Analisi funzionale di un promotore Heat Shock di girasole e sue applicazioni biotecnologiche
Author(s)
De Pascali, Mariarosaria
Date Issued
March 21, 2011
Type
Doctoral Thesis
Abstract
Plants respond to environmental stimuli, such as heat shock, by re-programming cellular
activity, mainly regulated at transcription level, that activate genes encoding specific
proteins (HSP). The object of this work is the analysis of genomic DNA region spanning
the two sunflower small HSP genes (HaHSP17.6a and HaHSP17.6b); these genes are
physically associated, arranged in tandem in head-to-head orientation and linked by a 3809
bp region. The regions encoding for these two genes show only slight structural differences,
in fact they differ for only 30 nucleotides, which give a eight aminoacid substitution in the
deduced aminoacidic sequence. However, they have a very different expression profile,
probabilis due to the fact that they are differently regulated, because of differences into
their promoters. By comparison of the intergenic region sequence with PLACE and
PlantCARE databases, it has been possible to definine the structure of this region
containing characteristic elements of plant HS promoters (TATA box, HSE, CCAAT box)
and other stress related cis-acting elements. These elements are present in different number
and positions in the upstream regions of the two genes and this could explain the
differences in their of HS response to different stresses. Functional analysis by progressive
deletions of the upstream region of the more responsive gene, HaHSP17.6b, was
performed in order to verify the functional role of the cis acting elements identified. Using
GFP as reporter gene different recombinant plasmids ware produced containing the
fragments obtained by progressive deletions of the region of interest. The recombinant
plasmid collection was used to carry out transient expression assays in N. tabacum
protoplasts, subjected to the various stresses.
The data obtained indicate that GFP gene expression is induced not only in response to
heat shock, but also under heavy metal, NaCl, cold and abscissic acid stresses. The higher
expression of GFP is obtained when it is under the control of the whole upstream region
(1682) of HaHSP17.6b. Howewer the transcription level does not decrease significantly
when constructs whit partial parts of the upstream region are used. Interestly the expression
level obtained whit HS promoter is comparable whit that of the CaMV35S strong promoter.
Thus, the HS promoter acts as a strong promoter but it has the ability to further increase
transcription activity in response to different environmental stimuli. This indicate that the
HaHSP17.6b promoter could be in the future utiliser as a powerful biotechnological tool
for instance to produce a biosensor for environmental monitoring.
Additional information
Dottorato di ricerca in Biotecnologie vegetali
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