昆虫学报 ›› 2021, Vol. 64 ›› Issue ( 2): 187-204.doi: 10.16380/j.kcxb.2021.02.006

• 研究论文 • 上一篇    下一篇

参与调控意大利蜜蜂工蜂中肠基因表达的东方蜜蜂微孢子虫miRNA的组学解析及其调控网络

范小雪1,#, 杜宇1,#, 张文德1, 王杰1, 蒋海宾1, 范元婵1, 冯睿蓉1, 万洁琦1, 周紫彧1, 熊翠玲1,2, 郑燕珍1,2, 陈大福1,2,3, 郭睿1,2,3,*   

  1. (1. 福建农林大学动物科学学院(蜂学学院), 福州 350002; 2. 福建农林大学蜂疗研究所, 福州 350002; 3. 福建农林大学蜂产品加工与应用教育部工程研究中心, 福州 350002)
  • 出版日期:2021-02-20 发布日期:2021-03-11

Omics analysis of Nosema ceranae miRNAs involved in gene expression regulation in the midgut of Apis mellifera ligustica workers and their regulatory networks#br#

FAN Xiao-Xue1,#, DU Yu1,#, ZHANG Wen-De1, WANG Jie1, JIANG Hai-Bin1, FAN Yuan-Chan1, FENG Rui-Rong1, WAN Jie-Qi1, ZHOU Zi-Yu1, XIONG Cui-Ling1,2, ZHENG Yan-Zhen1,2, CHEN Da-Fu1,2,3, GUO Rui1,2,3,*   

  1.  (1. College of Animal Sciences (College of Bee Science), Fujian Agriculture and Forestry University, Fuzhou 350002, China; 2. Apitherapy Research Institute, Fujian Agriculture and Forestry University, Fuzhou 350002, China; 3. Engineering Research Center of Processing and Application of Bee Products, Ministry of Education, Fujian Agriculture and Forestry University, Fuzhou 350002, China)
  • Online:2021-02-20 Published:2021-03-11

摘要:

【目的】本研究结合前期已获得的miRNA和mRNA组学数据对东方蜜蜂微孢子虫Nosema ceranae的差异表达miRNA(differentially expressed miRNA, DEmiRNA)靶向意大利蜜蜂Apis mellifera ligustica工蜂中肠的mRNA和差异表达mRNA(differentially expressed mRNA, DEmRNA)进行生物信息学预测、数据库注释和调控网络分析,旨在解析东方蜜蜂微孢子虫对意大利蜜蜂工蜂中肠的基因表达调控。【方法】比较东方蜜蜂微孢子虫感染7 d(AmT1)和10 d(AmT2)的意大利蜜蜂工蜂中肠和未感染中肠(分别为AmCK1和AmCK2)的mRNA数据,筛选意大利蜜蜂工蜂的显著性DEmRNA;通过比较侵染AmT1和AmT2的东方蜜蜂微孢子虫(分别为NcT1和NcT2)和东方蜜蜂微孢子虫纯净孢子(NcCK)的miRNA数据筛选出东方蜜蜂微孢子虫的DEmiRNA。利用TargetFinder软件预测东方蜜蜂微孢子虫DEmiRNA靶向的意大利蜜蜂工蜂中肠DEmRNA。利用相关生物信息学工具对上述意大利蜜蜂工蜂中肠靶DEmRNA进行GO和KEGG数据库注释。根据KEGG数据库注释信息筛选出意大利蜜蜂工蜂中肠免疫防御和能量代谢通路相关DEmRNA,并构建和分析上述DEmRNA与相应的东方蜜蜂微孢子虫DEmiRNA之间的调控网络。【结果】NcCK vs NcT1比较组中东方蜜蜂微孢子虫的77条显著上调miRNA和52条显著下调miRNA可分别靶向AmCK1 vs AmT1比较组中意大利蜜蜂工蜂中肠的118条显著下调mRNA和135条显著上调mRNA,这些mRNA可分别注释到31和25个GO条目,以及113和107条KEGG通路。NcCK vs NcT2比较组中东方蜜蜂微孢子虫的52条显著上调miRNA和49条显著下调miRNA可分别靶向AmCK2 vs AmT2比较组中意大利蜜蜂工蜂中肠的97条显著下调mRNA和210条显著上调mRNA,这些mRNA可分别注释到27和30个GO条目以及97和127条KEGG通路。NcCK vs NcT1和NcCK vs NcT2比较组中的11条共同显著上调miRNA和19条共同显著下调miRNA分别靶向AmCK1 vs AmT1和AmCK2 vs AmT2比较组的6条共同显著下调和14条共同显著上调mRNA,可分别注释到7和10个GO条目以及0和9条KEGG通路。NcCK vs NcT1和NcCK vs NcT2比较组中东方蜜蜂微孢子虫的DEmiRNA可靶向AmCK1 vs AmT1和AmCK2 vs AmT2比较组中意大利蜜蜂工蜂中肠的氧化磷酸化和硫代谢等能量代谢通路相关DEmRNA,以及胞吞作用、黑色素生成、溶酶体、自噬、Toll样受体信号通路、细胞凋亡、Ras信号通路、泛素介导的蛋白水解和MAPK信号通路等免疫防御通路相关DEmRNA。进一步分析发现,miR-216-x, miR-5119-y, bantam-y和miR-8-y在NcCK vs NcT1和NcCK vs NcT2比较组中皆显著上调表达,且靶向意大利蜜蜂工蜂中肠的溶酶体、黑色素生成、泛素介导的蛋白水解、MAPK信号通路及Ras信号通路等免疫防御通路相关的显著下调mRNA。【结论】在侵染过程中,东方蜜蜂微孢子虫的DEmiRNA对意大利蜜蜂工蜂的基因表达具有广泛的潜在影响,东方蜜蜂微孢子虫可能通过上调部分miRNA跨界调控意大利蜜蜂工蜂的免疫防御以促进侵染,通过下调部分miRNA跨界调控意大利蜜蜂工蜂的能量代谢以加强能量窃取并促进增殖。

关键词: 东方蜜蜂微孢子虫, 意大利蜜蜂, 微小RNA, 免疫防御, 能量代谢, 基因表达调控, 中肠

Abstract: 【Aim】 Based on the previously gained miRNA and mRNA omics datasets, bioinformatic prediction, database annotation and regulatory network analysis of differentially expressed mRNAs (DEmRNAs) in the midgut of Apis mellifera ligustica workers targeted by differentially expressed miRNAs (DEmiRNAs) of Nosema ceranae were conducted in this study, aiming to uncover the regulation of gene expression in the midgut of A. m. ligustica by N. ceranae. 【Methods】 Significant DEmRNAs of A. m. ligustica workers were screened by comparison of miRNA data from the midgut of A. m. ligustica workers at 7 d (AmT1) and 10 d (AmT2) post N. ceranae infection and the corresponding uninfected midguts (AmCK1 and AmCK2, respectively), while significant DEmiRNAs of N. ceranae were screened through comparison of miRNA data from N. ceranae infecting the midgut of A. m. ligustica workers (NcT1 and NcT2, respectively) and clean fungal spores (NcCK). DEmRNAs in the midgut of A. m. ligustica workers targeted by N. ceranae DEmiRNAs were predicted using TargetFinder software. GO and KEGG database annotations of the aforementioned target DEmRNAs in the midgut of A. m. ligustica workers were performed with related bioinformatic tools. Following KEGG database annotations, DEmRNAs in the midgut of A. m. ligustica workers relative to immune defense and energy metabolism were filtered out, followed by construction and investigation of the regulatory networks between the abovementioned DEmRNAs in the midgut of A. m. ligustica workers and the corresponding N. ceranae DEmiRNAs. 【Results】 In the NcCK vs NcT1 comparison group, 77 significantly up-regulated miRNAs and 52 significantly down-regulated miRNAs of N. ceranae could respectively target 118 significantly down-regulated mRNAs and 135 significantly up-regulated mRNAs in the midgut of A. m. ligustica workers in the AmCK1 vs AmT1 comparison group, and these mRNAs could be annotated to 31 and 25 GO terms and 113 and 107 KEEG pathways, respectively. In the NcCK vs NcT2 comparison group, 52 significantly up-regulated miRNAs and 49 significantly down-regulated miRNAs of N. ceranae could respectively target 97 significantly down-regulated mRNAs and 210 significantly up-regulated mRNAs in the midgut of A. m. ligustica workers in the AmCK2 vs AmT2 comparison group, and these mRNAs could be annotated to 27 and 30 GO terms and 97 and 127 KEGG pathways, respectively. Moreover, 11 shared significantly up-regulated miRNAs and 19 shared significantly down-regulated miRNAs in the NcCK vs NcT1 and NcCK vs NcT2 comparison groups could respectively target six shared significantly down-regulated mRNAs and 14 shared significantly up-regulated mRNAs in the midgut of A. m. ligustica workers in the AmCK1 vs AmT1 and AmCK2 vs AmT2 comparison groups, which could be annotated to 7 and 10 GO terms and 0 and 9 KEEG pathways, respectively. DEmiRNAs in the NcCK vs NcT1 and NcCK vs NcT2 comparison groups could target DEmRNAs associated with energy metabolism pathways including oxidative phosphorylation and sulfur metabolism, and immune defense pathways such as endocytosis, melanogenesis, lysosome, autophagy, Toll-like receptor signaling pathway, apoptosis, Ras signaling pathway, ubiquitin-mediated proteolysis, and MAPK signaling pathway in the midgut of A. m. ligustica workers in the AmCK1 vs AmT1 and AmCK2 vs AmT2 comparison groups. Further analysis indicated that miR-216-x, miR-5119-y, bantam-y and miR-8-y were all significantly up-regulated in both the NcCK vs NcT1 and NcCK vs NcT2 comparison groups, and targeted several significantly down-regulated mRNAs relative to immune defense pathways such as lysosome, melanogenesis, ubiquitin-mediated proteolysis, MAPK signaling pathway and Ras signaling pathway in the midgut of A. m. ligustica workers. 【Conclusion】 DEmiRNAs of N. ceranae may exert extensive effects on gene expression in A. m. ligustica workers during the infection process. N. ceranae is likely to enhance its infection through cross-kingdom regulation of immune defense of A. m. ligustica workers via up-regulating some miRNAs, and increase energy stealing and proliferation by cross-kingdom regulation via down-regulating some miRNAs.

Key words: Nosema ceranae, Apis mellifera ligustica, microRNA, immune defense, energy metaboliam, gene expression regulation, midgut