| Plant non-specific lipid transfer proteins (nsLTPs) are small, soluble, basic proteins, which widely distribute in plant organs, such as embryo, cotyledon, stem, leaf, fruit and flower, and mostly concentrated in the skin and peripheral cells of plant organs and abscission zone. nsLTPs present in high amounts in higher plants, accounts for as much as4%of the total soluble proteins. nsLTPs involve in many biological process such as membrane biogenesis and regulation of the intracellular fatty acid pools, cutin formation, embryogenesis, defense reactions against phytopathogens, symbiosis, and the adaptation of plants to various environmental conditions. Plant nsLTPs are also a potential panallergen in Rosaceae nsLTPs (peach, apple, apricot) and those from different plant sources (mugwort, chestnut) due to common epitopes shared by nsLTPs from these foods and pollens, which accounting for the frequent clinical cross-reactivity between them.Peach [Prunus persica (L.) Batsch] origins from the western part of China, where has the richest peach germplasm and the largest production and cultivation area. Peach is also an important food allergen resource frequently reported in the Mediterranean area and northern part of China. Several studies have demonstrated that the nsLTPs have different expression pattern in peach cultivars. Our study focused on the genetical diversity of nsLTPs gene from different peach cultivars and peach wild relatives in order to clarify the factors affecting the expression differences. Seven monoclonal antibodies were produced and a monoclonal antibody based sandwich ELISA was established to determine the protein amount in peach cultivars. The peel and pulp from eight peach cultivars were measured, and the main results from our study were as follow:1. The Pru p3encoding gene was completely conserved among peach cultivars and no mutant site were found in the ORF region. Eighteen mutant sites were found between peach and the three peach wild relatives including sixteen point mutants and two Indels, ten of which were in the first exon region, eight were in the intron region. Take the number of mutant site into account, Prunus davidiana is much distant away from peach. And at the amino acid level, seven mutants were found between Prunus davidiana and peach, while two between Prunus mira and peach, and only one between Prumus kansuensis and peach. The analysis results of the evolutionary relationship among Rosaceae fruit showed that Prunus persica, Prunus kansuensis and Prunus mira were clustered into the same group while Prunus davidiana and Prunus dulcis were grouped together. Three different consensus sequences, upLTP1-a, upLTP1-b and upLTP1-c, were got from the upstream region of the nsLTP gene in peach cultivars and the peach wild relatives. Most of the peach cultivars from the northern part of China are heterozygous and have the type upLTP1-b, while Prunus mira, Prunus davidiana and Prunus kansuensis only have type upLTP1-b. The bioinformatics analysis of the promoter region showed us many transcript regulation elements like TATA-box and CAAT box and light response element and so on.2. Three different allele sequences, upLTP1-a, upLTP1-b and upLTP1-c, were obtained from50Prunus accessions on the basis of the upstream region of the LTP1-encoding gene and three allele specific markers were derived according to the polymorphic sites. These markers were used to test316peach cultivars, most of which clustered within the three main subpopulations of peach,’Yu Lu’,’Hakuho’ and landraces. The genotypic frequencies and allele frequencies in the oriental peach cultivars in these subpopulations except for’Yu Lu’ were found to be in Hardy-Weinberg equilibrium (P<0.05). The dominant alleles were upLTPl-a in the’Yu Lu’ subpopulation and upLTP1-c in the’Hakuho’ subpopulation,allele upLTP1-b and allele upLTP1-c had the highest rates in the landraces, and the dominant allele in the three peach wild relatives was allele upLTP1-b. Furthermore, we found many TATA-box, CAAT box and light-responsive elements in the upstream region.3. The BALB/c mice were immunized with recombinant Pru p3and seven monoclonal antibodies (4-1,4-6, A7-1, A7-2,6-F7,11-B and B10-5) were produced from the immunization. The titer of these antibodies are1:20000,1:10000,1:60000,1:20000,1:20000,1:2000and1:20000, respectively. And the isotype of4-1and4-6are IgG2a(K chain) and the rest five is IgGl(K chain). The western blotting analysis showed all of these antibodies can bind peach lipid transfer protein.4. A monoclonal antibodies-based sandwich ELISA were established with mAb B10-5as capture mAb (bound to the solid phase) and biotinylated mAb4-1as the detector, which shows high accuracy,resolution,reproducity and stability. And the specificity of this measurement was confirmed using peach and other fruit nsLTPs, which showed that these mAbs not only bind to peach nsLTP, but also to other stone fruit, such as apricot, plum and cherry, but the binding activity to the latter was much lower.5. The Pru p3level in the peel and pulp of eight peach cultivars were determined with the established sandwich ELISA while purified natural Pru p3was chosed as standard. The results indicated that in the same peach cultivar, the Pru p3content in peel is much higher than the pulp. And significant differences were found among peach cultivars. Among the eight peach cultivars measured in this study,’Hongshanhu’has the highest Pru p3.01amount of9.05μg/g, while the lowest one is ’Yanfeng’ and the amount was1.55μg/g. The average Pru p3amount in the peel among the eight cultivars was6.6μg/g. The Pru p3in the pulp ranged from0.07μg/g to0.77μg/g with the highest ones were ’Qiufen’ and ’Jinxiuhuangtao’... |
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