基于转录组测序的芒果过敏原基因鉴定及表达分析

Identification and expression analysis of mango allergens according to transcriptome sequencing

【目的】基于芒果果实和叶片的转录组测序结果,筛选出潜在过敏原基因,并分析其在不同组织中的表达模式,为选育低过敏性的芒果品种提供理论依据。【方法】提取芒果果实和叶片总RNA,构建cDNA文库,利用Illumina HiSeq 4000进行转录组测序(测序读长为150 bp),从原始数据中筛选获得Clean reads,并利用Trinity进行组装,得到Unigenes,将其提交至NR数据库和Pfam数据库进行功能注释,从而筛选潜在的过敏原基因。通过实时荧光定量PCR(qRT-PCR)测定过敏原基因在不同组织中的表达情况。通过Cluspro分析果实中高表达过敏原与IgE抗体结合的表位区域。【结果】经组装获得230242条Transcripts和99328条Unigenes,二者的N50分别为2278和1183 bp,说明组装完整性较好。通过NR数据库和Pfam数据库共筛选到66个潜在的芒果过敏原基因,主要被注释为花粉过敏原、Bet v家族和NADPH-依赖FMN还原酶等,其中有6个不表达,其余60个均表达,其中有24个主要在果实中表达,其他36个基因在叶片中高表达。qRT-PCR检测结果显示,9个过敏原基因在芒果叶片和果实中表达量检测结果与RNA-Seq转录组表达分析结果基本相符;除几丁质酶基因c64821.graph_c0在叶片中表达量显著高于花和果实(P<0.05,下同)外,其他过敏原基因在花中的表达量显著高于叶片和果实。在RCSB PDB数据库搜索获得在果实中高表达的过敏原c61327.graph_c0的同源蛋白结构域(PDB:4Z8W),其与IgE结合表位的氨基酸序列为74-SFRQEVKTEKHGEFKVHLPFSVSEHV-99。不同物种过敏原蛋白的结合表位氨基酸序列具有高度保守性。【结论】不同芒果过敏原基因具有组织表达特异性,大部分基因在花中高表达,说明对食用芒果后产生过敏反应的人群应避免触摸芒果花等组织。c61327.graph_c0与其他物种的同源过敏原产生交叉过敏反应。

【Objective】The potential allergen genes were determined and the expression profiles in different tissues were evaluated using transcriptome sequencing of mango leaves and fruits to provide a theoretical basis for breeding lower allergenic mango cultivars.【Method】The total RNA was extracted from mango fruits and leaves.Transcriptome sequen-cing(150 bp reads)using HiSeq 4000 was performed after the cDNA libraries construction.Unigenes were obtained with Trinity assemble of clean reads.The unigenes related to potential allergen genes were annotated using NR and Pfam data-bases.The expression levels of allergen genes in different tissues were determined using real-time fluorescence quantita-tive PCR(qRT-PCR).The epitopes of the allergen highly expressed in fruits combined with IgE antibody were obtained by Cluspro analysis.【Result】A total of 230242 transcripts and 99328 unigenes were obtained.The N50 lengths of transcripts and unigenes were 2278 and 1183 individually,indicating the high assembly quality.Identified 66 potential allergen genes using NR and Pfam databases,including pollen allergens,Bet v family and NADPH-dependent FMN reductase.Six aller-gens showed no expression,60 showed expression.There were 24 allergens and 36 allergens highly expressed in fruits and leaves,respectively.The qRT-PCR test showed that the expression of nine allergen genes in mango leaves and fruits was largely consistent with the RNA-Seq transcriptome expression analysis.Except that the expression of chitinase gene c64821.graph_c0 in leaves was significantly higher than flowers and fruits(P<0.05,the same below),the others were sig-nificantly expressed in flowers than leaves and fruits.The allergen c61327.graph_c0 highly expressed in fruit was used as the template to obtain homologous protein domain(PDB:4Z8W)by searching in the RCSB PDB database,the amino acid sequences combined with IgE epitope was 74-SFRQEVKTEKHGEFKVHLPFSVSEHV-99.The amino acid sequences of binding epitopes of allergen protein from different species were highly conserved.【Conclusion】Different mango aller-gen genes have tissue expression specificity.Most genes are highly expressed in flowers,suggesting that people with aller-gic reactions after eating mango should avoid touching mango flowers.C61327.graph_c0 has cross-allergenic reactions with homologous allergens of other species.