意大利蜜蜂幼虫肠道内球囊菌及其纯培养的高表达基因差异分析

Highly-expressed gene differences between Ascosphaera apis stressing the larval gut of Apis mellifera ligustica and the pure culture of Ascosphaera apis

【背景】球囊菌是一种典型的蜜蜂真菌性病原,特异性地侵染蜜蜂幼虫。目前,有关球囊菌在侵染过程中的基因表达规律的信息极为有限。【目的】通过深入分析胁迫意大利蜜蜂(意蜂)幼虫肠道的球囊菌及其纯培养的高表达基因(HEGs)差异,探索球囊菌在胁迫意蜂幼虫肠道后期与胁迫前的基因表达规律。【方法】利用RNA-Seq技术对球囊菌胁迫的意蜂6日龄幼虫肠道(Aam T)及球囊菌纯培养(AaCK)进行深度测序,根据FPKM值筛选得到球囊菌的HEGs,进而通过GO(Gene ontology)及KEGG(Kyoto Encyclopedia of Genes and Genomes)富集分析、Venn分析对上述HEGs进行功能预测和生物学意义挖掘。【结果】AaCK与Aam T的转录组测序共得到105 447 578条原始读段(Raw reads),经过滤得到88 466 344条有效读段(Clean reads),两端平均Q20为97.50%,平均Q30为93.81%。GO富集分析结果显示,AaCK的HEGs富集于26个GO terms,基因富集数最多的是细胞(22 Unigenes),其次是细胞组件(22 Unigenes)和代谢过程(21 Unigenes);Aam T的HEGs富集于22个GO terms,基因富集数最多的是催化活性(23 Unigenes),其次是细胞进程(18 Unigenes)和代谢过程(18 Unigenes)。KEGG代谢通路(Pathway)富集分析显示,AaCK的HEGs富集在109个Pathways上,基因富集数最多的是核糖体(179 Unigenes),其次是氨基酸生物合成(70Unigenes)和碳代谢(62 Unigenes);Aam T的HEGs富集在114个Pathways上,基因富集数量最多的是核糖体(178 Unigenes),其次是碳代谢(116 Unigenes)和氧化磷酸化(112 Unigenes)。Venn分析结果显示,AaCK与Aam T共有的HEGs有260个,二者特有的HEGs分别为2 161个和4 445个。【结论】提供了胁迫意蜂6日龄幼虫肠道的球囊菌及其纯培养的HEGs表达谱,揭示了球囊菌在胁迫前和胁迫后期的基因表达规律,也为阐明球囊菌致病的分子机理提供了有益的信息和线索。

[Background] Ascosphaera apis is a special fungal pathogen that specially infect honeybee larvae. Currently, information related to gene expression of A. apis during the infection process is limited. [Objective] This study was designed to analyze highly-expressed genes （HEGs） differences between A. apis stressing the 6-day-old larval gut of Apis mellifera ligustica and the pure culture ofA. apis, and to explore the gene expression ofA. apis during the late stage of stress and the pure culture ofA. apis. [Methods] In our study, the 6-day-old larval gut ofA. m. ligustica under the stress ofA. apis and the pure culture ofA. apis were sequenced using RNA-Seq, and the HEGs were obtained based on FPKM value, followed by function prediction and exploration of biological significance via GO （Gene ontology） and KEGG （Kyoto Encyclopedia of Genes and Genomes） pathway enrichment as well as Venn analyses. [Results] A total of 105 447 578 raw reads were produced from RNA-Seq, and 88 466 344 clean reads with a mean Q20 of 97.50% and a mean Q30 of 93.81% were obtained after filtration. GO enrichment analysis showed that the HEGs of AaCK were enriched in 26 GO terms, among them the cell （22 unigenes）, cell part （22 unigenes） and metabolism （21 unigenes） were mostly enriched; the HEGs of AamT were enriched in 22 GO terms, and the mostly enriched ones were the catalytic activity （23 unigenes）, cell processes （18 unigenes） and metabolic processes （18 unigenes）. KEGG pathway enrichment analysis showed that the HEGs of AaCK were involved in 109 pathways, among them the largest group was ribosome （179 unigenes） followed by biosynthesis of amino acids （70 unigenes） and carbon metabolism （62 unigenes）; the HEGs of AaCK were involved in 114 pathways, and the mostly enriched one was ribosome （178 unigenes） followed by carbon metabolism （116 unigenes） and oxidative phosphorylation （112 unigenes）. Furthermore, Venn analysis suggested that there are 260 shared HEGs, while 2 161 and 4 445 HEGs were specially expressed in AaCK and AamT, respectively. [Conclusion] Findings in the present study can offer the expression profiles of HEGs of A. apis stressing the 6-day-old larval gut of A. m. ligustica and the pure culture of A. apis, reveal the gene expression rules of A. apis before stress and A. apis during the late stage of stress, and provide helpful information for uncovering molecular mechanisms regulating the pathogenesis ofA. apis.