Proteomics Analysis And Potential Allergen Genes Excavation Of Lily Pollen

Liliunz spp.is the perennial herb bulbous flower of Liliaceae.The lily has beautiful colors and pleasant aromas,and is the main cut flower in the world.Lily has the beautiful meaning of "a hundred years of harmony and a consensus of all things",which is loved by consumers.Lily anthers contains huge amount of pollen,and the pollen emitted in large quantities not only pollutes the petals but also affects the ornamental value.At the same time,some pollen will contact the skin and mucous membranes of the human body,that will causing allergic symptoms such as rhinitis,skin swelling,and severe shock.Pollen allergies affect the life quality of susceptible people seriously.With the continuous improvement of people’s living standards,cut flower lily is gradually popular in the family.To a certain degree,pollen allergy has limited the application of cut flower lily to the market.Therefore,the analysis and identification of potential allergens in lily pollen,will have important significance for the follow-up prevention and treatment of lily pollen allergy.In order to explore the potential allergens in lily pollen,and identify genes related to lily pollen allergens.In this study,Oriental lily ’Siberia’ was used as the experimental material.The morphological characteristics of lily pollen at different developmental stages were compared and analyzed.The transcriptome and proteomics were sequenced from early mononuclear pollen and mature pollen.The differences in genes and proteins between the two stages were analyzed for mining transcriptome data.Predicting potential allergen genes of lily pollen were used by bioinformatics method.The candidate genes were cloned and analyzed.The main results are as follows:(1)The development of lily pollen is mainly divided into 6 stages:sporulation stage,mother cell stage,meiosis stage,tetrad stage,early mononuclear stage and mature pollen stage through histological and cytological studies.We clarified the correlation between lily bud and anther morphological characteristics with pollen development period.We found early mononuclear pollen began to form pollen walls,and the reticular structure of the pollen walls was observed.The pollen walls were significantly thickened at mature pollen stage and the germination groove was observed.(2)In order to research and classify lily pollen proteins,then screened the allergen proteins.We performed TMT proteome sequencing on mononuclear early and mature’Siberian’ pollen.The proteins function were annotated by GO,KEGG,COG and IPR databases,and the numbers were 6301,10,636,4844 and 8911,respectively.The total number of significantly different proteins is 2328,of which 886 proteins are up-regulated and 1442 proteins are down-regulated.Screening for differential proteins in lily pollen proteome data,it was found that the pollen wall synthesis-related proteins has high relative expression in early mononuclear pollen.Many proteins in mature pollen of lily are annotated as allergen-related proteins,of which c32116_g1 and c40755_g2 are pan-allergen proteins profilin,c31533_g1 and c24431_g1 are annotated as Ph1 p 7 in the polcalcin family.The expression of these proteins is significantly higher in mature pollen than in early mononuclear.(3)High-throughput transcriptome sequencing was performed on lily pollen to obtain 19.04Gb data.The genes function were annotated by NCBI,Nr,Nt,Pfam,KOG,Swiss-Prot,KEGG and GO databases.22835 differentially expressed genes(DEGs)were screened,of which 8390 genes were up-regulated and 14445 genes were down-regulated.Analysis of lily pollen transcriptomes identified DEGs associated with pollen allergens,such as Profilin,Ph1 p 7(Polcalcin),Ole e 1 and Ph1 p 11.Combined with proteomics analysis results,it was found that pollen-sensitized proteins had a significant increase in both transcription and protein levels.Phylogenetic tree analysis of allergen genes showed that c32116_g1 and c24431_gl had the highest homology with the identified profilin and polcalcin family allergens,respectively.Lily pollen has potential allergen genes.(4)Two pan-allergen genes LoProfilin and LoPolcalcin were isolated from Lily’Siberia’.Their open reading frames are 396 bp and 246 bp,encoding 131 and 81 amino acids,respectively.Amino acid sequence and structure alignment showed that the protein sequences of LoProfilin and LoPolcalcin were highly conserved.Subcellular localization analysis showed that LoProfilin and LoPolcalcin were distributed throughout the tobacco cells.The tertiary structure prediction analysis showed that LoProfilin and LoPolcalcin had the typical spatial structure of profilin and polcalcin family allergens,suggesting that they may be allergens in lily pollen.Real-time quantitative PCR analysis showed that LoProfilin was much more expressed in pollen than in other tissues.In eight different developmental stages of pollen,the expression of LoProfilin continued to increase,reaching the highest level at the mature stage 3.LoPolcalcin is specifically expressed in mature pollen of lily,and its expression increases during maturity,it is not expressed in other stages.This study provides a basis for verifying the existence of potential allergens in lily pollen and lays the foundation for the study of lily pollen allergens.