Proceedings of the XLV Italian Society of Agricultural Genetics - SIGA Annual Congress

Salsomaggiore Terme, Italy - 26/29 September, 2001

ISBN 88-900622-1-5

Poster Abstract

ISOLATION AND CHARACTERIZATION OF THREE HOMOEOLOGUS GENE SEQUENCES CODING FOR PROTEIN DISULFIDE ISOMERASE (PDI) IN HEXAPLOID WHEAT

CIAFFI M.*, PAOLACCI A.R.*, DOMINICI L.*, ZANOTTI A.*, CENCI A.**, PORCEDDU E.*

* Dipartimento di Agrobiologia ed Agrochimica, Università degli Studi della Tuscia, Via S. Camillo de Lellis, 01100 Viterbo, Italy

ciaffi@unitus.it

** Dipartimento Biologia e Chimica Agroforestale e Ambientale, Università degli Studi di Bari, Italy

PDI, hexaploid wheat, gene structure, gene promoter, inverse PCR

Protein Disulfide Isomerase (PDI) is an abundant protein in the lumen of the endoplasmic reticulum (ER), which is regarded as the in vivo catalyst for disulfide bond formation during the biosynthesis of various secretory and cell surface proteins. In addition to their known role as redox catalyst and isomerase, PDI has revealed some additional functions such as peptide binding, cell adhesion and perhaps chaperone activities. Plant genomic DNA sequences encoding for proteins belonging to the PDI family have been so far isolated only for Arabidopsis, whereas PDI or PDI-like cDNA sequences have been cloned and sequenced from species such as alfalfa, barley, common wheat, maize, castor bean and tobacco. PDI gene sequences are located in all the three genomes of the hexaploid wheat T. aestivum, one in each 4AL, 4DS, 4BS and 1BS chromosome arms (Ciaffi et al., 1999, Theor. Appl. Genet. 98: 405-410). The organisation of a durum wheat genomic sequence located in chromosome 4A has been recently described (Ciaffi et al., 2001, Gene: 265: 147-156). Alignment of the nucleotide sequences of the genomic and cDNA clones allowed to demonstrate that the isolated PDI genomic sequence was composed of 10 exons and its whole extent was about 3.5 kb. Analysis of PDI transcription levels showed that mRNAs were constitutively present in several wheat tissues, but they were expressed at very low levels in seedlings, roots, leaves and florets and at very high levels in developing caryopses, where the transcript levels remained high for 17 days after anthesis, then decreased (Ciaffi et al., 2001, Gene: 265: 147-156).

In order to isolate the four genes detected in common wheat, genomic sequences from T. aestivum cv Chinese Spring were amplified using three different combinations of primer pairs, which amplified the entire coding region plus short segments of the untraslated 3’ flanking sequences. For one of the three primer combinations analyses of the PCR outputs by polyacrylamide-gels showed the presence of three distinct amplification products. Nulli-tetrasomic analysis carried out using this primer pair indicated that the three amplified fragments corresponded to gene sequences located in group-4 homoeologous chromosomes. The three distinct PCR products were extracted from the poly-acrylamide gel, cloned and completely sequenced. The differences in the structural organisation of the three genomic sequences have been observed and discussed. At the same time we started the screening of a BAC library from T. durum cv. Langdon to identify clones containing DNA sequences encoding for proteins belonging to the PDI family.

The inverse PCR (IPCR) technique provides an alternative approach to classic methods which were used for the isolation of the promoter of the wheat PDI gene. The IPCR strategy was based on the PDI gene sequence located in 4A chromosome (Ciaffi et al., 2001, Gene: 265: 147-156). Two oligonucleotide primers were designed at the 5’ end of the PDI gene (respectively, 20 bp and 704 bp from the translation codon ATG) towards the unknown sequence and a BglII site located in the third exon (806 bp from the ATG) was chosen for the circularisation of the wheat genomic DNA. The analysis of PCR reaction by agarose gel showed a single amplified product of about 1.5 kb, which was purified and cloned into the pGEMT vector. Sequence analysis of the IPCR clone did not reveal any change in the 102 bp region overlapping the wheat PDI gene sequences located in the 4A chromosome, indicating that the IPCR clone contained a sequence of about 700 bp upstream of the coding region.

Future studies should focus on the isolation and functional analysis of promoter regions from different wheat PDI genes in order to elucidate the regulatory mechanisms controlling their spatial and temporal specific expression.