意大利蜜蜂工蜂中肠发育过程中的差异表达环状RNA及其调控网络分析

Analysis of Differentially Expressed Circular RNAs and Their Regulation Networks During the Developmental Process of Apis mellifera ligustica Worker's Midgut

【目的】环状RNA(circular RNA,circRNA)在可变剪接、转录调控和来源基因的表达调控等方面具有重要功能。本研究旨在探究意大利蜜蜂(Apis mellifera ligustica,简称意蜂)工蜂中肠发育过程中circRNA的表达谱及其发育过程中的差异表达circRNA(differentially expressed circRNA,DEcircRNA),进而在转录组水平探究DEcircRNA在中肠发育中的作用。【方法】基于前期获得的意蜂7和10日龄工蜂中肠样品(Am7和Am10)的全转录组数据,利用find_circ软件从质控后的数据中预测circRNA。采用RPM算法归一化处理得到circ RNA的表达量。利用DEGseq软件对circ RNA进行差异分析,按照差异倍数(fold change)≥2、P<0.05及错误发现率(false discovery rate,FDR)<0.05条件筛选DEcircRNA。通过BLAST比对GO和KEGG数据库,对DEcircRNA的来源基因进行功能和代谢通路注释。利用TargetFinder软件预测DEcircRNA-miRNA及DEcircRNA-miRNA-mRNA调控网络,通过Cytoscape v.3.2.1软件对调控网络进行可视化。通过实时荧光定量PCR(RT-qPCR)验证测序数据的可靠性。【结果】意蜂中肠各样品比对上参考基因组的短序列读段数平均为19 616 356条。Am7与Am10的组内Pearson相关系数均≥0.950。共预测出256个DEcircRNA,包括105个上调circRNA和151个下调circRNA。Novel_circ_009675和novel_circ_013879分别在Am7和Am10中高量表达。DEcircRNA的来源基因可注释到包括结合、单组织进程及细胞进程在内的32个GO条目,其中分别有35、35和7个来源基因注释到催化活性、代谢进程和应激反应。上述来源基因还可注释到35条KEGG代谢通路,其中分别有5、5和4个来源基因注释到Hippo信号通路、内吞作用和吞噬体;进一步分析发现分别有1、2和2个来源基因注释到磷酸肌醇代谢、淀粉和蔗糖代谢和半乳糖代谢等物质代谢通路,5、4、3、1和1个来源基因注释到内吞作用、吞噬体、溶酶体、泛素介导的蛋白水解和MAPK信号通路等免疫通路。上述结果表明相应的DEcircRNA广泛参与意蜂工蜂中肠的生长发育、新陈代谢和免疫防御。DEcircRNA-miRNA调控网络分析结果显示,141个DEcirc RNA可靶向结合107个mi RNA,其中多数仅能结合1—2个mi RNA,但novel_circ_011577和novel_circ_010719结合的靶miRNA数可达32和28个;此外,mir-136-y、ame-miR-6001-3p及mir-136-y结合的circRNA数最多,分别为15、14和14个,表明相应的DEcircRNA可作为竞争性内源RNA在意蜂中肠发育过程发挥作用。进一步构建DEcircRNAs-ame-miR-6001-3p-mRNA调控网络,分析结果显示14个DEcircRNA可共同靶向结合ame-miR-6001-3p,表明它们可能通过调控ame-miR-6001-3p对意蜂中肠干细胞的分裂和分化进行间接调控。随机选取6个DEcircRNA进行RT-qPCR验证,结果显示5个DEcircRNA的表达量变化趋势与测序结果一致,证实了本研究测序结果的可靠性。【结论】通过对意蜂工蜂中肠发育过程中的DEcircRNA深入分析,提供了circRNA在意蜂工蜂中肠发育过程中的表达谱和差异表达信息,揭示了DEcircRNA在中肠发育过程中的作用,为中肠发育相关的关键circRNA的筛选和功能研究打下了基础。

【Objective】Circular RNA(circ RNA) plays a primary role in alternative splicing, transcription regulation and expression regulation of parental gene. The objective of this study is to investigate the profile expression of circRNAs and differentially expressed circRNAs(DEcircRNAs) during the developmental process of the midguts of Apis mellifera ligustica workers, and to explore the role of DEcircRNAs in the development of midgut at the transcriptional level. 【Method】 On basis of the whole transcriptome data from 7-and 10-day-old worker’s midguts of A. m. ligustica(Am7 and Am10), findcirc software was used to predict circRNAs based on the filtered sequencing data. The circ RNA expression level was normalized by RPM algorithm. Differential expression analysis for circ RNAs was conducted via DEGseq software following standards fold change≥2.0, P<0.05 and false discovery rate(FDR)<0.05. Source genes of DEcircRNAs were annotated to GO and KEGG databases to gain function and pathway annotations by using BLAST. DEcirc RNAs-mi RNAs and DEcirc RNAs-mi RNAs-m RNAs networks were predicted with Target Finder software and visualized using Cytoscape v.3.2.1 software. RT-q PCR was conducted to verify the reliability of sequencing data.【Result】 On average, 19 616 356 anchors reads were obtained from each A. m. ligustica worker’s midgut sample. Pearson correlations between different biological repeats within Am7 and Am10 groups were ≥0.950. In total, 256 DEcircRNAs including 105 up-regulated circRNAs and 151 down-regulated circ RNAs were predicted. Novelcirc009675 and novelcirc013879 were highly expressed in Am7 and Am10, respectively. Source genes of DEcircRNAs could be annotated to 32 GO terms including binding, single-organism process and cellular process, among them 35, 35 and 7 source genes were involved in catalytic activity, metabolic process and stress response. Additionally, these source genes could be annotated to 35 KEGG pathways, in which 5, 5 and 4 source genes were associated with Hippo signaling pathway, endocytosis and phagosome, respectively; further investigation showed that 1, 2 and 2 source genes could be annotated to material metabolisms such as phosphoinositol metabolism, starch and sucrose metabolism and galactose metabolism; 5, 4, 3, 1 and 1 source genes could be annotated to immune signaling pathways including endocytosis, phagosome, lysosome, ubiquitin-mediated proteolysis and MAPK signaling pathway, respectively. These results suggested that the corresponding DEcirc RNA was involved in the development, metabolism and immune defense of the midgut of A. m. ligustica. DEcirc RNA-mi RNA regulation network analysis showed that 141 DEcirc RNAs could link to 107 mi RNAs, most of these DEcirc RNAs could only bind to 1-2 mi RNAs, but novelcirc011577 and novelcirc010719 could respectively bind to 32 and 28 miRNAs. In addition, the number of DEcircRNAs combined with mir-136-y, ame-mi R-6001-3 p and mir-136-y was the highest(15, 14 and 14, respectively), which indicated that the corresponding DEcirc RNA could play roles during the developmental process of A. m. ligustica worker’s midgut as competing endogenous RNAs. Furthermore, DEcirc RNAs-ame-mi R-6001-3 p-m RNA network was constructed and analyzed, and the result indicated that 14 DEcirc RNAs could jointly link to ame-mi R-6001-3 p, implying they were likely to indirectly regulate division and differentiation of stem cells in A. m. ligustica worker’s midgut via regulation of ame-miR-6001-3 p. Six DEcircRNAs were randomly selected for RT-qPCR assay, the result showed the alteration trend of expression levels of 5 DEcircRNAs was consistent with that of the sequencing data, which proved the reliability of trancriptome data.【Conclusion】Through the deep investigation of DEcircRNAs during the developmental process of A. m. ligustica worker’s midgut, the expression profile and differential expression information of circRNAs in the development of midgut of worker bee were provided, and the role of DEcircRNAs in the development of midgut was revealed. It provides a basis for the screening and functional study of key circRNAs associated with the development of the midgut.