中华蜜蜂工蜂中肠响应东方蜜蜂微孢子虫胁迫的高表达基因分析

Analysis of highly expressed genes in Apis cerana cerana workers' midguts responding to Nocema ceranae stress

摘要:

为探究中华蜜蜂（Apis cerana cerana，简称中蜂）工蜂中肠响应东方蜜蜂微孢子虫（Nosema ceranae）胁迫的高表达基因（highly expressed gene, HEG）的作用，利用RNA-seq技术对正常中蜂工蜂中肠7和10 d（Ac7CK和Ac10CK）、N. ceranae胁迫7和10 d的中蜂工蜂中肠（Ac7T和Ac10T）进行高通量测序、生物信息学分析和反转录PCR验证（Reverse transcription-PCR, RT-PCR）. 共得到1809736786条原始读段，经质控后得到1562162742条有效读段，Q30均达93.34%及以上，组内各样品Pearson相关性均在0.8728及以上. 按照FPKM值＞15的标准分别从Ac7CK、Ac7T、Ac10CK和Ac10T中分别筛选出3163、3312、3305和2426个HEG. Venn分析结果显示，上述4组的共有HEG为2074个，特有HEG分别为89、283、156和78个. GO分类结果显示，Ac7T和Ac10T的特有HEG分别涉及35和28个功能条目，注释基因数最多的皆为细胞进程. 代谢通路分析结果显示Ac7T和Ac10T的特有HEG可分别富集在39和37条代谢通路，富集基因数最多的分别是核糖体和内质网蛋白加工. 进一步分析发现中蜂工蜂中肠响应N. ceranae胁迫的过程中，宿主的物质和能量代谢、细胞和体液免疫均受到不同程度的激活，二者间存在密切的相互作用. 从共有HEG中随机选取8个进行RT-PCR验证，均能成功扩增出目的条带，说明本研究中的HEG真实存在. 研究结果揭示了中蜂工蜂中肠响应N. ceranae胁迫的HEG表达谱信息及潜在作用，为解析中蜂响应N. ceranae胁迫的应答机制、中蜂-N. ceranae互作机制提供了有益的信息和线索.

Abstract:

This study aims to investigate the role of highly expressed genes (HEGs) in the midguts of Apis cerana cerana workers responding to Nocema ceranae stress, normal 7- and 10-old day workers' midguts (Ac7CK and Ac10CK) and N. ceranae-stressed 7- and 10-old day workers' midguts (Ac7T and Ac10T) were analyzed using RNA-seq, bioinformatics and RT-PCR. 1809736786 raw reads were produced, and 1562162742 clean reads with Q30 ≥93.34% were gained after quality control; Pearson correlations between every sample within each group were above 0.8728. Based on the standard of FPKM value >15. 3163, 3312, 3305 and 2426 HEGs were screened out from Ac7CK, Ac7T, Ac10CK and Ac10T, respectively. Venn analysis showed there were 2074 shared HEGs in aforementioned four groups, and the number of specific HEGs were 89, 283, 156 and 78, respectively. GO classifications indicated specific HEGs in Ac7T and Ac10T were repsectively associated with 35 and 28 GO terms, among them cellular process was the largest one. Moreover, KEGG pathway enrichment analysis demonstrated specific HEGs in Ac7T and Ac10T were repsectively enriched in 39 and 37 pathways, and ribosome and protein processing in endoplasmic reticulum were the mostly abundant. Further exploration showed host material and energy metabolisms as well as cellular and humoral immune were activated to various degrees; additionally, close interactions existed between A. cerana cerana and N. ceranae. Finally, eight shared HEGs were randomly selected for RT-PCR validation, and signal bands were sucessfully amplified from them, indicative of the authenticity of HEGs in the study. These findings not only reveal the expression profile and putative role of host HEGs during the responses of A. cerana. cerana to N. ceranae stress, but also offer beneficial and valuable information for uncovering the molecular mechanisms regulating of host stress responses and A. cerana cerana-N. ceranae interactions.