【作者】 谢再东；

【导师】 沈卫德；

【作者基本信息】 苏州大学 ， 遗传学， 2010， 硕士

【摘要】 家蚕是完全变态昆虫,一生中要经过卵、幼虫、蛹和成虫4个形态完全不同的发育阶段。对卵期胚胎发育不同时期的蛋白质时间和空间的表达差异已经有大量的研究,但对家蚕卵期和蚁蚕性别差异性表达的研究报道很少。本研究利用双向电泳和质谱相结合的蛋白质组学的研究方法利用家蚕卵色限性品种(W-1)对催青后期胚胎和蚁蚕进行雌雄性别差异蛋白质的研究,找出性别生理差异蛋白,结合家蚕数据库进行质谱鉴定,进一步发现性别会影响家蚕胚胎发育过程中器官和组织蛋白质的合成,获得的主要结果如下:1.W-1蚕卵不同发育阶段的形态图用氢氧化钾脱壳法成功解剖了家蚕品种W-1雌雄卵各个时期的胚胎,采用倒置显微镜拍摄了照片。为后续实验材料做了充分的前期准备。并且将雌雄胚胎分别解剖出来。2家蚕品种W-1己3期雌雄卵可溶性蛋白质表达谱的研究2.1家蚕品种W-1己3期雌雄卵可溶性蛋白质双向电泳图谱的建立用蛋白质双向电泳技术对己3期家蚕卵可溶性蛋白进行了蛋白质组分的比较分析。结果显示:雄卵和雌卵分别检测到317和335个斑点,能互相匹配的蛋白点273对。其中雄卵特异斑点44个,雌卵特异斑点62个。这些差异蛋白的出现,显示雌雄蚕卵可溶性蛋白质组分存在差异。2.2家蚕品种W-1己3雌雄卵可溶性蛋白表达谱差异蛋白的质谱鉴定特征蛋白进行了基质辅助质量飞行时间质谱(MALDI-TOF MS)分析,已鉴定的蛋白包括家蚕未知的角蛋白、原肌球蛋白同源体5、Sqd基因编码的RNA结合蛋白、转胶蛋白、热激蛋白20.4、原肌球蛋白1和与果蝇同源的Tm1蛋白。进一步说明性别会影响胚胎器官和组织蛋白质的合成。3家蚕品种W-1己4期雌雄卵蛋白质双向电泳图谱比较分析用蛋白质双向电泳技术对己4期家蚕卵蛋白进行了蛋白质组分的比较分析。结果显示:雄性蚕卵蛋白样品中共检测到486个蛋白斑点,雌性蚕卵蛋白样品中共检测到427个蛋白斑点,雌雄能匹配的蛋白斑点有380对,匹配率达到81.29%。其中雄性特异蛋白斑点106个,占总蛋白斑点数的21.81%。而雌性特异蛋白斑点47个,占总蛋白斑点数的11.01%。4家蚕品种W-1己5期雌雄卵蛋白质双向电泳图谱比较分析对胚胎己5期雌雄卵蛋白质用双向电泳的技术和方法进行蛋白组分的比较分析。结果显示:雄性蚕卵蛋白样品中共检测到471个蛋白斑点,雌性蚕卵蛋白样品中共检测到432个蛋白斑点,雌雄能匹配的蛋白斑点有395对,匹配率达到84.27%。其中雄性特异蛋白斑点76个,而雌性特异蛋白斑点37个。5家蚕卵色限性品种(W-1)雌雄蚁蚕蛋白质表达谱的研究5.1家蚕品种W-1雌雄蚁蚕蛋白质双向电泳图谱的建立应用双向电泳和图像分析技术,研究家蚕品种W-1雌雄蚁蚕的总蛋白的差异。结果显示:雄蚁和雌蚁分别检测到478和444个蛋白点,能相互匹配的蛋白点为410对,占88.57%,雄蚁特异斑点有68个,雌蚁有34个。这些差异蛋白的出现,显示雌雄蚁蚕蛋白质组分存在差异。5.2家蚕品种W-1雌雄卵蚁蚕表达谱差异蛋白的质谱鉴定特征蛋白进行了基质辅助质量飞行时间质谱(MALDI-TOF MS)分析,已鉴定的蛋白包括血浆蛋白30K,热激蛋白21.4,低分子量30K蛋白脂蛋白质PBMHP-12,肌球蛋白轻链2,推测的蛋白质和与果蝇同源的蛋白GM17246。进一步说明性别会影响胚胎器官和组织蛋白质的合成。更多还原

【Abstract】 Silkworm is a complete metamorphosis insect, In their lifetime they go through four different developmental stages:egg, larvae, pupa and adult .Protein of eggs in different periods have been studied. But protein of gender differences of eggs and newly-hatched larvae in Bombyx mori have little report .In this study, we identify different protein on eggs at later stages and the newly-hatched larvae combining two-dimensional electrophoresis and mass spectrometry using egg color-limited Bombyx mori variety (W-1),Furtherly we find that gender of the Bombyx mori have an influence on organs and tissues during embryonic development. Main results obtained are as follows:1 male and female embryonic morphology of egg color-limited Bombyx mori variety,W-1,in different periodsmale and female embryos of egg color-limited Bombyx mori variety,W-1 in different periods were successfully dissected with potassium hydroxide.Then prepare for the follow-up experiments.And for the first time male and female embryos are presented separately.2 The study of expression profiling of dissolvable protein of male and female eggs of the period of ji3 in Bombyx mori variety,W-12.1 The establishment of patterns of dissolvable protein of male and female eggs of the period of ji3 in Bombyx mori variety,W-1 by two dimensional polyacrylamide gel electrophoresiswe compared the dissolvable protein of male and female eggs of the period of ji3 by using two-dimensional electrophoresis technology. The results showed that 317 and 335 protein spots were detected in dissolvable proteins of male and female egg respectively. Comparison analysis showed that 273 of the total protein spots were matched between male and female egg, and 44 and 62 specific protein spots were found in male and female egg respectively. The results indicated that dissolvable protein of male and female eggs of the period of ji3 in silkworm are different between male and female.2.2 the identification of different proteins from patterns of dissolvable protein of male and female eggs of the period of ji3 in Bombyx mori variety,w-1 by mass spectrometryThe characteristic protein spots were excised to identify by MALDI-TOF MS.The result show that proteins identified were putative cuticlar protein, tropomyosin isoform 5, Bmsqd-1, transgelin , heat shock protein 20.4 ,tropomyosin 1 and Tm1 protein similar to Drosophila.It furtherly show that gender has an important influence on the formation of embryonic organs and tissues.3 Anaylsis of patterns of protein of male and female eggs of the period of ji4 in Bombyx mori variety,W-1 by two dimensional polyacrylamide gel electrophoresiswe compared the protein components of male and female eggs of the period of ji4 by using two-dimensional electrophoresis technology. The results showed that 486 and 427 protein spots were detected in proteins of male and female egg respectively. Comparison analysis showed that 380 of the total protein spots were matched between male and female egg, the matching rate is 81.29% and 106 and 47 specific protein spots were found in male and female egg respectively.4 Anaylsis of patterns of protein of male and female eggs of the period of ji5 in Bombyx mori variety,W-1 by two dimensional polyacrylamide gel electrophoresiswe compared the protein components of male and female eggs of the period of ji5 by using two-dimensional electrophoresis technology. The results showed that 471and 432 protein spots were detected in proteins of male and female egg respectively. Comparison analysis showed that 395 of the total protein spots were matched between male and female egg, the matching rate is 84.27% and 76 and 37 specific protein spots were found in male and female egg respectively. 5 The study of protein expression profiling of the male and female newly-hatched larvae in Bombyx mori variety,W-15.1 The establishment of protein patterns of male and female newly-hatched larvae in Bombyx mori variety , W-1 by two dimensional polyacrylamide gel electrophoresiswe compared the protein components of male and female newly-hatched larvae by using two-dimensional electrophoresis technology. The results showed that 478 and 444 protein spots were detected in dissolvable proteins of male and female egg respectively. Comparison analysis showed that 410 of the total protein spots were matched between male and female egg, the matching rate is 88.57% and 68 and 34 specific protein spots were found in male and female egg respectively.5.2 the identification of different proteins from patterns of male and female newly-hatched larvae in Bombyx mori variety,w-1 by mass spectrometryThe characteristic protein spots were excised to identify by MALDI-TOF MS.The result show that proteins identified were major plasma protein 30K, heat shock protein 21.4, low molecular 30kD lipoprotein PBMHPC-21, myosin light chain 2,putative protein CLK_A0112and GM17246 protein similar to Drosophila.It furtherly show that gender has an important influence on the formation of embryonic organs and tissues.更多还原