【作者】 萧小鹃；

【导师】 刘选明； 林辰涛；

【作者基本信息】 湖南大学 ， 分析化学， 2008， 博士

【摘要】 蛋白质组学研究在功能基因组时代发挥着越来越重要的作用。蛋白质组研究旨在研究基因组编码的整套蛋白质的结构和功能,从而补充转录组的研究。目前,基因突变引起的蛋白质表达变化是蛋白质组学研究的重要内容。拟南芥和水稻是植物分子生物学研究中两种重要的模式植物。本文以新筛选的拟南芥突变体和实验室已有的水稻突变体为材料,利用蛋白质组学方法分别研究了它们发育突变体中蛋白质表达谱的变化,获得了以下主要研究结果。(1)拟南芥发育突变体的筛选研究以拟南芥蓝光受体双突变体cry1cry2为实验材料,通过激活标签技术构建了T-DNA插入突变体库,并从中筛选到6个与开花时间有关和2个矮化的发育突变体。采用质粒拯救(plasmid rescue)、IPCR(inverse PCR,反向PCR)和TAIL-PCR(Thermal asymmetric interlaced-PCR,热不对称巢式PCR)等方法,分析得出了这些发育突变体T-DNA插入位点的基因组侧翼序列。通过RT-PCR方法分析了突变体T-DNA插入位点两侧基因的表达情况,结果表明突变体表型的变化可能是T-DNA插入后35S强启动子引起附近的基因表达量增加造成的,从而进一步证实了这些发育突变体的突变分子基础。通过筛选发育突变体和鉴定相关基因,为深入研究植物光周期开花调控以及下胚轴伸长抑制的机理奠定了基础。(2)水稻和拟南芥育性突变体的蛋白质组学研究通过双向电泳、MALDI-TOF质谱、MALDI-TOF-TOF质谱等技术比较分析了水稻株1S不育和可育条件下花粉母细胞形成期,减数分裂期以及花粉内容物充实期(第Ⅴ、Ⅵ、Ⅶ期)的幼穗蛋白的动态变化过程。结果发现水稻株1S在不育和可育条件下,分别在幼穗第Ⅴ期有43个差异蛋白质点,第Ⅵ期有32个差异蛋白质点,第Ⅶ期有23个差异蛋白质点。选取其中50个蛋白用于质谱鉴定,最后有20个差异蛋白质点得到了鉴定,分析后发现这些蛋白主要参与了糖代谢,能量代谢,蛋白质的生物合成,细胞壁形成和胁迫响应等与花粉发育相关的过程。通过RT-PCR方法对其中3个蛋白质对应基因进行表达分析,结果发现其中1个蛋白质的表达与相应mRNA水平的表达相一致,另外2个蛋白质的表达与其mRNA水平的表达不一致,表明后2个蛋白质对应基因可能由于基因表达存在翻译后修饰。我们共得到了8个与育性相关的蛋白,其中UDP-葡糖醛酸脱羧酶、咖啡酰辅酶A-氧-甲基转移酶和OSJNBa0060D06.10蛋白与育性转换紧密关联。这些分析结果表明温度控制水稻的育性是通过共调节相关基因的表达而实现的,初步从蛋白质水平阐述了温敏核不育水稻育性转换的分子机理。选取筛选到的拟南芥晚开花突变体scc106-D和对照材料cry1cry2作为研究对象,比较了两者生长在白光下的蛋白质水平差异,通过质谱分析最后鉴定得到了22个差异蛋白质点,并发现其中17个蛋白在scc106-D突变体中同时下调,而另有3个蛋白仅在cry1cry2中表达。分析鉴定得到的差异蛋白发现,它们主要参与了碳水化合物代谢、能量代谢、硫代谢、光合作用、光呼吸、胁迫响应和蛋白质的合成等过程,其中光合蛋白占36%,能量相关蛋白占14%。结果说明光合速率下降和能量降低可能是使突变体发育迟缓,开花延迟,育性降低,下胚轴变短的主要原因。这些结果提示,T-DNA插入后35S强启动子引起附近的基因表达量增加,在光作用下调节相关基因的表达从而导致突变体表型的变化。(3)水稻和拟南芥矮化突变体的蛋白质组学研究采用双向电泳技术分离水稻株1S、矮秆突变体SV14和湘陵628S的茎蛋白,通过质谱鉴定得到了33个差异蛋白质点。其中在矮秆突变体SV14中27个蛋白点下调,1个仅在SV14中表达,2个仅在株1S中表达:在湘陵628S中25个蛋白点下调,1个仅在湘陵628S中表达,3个仅在株1S中表达。通过分析可以将这些差异蛋白分为两大类:第一类是调节性蛋白,如咖啡酸三氧转甲基酶,热激蛋白70,醛酮变位酶I,肌动蛋白等,它们主要参与蛋白质的合成和加工、胁迫响应、细胞骨架稳定等过程;第二类是功能性蛋白,如抗坏血酸过氧化物酶,细胞质苹果酸脱氢酶,细胞质磷酸甘油酸激酶,S-腺苷蛋氨酸合成酶,ATP合酶等,它们则参与氧化还原平衡、光合作用、光呼吸和各种代谢等过程。调节性蛋白丰度的下调可能引起了水稻发育的迟缓,功能性蛋白活性和丰度的改变,则主要破坏水稻植株细胞内的代谢平衡,两者共同作用从而最终导致茎变短和植株变矮。该结果从蛋白质表达水平阐述了水稻植株矮化的内在原因,为进一步开展水稻矮秆分子育种奠定了基础。选取筛选到的矮化突变体scc8-D和对照材料cry1cry2作为研究对象,比较了两者生长在白光下的蛋白质水平的差异。通过质谱分析共鉴定得到了22个差异蛋白质点,其中有16个蛋白在scc8-D突变体中都是下调的,同样有3个蛋白仅在cry1cry2中表达。经分析发现这些差异蛋白可能参与了碳水化合物代谢、光合作用、光呼吸、胁迫响应、蛋白质的合成和氧化还原平衡等过程,其中光合蛋白占32%。此结果进一步说明调节性蛋白和功能性蛋白丰度的表达受到光调控从而抑制植物下胚轴的伸长。更多还原

【Abstract】 Proteomics study is playing an increasingly important role in the functional genomics era. Proteome studies aim at the complete set of proteins encoded by the genome and thus complement the transcriptome studies. At present, the change at the protein expression level caused by gene mutation is one of the most important parts in proteome studies.Rice and Arabidopsis are two important model plants. In the present study, we analyzed the difference at the protein expression level in rice development mutants and Arabidopsis development mutants screened. The results of this study were listed as follows:(1) The screening of Arabidopsis development mutantsWe used Arabidopsis mutant cry1cry2 as material in this paper and constructed a library of arabidopsis mutants by activation tagging. Through screening and observation, some mutants with visible morphological phenotypic variations were obtained, including six mutants related to flowering time and two dwarf mutants. Plasmid rescue, inverse PCR (IPCR) and Thermal asymmetric interlaced-PCR (TAIL-PCR) were used to identify the flanking genomic sequences of mutated target genes. At the same time, the semi-quantitative RT-PCR was used to analyse expression of the T-DNA insertion flanking genes.We conclude that phenotypic variations in mutants were caused by the increased expression of T-DNA insertion flanking genes.The result laid the foundation for further study of regulation of flowering time and inhibition of hypocotyl elongation.(2) Proteome analysis of rice and Arabidopsis sterile mutantsWe used proteomic analysis to investigate the changing patterns of young panicle proteins during different developmental stages [the formation stage of pollen mother cell(Ⅴ), the stage of meiotic division of pollen mother cell(Ⅵ), the filling stage of pollen (Ⅶ)] under sterile and fertile conditions. Proteins were extracted from the spikelets samples and separated by 2-D gel electrophoresis (2-DE). Image acquisition and analysis, under sterile and fertile conditions, 43 protein spots were differently expressed in Zhu-1S during No.Ⅴstage; 32 protein spots were differently expressed in Zhu-1S during No.Ⅵstage; 20 protein spots were differently expressed in Zhu-1S during No.Ⅶstage. Based on spot quantity and quality, 50 protein spots were analyzed by matrix associated laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) and 20 spots were identified. Most of these proteins are closely associated with sugar metabolism, energy metabolism, protein biosynthesis, cell wall formation and stress responses, which are essential cell activities to the pollen development. The expression of genes corresponding to three protein spots was analyzed by semi-quantitative RT-PCR. One of them the mRNA and protein levels was correlated well confirmed, but the other two in some instance, the level of mRNAs and the expression level of the related proteins were inconsistent. This difference could be affected by post-translational modifications. We identified 8 proteins related to fertility, of which UDP-glucuronic acid decarboxylase, putative caffeoyl-coA O-methyl- transferase 1 and OSJNBa0060D06.10 were closed to fertility change. The results indicate that temperature control of rice fertility is co-regulated by many other related genes and elucidate the molecular mechanism of thermo-sensitive genic male-sterile rice fertility change at the protein level.To study the difference at the protein expression level of the cry1cry2 and scc106-D, we used a proteome approach based on 2-DE to compare their protein patterns. The 22 different protein spots were identified by MALDI-TOF-TOF-MS. There are 17 protein spots were decreased in scc106-D and 3 protein spots only appeared in cry1cry2. The identified proteins are involved in several processes, i.e. protein biosynthesis, stress responses, photosynthesis, photorespiration and metabolisms of carbon, sulfur and energy. Most proteins showed enhanced degradation in scc106-D, especially 36% photosynthetic proteins such as Rubisco large subunit and 14% proteins related with energy. The decrease of photosynthesis rate and energy may be the cause of dwarf, sterile, later flower of mutant. The results indicate that light control of phenotypic variations in mutants caused by the increased expression of T-DNA insertion flanking genes is co-regulated by many other related genes.(3) Proteomic analysis of rice and Arabidopsis dwarf mutantsThe stem of thermo-sensitive genic male-sterile rice Zhu-1S and its dwarfing variant SV14 and H628S were separated by 2-DE. 33 differential spots were identified by MALDI-TOF-TOF-MS. There were 27 protein spots were decreased in SV14 and 2 protein spots only appeared in Zhu-1S. There were 25 protein spots were decreased in H628S and 3 protein spots only appeared in Zhu-1S. The identified proteins were divided into two following categories: one is regulatory proteins, for example, caffeic acid 3-O-methyltransferas, HSP70, glyoxalase I , actin, and so on. They are involved in protein biosynthesis and processing, cellular structural organization and stress responses. The other is function proteins, for instance, ascorbate peroxidase, cytoplasmic malate dehydrogenase, cytosolic phosphoglycerate kinase, S-adenosyl methionine synthetase, ATP synthase, and so on. They are involved in photosynthesis, photorespiration, redox homeostasis, and metabolisms. The regulatory proteins promoted the devolopment of stem and their decrease impacted the stem elongation. The decrease of function proteins destroyed the metabolism homeostasis in rice and impacted the stem elongation. The results elucidate the molecular mechanism of rice dwarf at the protein level and lay the foundation for further study of molecular breeding of dwarf rice.To study the difference at the protein expression level of the cry1cry2 and scc8-D, we used a proteome approach based on 2-DE to compare their protein patterns. The 22 different protein spots were identified by MALDI-TOF-TOF-MS. There are 16 protein spots are decreased in scc8-D and 3 protein spots only appeared in cry1cry2. The identified proteins are involved in several processes, i.e. redox homeostasis, stress responses, photosynthesis, photorespiration, and metabolisms of carbon. 32% proteins showed enhanced degradation in scc8-D, especially the photosynthetic proteins such as Rubisco large subunit. The results suggest that the inhibition of hypocotyl elongation was caused by the decreased expression of regulatory proteins and function proteins, which is under light control.更多还原