Key Laboratory of Pollinating Insect Biology, Ministry of Agriculture/Institute of Apicultural Research, Chinese Academy of Agricultural Science, Beijing, China.
J Proteome Res. 2011 Sep 2;10(9):4263-80. doi: 10.1021/pr200473a. Epub 2011 Aug 3.
Despite their similar genetic makeup, honeybee (A. mellifera) queens and workers show alternative morphologies driven by nutritional difference during the larval stage. Although much research have been done to investigate the causes of honeybee caste polymorphism, information at subcellular protein levels is limited. We analyzed queen- and worker-destined larvae mitochondrial proteome at three early developmental stages using combinations of differential centrifugation, two-dimensional electrophoresis, mass spectrometry, bioinformatics, and quantitative real time PCR. In total, 67, 69, and 97 protein spots were reproducibly identified as mitochondrial proteins at 72, 96, and 120 h, respectively. There were significant qualitative and quantitative protein expression differences between the two castes at three developmental stages. In general, the queen-destined larvae up-regulated large proportions of proteins at all of the developmental stages and, in particular, 95% at 72 h. An overwhelming majority of the queen larvae up-regulated proteins were physiometabolic-enriched proteins (metabolism of carbohydrate and energy, amino acid, and fatty acid) and involved in protein folding, and this was further verified by functional enrichment and biological interaction network analyses as a direct link with metabolic rates and cellular responses to hormones. Although wide-ranging mitochondrial proteomes participate to shape the metabolic, physiologic, and anatomic differences between the two castes at 72 h, physiometabolic-enriched proteins were found as the major modulators of the profound marking of this caste differentiation. Owing to nutritional difference, prospective queen larvae showed enhanced growth, and this was manifested through the overexpression of metabolic enzymes. Differently from similar studies targeting the causes of honeybee caste polymorphism, this subcellular level study provides an in-depth insight into mitochondrial proteins-mediated caste polymorphism and greatly improves protein coverage involved during honeybee caste determination. Hence, it is a major step forward in the analysis of the fundamental causes of honeybee caste pathway decision and greatly contributes to the knowledge of honeybee biology. In particular, the consistency between the 22 proteins and mRNA expressions provides us important target genes for the reverse genetic analysis of caste pathway modulation through RNA interference.
尽管它们的遗传组成相似，但在幼虫阶段由于营养差异，蜜蜂（A. mellifera）蜂王和工蜂表现出不同的形态。尽管已经有很多研究致力于探讨蜜蜂品级多态性的原因，但在亚细胞蛋白质水平上的信息是有限的。我们使用差速离心、二维电泳、质谱、生物信息学和定量实时 PCR 的组合，在三个早期发育阶段分析了蜂王和工蜂幼虫的线粒体蛋白质组。总共在 72、96 和 120 h 时分别可重复性地鉴定出 67、69 和 97 个蛋白质斑点为线粒体蛋白质。在三个发育阶段，两种品级之间存在显著的蛋白质表达的定性和定量差异。一般来说，蜂王幼虫在所有发育阶段都上调了很大比例的蛋白质，尤其是在 72 h 时上调了 95%。蜂王幼虫上调的绝大多数蛋白质是生理代谢富集蛋白（碳水化合物和能量代谢、氨基酸和脂肪酸代谢），并涉及蛋白质折叠，这通过功能富集和生物相互作用网络分析进一步证实，这与代谢率和细胞对激素的反应直接相关。尽管广泛的线粒体蛋白质组参与塑造了 72 h 时两种品级之间的代谢、生理和解剖差异，但生理代谢富集蛋白是这种品级分化的主要调节因子。由于营养差异，未来的蜂王幼虫表现出增强的生长，这表现在代谢酶的过度表达上。与针对蜜蜂品级多态性原因的类似研究不同，这项亚细胞水平的研究深入了解了线粒体蛋白介导的品级多态性，并大大提高了参与蜜蜂品级决定过程的蛋白质覆盖度。因此，这是分析蜜蜂品级途径决定的根本原因的重要一步，并为蜜蜂生物学知识做出了重要贡献。特别是，22 个蛋白质和 mRNA 表达之间的一致性为我们提供了通过 RNA 干扰调节品级途径的反向遗传分析的重要靶基因。
