二化性家蚕卵低温和高温催青胚胎期蛋白质表达差异分析
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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。
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.
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。
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.
引文
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[2]谢立群,蒋明星,张孝羲.烟青虫滞育特征的研究[J].植物保护学报, 1997, 24 (3):199-203
[3] Saunders D S. Under-sized larvae from short-day adults of the blow fly Calliphora vicina side-step the diapause programme [J]. Physiol Entomol, 1997, 22:249-255
[4] Bell C H. Factors governing the induction of diapause in Ephestia elutella and Plodia interpunctella [J]. Physiol Entomol, 1976, 1:83-91
[5] Taleda M. Photoperiodic time measurement and related physiological mechanisms in insects and mites [J]. Annu Rev Entomol, 1997, 42:323-349.
[6]张作法,李有贵,时连根,等.家蚕化性及其变化的调控[J].生物学通报, 2004, 39(12):11-13
[7] Watanabe K. Studies on the voltinism in Bombyx [J]. Dep. Agric. Kyushu Imperial Univ. 1924, 4: 1-93
[8]宋志光.现行家蚕品种化性和眠性的控制[J].蚕学通讯, 2006, 26(3):23-26
[9]付中华.家蚕不越年卵的发生与防止[J].北方蚕业, 2003, 24(96):39-40
[10]黄君霆.家蚕滞育分子机制的研究[J].蚕业科学, 2003, 29 (1):1-6
[11] Katagiri N, Imai K, Yamashita O. Multiple gene expression upregulated by diapause hormone in developing ovaries of the silkworm , Bombyx mori [J]. J Insect Biotech Sericol, 2001, 70:113-120.
[12] Hasegawa K. Studies on the voltinism in the silkworm, Bombyx with special reference to organs containing determination of voltinism [J]. Proc. Jap. Acad, 1951, 27:667-671
[13] Fukuda S. The production of diapause eggs by transplanting the suboesophageal ganglion in the silkworm [J]. Proc.Jap.Acad, 1951, 27: 672-676
[14] Yamashita O. Diapause hormone of the silkworm, Bombyx mori: structure, gene expression and function [J]. J Insect Physiol, 1996, 42:669-679
[15] Hasegawa K. The diapause hormone of the silkworm, Bombyx mori [J]. Nature, 1957, 179:1300-1 301
[16] Horie Y, Kanda T, Mochida Y. Sorbitol as an arrester of embryonic development in diapausing eggs of the silkworm, Bombyx mori [J]. J Insect Physiol, 2000, 46:1009-1016
[17] Xu W H ,Saito Y, Yamashita O. Molecular characterization of the gene encoding the precursor protein of diapause hormone and pheromone biosynthesis-activating neuropeptide of the silkworm,Bombyx mori and its distribution in some insects [J]. Biochem Biophys Acta, 1995, 1261:83-89
[18] Saito H, Takeuchi Y, Takeda R, et al. The core and complementary sequence responsible for biological activity of the diapause hormone of the silkworm, Bombyx mori [J]. Peptides, 1994, 15:1173-1178
[19] Suwan S, Isobe M, Yamashita O, et al. Silkworm diapause hormone, Structure activity relationships indispensable role of C-terminal amide [J]. Insect Biochem. Molec.Biol, 1994, 24:1001-1007
[20] Watanabe K, Hull J. J, Niimi T, et al. FXPRL-amide peptides induce ecdysteroidogenesis through a G-protein coupled receptor expressed in the prothoratic gland of Bombyx mori [J]. Mol. Cell. Endocrinol,2007, 273:51-58
[21] Xu W.H, Zhang Y.Q, Wang Y, et al. Molecular mechanism of diapause in insect. I. expression of diapause hormone gene and incubation temperature in embryonic stage of Bombyx mori [J]. Science in China (Ser.C), 1998, 41(3):344-350
[22]徐卫华.家蚕滞育的分子机理-2蛹期滞育激素的表达与滞育决定[J].遗传学报, 1999, 26(2):107-111
[23]徐卫华.家蚕滞育激素-性信息素合成激活肽基因表达的调节[J].中国生物化学与分子生物学报, 1998, 14(5):557-561
[24] Morita A, Niimi T, Yamashita O. Physiological differentiation of DH-PBAN-producing neurosecretory cells in the silkworm embryo [J]. Journal of Insect Physiology, 2003, 12(49):1093-1102
[25] Norio Kitagawa, Kunihiro Shiomi, Kunio Imai, et al. Establishment of a sandwich ELISA system to detect diapause hormone, and developmental profile of hormone levels in egg and subesophageal ganglion of the silkworm, Bombyx mori [J]. Zoological Science, 2005, 22:213-221
[26] Norio Kitagawa, Kunihiro Shiomi, Kunio Imai, et al. Diapause hormone levels in subesophageal ganglia of Uni-Bi and Poly-voltine races during pupal-adult development of Bombyx mori, and the effects of ouabain, an inhibitor of Na+-K+ ATPase, on the hormone levels [J]. Journal of insect Biotechnology and sericology, 2005, 74:57-62
[27] TianY.Z, Le K, Zhi F.Z, et al. Identification of a POU factor involved in regulating the neuron-specific expression of the gene encoding diapause hormone and pheromone biosynthesis activating neuropeptide in Bombyx mori [J]. Biochemical Journal, 2004, 380:255-263
[28] Yamashita O, Isobe M, Imai K, et al. Serum albumin as an effective carrier for diapause hormone of the silkworm, Bombyx mori [J]. Appl. Entomol. Zool, 1980, 15:90-95
[29] Yamashita O, Hasegawa K. Oocyte age sensitive to the diapause hormone from the standpoint of glycogen synthesis in the silkworm, Bombyx mori [J]. J. Insect Physiol, 1970, 16:2377-2383
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