Table 1.
Primer sequences and amplicon characteristics for Citrus sinensis genes used in semi-quantitative and qRT-PCR.
Table 2.
Primer sequences and amplicon characteristics of Triticum aestivum (wheat) and Malus domestica (apple) cross-allergens.
Table 3.
Clinical features of the three patients with citrus fruit allergy.
Table 4.
Pollinosis patients' clinical features.
Figure 1.
Allergens and phospholipase expression analysis in Citrus clementine and Triticum aestivum pollens.
The cDNAs obtained from mRNA of Citrus c. (Cit) and Triticum a. (Tri) pollens ungerminated (UGP) and germinated (GP) have been tested for the different allergens (Cit s 1, Cit s 2, Cit s 3, Tri a 12 and Tri a 14) and phospholipase isoforms (PLA2 α and PLA2 β) through semi-quantitative RT-PCR after 40 cycles. EF-1 expression was used as reference gene (A). Lanes: Citrus clementine pollen ungerminated: Cit UGP; Citrus clementine pollen germinated: Cit GP; Triticum aestivum pollen ungerminated: Tri UGP; Triticum aestivum pollen germinated: Tri GP. Densitometries of the PCR bands revealed through semi-quantitative RT-PCR (B). Data are expressed as allergen/reference gene ratio ± SD. EF-1 expression was used as reference gene. The cDNAs of Citrus clementine UGP and GP have been tested for two different apple allergens (Mal d 3.02 and Mal d 4.02), and one isoform of apple secretory phospholipase (PLA 2.23) through semi-quantitative RT-PCR after 40 cycles (C). Logarithmic histogram (LOG10) of the expression levels of the different allergens (Cit s 1, Cit s 2, Cit s 3) and phospholipase isoforms (PLA2 α and PLA2 β) obtained through qRT-PCR in samples of Citrus c. pollen (Cit UGP and Cit GP) after 40 cycles of reaction (D). Data were calculated on the basis of the quantitative-comparative Ct (ΔΔCt) method normalized to the geometric mean of two housekeeping genes (EF-1 and UBQ 10), chosen according to the geNorm software. Values are the mean (n = 9) ± SD. Different letters (a–a', b–b', c–c', d–d') indicate means that are significantly different in allergens pairwise comparison (between bars marked by the same letter, i.e. a and a', b and b' etc.); * Statistical significance compared different allergens in the same sample (Tri GP); § Statistical significance compared the same allergen among the different samples (p≤0.05 with the Student's t-test [two-tailed distribution, two-sample equal variance]).
Figure 2.
ClustalΩ multiple sequence alignments of allergen sequences annotated in UniProtKB database.
Identity degree values expressed as multiple sequence alignment percentages of Triticum aestivum (PROF_WHEAT, Uniprot ID P49232-34, B6EF35), Phleum pratense (PROF_PHLPR, Uniprot ID P35079, O24650, O24282) and Cynodon dactylon (PROF_CYNDA, Uniprot ID O04725) profilin proteins (A). Identity percentages obtained from Citrus sinensis Cit s 1 (GLP), Cit s 2 (PROFILIN) and Cit s 3 (nsLTP) multiple sequence alignments (B). First column: Citrus sinensis GLP (GLP_CITSI, Uniprot ID P84159) with those for Triticum aestivum (GER_WHEAT, Uniprot ID P15290, B9VR55, C3UZE8, P26759, Q9SM34, Q9LD27, Q70PK0) and Betula pendula (GLP_BETPN, Uniprot ID P85352-54, P85336). Second column: Citrus sinensis PROFILIN (PROF_CITSI, Uniprot ID P84177) with those of Triticum aestivum (PROF_WHEAT, Uniprot ID P49232-34, B6EF35), Phleum pratense (PROF_PHLPR, Uniprot ID P35079, O24650, O24282), Cynodon dactylon (PROF_CYNDA, Uniprot ID O04725), Artemisia vulgaris (PROF_ARTVU, Uniprot ID Q8H2C8-9), Betula pendula (PROF_BETPN, Uniprot ID A4K9Z8, P25816), Parietaria judaica (PROF_PARJU, Uniprot ID Q9XG85, Q9T0M8), Olea europaea (PROF_OLEEU, Uniprot ID O24169-71), Arachis hypogaea (PROF_ARAHY, Uniprot ID Q9SQI9, Q5XXQ5, D3K177), Malus domestica (PROF_MALDO, Uniprot ID Q9XF40-42), Corylus avellana (PROF_CORAV, Uniprot ID A4KA40-45, Q9AXH4-5). Third column: Citrus sinensis nsLTP (NLTP_CITSI, Uniprot ID Q8L5S8, Q6EV47) with those of Triticum aestivum (NLTP_WHEAT, Uniprot ID Q5NE27-31, Q2PCB0, Q2PCB7-8, Q2PCD1-2, Q84N29), Artemisia vulgaris (NLTP_ARTVU, Uniprot ID P0C088, C4MGH0-2, C4MGG9), Parietaria judaica (NLTP_PARJU, Uniprot ID P43217, P55958, O04403-4, Q40905), Olea europaea (NLTP_OLEEU, Uniprot ID B2BGS3), Arachis hypogaea (NLTP_ARAHY, Uniprot ID B6CEX8, B6CG41), Malus domestica (NLTP_MALDO, Uniprot ID Q5GLH0, Q9M5X7, Q5J026), Corylus avellana (NLTP_CORAV, Uniprot ID Q9ATH2). Data are presented as range of percentage identity values as it has been considered all the allergen isoforms annotated and reviewed in UniProtKB database.