【作者】 孟令波；

【导师】 杨传平； 王柏臣；

【作者基本信息】 东北林业大学 ， 林木遗传育种， 2008， 博士

【摘要】 种子萌发是植物生长发育过程中的早期阶段,是其个体发育中最为关键的时期。这一发育过程是一系列基因表达的综合体现,涉及到一系列复杂的生理生化代谢反应和诸多信号分子的传导。对这一过程的分子机制的探讨,一直是现代植物分子生物学研究的重要课题。蛋白质组学的出现和发展,为研究种子萌发机理提供了有效的手段。目前种子萌发的蛋白质组学研究主要集中在拟南芥、水稻等少数测序的模式植物上,作为重要的遗传学模式植物的玉米在此方面的研究还缺少相关报道。开展玉米(Zea Mays)种子萌发的蛋白质组学分析工作,不仅对探讨玉米(Zea Mays)萌发的机理,完善种子生物学研究,解决林木种子萌发难题有非常重要的意义,而且对于深入理解复杂基因组植物的分子遗传学特征及指导其育种研究也具有重要价值。另外,为没有完成或根本未进行基因组测序的重要生物进行前瞻性蛋白质组研究,进而推动其基因组研究提供新的思路和经验。本文从三个方面进行研究,结果如下:1.采取基于2-DE电泳、考马斯亮蓝染色、质谱鉴定的经典蛋白质组学的方法,对玉米种子萌发过程中胚和胚乳的蛋白质变化分别进行分析。在胚中鉴定到96个萌发中发生变化的蛋白质点,胚乳中鉴定到33个。对此分析,得到的结论是:胚的吸水对于种子的萌发是第一事件,静态的玉米种子在众多细胞发生吸胀作用后,其蛋白质组发生了巨大的变化;不同功能蛋白质的变化模式,可以反映玉米种子不同组织在萌发过程中的生化反应和代谢过程的特点;玉米种子萌发,蛋白质水平上的变化先于转录水平上的变化,而且胚的蛋白质变化模式与胚乳中存储产物的分解没有关系;胚中贮藏蛋白的降解和胚乳中的贮藏蛋白的降解具有不同的变化模式,胚中的蛋白质组的变化可能早于胚乳中的蛋白质组变化;0h到24h的快速吸水期、24h到42h的种子萌发期、48h到60h的胚根伸长后期,这3个阶段是种子萌发的关键时期。我们推测了,LEA(胚胎发生晚期丰富)蛋白在萌发前期的降解对激活干种子的新陈代谢具有重要的作用,消除种子中的ABA(脱落酸)影响是萌发中的重要事件。通过此项研究,使我们了解了玉米种子的不同组织细胞在萌发过程中的蛋白变化情况,揭示了它们在萌发过程中生理生化变化和相互间作用的分子机制。2.基于第一方面的研究结果,我们采用经典蛋白质组学的方法,探讨了在萌发过程中不同程度的NaCl胁迫对玉米种子胚和胚乳蛋白质变化的影响。根据不同浓度的NaCl溶液对玉米种子萌发抑制程度,确定玉米种子萌发NaCl的伤害阈值和存活阈值分别为0.1 mol/L、0.2 mol/L,分别用此浓度处理玉米种子。蛋白质组学分析表明,NaCl胁迫下种子贮存物质活化的延迟和能量代谢的紊乱是抑制种子萌发的主要原因;NaCl胁迫引起的生理干旱和离子毒害,导致许多保护蛋白和相关蛋白的表达,并参与大规模的防御代谢途径,但这些途径只在低盐浓度下(NaCl伤害阈值以下)起作用,在高盐浓度(NaCl存活阈值)这些途径也受到伤害;NaCl对种胚的蛋白质影响比胚乳大。此项研究使我们从时空变化的角度,更加深入、全面的理解盐胁迫的对玉米种子萌发伤害机制及其对盐胁迫的适应机制。3.为了更好的理解可逆蛋白磷酸化在种子萌发中的作用,我们研究了萌发的玉米种子胚的磷酸化蛋白质组。通过强阳离子柱结合鸟枪法共鉴定了776个蛋白质,其中包括39个磷酸激酶,16个磷酸酯酶以及含有36个体内磷酸化位点的33个磷酸化蛋白质。除了先前报道的HSP22磷酸化位点外,本试验所鉴定的其它磷酸化位点均未见文献报道。通过QRT-PCR测定,发现在种子萌发期间15个激酶基因的转录是明显上调的,萌发后4个磷酸酶基因转录是上调的。这些结果表明在玉米种子萌发过程中,可逆蛋白质磷酸化及其所调控的复杂网络系统被激活。而且在所鉴定的磷酸化蛋白中至少有三分之一是作为重要的生物学元件在此过程中发挥关键作用。例如DNA修复、基因转录、RNA剪接、蛋白质翻译等。这充分说明蛋白质磷酸化在种子萌发过程中发挥着重要的作用。据我们所知,本试验是首次在单子叶植物中进行磷酸化蛋白质组学研究,为进一步揭示种子萌发及苗期发育的分子生物学机制奠定坚实的基础。更多还原

【Abstract】 Seed germination is the early stage of growth and development of plants and key period of individual development.This process is comprehensive expressions of genes.A series of complex physiological and biochemical responses and a number of molecular signal transductions are involved in this process.It is one of the most important issues of modern plant molecular biology to study the molecular mechanisms of seed germination.The emergence and progress of proteomics provide a good means for studying the mechanisms of seed germination.At present,few proteomic works have been performed on maize(Zea Mays) being an important food and energy crops except on model plant Arabidopsis,rice and other minority of model plants.The proteomics research work on maize seed germination has very important significant not only for exploring the mechanism of germination and improving seed biology research but also for deeply understanding characteristics of molecular genetics of complex genomic plants.It also has great value for guiding breeding research work. Furthermore,it provides new ideas and experiences for prospective proteome study and further promoting genome research of important biology which genome sequencing has not been completed or even has not been done.This work was carried out in three aspects and following results were obtained.1.The study was carried out based on classical methods of proteomics- 2-DE electrophoresis,stained Coomassie Brilliant Blue and MS identification,protein changes in embryo and endosperm were analyzed respectively during maize seed germination.96 proteins spots in embryo were detected and showed variations during seed germination.And 33 protein spots in endosperm were identified.This analysis showed that it was the first event to absorb water by seed.After many cells of static corn seeds imbibed,their proteome had undergone tremendous changes.The changes of different functional proteins could indicate reactions and metabolic characteristics of different tissues during germination.Here,the results indicated that changes of protein were in advance of the changes of transcription.Moreover the changes of protein in embryo were not related to decomposition of storage substance in endosperm. Degradation of storage protein in embryo had different patterns compared with those in the endosperm.The change of proteins in embryo could be earlier than that in endosperm.Rapid water uptake is from Oh to 24h,seed germination is from 24h to 42h and later radical elongation is from 48h to 60h.The three stages were the key periods for seed germination.It was suggested that degradation of protein LEA in early stage of germination played an important role in activating metabolism of dry seeds.It was an important event to eliminate impact of ABA in seed during germination.Therefore,this research work shows protein changes in cells of different tissues during maize seed germination it also reveals molecular mechanisms of their physiological and biochemical changes and interactions in seed germination.2.Based on results of first aspect,classical proteomics methods were conducted to study effect of different concentrations of NaCl on protein changes in embryo and endosperm during maize seed germination.Damage threshold and survival threshold values of NaCl were 0.1 mol/L and 0.2 mol/L according to inhibition of different concentrations of NaCl solution on maize seed germination.Seeds were treated by these concentrations respectively.Proteomics analysis showed that the delaying of storage mobilization and disorder of decomposition of the energy metabolism were the main reasons for inhibition of seed germination under NaCl stress. NaCl stress resulted in physiological drought and ion poisoning,which led to expressions of related proteins and protection proteins.These proteins were involved in large-scale defending metabolic pathways.However,these pathways could work under low NaCl concentrations (below threshold values).They would be injured under high NaCl concentrations(survival threshold values).In addition,we found that the effect of NaCl on embryo was stronger than that on endosperm.This research work made us deeply understand temporal and spatial changes of toxicological and adaptation mechanisms of maize seed to salt stress.3.In order to better understand the role of reversible protein phosphorylation played in seed germination,we initiated a phosphoproteomic investigation of the embryo of germinated maize seed.A total of 776 proteins including 39 kinases,16 phosphotases,and 33 phosphoproteins containing 36 precise in vivo phosphorylation sites were identified.Except for the phosphorylation site on HSP22 which was reported previously,all the phosphorylation sites identified were never documented.Assayed with QRT-PCR,the transcripts of 15 kinase genes were found to be dramatically up-regulated during seed germination and those of 4 phosphotase genes were up-regulated after germination,indicating that reversible protein phosphorylation occurs and complex regulating networks are activated during this period. Among these phosphoproteins,at least one third is key components involved in the important biological processes which are related to seed germination,such as DNA repairing,gene transcription,RNA splicing,protein translation,indicating that protein phosphorylation plays important roles in seed germination.As far as we know,this is the first phosphoproteomic study on monocot plant and it will lay a strong foundation for further study of the molecular mechanisms of seed germination and the consecutive seedling development.更多还原