【作者】 陈媛媛；

【导师】 谈建中；

【作者基本信息】 苏州大学 ， 园林植物与观赏园艺， 2013， 硕士

【摘要】 果桑栽培已经成为现代蚕桑产业发展的新领域，有关桑雌花与桑椹生长发育的研究也因此受到越来越多的关注。为了探讨桑树雌花发育及其调控的分子机理，本研究以果桑品种“大10”和“SG01”为试验材料，使用差异蛋白质组学研究技术，分析了不同开花期的雌花、雌花不同部位、以及不同品种雌花和成熟桑椹蛋白质的表达特征，鉴定获得了桑雌花发育相关的差异蛋白组分，并分析了差异表达蛋白质的功能，主要研究结果如下：（1）采用SDS-PAGE和质谱技术，对“大10”不同开花期雌花的蛋白质进行了分离，结果检测到了差异表达的蛋白质组分；在SDS-PAGE分析的基础上，进一步采用双向电泳和质谱技术进行分析。结果表明，随着雌花的开放，蛋白斑点数目呈现先增加后减少的变化趋势，即盛花期检测到的蛋白斑点数最多，为471±4个，始花期和末花期分别检测到蛋白斑点数为450±15和446±14个。从雌花蛋白质2-DE凝胶中，选取25个分离清晰的差异表达蛋白质进行质谱分析，结果成功鉴定了其中的21个组分。通过生物学分析，这些蛋白组分在转录和翻译、细胞代谢、氧化还原、细胞构成、植物应答防御等生命活动过程方面的差异表达与桑树雌花的正常开放关系密切。（2）采用双向电泳和质谱技术对“大10”盛花期雌花的花柱（带柱头）、子房两个不同部位的蛋白质组分进行了分离和鉴定。结果表明，桑雌花不同部位的蛋白质表达谱存在着显著差异，从蛋白质2-DE凝胶中，选取28个分离良好的差异表达蛋白质进行质谱分析，结果成功鉴定了其中的26个。通过生物学分析，这些蛋白组分主要涉及胁迫应答反应、次生代谢途径、氧化还原反应以及蛋白质的生物合成和代谢等。说明桑树花器官不同部位蛋白组分的差异表达可能与花粉萌发、花粉管生长或其它异源物质的侵入密切相关。（3）以两个果桑品种“大10”和“SG01”为材料，采用双向电泳技术对授粉后雌花和成熟桑椹的蛋白质组分进行了分离。结果表明，不同品种间授粉后的雌花蛋白质表达存在较大差异。从品种间差异表达在2倍及2倍以上的斑点中，选取13个表达量较多且分离良好的蛋白质进行质谱分析，结果成功鉴定了其中的9个。通过生物信息学分析，这些蛋白组分参与了蛋白的合成与分解代谢、氨基酸代谢、糖代谢以及植物抗逆等。其次，不同品种间成熟桑椹的蛋白质表达谱存在着极显著差异，从品种间特异表达的蛋白斑点中选取20个表达量较多且分离良好的蛋白质进行质谱分析，5个获得成功鉴定。通过生物信息学分析，这些蛋白组分主要涉及植物应答反应、细胞构成、蛋白的合成与分解代谢等。研究结果说明不同果桑品种间发育相关蛋白质组的表达存在差异，并且从雌花发育到果实成熟的过程中，品种间的差异变得更为显著。综上所述，桑雌花发育过程中涉及到多种蛋白质的差异表达，这些蛋白功能分别涉及基因的转录和翻译、物质与能量代谢、氧化还原反应、细胞构成及应答反应等多种生命现象，对桑花器官、桑椹发育及其调控具有积极意义，为开展果桑分子育种研究提供了新的信息。更多还原

【Abstract】 Fruit mulberry cultivation has become a new field of the modern development ofsericulture industry, the growth and development of mulberry’s pistillate flower andfruit have already been gained more and more attention. In order to explore themolecular mechanism of pistillate flowering development and regulation, the proteomeexpression was investigated by using pistillate flowers from different florescence andposition, pistillate flowers after pollination and ripe fruit of mulberry varieties named“Da10”and “SG01”. Through these studies, we realized the function of different proteincomponents connected with the molecular genetics of pistillate flowers. The resultswere as follows:(1) Based on the SDS-PAGE technology and mass spectrometry, the differentbands protein components were detected from different florescence of mulberry’spistillate flower of “Da10”. In the further study with IPG-IEF/SDS-PAGE analysis, wefound the general numbers of protein increased and then decreased. And the quantity ofprotein components which were the utmost luxuriance of pistillate flowers achieved471±4, while starting and termination respectively achieves450±15and446±14. Result21protein components which were identified from25spots was clearly isolated in2-DE of pistillate flowers by using mass spectrometry techniques. Much has beenlearned from classical biochemical approaches about the expression of transcription andtranslation, cell metabolism, oxidation-reduction, cellulosity, and plant defenses wereclosely related to the normal blooming of pistillate flower in mulberry.(2) We chose the two-dimensional electrophoresis and mass spectrometry to isolateand identify the protein components from the style (with the stigma) and the ovary wasin its utmost luxuriance of pistillate flower of “Da10”. The results showed that theproteome from different position of pistillate flower had significant differentialexpression.28better separations of differentially expressed protein spots were selectedto be analyzed by mass spectrometry from2-DE, and26of them were successfully identified. Much has been learned from classical biochemical approaches about theseprotein components mainly related to plant stress responses, secondary metabolites,oxidation-reduction reaction, and biosynthesis and anabolism of protein, The resultsshowed that differentially expressed of the protein components were closely related topollen germination, growth of the pollen tube and the intrusion of other xenobiotic.(3) Moreover, we chose the two-dimensional electrophoresis to isolate the proteincomponents from pistillate flowering and had already pollinated and ripe fruit of “Da10” and “SG01”. The proteome from different varieties of pistillate flowers with alreadypollinated had significant differential expression.9protein components were identifiedfrom the expression difference in13proteins reached more than two times in differentvarieties by mass spectrometry. Much has been learned from classical biochemicalapproaches about these protein components mainly related to the synthesis andcatabolism of protein, amino acid metabolism, carbohydrate metabolism and plant stressresistances. Secondly, we found the proteome from different varieties of ripe fruit hadsignificant differential expression too.5protein components were identified from theexpression difference in20proteins by using mass spectrometry. Much has been learnedfrom bioinformation analysis approaches about these protein components mainly relatedto plant responses, cell composition, protein synthesis and catabolism. The resultsindicated that the protein components were significant different in varieties, and thedifferences get more obvious after pistillate flowers developmental into fruit.Above all, the development of pistillate flower involved in kinds of proteins whichrelated to a lot of phenomenon of life, gene transcription and translation, both matterand energy metabolism, oxidation-reduction reaction, cell composition and plantresponses. The discovery was fully meaningful for development and regulation, and it’sevident to me that it provided new information about studying the molecular breeding ofmulberry.更多还原