Molecular mechanisms of queen bee longevity

描述（由申请人提供）：我们建议使用蜜蜂作为模型，以确定自然界中存在哪些可以通过操纵来延长寿命的机制。蜂王是高度繁殖活跃的，但通常寿命比工蜂长10倍。以衰老的自由基损伤理论为基础，我们将：1)确定蜂王寿命的差异是否与编码抗氧化剂、电子链蛋白或两者的基因表达差异有关。我们有25个与寿命相关的基因完整或接近完整的序列：15个抗氧化基因，8个线粒体基因和2个信号转导基因；12个将用实时定量RT-PCR进行分析，其余的将在Cdna微阵列上进行分析（如下图）。在选定的情况下，分析将辅以蛋白质测量。初步结果表明，基因表达存在显著差异，尤其是在生命早期。测量ATP的产生和对ROS损伤的影响将检验这些差异的功能意义；2)确定一些工蜂后与转基因蝇基因表达差异的因果基础。在合作者J. Tower的实验室中，将选择具有一些最强的蜂后差异的三个基因并制造转基因果蝇（上调和下调）。合作者K. Hughes将研究它们特定年龄的生存、繁殖和发育速度。休斯还将研究长寿命和短寿命的选定果蝇系，以确定选择是否作用于一些相同的基因，这些基因在蚁后和工蜂中有所不同；3)进行微阵列调查，以确定在蜂王和工蜂之间表达差异的其他基因，以确定哪些相关途径与Aim 1中研究的特定基因的差异有关。我们将使用我们最近开发的cDNA微阵列，它代表了大约6000种不同的蜜蜂基因，包括额外的抗氧化和呼吸相关基因，以及编码不同热休克蛋白的基因。这项研究的主要意义在于，它将确定促进长寿的自然发生的分子机制。随着2003年晚些时候基因组测序的完成，蜜蜂作为衰老模型的价值将显著增强。

DESCRIPTION (provided by applicant): We propose to use the honey bee as a model to determine which of the mechanisms that can be manipulated to increase lifespan actually have been in nature. The queen bee is highly reproductively active but typically lives 10-fold longer than does the worker bee. Using the free radical damage theory of aging as a foundation, we will: 1) Determine whether queen-worker longevity differences are associated with differences in expression of genes encoding antioxidants, electron chain proteins, or both. We have complete or near complete sequence for 25 genes related to longevity: 15 antioxidant, 8 mitochondrial, and 2 signal transduction genes; 12 will be analyzed with real-time quantitative RT-PCR and the rest are on a Cdna microarray (below). Analyses will be supplemented with protein measures in selected cases. Preliminary results indicate striking differences in gene expression, especially early in life. Measurements of ATP production and effects of ROS damage will test the functional significance of the differences; 2) Determine the causal basis of some of the queen-worker differences in gene expression with transgenic flies. Three of the genes with some of the strongest queen-worker differences will be selected and transgenic flies made (up- and downregulation) in the laboratory of collaborator J. Tower. Collaborator K. Hughes will study their age-specific survival and reproduction, and developmental rate. Hughes will also work with long- and-short lived selected lines of flies to determine whether selection acted on some of the same genes that differ in queens vs. workers; and 3) Conduct a microarray survey to identify additional genes that differ in expression between queens and workers, to determine what related pathways are affected in association with differences in the specific genes studied in Aim 1. We will use our recently developed cDNA microarrays which represent ca. 6000 different bee genes, including additional antioxidant and respiration-related genes, and genes encoding different HSPs. The principal significance of this research is that it will identify naturally occurring molecular mechanisms promoting longevity. The value of the bee as an aging model will be enhanced significantly with the expected completion of genome sequencing later in '03.