【作者】 邵媛媛；

【导师】 毕玉平； 柳展基；

【作者基本信息】 山东师范大学 ， 发育生物学， 2010， 硕士

【摘要】 植物在其生长过程中经常会遭受到不同程度的生物和非生物逆境胁迫,这些胁迫严重影响植物的生长发育,并对农业生产造成极大的负面影响。花生是重要的油料作物和经济作物。我国是世界上种植花生的主要国家之一,花生产区主要分布于干旱和半干旱地区。干旱严重影响花生的产量和品质,是花生生产的首要限制因子。我国有70%的花生面积受到不同程度的干旱危害,平均减产20%以上,造成巨大的经济损失。因此,解决干旱对花生生产的影响是当务之急。蛋白质组学可以研究许多基因组水平上无法体现的层面,如:基因表达的翻译水平、翻译后的各种蛋白质修饰以及蛋白质的相互作用等。近年来,植物蛋白质组学的研究日益增多,主要集中在拟南芥、水稻等模式植物上,研究范围包括在非生物因子的环境胁迫条件下的生理变化,如干旱、高盐、缺氧、ABA处理和机械损伤等,生物因子如菌根真菌以及与植物共生关系的根瘤菌对植物生长的影响。还有植物组织器官如根、茎、叶、花、果实、种子发育过程中的蛋白质变化。此外植物蛋白质组学研究已经深入到亚细胞水平,即研究在一个细胞器内表达的蛋白质组,这些细胞器包括叶绿体、线粒体、内膜系统等,而且还涉及植物群体遗传方面,包括基因多样性和表型多样性之间的关系,基因突变引起的蛋白质表达变化,植物激素的信号传导和作用机理等方面。花生是重要的油料作物,具有很高的营养价值,为异源四倍体植物,基因组庞大(约2.8×109 bp),其蛋白质组学的研究尚处于起步阶段。本研究以花生品种鲁花14为实验材料,PEG6000模拟干旱环境,提取叶片可溶性蛋白进行双向电泳分析。在三氯乙酸/丙酮沉淀法的基础上,对样品抽提、样品溶解、胶条pH范围选择、蛋白上样量、SDS-聚丙烯酰胺凝胶电泳等关键步骤进行改进,获得了蛋白点较多、重复性好的双向电泳图谱。花生叶片蛋白质双向电泳选用pH 4-7的IPG胶条,采用考马斯亮蓝染色法,在上样量为280μg时,得到效果较佳的电泳图谱。2-DE图谱上蛋白点数量约480个,图像清晰,蛋白点无明显拖尾现象,这为开展花生叶片蛋白质组研究奠定了基础。经过比对分析,发现了40个差异蛋白点,其中有17个上调表达,23个下调表达。然后利用质谱技术(MALTI-TOF MS)进行鉴定,并通过数据检索,得到了5个与干旱胁迫相关的差异蛋白质。更多还原

【Abstract】 Plant is often exposed to biotic and abiotic stresses of varying degrees in growth process which affected plant growth and development badly and cause huge negative effect to agriculture. Peanut is an important oil and cash crop. China is one of the most vital peanut-cultivated countries in the world. Peanut distributes mainly in arid and semi-arid regions. Drought significantly affects the yield and quality of peanut. It is reported that 70% of peanut area in China are in various degree of drought and the average decrease in yield was more than 20%. Therefore, it is essential to resolve the negative effect of drought stress to peanut production.The proteomics can be implied in many fields such as the protein expression level,the sequence of amino acids, the post-translational modification, the protein location and the protein-protein interaction, which cannot be solved by genomics. The proteome of many plants, such as Arabidopsis, rice and corn, has been studied. The physiological changes under various stresses, such as drought, high salt content, hypoxia, ABA treatment, and mechanical damage, have been investigated. Influence on plant growing by biologic factors such as mycorrhizal fungi and plant accompanied rhizobium, changes of protein in the growing process of plant’s tissue such as root, stem, leafe, fruitage, and seed. Proteomics has also extended its studying field to sub-cell level, meaning to study proteome inside organelles including chloroplast, mitochondria, Internal membrane system etc. Some other fields are also studied in Proteomics, such as the relationship between genotypic diversity and phenotypic diversity, protein diversification caused by gene mutation, mechanism of phytohormone and so on.The results show that both quantitative and qualitative changes of many proteins are induced under these stresses. Moreover, the study of plant proteomics can be conducted at the subcellular level.Peanut is an important oil crop and possess high nutritional value, it’s a kind of allotetraploid plant. The study of peanut at the genomic level, however, is limited due to its huge genome. The peanut proteomics is also at an initial stage.In the present study, leaf soluble proteins were extracted from a kind of peanut (Luhua 14) and were analyzed by dielectrophoresis. Well reproduced dielectrophoresis spectra with a large number of protein spots were obtained on the base of precipitation method using trichloroacetic acid and acetone as solvents. Sample extraction, protein dissolution, range of pH, protein amount, SDS-polyacrylamide gel electrophoresis and other key steps were improved. Better dielectrophoresis spectra were obtained when 280μg of protein was used and the pH of IPG adhesive tape was 4-7. About 480 protein spots were observed clearly from the 2-DE spectrum while no obvious tailing phenomena was observed. 40 protein diversity were found according to analysis and comparation, 17 of them are up-regulation of experssion and 23 of them are down-regulation of experssion.Five differential proteins corresponding to drought stress were confirmed through MALTI-TOF MS measurements and data retrieval. Although the results caused by in-gel digestion are unsatisfactory, the presented in this study contributes to the peanut leaf proteomics.更多还原