【作者】 刘培刚；

【导师】 李卫国； 王永强；

【作者基本信息】 山东农业大学 ， 特种经济动物饲养， 2010， 硕士

【摘要】 动植物的有性生殖是自然界普遍的现象,是繁殖后代的主要方式。在自然界中,有一部分动植物是由单性生殖来繁殖后代的,其中孤雌生殖是其中的一种形式,主要发生在无脊椎动物、低等脊椎动物和许多孢子、被子植物中,在鱼类、两栖类和爬行类等脊椎动物中也有发生。家蚕是鳞翅目昆虫的模式生物,也是重要的经济昆虫,主要是通过有性生殖来繁殖后代。家蚕属偶发性的孤雌生殖,在自然条件下孤雌生殖发生率和孵化率很低,在实验室研究中没有得到广泛地应用。但应用人工孤雌生殖方法,可有效提高其发生率、孵化率水平,各国学者就家蚕孤雌生殖人工诱导做了大量的研究工作,发现了一些有效可行的方法。其中,俄罗斯Astaurov等建立的“温汤处理”方法简便、安全和有效,被许多学者采用并沿用至今。同时,各国学者还从遗传分析角度、细胞和分子水平开展了家蚕孤雌生殖的相关研究。虽然前人在家蚕孤雌生殖研究方面做了大量的工作,但是家蚕孤雌生殖的发生相关机制还没有完全阐明。为了进一步了解家蚕孤雌生殖的发生机制和相同基因型下孤雌生殖发生率、孵化率的可选择性机理,本论文以高发生率、孵化率的孤雌生殖系(无14)与其亲本有性生殖系(54A)为材料,主要从蚁蚕、5龄幼虫不同时期的血液和卵巢等3个方面开展了比较蛋白质组学研究,取得如下主要结果:1、利用双向电泳、生物质谱和生物信息学技术对家蚕孤雌生殖系无14与其亲本有性生殖系54A的蚁蚕蛋白质组成进行了比较分析。通过分析发现:较其亲本有性生殖系54A,孤雌生殖系无14蚁蚕有19个蛋白表达量上调在2倍以上,其中有2个蛋白特异表达,有25个蛋白表达量下调在2倍以上,其中有3个蛋白缺失。从上述44个差异蛋白中选取9个界限清楚、表达量较高的蛋白进行MALDI-TOF/MS分析,对获得肽指纹图谱经软件搜索相关蛋白质数据库,其中8个蛋白具有可信结果,包括表皮角蛋白-2、β-肌动蛋白、肌肉蛋白-20、A1型肌动蛋白、RR-表皮蛋白-19、翻译控制肿瘤蛋白和谷胱甘肽S-转移酶等,推测这些蛋白可能与家蚕孤雌生殖系的胚胎发育调节相关。2、为了探明家蚕孤雌生殖系与其亲本有性生殖系5龄不同发育阶段血液蛋白的表达差异,以孤雌生殖系无14和亲本有性生殖系54A的血液为材料,借助蛋白质组学技术进行了比较分析。结果显示:有5个蛋白在54A和无14不同发育阶段的血液中持续存在差异,1个蛋白在54A 5龄后期血液中特异表达。通过对这6个蛋白进行MALDI-TOF/MS分析,并对获得肽指纹图谱经软件搜索相关蛋白质数据库,获得了这些蛋白的相关信息,其中2个蛋白具有可信结果,分别为血液保幼激素结合蛋白与胰蛋白酶抑制剂-8A,推测这2个蛋白可能与家蚕孤雌生殖发育过程的调节相关。3、利用双向电泳、生物质谱和生物信息学技术对家蚕孤雌生殖系无14与其亲本有性生殖系54A 5龄不同时期的卵巢蛋白质组成进行了比较分析,发现了有5个蛋白在5龄不同时期的卵巢中持续存在差异,1个蛋白在54A5龄后期卵巢中特异表达。对这6个蛋白进行MALDI-TOF/MS分析,对获得肽指纹图谱经软件搜索相关蛋白质数据库,其中4个蛋白获得了可信结果,包括细胞维甲酸结合蛋白、肌动蛋白解聚因子-1和热激蛋白HSC70,推测这些蛋白可能与家蚕孤雌生殖发育过程的调节相关。利用双向电泳技术对两品系的蚁蚕、幼虫不同时期的血液和卵巢的蛋白质组成进行了比较分析,发现了一些差异蛋白。通过对差异蛋白的质谱鉴定和生物信息学分析,在蛋白水平了解了家蚕孤雌生殖系与其亲本有性生殖系在发育过程中存在的生理调节和适应上的差异,进一步探讨了家蚕孤雌生殖发生的相关机理。另外,两品系组织器官存在的其他差异蛋白的功能有待今后进一步研究。更多还原

【Abstract】 Sexual reproduction of animals and plants was wildly distributed in nature and was the mian reproductive way. In nature,a part of animals and plants reproduced by parthenogenesis. Parthenogenesis occurs mainly in the Invertebrata,Lower vertebrates, Spores and of the angiosperms, fish, amphibians and reptiles, etc have also occurred.As a model organism of Lepidoptera and a important economic insect, silkworm reproduced mainly by Sexual reproduction. Partheno- genesis of silkworm is sporadic,the parthenogenetic incidence rate, hatchability of silkworm is very low in nature, therefore there are not wildly application in experiment.Parthenogenetic incidence rate, hatchabilityof silkworm increased after the found of aritificial parthenogenesis(AMP), a lot of works which are about the induce method of aritificial parthenogenesis have done by scholar of diffirent country, and some good induce methods have also been found. The which was established by Astaurov was convenient, safe and effective,and was followed by another scholar so far.There were also some reserchs on parthenogenesis in genetic,celler and molecular level.While a lot of studies about silkworm parthenogenesis have been done, the mechanism of it has not been explained completely. To gain an insight into the causing mechanism silkworm parthenogenesis deeply and selective mechanism, we employed comparative proteomic approach mainlyin new-hatched silkworm, hemolymph and ovary which are from parthenogenetic line wu 14 with high incidence rateand hatchability and parent of amphigenetic line 54A with low incidence rateand hatchability. The main results as follow:1.The new-hatched silkworm proteins of Bombyx mori partheno- genetic line wu 14 and parent of amphigenetic line 54A were compared by two dimensional electrophoresis(2-DE), matrix assisted laser desorption ionization and time of fight mass spectrometry (MALDI-TOF/MS) and bioinformatics analysis.Compared with parent of amphigenetic line 54A, tthere are 19 up-ragulated proteins which included 2 specific express proteins and 25 down-ragulated proteins which included 3 specific delect proteins in parthenogenetic line wu 14. 9 proteins which express highly and are clear and were choosed from above 44 different proteins were analysised by MALDI-TOF/MS, and research the protein data bank by software on the basis of obtained Peptide mass fingerprinting(PMF),and 8 proteins have credible result. This proteins include epidermal cytokeratin 2, muscular protein 20, odorant binding protein, glutathione S-transferase translationally con- trolled tumor protein homolog , cuticular protein RR-1 ,cuticular protein RR-1, beta-actin actin, muscle-type A1 . It could be speculated this 8 proteins might be associated with the regulation of lavare growth of parthenogenetic line.2.To gain an insight into the mechanism of silkworm partheno- genesis, the hemolymph proteins of Bombyx mori parthenogenetic line wu 14 and parent of amphigenetic line 54A were compared by promotemic techniques. 5 different peoteins whose difference expressed continually and 1 protein which express specificly in 54A were found .This 6 different proteins were analysised by MALDI- TOF/MS,and research the protein data bank by software on the basis of obtained Peptide mass fingerprinting(PMF), and 2 proteins have credible result,include hemolymph juvenile hormone binding protein, Chymotrypsin inhibitor-8A. It could be speculated this 2 proteins might be associated with the growth regulation of parthenogenetic line.3.The ovary silkworm proteins in the 5th instar larvae of Bombyx mori parthenogenetic line wu 14 and parent of amphigenetic line 54A were compared by two dimensional electrophoresis(2-DE), matrix assisted laser desorption ionization and time of fight mass spectrometry (MALDI-TOF/MS) and bioinformatics analysis, and find 5 different peoteins whose difference expressed continually and 1 protein which express specificly in 54A. This 6 different proteins wThe ovary silkworm proteins in the 5th instar larvae of Bombyx mori parthenogenetic line wu 14 and parent of amphigenetic line 54A were compared by two dimensional electrophoresis(2-DE), matrix assisted laser desorption ionization and time of fight mass spectrome- try (MALDI-TOF/MS) and bioinformatics analysis, and find 5 diffe- rent peoteins whose difference expressed continually and 1 protein which express specificly in 54A. This 6 different proteins were analysised by MALDI-TOF/MS, and research the protein data bank by software on the basis of obtained Peptide mass fingerprinting(PMF) , and 4 proteins have credible result, include cellular retinoic acid binding protein, actin-depolymerizing factor, heat shock cognate protein 70. It could be speculated this 4 proteins might be associated with the growth regulation of parthenogenetic line.The proteins of new-hatched silkworm, hemolymph and ovary in the 5th instar larvae at various days in two lines were compared by two dimensional electrophoresis(2-DE), and a few different spots were found. The differences of physiologic accormmodation and adapt inthe development of Bombyx mori parthenogenetic line and parent of Amphigenetic line were got across in protein level by the analysis of different spot though matrix assisted laser desorption ionization and time of fight mass spectrometry(MALDI-TOF/MS) and bioinformatics analysis, and gained an insight into the mechanism of silkworm parthenogenesis. In addition, the function of the another different spots in different organs of two lins need research later on .更多还原