王浆高产蜜蜂咽下腺磷酸化蛋白质组分析

doi:10.3864/j.issn.0578-1752.2013.23.023

摘要/Abstract

摘要： 【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。

关键词: 哺育蜂 , 浆蜂 , 咽下腺 , IMAC , SCX , 磷酸化蛋白质组

Abstract: 【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.

Key words: nurse bee , high producing royal jelly bee , hypopharyngeal gland , IMAC , SCX , phosphoproteome

鲁小山12, 韩宾2, 张兰2, 冯毛2, 房宇2, 李荣丽2, 周天娥12, 李建科2. 王浆高产蜜蜂咽下腺磷酸化蛋白质组分析[J]. 中国农业科学, 2013, 46(23): 5050-5057.

LU Xiao-Shan-12, HAN Bin-2, ZHANG Lan-2, FENG Mao-2, FANG Yu-2, LI Rong-Li-2, ZHOU Tian-E-12, LI Jian-Ke-2. Phosphoproteome Analysis of Hypopharyngeal Glands of High Royal Jelly Producing Bee (Apis mellifera L.)[J]. Scientia Agricultura Sinica, 2013, 46(23): 5050-5057.

0
/ / 推荐

导出引用管理器 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2013.23.023

https://www.chinaagrisci.com/CN/Y2013/V46/I23/5050

参考文献

[1]Huang Z Y, Otis G W, Teal P E A. Nature of brood signal activating the protein synthesis of hypopharyngeal gland in honey bees, Apis mellifera (Apidae: Hymenoptera). Apidologie, 1989, 20(6): 455-464.

[2]Evans J D, Wheeler D E. Differential gene expression between developing queens and workers in the honey bee, Apis mellifera. Proceedings of the National Academy of Sciences of the United States of America, 1999, 96(10): 5575-5580.

[3]Viuda-Martos M, Ruiz-Navajas Y, Fernández-López J, Pérez-Álvarez J. Functional properties of honey, propolis, and royal jelly. Journal of Food Science, 2008, 73(9): R117-R124.

[4]李建科, 陈盛禄, 钟伯雄, 苏松坤. 西方蜜蜂咽下腺与繁殖力的发育遗传研究. 中国畜牧杂志, 2003, 39(6): 9-11.

Li J K, Chen S L, Zhong B X, Su S K. Genetic analyses for developmental behavior of reproductive ability and hypopharyngeal gland in Western honeybees (Apis melliffera L.). Chinese Journal of Animal Science, 2003, 39(6): 9-11. (in Chinese)

[5]胡福良, 陈盛禄, 林雪珍, 苏松坤, 杜芝兰. 意大利蜂工蜂咽下腺细胞超微结构与分泌活性. 浙江农业大学学报, 1997, 23(1): 75-80.

Hu F L, Chen S L, Lin X Z, Su S K, Du Z L. Study on the ultrastructure of hypopharyngeal galnd cells and secretory activity of worker honey bee (Apis mellifera L.). Journal of Zhejiang Agricutural Univercity, 1997, 23(1): 75-80. (in Chinese)

[6]冯毛, 李建科. 王浆高产蜜蜂和原种意大利蜜蜂咽下腺发育蛋白质组分析. 中国农业科学, 2009, 42(2): 677-687.

Feng M, Li J K. Proteome analysis of the development of hypopharyngeal gland of high royal jelly producing bees and native Italian bees. Scientia Agricultura Sinica, 2009, 42(2): 677-687. (in Chinese)

[7]Mann M, Jensen O N. Proteomic analysis of post-translational modifications. Nature Biotechnology, 2003, 21(3): 255-261.

[8]Li J K, Feng M, Begna D, Fang Y, Zheng A J. Proteome comparison of hypopharyngeal gland development between Italian and royal jelly producing worker honeybees (Apis mellifera L.). Journal of Proteome Research, 2010, 9(12): 6578-6594.

[9]Li J K, Wang T, Zhang Z H, Pan Y H. Proteomic analysis of royal jelly from three strains of Western honeybees (Apis mellifera). Journal of Agricultural and Food Chemistry, 2007, 55(21): 8411-8422.

[10]Alonso A, Sasin J, Bottini N, Friedberg I, Osterman A, Godzik A, Hunter T, Dixon J, Mustelin T. Protein tyrosine phosphatases in the human genome. Cell, 2004, 117(6): 699-711.

[11]Zolnierowicz S, Bollen M. Protein phosphorylation and protein phosphatases. The EMBO Journal, 2000, 19(4): 483-488.

[12]Porath J. IMAC-immobilized metal ion affinity based chromatography. TrAC Trends in Analytical Chemistry, 1988, 7(7): 254-259.

[13]Thingholm T E, Jensen O N, Larsen M R. Analytical strategies for phosphoproteomics. Proteomics, 2009, 9(6): 1451-1468.

[14]张兰, 李建科, 吴黎明. 王浆高产蜜蜂 (Apis mellifera L.)卵期发育蛋白质组学分析. 中国农业科学, 2007, 40(6): 1276-1287.

Zhang L, Li J K, Wu L M. Profile analysis of the proteome of the eggs of the higher royal jelly producing bees (Apis mellifera L.). Scientia Agricultura Sinica, 2007, 40(6): 1276-1287. (in Chinese)

[15]Knecht D, Kaatz H H. Patterns of larval food production by hypopharyngeal glands in adult worker honey bees. Apidologie, 1990, 21(5): 457-468.

[16]Liljas A, Gudkov A T. The structure and dynamics of ribosomal protein L12. Biochimie, 1987, 69(10): 1043-1047.

[17]Uchiumi T, Kominami R. Binding of mammalian ribosomal protein complex P0· P1· P2 and protein L12 to the GTPase-associated domain of 28 S ribosomal RNA and effect on the accessibility to anti-28 S RNA autoantibody. The Journal of Biological Chemistry, 1997, 272(6): 3302-3308.

[18]Uchiumi T, Honma S, Nomura T, Dabbs E R, Hachimori A. Translation elongation by a hybrid ribosome in which proteins at the GTPase center of the Escherichia coli ribosome are replaced with rat counterparts. The Journal of Biological Chemistry, 2002, 277(6): 3857-3862.

[19]Santos C, Ballesta J P. Ribosomal protein P0, contrary to phosphoproteins P1 and P2, is required for ribosome activity and Saccharomyces cerevisiae viability. The Journal of Biological Chemistry, 1994, 269(22): 15689-15696.

[20]Hamel E, Koka M, Nakamoto T. Requirement of an Escherichia coli 50 S ribosomal protein component for effective interaction of the ribosome with T and G factors and with guanosine triphosphate. The Journal of Biological Chemistry, 1972, 247(3): 805-814.

[21]Hamman B D, Oleinikov A V, Jokhadze G G, Traut R R, Jameson D M. Rotational and conformational dynamics of Escherichia coli ribosomal protein L7/L12. Biochemistry, 1996, 35(51): 16672-16679.

[22]Santos C, Ballesta J P. The highly conserved protein P0 carboxyl end is essential for ribosome activity only in the absence of proteins P1 and P2. The Journal of Biological Chemistry, 1995, 270(35): 20608-20614.

[23]Saenz-Robles M T, Remacha M, Vilella M D, Zinker S, Ballesta J P. The acidic ribosomal proteins as regulators of the eukaryotic ribosomal activity. Biochimica et Biophysica Acta (BBA)-Gene Structure and Expression, 1990, 1050(1): 51-55.

[24]Nusspaumer G, Remacha M, Ballesta J P. Phosphorylation and N-terminal region of yeast ribosomal protein P1 mediate its degradation, which is prevented by protein P2. The EMBO Journal, 2000, 19(22): 6075-6084.

[25]Remacha M, Santos C, Bermejo B, Naranda T, Ballesta J. Stable binding of the eukaryotic acidic phosphoproteins to the ribosome is not an absolute requirement for in vivo protein synthesis. The Journal of Biological Chemistry, 1992, 267(17): 12061-12067.

[26]Lieberman K R, Noller H F. Ribosomal protein L15 as a probe of 50 S ribosomal subunit structure. Journal of Molecular Biology, 1998, 284(5): 1367-1378.

[27]Franceschi F J, Nierhaus K H. Ribosomal proteins L15 and L16 are mere late assembly proteins of the large ribosomal subunit. Analysis of an Escherichia coli mutant lacking L15. The Journal of Biological Chemistry, 1990, 265(27): 16676-16682.

[28]Soung G Y, Miller J L, Koc H, Koc E C. Comprehensive analysis of phosphorylated proteins of Escherichia coli ribosomes. Journal of Proteome Research, 2009, 8(7): 3390-3402.

[29]Hill W E. The Ribosome: Structure, Function, and Evolution. American Society for Microbiology, 1990.

[30]Graham J M, Ambrose J T, Langstroth L. The Hive and the Honey Bee: A New Book on Beekeeping Which Continues the Tradition of “Langstroth on the Hive and the Honeybee”. USA: Dadant, Hamilton, Ill., 1992.

[31]Garcia L, Saraiva Garcia C H, Cala?bria L K, da Cruz G C N, Sa?nchez Puentes A, Ba?o S N, Fontes W, Ricart C A, Salmen Espindola F, Valle de Sousa M. Proteomic analysis of honey bee brain upon ontogenetic and behavioral development. Journal of Proteome Research, 2009, 8(3): 1464-1473.

[32]Tamura S, Amano S, Kono T, Kondoh J, Yamaguchi K, Kobayashi S, Ayabe T, Moriyama T. Molecular characteristics and physiological functions of major royal jelly protein 1 oligomer. Proteomics, 2009, 9(24): 5534-5543.

[33]Kamakura M. Royalactin induces queen differentiation in honeybees. Nature, 2011, 473(7348): 478-483.

[34]Schmitzova J, Klaudiny J, Albert Š, Schröder W, Schreckengost W, Hanes J, Judova J, Šimúth J. A family of major royal jelly proteins of the honeybee Apis mellifera L. Cellular and Molecular Life Sciences, 1998, 54(9): 1020-1030.

[35]Thompson G J, Kucharski R, Maleszka R, Oldroyd B P. Towards a molecular definition of worker sterility: differential gene expression and reproductive plasticity in honey bees. Insect Molecular Biology, 2006, 15(5): 537-644.

[36]Birrell G W, Earl S T, Wallis T P, Masci P P, de Jersey J, Gorman J J, Lavin M F. The diversity of bioactive proteins in Australian snake venoms. Molecular & Cellular Proteomics, 2007, 6(6): 973-986.