中华蜜蜂Orco嗅觉受体基因的克隆、表达及亚细胞定位

【目的】克隆鉴定中华蜜蜂Apis cerana cerana的Orco嗅觉受体基因， 并对其在工蜂触角上进行免疫荧光定位。【方法】利用RT-PCR技术克隆中华蜜蜂Orco基因， 并对其编码的氨基酸序列进行生物信息学分析， 使用Real-time PCR技术鉴定其在中华蜜蜂不同发育时期及不同组织的表达谱； 利用免疫荧光定位技术在中华蜜蜂工蜂触角中对Orco进行亚细胞定位。【结果】获得中华蜜蜂Orco基因的全长cDNA序列， 命名为AcerOrco (GenBank登录号： JF968610.1)， 其全长为1 434 bp， 编码477个氨基酸， 预测其含7个跨膜结构以及4个位于细胞膜外的亲水区。表达谱分析显示， AcerOrco在卵、 幼虫和蛹期呈低丰度表达， 1日龄及内勤蜂时期主要在触角和足中表达， 且在1日龄的触角中表达量最高； 采集蜂时期的触角、 头(去除触角)、 胸、 腹和翅中均有较高丰度的表达。亚细胞定位结果显示， AcerOrco不仅在采集蜂触角鞭节上大量表达（尤其在触角鞭节第1亚节中表达量较高）， 而且常成对出现， 并且发现AcerOrco可能主要在触角毛形感器的外部神经元(outer dendrite, OD)以及板形感器的树突神经元中表达。【结论】成功克隆了AcerOrco基因全长， 获得了其表达谱， 且将其定位于工蜂采集蜂的触角感器神经元上， 最终推测AcerOrco与中蜂嗅觉发育和触角感器功能密切相关。

Cloning, expression and subcellular localization of the olfactory co-receptor Orco gene in the Chinese honeybee, Apis cerana cerana (Hymentoptera: Apidae)

【Aim】 To clone and identify the olfactory receptor co-receptor (Orco) gene in the Chinese honeybee, Apis cerana cerana, and locate its proteins in the worker’s antennae. 【Methods】 The olfactory receptor Orco gene from honeybee A. cerana cerana was cloned by RT-PCR, and the expression of this gene at different developmental stages and in different tissues of the honeybee were profiled by Real-time PCR. The putative amino acid sequence was analyzed by bioinformatics methods, and the sub-cellular localization of Orco in the worker’s antennae was detected by immunofluorescence. 【Results】 The full-length cDNA sequence of olfactory receptor Orco was obtained from A. cerana cerana, and named as AcerOrco (GenBank accession no. JF968610.1). Its open reading frame is 1 434 bp in length, encoding 477 amino acid residues. Homology analysis showed that the amino acid sequences of AcerOrco contain seven-transmembrane domains and four hydrophilic regions outside the membrane. The expression profiling at different developmental stages showed that AcerOrco was expressed in the egg, larval and pupal periods at very low levels. The expression profiling in different tissues revealed that AcerOrco was highly expressed in the antennae and legs in nurse bee, with the highest level in the antennae of the 1 day-old worker, while in forager it was highly expressed in antennae, head (without antennae), thorax, abdomen and wings. Subcellular localization displayed that AcerOrco was largely expressed in pairs in the antenna of forager, especially in the 1st flagellar segment of antenna. Furthermore AcerOrco was seemingly to be mainly expressed in the outer dendrite (OD) neurons of sensilla trihodea and the dendrites of sensilla placodea in antennae. 【Conclusion】 The full-length olfactory receptor gene AcerOrco and its expression profiles were obtained, and the encoded protein in the forager antenna of worker honeybee was successfully localized using immunofluorescence. It is inferred that AcerOrco is closely related with the olfactory development and functions of antennal sensilla in the honeybee.