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

Giardini Naxos, Italy - 18/21 September, 2002

ISBN 88-900622-3-1

Poster Abstract - 3.32

cloning and characterization of the promoter sequences of three homoeologous genes coding for PDI in wheat

ZANOTTI A., DOMINICI L., PAOLACCI A.R., CATARCIONE G., TANZARELLA O.A., CIAFFI M.

Dipartimento di Agrobiologia e Agrochimica, Università degli Studi della Tuscia, Via S. Camillo De Lellis, 01100 Viterbo

ciaffi@unitus.it

promoters, PDI, wheat, inverse PCR, homoeologous sequences

PDI (Protein Disulfide Isomerase) is an abundant protein resident in the lumen of the ER (endoplasmic reticulum). It is considered the in vivo catalyst for disulfide bond formation during the biosynthesis of several secretory and cell surface proteins. In addition to its most acknowledged role as redox catalyst and isomerase, PDI owns further functions, such as peptide binding, cell adhesion and possibly chaperone activities. Gene sequences coding for PDI were located in chromosome arms 4AL, 4DS, 4BS and 1BS of all the three genomes of hexaploid wheat by aneuploid analysis (Ciaffi et al., 1999, Theor. Appl. Genet. 98:405-410). The PDI genomic and cDNA sequences of the gene located in 4A chromosome of durum wheat have been cloned and sequenced. The genomic sequence was about 3.5 kb long and included ten exons. Northern analysis of mRNA frome different tissues showed a very low expression in seedling, roots, leaves and florets and a very high level of expression in developing caryopses (Ciaffi et al., 2001, Gene 265:147-156). The genomic sequences of the PDI genes located in 4A, 4B and 4D chromosomes of Triticum aestivum cv. Chinese Spring were amplified by PCR using three different combinations of primer pairs, cloned, sequenced and compared.

The promoter sequence of the PDI gene located in 4A chromosome of durum wheat cv. Langdon was cloned using the inverse PCR (IPCR) technique. The sequence analysis showed that the fragment cloned by IPCR included about 1.3 kb located upstream of the coding sequence. The promoter sequences of the PDI genes located in 4A, 4B and 4D chromosomes of Chinese Spring were cloned through PCR amplification using three primer pairs. One of the primers, the same for all the three pairs, was designed on the basis of a sequence in the distal region of the previously cloned promoter of the durum wheat 4A gene, whereas the second primer of each pair was chosen within regions of the transcribed sequences which were specific for each of the three homoeologous genes. The alignment of the three promoter sequences, of about 1300 bp, showed a high homology, with an identity of 89%. The level of sequence conservation was higher within 700 bp upstream of the coding sequence, with an identity of more than 93%, whereas it was lower in the distal 640 bp, which had an identity of about 80%. The search for consensus sequences found a putative TATA box located at –79 from the translation start codon, and several CAAT boxes. Additional cis-acting elements were found searching the plant promoter database PlantCARE, in particular some motifs involved in seed-specific expression: AACA, GCN4, prolamin box, RY element, Skn-1. Promoter analysis, based on loss/gain-of-function in transgenic plants transformed with a reporter gene controlled by different regions of the cloned sequences, will be performed in order to define the specific promoter elements necessary to confer endosperm-specific expression. The specific expression in different wheat tissues (roots, coleoptiles, spikelets, leaves and immature caryopses) of the three cloned PDI genes, located on 4A, 4B and 4D chromosomes, will be analysed by RT-PCR using gene-specific primers. A further long term goal will be the cloning, characterization and expression analysis of different gene sequences of the PDI family in wheat. The plant genes belonging to the PDI family have been classified into four different groups on the basis of sequence homology, number and position of thioredoxin domains and presence/absence of the KDEL signal. However, by comparing the PDI-like sequences existing in databases, we identified five different groups of genes. Five group of PDI-like genes were also recognized within the database of EST sequences of wheat.