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意大利蜜蜂气味受体蛋白AmelOr5生物信息学分析及基因表达特性

Bioinformatics Analysis and Gene Expression Characteristics of the Odorant Receptor Protein AmelOr5 in Apis mellifera ligustica

    摘要
  • 摘要:
    目的 对意大利蜜蜂（Apis mellifera ligustica）气味受体基因AmelOr5序列、编码蛋白结构及组织表达特征进行分析，为进一步开发其功能提供研究依据。
    方法 从美国国家生物技术信息中心（National Center for Biotechnology Information，NCBI）获取AmelOr5参考转录本（NM_001242959.1）及蛋白序列（NP_001229888.1），采用ORFfinder验证开放阅读框（open reading frame, ORF）并统计序列组成，BLASTn检索同源序列；利用ProtParam、ProtScale、SignalP、TMHMM、CELLO、NetNGlyc、DictyOGlyc、NetPhos、SOPMA、SWISS-MODEL以及CD-search等工具预测理化性质与结构特征；以采集工蜂为材料（n=3），通过实时荧光定量PCR（quantitative real-time PCR, qRT-PCR）检测触角、头部（不含触角）、胸部、腹部、足部和翅部的AmelOr5信使RNA（messenger RNA，mRNA）表达水平。
    结果 AmelOr5转录本全长1418 bp，编码序列（coding sequence, CDS）为1191 bp，鸟嘌呤和胞嘧啶（guanine-cytosine, GC）含量为37.45%，ORF完整编码396个氨基酸，翻译序列与参考序列数据库（Reference Sequence, RefSeq）注释蛋白一致性为100%；BLASTn结果显示其在蜂属近缘物种中具有较高保守性，比对覆盖度为100%，序列一致性为93.53%~95.63%。AmelOr5蛋白理论分子量45.48 kDa、理论等电点pI为9.01，不稳定系数28.67，预测不含信号肽；TMHMM预测含6个跨膜螺旋且主要定位于细胞膜；预测存在31个磷酸化位点、3个高置信度N-糖基化位点和1个潜在O-糖基化位点；二级结构以α-螺旋为主（64.14%），CD-search注释73~386 aa区段为7tm_6结构域。qRT-PCR显示AmelOr5在多组织中表达，其中足部表达水平最高。
    结论 AmelOr5编码蛋白预测为结构较稳定的多跨膜蛋白，AmelOr5基因在采集工蜂足部表达水平较高，提示其可能参与蜜蜂足部相关化学感受信号的识别与转导过程，具体配体谱及生理功能仍需进一步验证。

     

    Abstract:
    Objective Sequence, structure, and expressions of Or5 in Apis mellifera ligustica were analyzed for further studies on its functions.
    Methods The reference transcript (NM_001242959.1) and protein sequence (NP_001229888.1) of olfactory receptor AmelOr5 in Italian honey bees (IHBs) were obtained from the National Center for Biotechnology Information, ORF verified using ORFfinder, and sequence composition determined with homologous sequences identified using BLASTn. Physicochemical properties and structure of the protein were predicted by ProtParam, ProtScale, SignalP, TMHMM, CELLO, NetNGlyc, DictyOGlyc, NetPhos, SOPMA, SWISS-MODEL, and CD-search. On foraging worker IHBs (n=3), AmelOr5 mRNA expressions in antennae, heads excluding antennae, thoraxes, abdomens, legs, and wings were detected by qRT-PCR.
    Results The full-length AmelOr5 transcript was 1 418 bp with a coding sequence of 1 191 bp and a guanine-cytosine content of 37.45%. The ORF encoded 396 amino acids, which was fully consistent with the RefSeq annotated protein. The BLASTn results showed high conservation in closely related Apis species, with 100% alignment coverage and 93.53%–95.63% sequence identity. The protein had a theoretical molecular weight of 45.48 kDa, an isoelectric point of 9.01, an instability index of 28.67, and no signal peptide. The TMHMM-predicted 6 transmembrane helices were basically localized in cell membrane. The predicted post-translational modifications included 31 phosphorylation sites, 3 high-confidence N-glycosylation sites, and one potential O-glycosylation site. The secondary protein structure was mainly α-helical (64.14%), and the CD-search annotated the 73–386 aa region as a 7tm_6 domain. The gene expression was found in multiple tissues with the highest in the legs.
    Conclusion AmelOr5 was a relatively stable multi-transmembrane protein. The gene was high on mRNA expression in the legs of foraging IHBs. It was postulated to be involved in detecting and transmitting chemical signals in that organ. Further studies are in order to verify the specific ligands and physiological functions of the gene.

     

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