王浆高产蜜蜂咽下腺磷酸化蛋白质组分析

Phosphoproteome Analysis of Hypopharyngeal Glands of High Royal Jelly Producing Bee(Apis mellifera L.)

【目的】通过对王浆高产蜜蜂(Apis mellifera L.)(浙江浆蜂)哺育蜂(6-12 d)咽下腺的磷酸化蛋白质组分析,以期探明蛋白质磷酸化修饰对王浆分泌的生物学意义。【方法】将哺育蜂咽下腺蛋白质液内酶切后,用固相金属离子亲和层析色谱法(IMAC)、强阳离子交换(SCX)及LC-MS/MS和生物信息学分析,对工蜂咽下腺磷酸化蛋白质组进行研究。【结果】在哺育蜂的咽下腺中,共鉴定117个蛋白质,其中6个蛋白质的6条磷酸肽段的8个位点发生磷酸化修饰。这些磷酸化蛋白质是王浆主蛋白1和7的前体、与蛋白翻译合成相关的60S酸性核糖体蛋白P0、P1、P2和60S核糖体蛋白L15。【结论】哺育蜂咽下腺核糖体蛋白发生的磷酸化修饰主要为促进王浆蛋白的高效合成,而磷酸化的王浆蛋白1和7可保证浆蜂在王浆高产的前提下仍保证其王浆具合理的钙磷比,提高营养价值,以满足蜂王产卵和幼虫发育的营养需求。研究结果在蛋白质磷酸化水平上为揭示浆蜂王浆的高产机理奠定了新的理论基础。

[ Objective ] High royal jelly producing bee （Apis mellifera L.） is the unique honeybee resource in China. However, the mechanism of high royal jelly producing has not been clearly addressed. In order to reveal the significance of protein phosphorylation in hypopharyngeal gland for royal jelly synthesis and secretion, the phosphoproteome of hypopharyngeal gland of nurse bee （6-12 day） was analyzed. [ Method ] IMAC （immobilized metal-affinity chromatography） phosphoprotein enrichment, SCX （strong cation exchange） separation, LC-MS/MS （liquid chromatography-mass/mass） identification and bioinformatics analysis were applied to analyze phosphoproteome of hypopharyngeal gland of nurse bee. [Result] Of the identified 117 proteins in the hypopharyngeal gland, 6 of them were phosphorylated on 6 phosphopeptides and assigned 8 phosphorylated sites. They were related to protein translation and synthesis, such as 60S acidic ribosomal proteins P0, P1, P2 and 60S ribosomal protein L15, and major royal jelly proteins 1 and 7 precursor. [Conclusion] The phosphorylation modifications occurred on ribosome proteins of hypopharyngeal gland mainly contribute to the high efficiency of synthesizing and secreting of royal jelly protein. The phosphorylation of royal jelly proteins 1 and 7 maintain the reasonable ratio of calcium to phosphorus of royal jelly with the increasing yield, thus meeting the nutrition demand of fertile egg-laying queens and developing larvae. Hence, the data obtained in this study will provide new knowledge to deeply understand the mechanism how high royal jelly producing bees could produce higher amount of royal jelly at the level of protein phosphorylation.