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Open Access Methodology article

A qRT-PCR assay for the expression of all Mal d 1 isoallergen genes

Giulia Pagliarani12*, Roberta Paris24, Paul Arens1, Stefano Tartarini2, Giampaolo Ricci3, Marinus JM Smulders1 and W Eric van de Weg1

Author Affiliations

1 Wageningen UR Plant Breeding, Plant Research International, Droevendaalsesteeg 1, Wageningen, PB, 6708, The Netherlands

2 Department of Fruit Tree and Woody Plant Sciences, University of Bologna, Viale Fanin 46, Bologna, 40127, Italy

3 Department of Paediatrics, University of Bologna, Via Massarenti 11, Bologna, 40138, Italy

4 Present address: Consiglio per la Ricerca e la sperimentazione in Agricoltura-Centro di Ricerca per le Colture Industriali, via di Corticella 133, Bologna, 40128, Italy

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BMC Plant Biology 2013, 13:51  doi:10.1186/1471-2229-13-51

Published: 23 March 2013

Abstract

Background

A considerable number of individuals suffer from oral allergy syndrome (OAS) to apple, resulting in the avoidance of apple consumption. Apple cultivars differ greatly in their allergenic properties, but knowledge of the causes for such differences is incomplete. Mal d 1 is considered the major apple allergen. For Mal d 1, a wide range of isoallergens and variants exist, and they are encoded by a large gene family. To identify the specific proteins/genes that are potentially involved in the allergy, we developed a PCR assay to monitor the expression of each individual Mal d 1 gene. Gene-specific primer pairs were designed for the exploitation of sequence differences among Mal d 1 genes. The specificity of these primers was validated using both in silico and in vitro techniques. Subsequently, this assay was applied to the peel and flesh of fruits from the two cultivars ‘Florina’ and ‘Gala’.

Results

We successfully developed gene-specific primer pairs for each of the 31 Mal d 1 genes and incorporated them into a qRT-PCR assay. The results from the application of the assay showed that 11 genes were not expressed in fruit. In addition, differential expression was observed among the Mal d 1 genes that were expressed in the fruit. Moreover, the expression levels were tissue and cultivar dependent.

Conclusion

The assay developed in this study facilitated the first characterisation of the expression levels of all known Mal d 1 genes in a gene-specific manner. Using this assay on different fruit tissues and cultivars, we obtained knowledge concerning gene relevance in allergenicity. This study provides new perspectives for research on both plant breeding and immunotherapy.

Keywords:
Apple allergy; OAS; Mal d 1; Bet v 1; PR-10; gene family; qRT-PCR