甜菜M14品系花器官特异表达蛋白质的研究

【作者】 曹洪祥；

【导师】 李海英； 陈思学；

【作者基本信息】 黑龙江大学 ， 生物化学与分子生物学， 2009， 硕士

【摘要】 植物无融合生殖是指不经精卵融合而以种子进行繁殖的一种特殊的生殖方式,它可以保存优异的基因型、固定杂种优势、改变育种生产程序,因此对无融合生殖的研究在农业生产和育种工作中具有深远的经济意义。目前关于植物无融合生殖的研究主要集中在基因组水平上,然而在基因组水平上所获取的基因表达的信息并不足以揭示该基因在细胞内的确切功能。因此要对生命复杂的活动有全面和深入的认识,直接对蛋白质的表达模式和功能模式进行研究就成为生命科学发展的必然趋势,从基因水平向蛋白质水平的深化已经成为生命科学研究的迫切需要和新的任务。甜菜M14品系是由栽培甜菜(Beta vulgaris L.)与四倍体白花甜菜(Beta corolliflora Zoss.)种间杂交,进一步回交获得的甜菜无融合生殖单体附加系,其染色体组除了含有18条栽培甜菜染色体外,还附加有白花甜菜第9号染色体,附加染色体的传递率高达96.5%,是研究无融合生殖极其难得的材料。本研究以具有无融合生殖特性的甜菜M14品系和正常栽培甜菜为研究对象,从蛋白质水平上对甜菜M14品系的无融合生殖特性进行了比较蛋白质组学研究,通过改进和优化甜菜花器官总蛋白的提取方法及双向电泳条件,建立了高质量的甜菜花器官总蛋白质的双向凝胶电泳技术平台,获得了图像清晰、分辨率高和重复性好的甜菜M14品系和正常栽培甜菜在不同pH梯度下的2-DE图谱。利用Imagemaster 2D Platinum 6.0图像分析软件对花器官总蛋白质双向电泳图谱进行比较分析,结果显示,在三次重复实验的甜菜M14 2-DE图谱上检测到了1109±48个蛋白质点,而在栽培甜菜2-DE图谱上检测到了1006±37个蛋白质点。通过定性和定量分析发现共有96个蛋白质点的表达发生显著的变化,其中在栽培甜菜中缺失而在甜菜M14中特异表达的蛋白质点18个,只在栽培甜菜中表达的蛋白质点5个;与对照组栽培甜菜相比,在甜菜M14中表达丰度减弱的蛋白质点46个,表达丰度增强的蛋白质点27个(P<0.05)。将96个差异表达的蛋白点进行质谱分析,其中72个蛋白点得到了成功的鉴定。使用BLASTx程序,将采用mRNA差异显示技术和抑制消减杂交技术已经获得的298个ESTs序列与质谱鉴定出的72个蛋白点进行比对,结果6个在甜菜M14和栽培甜菜中表达量差异的蛋白质点、2个在甜菜M14中特异表达的蛋白质点与ESTs序列具有显著性匹配(E<1e-7)。根据Bevan等建立的蛋白质功能分类体系,对已经鉴定的差异表达蛋白质进行功能分析,结果表明,这些差异表达的蛋白质分别属于代谢相关蛋白、能量相关蛋白、蛋白质合成相关蛋白、胁迫和防御相关蛋白等十大类。这些差异表达蛋白的鉴定为全面、深入揭示甜菜M14品系无融合生殖特性的分子机制和蛋白质表达特性奠定了良好的基础。更多还原

【Abstract】 Apomixis is an asexual reproductive process by which plants produce seed without fertilization through female syngamy that produces embryos genetically identical to the maternal parent. It preserves heterozygosity vigor , maintains superior genotypes and changes the breeding production process by parthenogenetic embryo development. Therefore, the research on apomixis has far-reaching economic significance in the agricultural production and breeding work.The plant apomixis research to date mainly focuses on the level of the genome, but the gene expressed information obtained from the level of the genome is not sufficient to reveal the exact function of the gene. Therefore, in order to have comprehensive and thorough understanding to the life complex activity, the research on protein expression patterns and functional mode of life sciences has become the inevitable trend of development.Apomictic monosomic addition line M14 of Beta corolliflora Zoss. was the progeny from the hybridization of Beta vulgaris L. and Beta corolliflora Zoss. in sugar beet. Constituted of the normal 18 Beta vulgaris L. chromosomes with the No.9 chromosome of Beta corolliflora Zoss., M14 was found having a chromosome transmission frequency 96.5%, thus M14 is a precious material for research of apomixis. This research take apomictic monosomic addition line M14 and the normal cultivation beet as materials, and was carried on differential proteomic analysis by two-dimensional gel electrophoresis technology. Through improving and optimizing the floral organs of sugar beet protein extraction method and two-dimensional gel electrophoresis conditions,a high resolution and reproducible 2-DE analysis systerm was established, and we have generated high resolution and reproducible 2-DE maps for two type floral organs of sugar beet with different pH range.Using Imagemaster 2D gel analysis software, 1109±48 spots and 1006±37 spots were reproducibly detected across three replicate gels of apomictic and sexual floral organs of sugar beet, respectively. Ninety-six protein spots showed reproducible and statistically significant changes in M14 compared to B. vulgaris. Of them, 18 were unique to apomictic floral organs and five were only presented in sexual floral organs. Of the 830 common spots, 27 were found to be up-regulated and 46 were down-regulated in apomictic floral organs compared to sexual floral organs when p-value of 0.05 was used as the threshold. Protein spots from the gels were subjected to in-gel digestion and MS analysis. A total of 72 protein spots were identified by MS and MASCOT database searching. All of the unique ESTs sequences obtained by SSH and DDRT-PCR experiments were compared to the protein sequences identified by proteomics using BlastX. With an E-value less than 1e-7 as a criterion for significance, six proteins which were differences in protein expression between M14 and B. vulgaris, and two proteins only expressed in M14 showed significant match.The identified proteins were classified into 10 groups according to the functional categories established by Bevan et al. The results showed that the differentially expressed proteins involved in a wide range of cellular processes, such as metabolism, energy, protein synthesis, stress and defense etc.Identification of the differentially expressed proteins provides a good foundation for comprehensive and in-depth study of the apomixis molecular mechanism, and specific protein expression pattern in monosomic addition line M14.更多还原