基于高通量筛选的蜜蜂生殖不相容性分子机制研究

Complete and partial reproductive isolation in honeybees (Ginus Apis) is at molecular level involved in postcopulatory-prezygotic and postzygotic incompatibilities. To understand molecular characteristics with reproductive isolation, two independent analyses are conducted via high-throughput approaches: first, proteomics approach is applied to detect postcopulatory -prezygotic incompatibilities of heterotypic seminal fluid proteins (accessory gland proteins) between two types of honeybees (Apis cerana population in Changbai Mountain area and Apis mellifera population in Northeast China); furthermore, a assay for determining protein affinity and a assay for assessing genome-wide microsattlite polymorphism are respectively applied to detect postzygotic incompatibilities of heterotypic reproductive cells (sperms and drone eggs) between two types of honeybees (Apis mellifera mellifera and Apis mellifera carnica) and postzygotic incompatibilities of heterotypic somatic cells (head and thorax tissues) between two types of honeybees (Apis mellifera mellifera and Apis mellifera carnica). Above research produce molecular replicate datasets for mechanisms underlying postcopulatory-prezygotic and postzygotic incompatibilities. Original and novel: Complete and partial reproductive isolation in honeybee (Ginus Apis) is involved at molecular level in postcopulatory -prezygotic and postzygotic incompatibilities. For this purpose, three high-throughput tools, two dimensional gel electrophoresis, bio-layer interferometry and integrated capillary electrophoresis chip are incorporated respectively in proteomic anslysis, assay for determining protein affinity and assay for assessing genome-wide microsattlite polymorphism.

蜜蜂生殖隔离，即，蜜蜂种间完全生殖隔离和蜜蜂种内部分生殖隔离，在分子水平涉及"交配行为后-受精卵前"和"受精卵后"生殖不相容性分子机制。为了揭示蜜蜂生殖隔离分子机制，本文首先进行长白山东方蜜蜂、东北黑蜂雄蜂精液介质（即，异型附腺分泌物）的高通量蛋白质组学分析；其次，进行欧洲黑蜂、卡尼鄂拉蜜蜂雄蜂生殖细胞（即，异型精子和卵子）的高通量蛋白质亲和作用分析，以及它们体细胞（即，异型头、胸部组织）的高通量基因组微卫星长度多态性分析。上述分析可以给出反映蜜蜂"交配行为后-受精卵前"和"受精卵后"生殖不相容性分子机制的基础数据。特色与创新：本文在分子水平将蜜蜂生殖隔离机制归纳为"交配行为后-受精卵前"和"受精卵后"生殖不相容性分子机制的基本问题；为此，分别进行了基于双向电泳技术的蛋白质组学分析，基于生物膜层干涉技术的蛋白质亲和作用分析，以及基于集成芯片毛细管电泳技术的基因组微卫星长度多态性分析。

蜜蜂种间完全生殖隔离和蜜蜂种内部分生殖隔离在分子水平涉及“交配行为后-受精卵前”和“受精卵后”生殖不相容性分子机制。为了揭示蜜蜂生殖隔离分子机制，本研究首先进行长白山东方蜜蜂、东北黑蜂雄蜂精液介质（异型附腺分泌物）的蛋白质组学分析，其次进行欧洲黑蜂、卡尼鄂拉蜜蜂雄蜂生殖细胞（异型精子和卵子）的蛋白质亲和作用分析和它们体细胞（异型头、胸部组织）的基因组微卫星长度多态性分析。通过分析性蛋白质组学方法和制备性蛋白质组学方法，我们从长白山东方蜜蜂、东北黑蜂雄蜂精液介质之间的差异表达蛋白质谱式中鉴定了乙酰辅酶A转移酶、葡萄糖-6磷酸脱氢酶、亮氨酸氨基肽酶、丝氨酸羧肽酶、微管蛋白质1-α链、昆虫载脂蛋白-Ⅱ和几丁质酶等7个候选蛋白质；通过解析生殖细胞之间的蛋白质相互作用程度，我们从欧洲黑蜂、卡尼鄂拉蜂雄蜂之间的精子、1-日龄卵蛋白质亲和反应动力学曲线类型中发现它们之间的蛋白质亲和力常数KD值未呈现统计学差异；通过解析体细胞之间的基因组微卫星位点PCR优势扩增产物，我们从欧洲黑蜂、卡尼鄂拉蜂雄蜂的头胸部组织微卫星位点PCR扩增产物微流体胶电泳图谱和重叠色谱图中发现了有差异的连锁谱图16个微卫星位点的等位基因分型。本研究的主要科学意义是将西方蜜蜂微卫星连锁谱图开发为西方蜜蜂DNA分型鉴定试剂盒