蜂王浆高产蜜蜂与意大利蜜蜂工蜂上颚腺磷酸化蛋白质组分析

doi:10.3864/j.issn.0578-1752.2017.23.018

摘要/Abstract

摘要： 【目的】工蜂上颚腺的主要生物学功能是分泌脂肪酸为蜂群提供营养，并参与报警外激素的合成。蜂王浆高产蜜蜂(浆蜂，Apis mellifera liguatica)和意大利蜜蜂(意蜂，Apis mellifera liguatica)是中国主要蜂种，但磷酸化蛋白质组调控其上颚腺的发育和功能的机理尚未开展研究。通过比较二者工蜂上颚腺磷酸化蛋白质组的差异，揭示磷酸化蛋白质组调控上颚腺发育及脂肪酸代谢机理。【方法】解剖浆蜂、意蜂工蜂出房蜂、哺育蜂(10日龄左右)、采集蜂头部，取其上颚腺，进行蛋白质提取、液内酶切，采用固相金属离子亲和层析色谱法(IMAC)进行磷酸化肽段富集，采用Zip-tip C18柱对肽段除盐，通过LC-MS/MS(液相色谱与二级质谱串联)对样品进行分析，首先根据MaxQuant和Persues软件对数据质量进行评估、主成分分析、表达谱聚类定量分析。然后利用PEAKS软件对质谱数据进行蛋白质定量和定性分析，根据定性和定量分析结果，对浆蜂和意蜂上颚腺磷酸化蛋白质组进行生物学进程和KEGG代谢通路富集的生物信息学分析比较。最后利用Scaffold PTM软件确定磷酸化位点、预测磷酸化肽段的基序类型。【结果】浆蜂3个时期分别鉴定到2 225、1 922、2 159个磷酸化蛋白，意蜂分别鉴定到1 740、1 592、1 682个磷酸化蛋白，浆蜂的磷酸化蛋白数目显著高于意蜂，说明浆蜂上颚腺的磷酸化调控网络较意蜂复杂。尽管它们上颚腺的磷酸化过程存在很大差异，但幼蜂、哺育和采集3个时期的磷酸化蛋白表达谱均相似，说明浆蜂和意蜂3个时期表达的磷酸化蛋白质执行类似的生物学功能来保障腺体的发育和分泌活动。对每个时期磷酸化蛋白质组比较，发现浆蜂和意蜂上颚腺3个发育时期的磷酸化蛋白质组均存在显著差异，其中哺育蜂时期二者的差异最大，浆蜂有87个磷酸化蛋白高表达，主要参与能量和脂肪酸代谢，说明其上颚腺的脂肪酸合成力加强(包括10-HDA)，满足蜂王浆产量提高的同时10-HDA含量增加，为其种群繁衍提供必要营养，而意蜂上调表达41个蛋白，主要参与能量代谢。浆蜂、意蜂各时期均识别到酸性、碱性和脯氨酸介导的3种类型motif；哺育蜂时期浆蜂特异识别到的motif对应激酶家族与细胞增殖相关，可能支持其上颚腺形态发育，与浆蜂分泌能力增强相关。【结论】浆蜂和意蜂在3个时期均通过加强不同的磷酸化蛋白质组来支撑上颚腺的发育及功能，其中最显著的差异在哺育蜂时期，浆蜂哺育蜂显著提高了脂肪酸合成力，保障其蜂王浆的基本功能。这在蛋白质修饰的水平上深入了解浆蜂蜂王浆高产的产浆生物学机理。

关键词: 浆蜂, 意大利蜜蜂, 上颚腺, 磷酸化蛋白质组

Abstract: 【Objective】The principal biological function of worker bees’ mandibular glands is to secrete fatty acids to provide the nutrition for the colony and participate in synthesis of alarm pheromone. High royal jelly producing bees (RJBs, Apis mellifera liguatica) and Italian bees (ITBs, Apis mellifera liguatica) are both major honeybee species in China, but the mechanism of regulating the development and function of mandibular glands by phosphoproteome is not reported yet. The objective of this study is to compare the differences of mandibular glands of phosphoproteome, reveal the mechanism of the regulation of mandibular glands by phosphoproteome and fatty acids metabolism.【Method】The mandibular glands of newly emerged, nurse, and foragers of RJBs and ITBs were collected, then after, mandibular gland proteins were extracted and digested by an enzyme. Phosphopeptides were enriched by IMAC (immobilized metal-affinity chromatography) and desalted by using Zip-tip C18 columns. Peptides from each of the samples were analyzed by LC-MS/MS (liquid chromatography-mass/mass). Furthermore, MaxQuant and Perseus bioinformatics tools were used to evaluate phosphoproteome quality. These bioinformatics tools were further utilized for principal component and profiling hierarchical clustering quantification analyses. In addition, PEAKS software was employed for protein quantification and quality analyses. Finally, biological processes and KEGG metabolic pathways in mandibular glands of RJB and ITB were compared. Phosphosites were determined and motifs were predicted by using Scaffold PTM bioinformatics tool.【Result】 The numbers of identified phosphoproteins of RJBs (2 225, 1 922, 2 159) were significantly higher than those of 1 740, 1 592, 1 682 in ITBs, illustrating that RJBs’ mandibular gland phosphorylation regulation network was likely more complicated than ITBs’. Although the phosphorylation process in mandibular glands of RJBs was significantly different from that in ITBs, a similar phosphoproteomes profiling across the gland development suggested that the phosphoproteins at distinct stages in RJBs and ITBs performed similar biological function to ensure the gland development and secretion activity. the glands of each stage in RJBs and ITBs had significant phosphoproteome differences, of which nurse bees were the most significantly diverged. Eighty-seven phosphoproteins were highly abundant in RJBs, and most of them were mainly related to energy and fatty acid metabolic process. These indicated the key roles of fatty acid metabolism in boosting the ability of fatty acid synthesis (including 10-HDA) in mandibular glands to prime the quantity of 10-HDA in royal jelly alongside the increased royal jelly production, which was essential for providing qualitative nutrition for the survival of population. Forty-one highly abundant phosphoproteins in ITBs were mainly related to energy metabolism. Three kinds of motifs were detected at each stage of glands in both RJBs and ITBs: acidic, basic and pro-directed. The unique kinase family recognized by motifs in nurse bees of RJBs was associated with cell proliferation, which might support gland morphological development and related to increased secretion capacity of RJBs.【Conclusion】RJBs and ITBs have shaped different phosphoproteome signatures to maintain mandibular gland development and function at each stage. The most profound divergence occurs during the nurse stage, which RJBs may increase the ability of fatty acid synthesis to ensure the basic function of royal jelly. The data help us gain a novel understanding of the molecular underpinnings to drive enhanced high royal jelly production in RJBs at the level of protein modification.

Key words: high producing royal jelly bee, Italian bee, mandibular gland, phosphoproteome

李爽，李建科. 蜂王浆高产蜜蜂与意大利蜜蜂工蜂上颚腺磷酸化蛋白质组分析[J]. 中国农业科学, 2017, 50(23): 4656-4670.

LI Shuang, LI JianKe. Comparative Analysis of Phosphoproteome Between Mandibular Glands of High Royal Jelly Producing Bees and Italian Bees [J]. Scientia Agricultura Sinica, 2017, 50(23): 4656-4670.

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