昆虫学报 ›› 2020, Vol. 63 ›› Issue (6): 688-696.doi: 10.16380/j.kcxb.2020.06.004

• 研究论文 • 上一篇    下一篇

低温胁迫下意大利蜜蜂预蛹的差异表达基因趋势分析

姚丹, 周姝婧, 周冰峰, 徐新建*, 朱翔杰*   

  1. (福建农林大学动物科学学院(蜂学学院), 福州 350002)
  • 出版日期:2020-06-20 发布日期:2020-07-02

Trend analysis of differentially expressed genes in prepupae of Apis mellifera ligustica under low temperature stress

YAO Dan, ZHOU Shu-Jing, ZHOU Bing-Feng, XU Xin-Jian*, ZHU Xiang-Jie*    

  1. (College of Animal Sciences (College of Bee Science), Fujian Agriculture and Forestry University, Fuzhou 350002, China)
  • Online:2020-06-20 Published:2020-07-02

摘要: 【目的】蜜蜂是典型的具有发育狭温性的全变态昆虫。本研究以对低温最敏感的意大利蜜蜂Apis mellifera ligustica预蛹为研究对象,通过低温胁迫不同时间的差异表达基因(differentially expressed genes, DEGs)趋势分析,探讨低温胁迫对蜜蜂发育影响的关键基因。【方法】对3日龄意大利蜜蜂封盖子预蛹进行20℃低温胁迫18 h(T18)和36 h(T36),以未经低温胁迫的预蛹为对照(CK),通过Illumina HiSeqTM平台进行转录组学测定。利用Short Time-series Expression Miner(STEM)软件对2个处理组与对照组比较共有的差异表达基因进行趋势分析,再进一步对显著富集趋势模式中富集的差异表达基因进行GO分类和KEGG pathway分析。利用RT-qPCR对随机挑选的5个DEGs的表达模式进行验证。【结果】对检测到1 062个T18 vs CK和T36 vs CK共有的DEGs进行趋势分析,发现3个显著的基因表达模式,包括2个上调表达模式(Profile 6,有539个基因;Profile 7,有271个基因),1个下调表达模式(Profile 1,有183个基因)。对3个显著富集趋势模式DEGs分别进行GO富集分析和KEGG pathway分析,在Profile 6中找到同时富集在FoxO信号通路和寿命调节信号通路上的胰岛素样肽基因ILP和叉头蛋白O基因FoxO持续上调,说明低温胁迫会影响蜜蜂预蛹蜕皮激素信号传递;在Profile 7中CREB结合蛋白基因CBP持续上调,蜜蜂预蛹受到低温胁迫会影响细胞发育;在Profile 1中细胞色素P450基因CYP450 306a1 (phm)和WNT1显著下调,说明化蛹时受到低温胁迫会影响蜕皮激素的合成。通过RT-qPCR分析挑选的5个基因的表达结果和高通量测序结果一致。【结论】通过对蜜蜂响应低温胁迫的差异表达基因趋势分析发现,蜜蜂胰岛素和蜕皮激素共同调控的FoxO可能是低温胁迫抑制蜜蜂化蛹的一个关键基因。本研究为探索低温胁迫影响蜜蜂发育变态的分子调控机制奠定了基础。

关键词: 意大利蜜蜂, 化蛹, 低温胁迫, 转录组学, 蜕皮激素

Abstract: 【Aims】 Honey bees are of the holometabolous insects, whose brood is typically stenothermic. In order to understand the key genes involved in the sensitivity of the Italian honey bee, Apis mellifera ligustica to cold stress, we conducted a trend analysis of differentially expressed genes (DEGs) of cold-sensitive prepupae in response to cold stress. 【Methods】 The 3 d-old post-capped prepupae were exposed to cold temperature (20℃) for 18 (T18) and 36 h (T36), respectively, with those unexposed to cold stress used as the control (CK), and then subjected to transcriptomic sequencing based on Illumina HiSeqTM. Short Time-series Expression Miner (STEM) software was applied to compare the shared DEGs among the three datasets. In addition, GO and KEGG pathway enrichment analyses were performed for DEGs with up- and down-regulation profiles. The expression profiles of five randomly selected DEGs were verified by RT-qPCR. 【Results】 Trend analysis showed that 1 062 DEGs shared by T18 vs CK and T36 vs CK were clustered into three significant profiles including two up-regulation profiles (Profile 6 covering 539 DEGs and Profile 7 covering 271 DEGs) and one down-regulation profile (Profile 1 covering 183 DEGs). GO and KEGG pathway enrichment analyses showed that insulin-like peptide gene (ILP) and forkhead box protein O gene (FoxO) were up-regulated in both FoxO signaling and longevity regulating pathways within Profile 6, suggesting that ecdysone signaling responds to cold stress during honey bee pupation. The CREB-binding protein gene (CBP) in Profile 7 was up-regulated, and CYP450 306a1 (phm) and WNT1 in Profile 1 were down-regulated, suggesting that these genes regulate cell development and ecdysone biosynthesis in response to low temperature. Besides, the expression profiles of the five selected genes based on RT-qPCR analysis were consistent with those based on the transcriptome. 【Conclusion】 The trend analysis of DEGs reveals that FoxO regulated by insulin signaling and ecdysone signaling in honey bees may act as a key gene that represses honey bee pupation in response to cold stress. Our data lay a foundation for exploring the molecular regulation mechanisms underlying metamorphosis in response to cold stress in honey bees.

Key words: Apis mellifera ligustica, pupation, low temperature stress, transcriptomics, ecdysone