【作者】 章扬；

【导师】 沈兴家；

【作者基本信息】 江苏科技大学 ， 生物化学与分子生物学， 2010， 硕士

【摘要】 滞育是昆虫度过不良环境条件的一种重要行为,滞育机理的查明可以为经济昆虫的开发利用和农业害虫的防治奠定基础。家蚕是研究滞育的模式昆虫之一,家蚕二化性品种的滞育性受上代胚胎期环境条件调控,在胚胎期进行高温(25℃)明催青,子代为滞育卵;低温(15℃)暗催青,子代为非滞育卵。查找家蚕胚胎期滞育关联蛋白,可为最终阐明家蚕滞育的分子机制提供实验依据。以家蚕二化性品种秋丰蚕卵为材料,分别在25℃自然光照和18℃黑暗条件下催青,提取胚胎发育不同阶段(戊1、戊3、己2、己3和己4期)蚕卵易溶性和难溶性蛋白以及蚁蚕蛋白,采用蛋白质双向电泳(2-DE)和图像分析技术,研究家蚕胚胎不同催青条件下不同发育阶段的蛋白质差异表达谱。前4个发育时期的蛋白双向图谱纵向变化不显著,至己4期时有大量酸性蛋白结构基因被激活表达,在己4期的易溶性和难溶性蛋白图谱中分别检测到5个和7个差异显著的蛋白点,在蚁蚕蛋白的双向图谱中没有发现差异蛋白点。对这些差异蛋白点分别进行基质辅助激光解析电离串联飞行时间质谱(MALDI-TOF/TOF MS)和基质辅助激光解析电离飞行时间质谱(MALDI-TOF MS)质谱鉴定,有8个蛋白点得到可信度较高的最佳匹配蛋白报告,共鉴定出卵特异蛋白(参与胚胎发育、分化)、卵黄原蛋白(参与胚胎发育、分化)、表皮蛋白和丙酮酸脱氢酶激酶(抑制丙酮酸氧化脱羧)4种蛋白。这些差异表达蛋白可能导致蚕卵胚胎中物质代谢和能量代谢的不同,据此推测低温和高温催青可能在蚕卵胚胎中诱导了不同的生理生化反应。更多还原

【Abstract】 The diapause ability is an important behavior for insects to live through adverse environmental conditions. Understanding the mechanism of diapause may help lay the foundation for the development and utilization of economic insects and the prevention and treatment of agricultural pests. Silkworm, Bombyx mori, is one of the model insects for studying diapause. The diapause ability of bivoltine silkworm eggs is controlled by the environmental conditions in the embryonic stage of the maternal generation. Study on diapause associated proteins in the embryonic stage provides the experimental evidence for elucidation of the molecular mechanism of diapause.The silkworm eggs of Qiufeng, a bivoltine silkworm strain, were incubated under 25℃and 18℃separately, and soluble proteins and insoluble proteins were prepared from the eggs of five different embryonic stages including the labrum stage, shortening stage, end reversal stage, teachea colouring stage and head pigmentation stage, respectively. And proteins of newly-hatched silkworm were extracted with a different method. The differential profiles of protein expression in different embryonic stages from low and high temperature incubation were obtained with two-dimensional electrophoresis (2-DE) and image analysis system.There were no magnificent longitudinal changes detected during the first four embryonic stages. However, a large amount of structural genes expressing acidic proteins were activated during the head pigmentation stage. No distinctive protein spots were detected from the first four embryonic stages and newly-hatched silkworms; however, five and seven distinctive protein spots were detected from head pigmentation stage in soluble proteins and insoluble proteins, respectively. In which eight spots were identified by mass spectrometry to have credible best match proteins including egg specific protein, vitellogenin, putative cuticle protein, and pyruvate dehydrogenase kinase. Egg specific protein and vitellogenin are involved in embryonic development and differentiation. Pyruvate dehydrogenase kinase inhibits the oxidative decarboxylation of pyruvate. These different proteins may result in different material metabolism and energy metabolism. It was therefore presumed that low and high temperature incubation might induce different physiological and biochemical responses in silkworm eggs.更多还原