马秀梅,杜亚丽,刘晋佳,姚慧敏,张宇昊,马卫华,姜玉锁,2022,中华蜜蜂OBP19的克隆与时空表达分析[J].环境昆虫学报,(2):391-401
中华蜜蜂OBP19的克隆与时空表达分析
Cloning and spatiotemporal expression analysis of OBP19 from Apis cerana cerana
  
DOI:
中文关键词:  中华蜜蜂  AcerOBP19  基因克隆  生物信息学  时空表达
英文关键词:Apis cerana cerana  AcerOBP19  gene cloning  bioinformatics analysis  spatiotemporal expression
基金项目:现代农业产业技术体系(蜜蜂)建设专项资金(CARS-44-KXJ23)
作者单位
马秀梅,杜亚丽,刘晋佳,姚慧敏,张宇昊,马卫华,姜玉锁 1.山西农业大学动物科学学院山西太谷 0308012.山西省农业科学院园艺研究所太原 030031 
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中文摘要:
      本研究克隆获得了中华蜜蜂Apis cerana cerana(简称中蜂)的气味结合蛋白(odor-binding proteins,OBPs)基因AcerOBP19的cDNA序列,预测其蛋白结构,明确该基因在不同发育时期各组织的表达水平。PCR扩增AcerOBP19基因的开放阅读框序列,应用生物信息学软件对其编码蛋白的理化特性、结构特征和系统进化进行分析;用qRT-PCR技术对AcerOBP19在中蜂不同发育时期(1、5、10、15、20、25日龄)各组织(触角、足、口器、毒腺和中肠)中mRNA的表达情况进行分析。获得了AcerOBP19的完整ORF序列,序列长为420 bp,共编码139个氨基酸;含有4个保守的半胱氨酸位点,属于Mins-C OBP亚家族;N-末端有一段含16个氨基酸的信号肽,无跨膜结构。该蛋白包含6个α螺旋和2个二硫键。系统进化树分析表明,AcerOBP19与大蜜蜂Apis dorsata OBPs、小蜜蜂Apis florea OBPs和西方蜜蜂Apis melifera OBPs的氨基酸序列一致性较高,表明它们之间在进化关系上更加同源,且AcerOBP19具有PBP-GOBP superfamily家族的保守结构域,是一种分泌蛋白。qRT-PCR结果表明AcerOBP19在中蜂不同发育时期的各组织中均有表达,其中,15日龄触角中的表达量极显著高于其它组织(P<0.01),在其它日龄中足的表达量极显著高于其它组织(P<0.01);在各个时期中口器和毒腺中有较高表达,中肠中呈微量表达。通过AcerOBP19组织表达谱结果,推测AcerOBP19不仅参与嗅觉识别机制,在味觉识别过程中也可能发挥作用。
英文摘要:
      The odor-binding protein (OBP) gene AcerOBP19 sequence of Apis cerana cerana (abbreviated as Chinese bee) was cloned, and its protein structure was predicted, then, the expression level of the gene in various tissues at different developmental stages was clarified. The open reading frame (ORF) sequence of AcerOBP19 gene was amplified by PCR with specific primers. The physicochemical properties, structural characteristics and phylogenetic tree of AcerOBP19 were analyzed by bioinformatics software; The mRNA expression of AcerOBP19 in various tissues (antennae, legs, mouthparts, poison gland and midgut) of A. cerana cerana at different developmental stages (1, 5, 10, 15, 20, 25 d) was analyzed by qRT-PCR. The ORF sequence of AcerOBP19 was successfully obtained, with a total length of 420 bp, encoding 139 amino acids; There was a signal peptide containing 16 amino acids at the N-terminal without transmembrane structure; It contained four conserved cysteine sites and belongs to the Mins-C OBP subfamily. The protein contained 6 α-helices and two disulfide bonds. The phylogenetic tree showed that AcerOBP19 had high amino acid sequence consistency with the Apis dorsata OBPs, Apis florea OBPs and Apis melifera OBPs, suggesting a more homology in their evolutionary relationship and that AcerOBP19 had a conserved domain of the PBP-GOBP superfamily, it was a secretory protein. Real-time PCR revealed that AcerOBP19 was expressed in all tissues of different developmental stages of A. cerana cerana. The expression level of AcerOBP19 in antennae of 15 days old was significantly higher than other tissues (P<0.01), and the expression level of AcerOBP19 in legs was significantly higher than other tissues in other stages (P<0.01). In other tissues, the expression level of mouthparts and poison glands was relatively high, and the expression level of midgut was low. The results of tissue expression profile suggested that AcerOBP19 participates in olfactory recognition, but in gustatory function.
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