【作者】 陈蕊红；

【导师】 张改生；

【作者基本信息】 西北农林科技大学 ， 作物遗传育种， 2008， 博士

【摘要】 小麦是世界上最重要的农作物，也是我国仅次于水稻的主要的粮食作物。小麦杂种优势的研究与利用是大幅度提高小麦产量和品质的重要途径，为了更好地利用小麦杂种优势为农业发展服务，小麦雄性不育和杂种优势构成机理的探讨多年来一直是本领域的科学焦点。蛋白质组学研究是功能基因组学研究的主要内容之一，双向凝胶电泳是进行蛋白质组学研究的关键技术之一。为了能从蛋白质水平来揭示小麦雄性不育的机理，本研究以质核互作型雄性不育系(S)-1376A及其保持系(A)-1376B为材料，采用双向凝胶电泳研究技术，对小麦花药全蛋白质组学及其线粒体蛋白质组学进行了系统深入的研究，获得如下重要结果：1．以小麦单核期花药为材料，首先对小麦花药蛋白质组双向电泳技术体系进行了优化，结果表明，以TCA-丙酮法提取小麦花药组织中的蛋白，用蛋白质裂解液Ⅱ[7 mol／L尿素、2 mol／L硫脲、4％CHAPS、2％TBP、65 mmol／L DTT、0．2％载体两性电解质(其中0．1％pH 3～10，0．1％pH4～6)]溶解蛋白，以pH4～7 17cm的IPG胶条进行双向凝胶电泳，在上样量为800μg，13％SDS-PAGE胶浓度下，蛋白质得到了更好的分离，2-DE图谱上可分辨出602-631个蛋白点，图谱质量最佳。2．以IEF／SDS-PAGE双向凝胶电泳技术，对小麦质核互作型雄性不育系(S)-1376A及其保持系(A)-1376B在花药发育的单核期、二核期蛋白质进行了差异蛋白质组学研究。在分子量9．0～100．0 kD、等电点4～7线性范围内，可识别约610个蛋白质点，PDQuest软件分析结果表明：在单核期不育系和保持系之间存在40个差异蛋白质点，二核期两者共有49个差异点；不育系在单核期和二核期两者之间存在有15个差异点，保持系在两个不同时期有16个差异点；不育系与保持系间在表达量上的差异蛋白质点以及不育系与保持系间特异表达或特异缺失的蛋白质点很可能与小麦雄性不育有关。3．对28个差异表达的蛋白质点采用基质辅助激光解吸分离飞行时间质谱进行肽指纹图谱分析(MALDI-TOF-MS)，并利用Mascot软件在NCBInr数据库搜索，鉴定出了19个蛋白质点，9个蛋白质点未得到鉴定，19个蛋白质点分别被鉴定为苏氨酸合酶，泛素结合酶E2，甘氨酸富集蛋白，半胱氨酸蛋白酶抑制剂，1，5-二磷酸核酮糖羧化酶／加氧酶小链克隆512，抗坏血酸过氧化物酶，磷酸丙糖异构酶，4个蛋白质点被鉴定为同一个蛋白即1，5-二磷酸核酮糖羧化酶／加氧酶小亚基，2个蛋白均为光系统Ⅱ放氧蛋白，假定的果糖-1，6-二磷酸醛缩酶，三个假定蛋白及一个功能未知的蛋白，鉴定的蛋白质点其功能涉及到物质能量，糖代谢，蛋白质降解，细胞防卫等代谢过程，小麦雄性不育的发生很可能与这些代谢体系相关。4．线粒体是植物细胞能量转换的重要器官，其在核酸，氨基酸、脂肪以及维生素、辅因子等生物合成方面也起着重要的作用，同时线粒体与植物雄性不育的发生也有着密切的关系。本研究采用差速离心及Percoll密度梯度离心法对小麦幼穗线粒体进行了分离，经过一次不连续密度梯度离心后，线粒体在23％及45％的Percoll层形成了一个浅黄色的线粒体环，酶活性及叶绿素含量监测结果表明，小麦幼穗线粒体主要有过氧化氢体，质体和叶绿体的污染，而作为对照的小麦黄化苗无叶绿体的污染，仅有少量的过氧化氢体及质体的污染。为了获得高纯度的线粒体，对分离的小麦幼穗线粒体再进行了一次28％Percoll自形成密度梯度离心，结果表明，分离的线粒体中无胞液的污染，仅有微量的过氧化氢体、叶绿体及质体的污染，线粒体的完整性也从88％升至90％，并保持有较高的活性。5．对分离纯化的线粒体蛋白进行了双向凝胶电泳，在pH3～10，13cm IPG，上样量80ug条件下，通过银染显色，可在双向电泳图谱上分辨出306～327个清晰的蛋白质点，获得了重复性高，分辨率较好的双向凝胶电泳(2-DE)图谱；对分离纯化的小麦雄性不育系(S)-1376A及保持系(A)-1376B单核期幼穗线粒体蛋白进行了线粒体蛋白质组比较分析，经过PDQuest8．0．1分析，共获得11个差异蛋白质点，其中5个在不育系中上调表达，2个下调表达；仅在不育系中特异表达的点1个，仅在保持系中表达的3个。对其中5个差异蛋白质点进行了基质辅助激光解吸分离飞行时间质谱进行肽指纹图谱分析(MALDI-TOF-MS)，并利用Mascot软件在NCBInr数据库搜索，其中spot 1，4蛋白质点被鉴定为锰型超氧化物歧化酶(Mn-SOD)，锰型超氧化物歧化酶是由核编码的存在于线粒体中的一种清除线粒体氧自由基抗氧化酶系，Mn-SOD在雄性不育系中的缺失很可能与小麦雄性不育的发生有关。更多还原

【Abstract】 Wheat (Triticum aestivum L.) is the most important grain crop in the world.Heterosisutilization of wheat are important approaches to increase wheat yielding and quality.To betterutilize the wheat heterosis to serving for agriculture development,the mechanism of malesterility are the field of research focus for many years.Proteomics is one of the importantresearch of the functional genomiese.To better understand the molecular mechanism onprotein level and find the crucial proteins which related to fertillity,we use thecytoplasmic-nuclear male sterility line (S)-1376A and its maintainer line (A)-1376B as theplant materials,the differential proteomic analyses of anther and the mitochondrial proteomeof spike in wheat are systematically studied,the important results are as follows:1.We firstly optimized the two-dimensional electrophoresis conditions on the uninucleateanther stage of wheat,the results showed that,the proteins extracted by the TCA-acetonemethod,dissolved protein with the lysis bufferⅡ,with pH 4～7 srips,800μg loading quantityand 13% concentrations of the SDS-PAGE gel,the proteins were well separated,total601-631 protein spots were detected on the 2-DE gels map.The optimized condition of 2-DEwas suitable for proteomic analysis of wheat anthers.2.Total anthers proteins were extracted from uninucleate and binucleate stage of(S)-1376A and (A)-1376B for IEF/SDS-PAGE.An higher repartability and better separatingprotein method from wheat anther for 2-DE was established.The results showed that about610 protein spots could be visualized on the two dimensional electrophoresis map bycoomassie brilliant blue staining within Mr 9.0～100.0kD and pH 4～7,and about 94 spotswere differential expressed between male-sterile line and its maintainer line by PDQuestsoftware.Among those differential proteins,about 49 spots and 40 spots were differentialexpressed between uninucleate anther stage and binucleate stage respectively;Meanwhilecomparing with the 2-DE maps of the total proteins from the binucleate anther stage anduninucleate stage,15 differential spots were detectable at male-sterile line;and 16 differentialspots were detectable at its maintainer line.These diffence proteins which up or down-regula-ted protein and specific expressed or not expressed proteins between male-sterile line and itsmaintainer line maybe related with cytoplasmic male sterility of wheat. 3.A total of 28 differentially expressed proteins were identified by matrix-assisted laserdesorption ionization time of flight mass spectrometry(MALDI-TOF-MS),19 of them wereidentified following NCBInr database queries,which are ubiquitin-conjugating enzymeE2,putative glycine-rich protein,ascorbate peroxidase,putative cysteine proteinase inhibitor,ribulose bisphosphate carboxylase small chain clone 512,triosephosphate isomerase,two ofthem which were identified is 23KD subunit of oxygen evolving system of photosystemⅡ,4proteins were identified with a same protein-ribulose-l,5-bisphosphate carboxylase/oxygen-ase small subunit,putative fructose 1-,6-biphosphate aldolase,three hypothetical proteins andone unkonwn protein.These proteins were involved in the process of energy metabolism,protein degradation,cell defense,regulating flower development.These results provide a cluefor elucidating the mechanism of CMS.4.Plant mitochondria play a primary role of energy-conversion in a cell system,and alsoperform many other secondary functions including synthesis of nucleotides,metabolism ofamino acids and lipids,biosynthesizing vitamins and cofactors,meanwhile,mitochondria alsoplay a unique role in expression of plant CMS.Isalation of high pure mitochondria fromwheat is one of the key techniques in CMS mitochondria proteomic analyse.Mitochondriafrom wheat young spike were isolated by a differential centrifugation and Percoll density-gradient method.The three-step discontinuous Percoll density gradient centrifugation resultedin the formation of a mitochondrial ring at the interface of the 23% and 45% Percoll layers.Determined by marker enzyme assays and chlorophyll content,the mainly contaminants inthe spike mitochondrial fraction were caused by peroxisomes,plastids and chloroplasts whileetiolated seedling was free of chloroplasts contamination but with a slight content ofperoxisomes,plastids.Improvement in purity was obtained by a 28 %(v/v) Percoll self-forming density gradient centrifugation on the mitochondrial fraction from spike,these cont-aminants were decreased to negligible amount and the integrity of mitochondria (88%) wasimproved to 90%,and the mitochondria still kept activity well after gradient centrifugation.5.The large sets of soluble protein of the spike mitochondria was extracted for IEF/SDS-PAGE,the results showed that about 326 protein spots could be visualized on the twodimensional electrophoresis map by silver staining,It indicated that the protocol of puremitochondria isolation was found to be reliable and suitable for proteomic analyses for thespecific materials.Compared the 2-DE maps of the male sterile line and the maintainer line,11 spots were differentially expressed,5 of them were up-regulated and 2 of them were down-regulated in the male sterility line of (S)-1376A,3 were specific expressed in the maintainerline of (A)-1376B,1 was specific expressed in male sterility line of (S)-1376A.The diffenceproteins which up or down-regulated protein and specific expressed between male-sterile line and its maintainer line maybe related with cytoplasmic-nuclear male sterility of wheat.A totalof 5 differentially expressed proteins were identified by matrix-assisted laser desorption ioni-zation time of flight mass spectrometry(MALDI-TOF-MS),only spot1,4 of them wereidentified following NCBInr database queries,are manganese superoxide dismutase(Mn-SOD),MnSOD is a nuclear-encoded antioxidant enzyme that localizes to the mitochondriaand is the major antioxidant defense enzyme in mitochondria to protect mitochondrialcomponents from superoxide liberated,the absence of this protein in male sterile linesuggested that the constitutive level of antioxidant capacity was impaired,which might berelated to the cytoplasmic sterility.更多还原