Abstract：
Phleum pratense is an allergenic grass that pollinates in spring in Pakistan. Databases Allergenonline.org and Allergen.org record ten P. pratense allergens and their isoforms. Phl P 1, Phlp 5, and Phl p 11 are major P. pratense-pollen allergens with demonstrated basophil activity and skin test reactivity. Little is known about P. pratense pollen adaptive variations in different climatic regions and pollen-associated microbial diversity. In this study, we collected P. pratense-pollen and soils in the spring season 2022. Samples were collected from three climatic regions in Pakistan (R1, R2 and R3) with differences in mean monthly air temperature, mean monthly precipitation and elevation. The morphology of pollen was observed by light microscopy, scanning electron microscopy (SEM), biochemical fingerprint analysis, and composition of pollen were investigated by fourier-transform infrared spectroscopy (FTIR). The pollen-associated bacterial populations were identified through a Biolog GEN III microplate system. The pollen water-soluble proteins were isolated and stabilized in phosphate buffer saline (PBS) and tested for allergenicity responses through dot blots and western blots analysis. The morphology study found difference in pollen biochemical composition. Biolog identified Brevibacterium epidermidis and Pantoea agglomerans from P. pratense pollen. Protein extract quantification and sodium dodecyl sulfate-poly acrylamide gel electrophoresis (SDS-PAGE) gel found decreased protein expression in R1 region pollen compared to R2 and R3 region pollen. Allergenicity studies found differential expression of beta-expansin and profilin allergens in pollen obtained from the three regions. Beta-expansin and profilin were suppressed in R1 pollen and expressed in compared to R2 and R3 pollen. This is the first study to identify B. epidermidis and P. agglomerans growth on P. pratense pollen. Variable allergen expression in P. pratense pollen has also been observed in different regions. Soil pH, an increase in mean monthly temperature and a decrease in mean monthly precipitation correlated with pollen biochemical composition, and reduced beta-expansin and profilin expression involved in pollen growth and development. The findings of this research are unique, which enhances basic knowledge and understanding of P. pratense-pollen associated microbiota and climate change impacts on the pollen allergen expression.
摘要：
Phleumpratense是一种过敏性草，在巴基斯坦春季授粉。Allergenonline.org和Allergen.org数据库记录了10种P.pratense过敏原及其亚型。PhlP1，Phlp5和Phlp11是主要的P.pratense-花粉过敏原，具有嗜碱性粒细胞活性和皮肤测试反应性。关于不同气候区域的P.pratense花粉适应性变化和花粉相关微生物多样性知之甚少。在这项研究中,我们在2022年春季收集了P.pratense花粉和土壤。样本是从巴基斯坦的三个气候区（R1，R2和R3）收集的，月平均气温不同，平均月降水量和海拔。通过光学显微镜观察花粉的形态，扫描电子显微镜（SEM），生化指纹分析,通过傅里叶变换红外光谱（FTIR）研究了花粉的组成。通过BiologGENIII微孔板系统鉴定花粉相关的细菌种群。将花粉水溶性蛋白分离并在磷酸盐缓冲液(PBS)中稳定,并通过斑点印迹和蛋白质印迹分析测试变应原性反应。形态学研究发现花粉生化组成存在差异。Biolog从P.pratense花粉中鉴定出表皮短杆菌和泛菌。蛋白质提取物定量和十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）凝胶发现，与R2和R3区域花粉相比，R1区域花粉中的蛋白质表达降低。致敏性研究发现，从三个区域获得的花粉中β-expansin和profilin过敏原的差异表达。与R2和R3花粉相比，R1花粉中的β-expansin和profilin被抑制，并在中表达。这是首次确定表皮芽孢杆菌和成团芽孢杆菌在P.pratense花粉上生长的研究。在不同地区也观察到了P.pratense花粉中可变的变应原表达。土壤pH值,月平均温度的升高和月平均降水量的减少与花粉生化成分相关，并减少参与花粉生长发育的β-expansin和profilin表达。这项研究的发现是独特的，这增强了对P.pratense-花粉相关微生物群和气候变化对花粉过敏原表达的影响的基本知识和理解。
