中华蜜蜂6日龄幼虫响应蜜蜂球囊菌胁迫的环状RNA应答

Circular RNA response of Apis cerana cerana 6-day-old larvae to Ascosphaera apis stress

摘要:

[目的] 蜜蜂球囊菌（Ascosphaera apis，简称球囊菌）专性侵染蜜蜂幼虫而导致白垩病，危害蜜蜂健康和养蜂生产。本研究旨在探究中华蜜蜂（Apis cerana cerana，简称中蜂）6日龄幼虫响应球囊菌胁迫的环状RNA（circular RNA，circRNA）差异表达谱及差异表达circRNA（differentially expressed circRNA，DEcircRNA）在宿主胁迫应答中的潜在功能。[方法] 利用去除线性RNA的circRNA-seq技术对正常和球囊菌侵染的中蜂6日龄幼虫肠道（AcCK和AcT）进行测序。利用find_circ软件鉴定circRNA，统计circRNA的长度和环化类型。根据|log₂（Fold change）|≥1和P≤0.05的标准筛选DEcircRNA。将DEcircRNA的来源基因比对Gene ontology（GO）数据库和Kyoto Encyclopedia of Genes and Genomes（KEGG）数据库，从而获得功能及通路（pathway）注释。随机挑选3个DEcircRNA进行RT-qPCR验证。[结果] AcCK和AcT的circRNA-seq分别得到76342570和68269362条原始读段（raw reads），经严格质控得到74524108和66974392条有效读段（clean reads），Q30分别为92.75%和94%，GC含量分别为54.31%和54.90%。比对上东方蜜蜂（Apis cerana）参考基因组的短序列读段（anchor reads）共计23648400条。AcCK和AcT中分别鉴定到805和702个circRNA，长度均介于201-1000 nt，数量最多的环化类型均为已注释外显子circRNA，但分布在不同长度、不同环化类型的circRNA数量存在差异。AcCK vs AcT比较组共有494个DEcircRNA，包括257个上调circRNA和237个下调circRNA；上调和下调幅度最大的circRNA分别为novel_circ_000123和novel_circ_000726。上述DEcircRNA的来源基因可注释到11条生物学进程相关条目，9条分子功能相关条目，9条细胞组分相关条目，以及73条通路。进一步分析发现，部分DEcircRNA的来源基因注释到7条细胞免疫通路和3条体液免疫通路。[结论] 中蜂6日龄幼虫响应球囊菌胁迫的过程中可能通过改变分布在不同长度和环化类型的circRNA数量，以及特异性表达一些circRNA和调节部分circRNA的表达量对病原产生应答；novel_circ_000027、novel_circ_000127、novel_circ_000312等DEcircRNA在宿主的胁迫应答过程中可能通过调控氧化磷酸化、细胞和体液免疫等通路发挥特殊作用。研究结果为深入理解中蜂幼虫对球囊菌的胁迫应答机制及二者的相互作用机制提供了新见解。

关键词:中华蜜蜂;幼虫;环状RNA;非编码RNA;蜜蜂球囊菌;免疫应答

Abstract:

[Objective] Ascosphaera apis exclusively infects honeybee larvae, leading to chalkbrood, a fungal disease damaging honeybee health and beekeeping industry. The objective of this study was to investigate the differential expression pattern of circular RNAs (circRNAs) and differentially expressed circRNAs (DEcircRNAs) involved in Apis cerana cerana 6-day-old larval response to Ascosphaera apis stress, and the putative role of DEcircRNAs in host A. apis-response. [Methods] Normal and A. apis-infected 6-day-old larval guts of A. c. cerana (AcCK and AcT) were sequenced using linear RNA-removed circRNA-seq technology. CircRNAs were identified using find_circ software, followed by summary of length and circulization type of circRNAs. DEcircRNAs were screened out following the standard of |log₂(Fold change)|≥1 and P≤0.05. Source genes of DEcircRNAs were annotated to Gene Ontology (Go) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases to gain function and pathway annotations. RT-qPCR was conducted to validate three randomly selected DEcircRNAs. [Results] Based on circRNA-seq, 76342570 and 68269362 raw reads were produced from AcCK and AcT, and 74524108 and 66974392 clean reads were obtained after strict quality control, with Q30 of 92.75% and 94% and GC content of 54.31% and 54.90%. In total, 23648400 anchor reads were mapped to the reference genome of Apis cerana. In AcCK and AcT, 805 and 702 circRNAs were identified; the lengths of these circRNAs were distributed among 201-1000 nt, and the most abundant circulization type was annotated exonic circRNA; however, the numbers of circRNAs distributed in various lengths and circulization types were different. There were 494 DEcircRNAs in AcCK vs AcT comparison group, including 257 up-regulated circRNAs and 237 down-regulated circRNAs, with novel_circ_000123 and novel_circ_000726 having the highest up-regulation and down-regulation levels. Source genes of the aforementioned DEcircRNAs were annotated to 11 biological process-associated terms, nine molecular function-associated terms, nine cellular component-associated terms, and 73 pathways. Further analysis demonstrated partial source genes were engaged in seven cellular immune pathways and three humoral immune pathways. [Conclusion] A. c. cerana 6-day-old larvae may response to A. apis stress by altering the numbers of circRNAs distributed in various lengths and circulization types, specifically expressing some circRNAs, and regulating the expression of partial circRNAs; several DEcircRNAs such as novel_circ_000027, novel_circ_000127 and novel_circ_000312 may play special roles in host A. apis-response via regulation of oxidative phosphorylation, cellular and humoral immune pathways. Our findings offered novel insights into further understanding molecular mechanisms underlying A. c. cerana larval response to A. apis and host-pathogen interaction.

Key words:Apis cerana cerana;larva;circular RNA;non-coding RNA;Ascosphaera apis;immune response