西方蜜蜂工蜂不同虫态发育的转录组学分析

Transcriptome analysis of development of different stages in Apis mellifera worker bees

  • 摘要: 【目的】基于转录组学对西方蜜蜂工蜂不同虫态间的差异表达基因(DEGs)进行筛选和功能注释分析,揭示与工蜂生长发育相关的信号通路,为深入解析工蜂生长发育的分子调控机理提供基础数据。【方法】以西方蜜蜂工蜂的3日龄幼虫、1日龄蛹和1日龄羽化工蜂3个虫态为研究对象,利用llumina NovaSeq 6000平台进行转录组测序,采用DESeq2筛选不同虫态样品间的表达差异基因,然后分别进行GO功能注释分析及KEGG信号通路富集分析,并通过实时荧光定量PCR进行验证。【结果】经转录组测序,在西方蜜蜂工蜂3日龄幼虫与1日龄蛹间筛选出4823个差异表达基因(51.86%上调,48.14%下调),在1日龄蛹与1日龄羽化工蜂间筛选出3295个差异表达基因(57.51%上调,42.49%下调),在3日龄幼虫与1日龄羽化工蜂间筛选出5267个差异表达基因(52.95%上调,47.05%下调)。GO功能注释分析结果显示,3日龄幼虫与1日龄蛹间的差异表达基因注释到43个GO功能条目,1日龄蛹与1日龄羽化工蜂间的差异表达基因注释到45个GO功能条目,3日龄幼虫与1日龄羽化工蜂间的差异表达基因注释到44个GO功能条目,主要涉及细胞过程、细胞部分及结合等。KEGG信号通路富集分析发现,3日龄幼虫与1日龄蛹间有2905个差异表达基因富集到332条KEGG信号通路上,其中17条KEGG信号通路呈显著富集,涉及核糖体、氧化磷酸化和昆虫激素生物合成等;1日龄蛹与1日龄羽化工蜂间有1644个差异表达基因富集到331条KEGG信号通路上,其中45条KEGG信号通路呈显著富集,涉及氧化磷酸化、生热作用和胰岛素分泌等;3日龄幼虫与1日龄羽化工蜂间有2958个差异表达基因富集到337条KEGG信号通路上,其中14条KEGG信号通路呈显著富集,涉及核糖体、蛋白酶体和胰岛素分泌等。6个随机挑选差异表达基因的实时荧光定量PCR检测结果与转录组测序结果相符,即转录组测序结果可靠。【结论】昆虫激素生物合成通路相关差异表达基因调控与西方蜜蜂工蜂各虫态JH滴度变化规律一致,氧化磷酸化信号通路则与各虫态的营养摄入和活动行为相关,而胰岛素分泌通路涉及各虫态的营养调控、脂肪体合成及细胞凋亡。可见,昆虫激素生物合成、胰岛素分泌和氧化磷酸化3种信号通路在西方蜜蜂工蜂幼虫、蛹和成虫的发育调控中发挥着重要作用。

     

    Abstract: 【Objective】To screen and functional annotation analysis of differentially expressed genes(DEGs)among different stages of Apis mellifera worker bees based on transcriptomics,reveal signaling pathways related to development of worker bees,so as to provide basic data for in-depth analysis of the molecular regulation mechanism of growth and development of worker bees.【Method】The 3-day-old larvae,1-day-old pupae and 1-day-old eclosion worker bees were takenas the research objects. Transcriptome sequencing was performed by Illumina NovaSeq 6000 platform,and screening of DEGs ment analysis were performed,and then real-time quantative PCR(qRT-PCR)verification was conducted.【Result】After transcriptome sequencing,4823 DEGs(51.86% up-regulated,48.14% down-regulated)were screened between 3-dayold larvae and 1-day-old pupae of A. mellifera worker bees. 3295 DEGs were screened between 1-day-old pupae and 1-day-old eclosion worker bees(57.51% up-regulated,42.49% down-regulated),5267 DEGs(52.95% up-regulated, 47.05% down-regulated)were screened between 3-day-old larvae and 1-day-old eclosion worker bees. The annotated GO function entries in the GO database of the three instar differential genes were 43(between 3-day-old larvae and 1-day-old pupae),45(between 1-day-old pupae and 1-day-old eclosion worker bees),and 44(between 3-day-old larvae and 1-dayold eclosion worker bees)respectively,mainly involving cellular process,cell part,binding,etc. KEGG signaling pathway enrichment analysis results showed that 2905 DEGs were enriched in 332 KEGG signaling pathways between 3-dayold larvae and 1-day-old pupae,of which 17 KEGG signaling pathways were significantly enriched,involving ribosomes,oxidation phosphorylation and insect hormone biosynthesis. 1644 DEGs were enriched in 331 KEGG signaling pathways between 1-day-old pupae and 1-day-old eclosion worker bees,of which 45 KEGG signaling pathways were significantly enriched,involving oxidative phosphorylation,thermogenesis and insulin secretion. 2958 DEGs were enriched in 337 KEGG signaling pathways between 3-day-old larvae and 1-day-old eclosion worker bees,of which 14 KEGG signaling pathways were significantly enriched,involving ribosomes,proteasomes and insulin secretion. The qRT-PCR results of 6 randomly selected DEGs were consistent with the transcriptome sequencing results,indicating that the transcriptome sequencing results were reliable.【Conclusion】The regulation of DEGs related to insect hormone biosynthesis pathway is consistent with the change rule of juvenile hormone(JH)titer in different stages of A. mellifera worker bees. The oxidative phosphorylation signaling pathway is related to the nutrient intake and activity behavior of different stages,and the insulin secretion pathway involves in the regulation of nutritional regulation,fat body synthesis and apoptosis of different stages. The results showsthat three signaling pathways of insect hormone biosynthesis,insulin secretion and oxidative phosphorylation play important roles in the developmental regulation of larvae,pupae and adult ofA. mellifera worker bees.

     

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