中华蜜蜂幼虫肠道响应球囊菌早期胁迫的转录组学

Transcriptome of Apis cerana cerana Larval Gut Under the Stress of Ascosphaera apis

【目的】白垩病是困扰养蜂生产的顽疾。目前,尚无利用二代测序技术研究中华蜜蜂(Apis cerana cerana,简称中蜂)幼虫白垩病的报道。本研究利用RNA-seq技术对健康(Ac CK)及球囊菌(Ascosphaera apis)胁迫的中蜂4日龄幼虫肠道(Ac T)进行深度测序,在转录组水平研究中蜂幼虫在球囊菌胁迫早期的胁迫应答。【方法】通过Illumina Hi Seq 2500平台对Ac CK和Ac T进行双端(PE125)测序,首先对测序数据进行质控和评估,利用edge R软件进行差异表达基因(DEG)分析,进而对DEGs进行GO富集分析及KEGG代谢通路富集分析,最后,利用实时荧光定量PCR(q RT-PCR)验证测序数据的可靠性。【结果】Ac CK和Ac T的转录组测序共得到188 457 338条原始读段(raw reads),经过滤得到182 088 448条有效读段(clean reads),两端Q20与Q30均在97.96%和94.97%以上,说明测序数据质量良好;主成分分析(PCA)结果显示第一与第二主成分可分别解释基因表达总体差异的75.8%和10.7%;DEG分析结果显示上调基因与下调基因的数量分别为344和239个;GO富集分析结果显示DEGs共富集在36个GO分类(term)上,其中基因富集数最多的细胞(106 unigenes)、细胞组件(106 unigenes)和代谢进程(104 unigenes);KEGG代谢通路富集分析结果显示上调与下调基因分别富集在72个和45个代谢通路上,其中上调基因富集数最多的是核糖体(72 unigenes)、碳代谢(16 unigenes)和糖酵解(14 unigenes),而下调基因富集数最多的是碳代谢(9 unigenes)、二羧酸代谢(8 unigenes)和氨基酸生物合成(7 unigenes),进一步分析表明中蜂幼虫肠道的部分细胞免疫对球囊菌的胁迫产生应答,而体液免疫不产生应答,宿主的代谢相关基因受到球囊菌的显著抑制。【结论】揭示了中蜂幼虫在球囊菌入侵早期的胁迫应答,为深入解析中蜂幼虫的胁迫应答机制提供了重要信息,也为在分子水平研究关键应答基因打下了基础。

【Objective】So far, no study on application of next-generation sequencing technology for the research of chalkbrood disease was reported. In the present research, RNA-seq technology was utilized to deep sequence of normal and Ascosphaera apis-treated 4th instar Apis cerana cerana larval gut to mine larvae＇s responses to A. apis challenge.【Method】Ac CK（un-treated group） and Ac T（A. apis-treated group） were sequenced on Illumina Hi Seq 2500 platform. After evaluation and filtration of raw data from RNA-seq, differentially expressed gene（DEG） analysis was performed using edge R software, further, Gene Ontology（GO） and KEGG pathway enrichment analyses were carried out, and finally, real-time quantitative PCR（q RT-PCR） was conducted to validate the RNA-seq data.【Result】 In total, RNA-seq produced 188 457 338 raw reads, and after filtration, 182 088 448 clean reads were obtained, Q20 and Q30 of each sample were above 97.96% and 94.97%, respectively, indicating that RNA-seq data in this research were with high quality. Principle component analysis（PCA） was performed on all genes level and the result showed PC1 and PC2 were able to account for 75.8% and 10.7% of the expressed genes＇ overall differences, respectively. DEG analysis result displayed that there were 344 up-regulated genes and 239 down-regulated genes in Ac CK VS Ac T. GO enrichment analysis result showed that the DEGs were enriched in 36 GO terms, among them, the mostly enriched ones were cell（106 unigenes）, cell part（106 unigenes） and metabolic process（104 unigenes）. KEGG pathway enrichment analysis result suggested that up-and down-regulated genes were enriched in 72 and 45 pathways, respectively, and the mostly enriched pathways for up-regulated genes were ribosome（72 unigenes）, carbon metabolism（16 unigenes） and glycolysis/gluconeogenesis（14 unigenes）, while the mostly enriched pathways for down-regulated genes were carbon metabolism（9 unigenes）, glyoxylate and dicarboxylate metabolism（8 unigenes） and amino acids biosynthesis（7 unigenes）. Further analysis demonstrated that the immune-related genes in A. c. cerana larval gut were activated, while the metabolism-related genes were greatly inhibited.【Conclusion】The findings of the study not only uncovered the A. c. cerana larval gut＇s responses to A. apis during the early stage of invasion, but also provided key information for clarifying the mechanism underlying the host＇s responses to A. apis, thus laying a foundation for functional investigation of key responding genes.