in stem tissue, with very low expression in floral organs. Semiquantitative RT-PCR also shows that the expression in stem tissues is also induced by wounding in epidermal layer of mature stem internode. The 5′ upstream region of the FAR6, derived from Arabidopsis, has been sequenced and analyzed for cis-acting regulatory elements important in controlling GUS gene expression in mutant line. Different 5′ deletion fragments of the upstream sequences were linked to the GUS reporter gene as transcriptional fusions and the expression patterns was histochemically analyzed in transgenic Arabidopsis plants. Sequences -304 bp upstream to the transcriptional start site were unable to drive GUS gene expression, whereas sequences -510 bp upstream to the transcriptional start site drive GUS expression at the base of the first internode in wounded stem. The addition of further upstream sequences (-510 to -958, -1400 or -1456) enhanced and extended the wound
inducible GUS expression throughout the mature stem. The mutant line along with the other plants harboring the various deletion constructs exhibited GUS expression in the anther tissues. Overall, the spatial and temporal regulation of gene expression pattern driven by FAR6 promoter suggest that the corresponding FAR6 gene (AT3G56700) may be involved in the defense response of wounding or pathogens that damage the plant mainly through the epidermis. This work provides a promoter element that could be exploited for the genetic engineering of the crop plants.
 
Date 2016-12-14T10:37:25Z
2016-12-14T10:37:25Z
2012
 
Type Thesis
 
Identifier http://krishikosh.egranth.ac.in/handle/1/90047
 
Format application/pdf
 
Publisher IARI, DIVISION OF MOLECULAR BIOLOGY AND BIOTECHNOLOGY