昆虫学报 ›› 2020, Vol. 63 ›› Issue (3): 266-277.doi: 10.16380/j.kcxb.2020.03.003

• 研究论文 • 上一篇    下一篇

意大利蜜蜂哺育蜂学习记忆相关基因的转录组学分析

高艳, 朱雅楠, 张威, 王雪妍, 李秋方, 苏松坤, 聂红毅*   

  1. (福建农林大学动物科学学院(蜂学学院), 福州 350002)
  • 出版日期:2020-03-20 发布日期:2020-04-16

Transcriptomic analysis of learning and memory-related genes in nurses of Apis mellifera ligustica (Hymenoptera: Apidae)

GAO Yan, ZHU Ya-Nan, ZHANG Wei, WANG Xue-Yan, LI Qiu-Fang, SU Song-Kun, NIE Hong-Yi*    

  1. (College of Animal Sciences (College of Bee Science), Fujian Agriculture and Forestry University, Fuzhou 350002, China)
  • Online:2020-03-20 Published:2020-04-16

摘要: 【目的】筛选与意大利蜜蜂Apis mellifera ligustica哺育蜂学习记忆密切相关的基因。【方法】在人工组建意大利蜜蜂蜂群中收集10日龄哺育蜂、21日龄哺育蜂、21日龄采集蜂,通过喙伸反应(proboscis extension reflex, PER)实验测定这3组样本之间学习记忆能力的差异。利用RNA-seq技术对具有学习能力和不具有学习能力的10日龄哺育蜂和21日龄哺育蜂脑中基因表达量进行全面分析,筛选出与哺育蜂学习记忆密切相关的差异表达基因(DEGs),并对这些差异表达基因进行GO和KEGG富集分析。qPCR检测随机选取的3个DEGs(上调基因TpnCⅢaMED23以及下调基因Pkc)在具有学习能力和不具有学习能力的10日龄哺育蜂和21日龄哺育蜂脑中的表达量。【结果】PER实验结果显示,经过5次训练后,意大利蜜蜂21日龄哺育蜂的学习能力显著高于10日龄哺育蜂的,而21日龄哺育蜂和21日龄采集蜂的学习能力无显著差异;同样地,21日龄哺育蜂的记忆能力显著高于10日龄哺育蜂的,而21日龄哺育蜂和21日龄采集蜂的记忆能力无显著差异。RNAseq分析筛选到88个与哺育蜂学习记忆密切相关的DEGs,其中18个上调表达,70个下调表达。GO富集结果显示,上调DEGs在生物学进程分类中富集的基因数最多,主要富集在信号转导、蛋白质加工修饰相关;下调DEGs也是在生物学进程分类中富集的基因数最多,主要富集在转录、信号转导、蛋白质生物合成相关,其中显著性富集在转录相关。KEGG富集结果显示,下调DEGs显著性富集在吞噬、光转导、AGERAGE信号通路。qPCR结果显示差异表达基因TpnCⅢa, MED23和Pkc在具有学习能力和不具有学习能力的10日龄哺育蜂和21日龄哺育蜂脑中的表达水平的变化趋势与RNA-seq数据中的变化趋势一致。【结论】PER实验表明日龄是影响意大利蜜蜂哺育蜂的学习和记忆能力的重要因素;同时本研究获得学习后哺育蜂脑部的基因表达变化趋势和富集分析,为深入研究哺育蜂学习记忆的分子机制奠定了重要的理论基础。

关键词: 意大利蜜蜂, 哺育蜂, 喙伸反应, 学习, 记忆, 基因, RNA-seq

Abstract: 【Aim】 This study aims to screen out the genes closely related to learning and memory in nurses of the Italian honey bee, Apis mellifera ligustica. 【Methods】 A. m. ligustica individuals at defined age (10-day-old nurses, 21-day-old nurses and 21-day-old foragers) in artificial bee colonies were collected, and the differences in learning and memory abilities of the three groups were determined using proboscis extension reflex (PER) assay. The gene expression levels in brains of the 10-day-old and 21-day-old nurses with and without learning ability were analyzed by RNA-seq technique, and the differentially expressed genes (DEGs) closely related to learning and memory were screened. The differentially expressed genes were subjected to GO and KEGG enrichment analysis. The expression levels of three DEGs randomly selected (the up-regulated genes TpnCⅢa and MED23, and down-regulated gene Pkc) in brains of the 10-day-old and 21-day-old nurses with and without learning ability were detected by qPCR. 【Results】 The results of PER assay showed that the learning ability of 21-day-old nurses of A. m. ligustica was significantly higher that of the 10-day-old nurses after they were trained five times, but the learning ability between the 21-day-old nurses and the 21-day-old foragers showed no significant difference. Similarly, the memory ability of the 21-day-old nurses was significantly higher than that of the 10-day-old nurses, but the memory ability between the 21-day-old nurses and 21-day-old foragers showed no significant difference. RNA-seq analysis demonstrated that 88 DEGs were closely related to learning and memory of nurses, with 18 genes up-regulated and 70 genes down-regulated. The results of GO enrichment showed that up-regulated DEGs were most enriched in the biological process classification, mainly in the related aspects of signal transduction, protein processing and modification. Down-regulated DEGs were also enriched in the biological process classification, mainly concentrated in the related aspects of transcription, signal transduction and protein biosynthesis, with significant enrichment in the related aspects of transcription. The KEGG enrichment showed that down-regulated DEGs were significantly enriched in phagosome, phototransduction, and AGE-RAGE signal pathway. The qPCR results showed that the expression trends of the differentially expressed genes TpnCⅢa, MED23 and Pkc in brains of the 10-day-old and 21-dayold nurses with and without learning ability were consistent with RNA-seq data. 【Conclusion】 The PER assay indicated that age is an important factor that affects the learning and memory abilities of A. m. ligustica nurses. This study obtained the change trends of gene expression and its enrichment analysis in the brain of nurses after learning train, providing a theoretical foundation for further research on the molecular mechanism of learning and memory of nurses.

Key words: Apis mellifera ligustica; nurses, proboscis extension reflex, learning, memory, gene, RNA-seq