Towards the Identification and Isolation of VvPNP
Date
2008-03
Authors
Kerr, Mark Stuart
Journal Title
Journal ISSN
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Publisher
Stellenbosch : University of Stellenbosch
Abstract
We have identified a novel plant natriuretic peptide (PNP) like gene in Vitis vinifera cultivars
of Chardonnay and Pinotage. The transcript of the gene was isolated from young leaves indicating
that the peptide may function within these organs of the plant. The 15 kDa peptide,
which we have putatively called Vitis vinifera plant natriuretic peptide (VvPNP), was expressed
in a bacterial system as a recombinant fusion protein. This protein shares sequence
similarity to other recognised natriuretic peptides, and was largely identified by using the
primary sequence of the well characterised plant natriuretic peptide (AtPNP-A) from the
model organism Arabidopsis thaliana. Previous studies have identified PNPs across a range
of plant species such as Dracena godseffiana, Hedera helix and Solanum tuberosum, where
the biological activity associated with water and solute homeostasis has been proven. Instrumental
to the functionality of these peptides is the conservation of two cysteine residues
which form a disulphide bridge, of no less than 23 amino acids apart, creating a secondary
ring structure. Alignment of the VvPNP and AtPNP-A primary structures indicates that the
two cysteine residues necessary for physiological function in AtPNP-A are in conserved positions
within VvPNP. Also 14 identical amino acids and 7 conservative amino acids aligned
within the active domain of the AtPNP-A molecule. The putative natriuretic peptide also
displays two diagnostic amino acids motifs characteristic in PNPs and one other molecule
CjBAp12, which is associated with citrus blight. On a nucleotide level the VvPNP contains a
100bp intron which is also found within the genomic sequence of AtPNP-A. Plant natriuretic
peptides have sequence similarities to expansins, molecules which directly modify the mechanical
properties of cell walls leading to turgor-driven cell extension. Although PNPs do
not contain a wall-binding domain, evidence does suggest an evolutionary relationship between
expansins and PNP molecules because of these similarities. The target site for PNPs
being the cell membrane and not the cell wall, and it is for this reason that PNPs affect protoplasts.
Although VvPNP shares some similarity with the domain organisation of expansins
it lacks the tryptophan rich C-terminal domain. This domain also makes expansins larger
( 25 kDa) than natriuretic peptides ( 14 kDa). Attempts to show functionality of the recombinant
fusion protein GST:VvPNP were unsuccessful thus far. Further inquiries into the
role that VvPNP plays in the homeostasis of grapevine are needed to elucidate the potential
enhancement for this important economic crop.
Description
Thesis (MSc (Genetics))--University of Stellenbosch, 2008.
Keywords
VvPNP, Natriuretic, Vitis, Dissertations -- Genetics, Theses -- Genetics, Grapes -- Genetics