›› 2015, Vol. 58 ›› Issue (12): 1291-1299.

• 研究论文 • 上一篇    下一篇

兰州熊蜂蜂王头部产卵相关microRNA生物信息学分析

孙冬婷1,2, 董捷2, 黄家兴2,*, 和绍禹1, 吴杰2,*   

  1. (1. 云南农业大学东方蜜蜂研究所, 昆明 650201;2. 中国农业科学院蜜蜂研究所, 农业部授粉昆虫生物学重点开放实验室, 北京 100093)
  • 出版日期:2015-12-20 发布日期:2015-12-20
  • 作者简介:孙冬婷, 女, 1988年12月生, 北京人, 硕士研究生, 研究方向为熊蜂发育生物学, E-mail: sundongting1221@126.com

Solexa sequencing and bioinformatics analysis of microRNA in the queen head of the bumblebee Bombus lantschouensis

SUN Dong-Ting1,2, DONG Jie2, HUANG Jia-Xing2,*, HE Shao-Yu1, WU Jie2,*   

  1. (1. Eastern Bee Research Institute, Yunnan Agricultural University, Kunming 650201,China; 2. Key Laboratory for Insect-Pollinator Biology of the Ministry of Agriculture, Institute of Apicultural Research, Chinese Academy of Agricultural Sciences, Beijing 100093,China)
  • Online:2015-12-20 Published:2015-12-20

摘要: 【目的】头部是熊蜂感觉和取食中心,也是生理行为的指挥中心。microRNA(miRNA)参与重要生理行为的调控。本研究初步探索了产卵和未产卵兰州熊蜂Bombus lantschouensis蜂王头部差异表达miRNA及其靶基因,以期探明蜂王头部miRNA在产卵过程中作用。【方法】以本土优良蜂种兰州熊蜂 B. lantschouensis 为材料,利用Solexa高通量测序技术对产卵和未产卵蜂王头部miRNA进行测序,运用生物信息学软件对miRNA及其靶基因进行预测;并应用qPCR技术验证极显著差异表达的miRNA。【结果】共获得兰州熊蜂产卵和未产卵蜂王头部miRNA的clean data数,分别为14 228 864和21 431 031 条reads;长度分析表明,在19~24 nt序列中22 nt序列数量最多,分别占产卵和未产卵蜂王序列的45.8%和45.1%。miRNA预测结果显示,共获得297个miRNA,其中270个为已知miRNA,有92个存在于蜜蜂中,其余178个分别存在于蜜蜂以外的其他物种中;另外27个为新的miRNA。在蜜蜂92个已知的miRNA中,在产卵蜂王头部中表达的共有91个,特异表达的有2个;在未产卵蜂王头部中表达的共有90个,特异表达的有1个。27个新的miRNA中,在产卵蜂王头部中表达的共有22个,特异表达的有2个;未产卵蜂王头部中表达的共有25个,特异表达的有5个。miRNA差异表达分析表明,在产卵与未产卵蜂王头部,共有8个已知miRNA表达量达到差异极显著水平(P<0.01)。qPCR结果表明,这8个表达差异极显著的miRNA均存在于熊蜂体内。在8个差异极显著的miRNA中,3个miRNA进行靶基因预测,发现它们共同调控9个靶基因,这些基因主要参与GTP结合和转录调控。【结论】本研究首次利用高通量测序技术鉴定了熊蜂头部组织的miRNA,并发现蜂王产卵与否受到miRNA差异表达的调控。结果为今后深入开展熊蜂miRNA功能验证及产卵调控机制研究奠定了基础。

关键词: 兰州熊蜂, 蜂王, miRNA, 高通量测序, 产卵, 生物信息学

Abstract: 【Aim】 The head is the centre controlling physiological behavior in bumblebees, and micro-RNAs (miRNAs) are involved in the regulation of this behavior. We compared the miRNA expression between the heads of egg-laying and non-egg-laying queens of the bumblebee Bombus lantschouensis so as to clarify the roles of miRNAs in queen head during oviposition.【Methods】 miRNAs in heads of egg-laying and non-egg-laying queens of B. lantschouensis were sequenced by Solexa. Bioinformatics software was used to identify the miRNA and its target genes. qPCR was used to verify the significantly differentially【Results】 Cleaned data showed 14 228 864 reads from heads of egg-laying queens and 21 431 031 reads from heads of non-egg-laying queens. The length of the reads ranged from 19 to 24 nt, and the 22 nt sequences of miRNAs were the most abundant in egg-laying and non-egg-laying queen heads, accounting for 45.8% and 45.1% of their samples, respectively. A total of 297 miRNA sequences were identified from the queen heads. Among them, 270 miRNAs were known and 27 were novel by comparison with the miRBase database. Of the known sequences, 92 miRNAs were known from the honeybee, and 178 miRNAs known from other animals. Within the known 92 miRNAs from the honeybee, there were 91 miRNAs in the head of egg-laying queens and 90 in the head of non-egg-laying queens. For the novel miRNAs, there were 22 miRNAs in the head of egg-laying queens and 25 in the head of non-egg-laying queens, respectively. Expression analysis showed that eight miRNAs reached significantly different expression levels in the heads of egg-laying and non-egg-laying queens (P<0.01). qPCR demonstrated that the eight significantly differentially expressed miRNAs existed in the bumblebee. The target genes for the top three miRNAs with significantly different expression levels were predicted, and it was found that nine target genes were co-regulated by the three miRNAs. All of the target genes are related to GTP binding and transcriptional regulation. 【Conclusion】 This is the first exploration of miRNA within bumblebees by high-throughput sequencing technology. Different miRNA expression levels are demonstrated between the heads of egg-laying and non-egg-laying queens. Our results may contribute to understanding the function of miRNAs and their roles in bumblebees.

Key words: Bombus lantschouensis, queen, miRNA, Solexa sequencing, egg-laying, bioinformatics analysis