Caratterizzazione strutturale e funzionale di geni codificanti per proteine della famiglia PDI in frumento tenero
Author(s)
D'Aloisio, Elisa Laura
Date Issued
September 10, 2008
Type
Doctoral Thesis
Abstract
The PDI (Protein Disulfide Isomerase) family includes several genes whose products are responsible for diversified metabolic functions, the proteins differ also for number and position of the active thioredoxin like sites, for presence/absence of other domains and of the KDEL signal of retention in the endoplasmic reticulum. The best characterised among the proteins encoded by this multigenic family is the classical PDI, which accomplishes several metabolic functions, the most important, which confers the name to the whole family, is due to the activity of the
thioredoxin like active domains and consists in disulfide bond formation and isomerization during the folding of secretory proteins. In plants the PDI family includes eight different phylogenetic classes, the gene products of five classes have two thioredoxin like active domains, whereas those of the other three classes own a single thioredoxin like domain. With the exception of the classical PDI, most available information on the other genes of this family concerns cloning and sequencing of cDNAs, but almost nothing is known on the regulation of their expression and the functions of their protein products. Some studies of molecular characterization, expression analysis and cell localisation in rice and maize have suggested the involvement of classical PDI in the assemblage and deposition of storage proteins in
these species. In wheat the likely involvement of classical PDI, as well as the potential participation of PDI-like proteins, in the storage protein folding and in the formation of high molecular weight protein aggregates makes their
study particularly interesting. In fact wheat flour quality is strongly affected by composition and structure of the storage proteins, which are largely determined by the formation of intra- and inter-molecular disulphide bonds. Isolation and
characterization in wheat of the three homoeologous gene sequences encoding classical PDI (TaPDIL1-1) and of their promoter sequences have been reported previously. Expression analyses showed that the transcripts of the genes coding
for this protein, although constitutively present in all the tested tissues, where particularly abundant in developing caryopses. These results are consistent with the identification of several conserved regulatory elements involved in
endosperm specific expression in the promoter sequences of the three homoeologous genes.
The aim of the present work was to identify and characterise new genes coding for PDI-like proteins in hexaploid wheat as well as to obtain molecular constructs and transgenic wheat lines for the functional analysis of an over-expressed gene coding for classical PDI and for the characterization of its promoter.
A cross search using PDI-like sequences of rice in the wheat EST databases “TIGR wheat gene index” and “HarvEST Wheat” identified nine sequences coding for PDI like proteins in wheat, whose full length cDNAs have been cloned. Phylogenetic analysis was based on the comparison of the deduced amino acid sequences of 52 proteins of the
PDI family including, in addition to the ten PDI and PDI-like sequences of wheat, sequences cloned from some plant species. The phylogenetic three, constructed using the neighbor-joining (NJ) method and evaluated through bootstrap analyses (PHYLIP version 3.6), allowed the assignment of the ten sequences of wheat to the eight phylogenetic groups identified in plants. Thus at least one gene has been cloned for each phylogenetic group. Seven subfamilies could be split into two major clades, whereas the eighth group was considered as outgroup, due to its high diversification from
the other subfamilies. The first major clade included the I (classical PDI), II and III phylogenetic groups, whose genes encode proteins containing two thioredoxin active domains, one at the N-terminal end and one at the C-terminal end, as well as the VII group, whose members conserve only the N-terminal active domain. On the basis of this shared feature it is possible to put forward the hypothesis of their common evolutionary origin from duplications of a single ancestral gene which took place in the common progenitor of monocots and eudicots. The second clade comprised the remaining three phylogenetic groups (IV, V e VIII), whose sequences show a much higher heterogeneity, which did not allow to infer any hypothesis on their origin and evolution. The search for conserved motives in the deduced amino acid sequences of the nine isolated genes, by comparison with sequences in different protein data bases, revealed a high level of structural similarity between the proteins encoded by the genes belonging to the same phylogenetic group. The comparison of the genomic organisations of three wheat PDI-like genes (TaPDIL2-1, TaPDIL4-1 e TaPDIL5-1) with their orthologous of rice and Arabidopsis showed a high level of conservation of their structural features (exon/intron
structure, exon length and position of the active sites) among the members of the same phylogenetic group. Most likely such conservation reflects the essential functional role of their encoded proteins. The chromosome location of the genes
encoding two wheat PDI-like proteins (TaPDIL4-1 and TaPDIL5-1) was determined through Southern analyses of DNA extracted from nulli-tetrasomic and ditelosomic lines of Chinese Spring. The three homoeologous gene sequences encoding TaPDIL4-1 were located in the short arm of the group 1 chromosomes, those encoding TaPDIL5-1 in the long
arm of the group 5 chromosomes. Northern analysis of TaPDIL2-1, TaPDIL4-1, TaPDIL5-1 and TaPDL1-1 (wheat classical PDI) detected different expression patterns in the analysed tissues.
Transgenic hexaploid wheat lines over-expressing the classical PDI, whose gene was put under the control of a strong endosperm specific promoter, were produced for the functional characterization of this gene. PCR analyses of a large part of the plants regenerated from bombarded embryos showed that some of them were transgenic for the overexpression construct. For a detailed characterization of the promoter role in the spatial and temporal regulation of the
expression of one of the genes encoding the PDI, the UidA reporter gene (GUS) was put under the control of four fragments obtained by progressive deletions of the 5’ end. The biolistic transformation experiments produced many
potentially transgenic plants, which will be analysed to verify the presence of the constructs carrying the PDI promoter fragments.
Further studies will be necessary to complete the molecular characterisation of this multigenic family in wheat.
The structural characterisation and a detailed analysis of expression patterns will be extended to the remaining six genes encoding PDI-like proteins. An exhaustive knowledge of the structural features and regulation of the PDI family genes will be useful to design the most suitable strategies for their functional characterization, in particular for the silencing of single genes or of gene groups through the RNA interference (RNAi) technology. The effect on the characteristics of seed storage proteins produced by the progressive knock-out of PDI and PDI-like genes will allow the understanding of their role in the formation of protein aggregates and will highlight possible functional redundancies.
Additional information
Dottorato di ricerca in Genetica agraria
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