中华蜜蜂气味受体基因AcOr10的克隆及表达分析

Cloning and expression analysis of olfactory receptors gene AcOr10 in the Chinese honeybee,Apis cerana cerana (Hymenoptera: Apidae)

【目的】中华蜜蜂Apis cerana cerana气味受体Or10是感受信息素的重要受体蛋白之一。本研究目的是克隆出该受体基因的序列,并分析该基因在雄蜂不同生理状态下触角和大脑中的表达特征,为该基因的功能研究提供理论依据。【方法】采集中华蜜蜂雄蜂触角,提取其总RNA,采用RT-PCR技术克隆中华蜜蜂Or10基因的序列。利用生物信息学软件分析该基因的氨基酸序列。通过荧光定量PCR技术(q PCR)分析其在巢门口未性成熟雄蜂、巢门口性成熟雄蜂、巢脾上未性成熟雄蜂、巢脾上性成熟雄蜂触角和大脑中的相对表达量。【结果】克隆到中华蜜蜂Or10基因(命名为Ac Or10)(Gen Bank登录号:MF693365)c DNA序列,长度为914 bp,编码区长738 bp,编码246个氨基酸,其蛋白质分子量为28.163 k D,理论等电点为5.50。系统发育树结果显示,中华蜜蜂Ac Or10与西方蜜蜂Apis mellifera Am Or10、小蜜蜂Apis florea Af Or4-like基因聚成一支。荧光定量PCR结果表明,巢门口性成熟雄蜂和巢脾上性成熟雄蜂触角中Ac Or10表达量显著高于巢门口未性成熟雄蜂和巢脾上未性成熟雄蜂触角中的表达量(P<0.05),但前两者以及后两者之间均差异不显著(P>0.05);巢门口性成熟雄蜂大脑中Ac Or10表达量显著高于巢门口未性成熟雄蜂、巢脾上未性成熟雄蜂和巢脾上性成熟雄蜂大脑中的表达量(P<0.05),但后三者之间差异不显著(P>0.05);巢门口未性成熟雄蜂、巢脾上未性成熟雄蜂和巢脾上性成熟雄蜂触角中Ac Or10的表达量都显著高于相同生理状态下大脑中Ac Or10的表达量(P<0.05),但巢门口性成熟雄蜂触角和大脑中Ac Or10的表达量差异不显著(P>0.05)。【结论】推测Ac Or10与中蜂雄蜂性成熟相关,出巢飞行对触角Ac Or10表达量影响较小,但引起其脑部变化,进而影响雄蜂的交尾行为。

【Aim】Olfactory receptor Or10 is one of the important receptor proteins for the perception of pheromone in the honeybee,Apis cerana cerana. The aim of this study is to clone the sequence of Or10 gene,and to analyze the expression levels of Or10 in drone antennae and brains across various physiological status,so as to provide the theoretical basis for further studies on the biological function of this gene. 【Methods】The drone antennae of A. cerana cerana were collected,from which the total RNA was extracted to clone the sequence of Or10. The relative expression levels of Or10 in antennae and brains of drones,which were sexually immature at hive entrance,sexually mature at hive entrance,sexually immature at frames in hive and sexually mature at frames in hive, were detected usingquantitative real-time PCR（ q PCR）. 【Results】The c DNA sequence of Or10 gene（ named as Ac Or10）（ Gen Bank accession no. ： MF693365） of A. cerana cerana was obtained,with the length of 914 bp and a coding region of 738 bp,encoding 246 amino acids. The encoded protein has the molecular weight of28. 163 k D and isoelectric point of 5. 50. The phylogenetic tree showed that Ac Or10 clustered with Or10 of A. mellifera and Or4-like of A. florea. The relative expression levels of Ac Or10 in antennae of sexually immature drones at hive entrance and sexually mature ones at frames in hive were significantly higher than those of sexually immature drones at hive entrance and sexually immature drones at frames in hive（ P〈0. 05）. However there was no significant difference in the relative expression level of Ac Or10 in antennae between sexually mature drones at hive entrance and mature ones at frames in hive（ P〈0. 05）. There was also no significant difference in the relative expression level of Ac Or10 in antennae between sexually immature drones at hive entrance and immature ones at frames in hive（ P〈0. 05）. The relative expression level of Ac Or10 in brains of sexually mature drones at hive entrance was significantly higher than those of drones sexually immature at hive entrance,sexually immature at frames in hive and sexually mature at frames in hive（ P〈0. 05）,while there was no significant difference among the latter three groups（ P〈0. 05）. The relative expression levels of Ac Or10 in antennae of drones sexually immature at hive entrance,sexually immature at frames in hive and sexually mature at frames in hive were all significantly higher than those in brains of drones with the same physiological status（ P〈0. 05）,but there was no significant difference in the expression level of Ac Or10 between antennae and brains of drones sexually mature at hive entrance（ P〉0. 05）. 【Conclusion】It is inferred that Ac Or10 may be related with sexual maturity of A. cerana cerana drones,and flight activities have little influence on the expression of Ac Or10 in antennae but cause the changes of brains,thereby affecting the mating behaviour of drones.