【作者】 李莉；

【导师】 张改生；

【作者基本信息】 西北农林科技大学 ， 种子工程， 2010， 硕士

【摘要】 杂种优势是生物界普遍存在的自然现象,作为世界第一大粮食作物,小麦有着明显的杂种优势,如何有效的利用其杂种优势,是我国现阶段提高小麦产量的一条重要途径。目前,小麦生产上杂种优势利用的途径主要有:(1)化学杀雄进行杂交制种;(2)采用雄性不育系进行杂交制种。但其雄性不育机理研究已成为小麦杂种优势利用中的一个科学难关仍在探索之中,近年将其焦点多集中在细胞核和线粒体中一些与育性相关因子的解析与研究。而本试验则以杀雄剂诱导的生理型雄性不育系ms(A)-西农1376、遗传型雄性不育系ms(S)-西农1376及其对应可育系MS(A)-西农1376单核早期的小花为试材,经差速离心结合蔗糖密度梯度离心技术先提纯小花完整叶绿体;然后,通过TCA-丙酮法沉淀蛋白,利用IEF/SDS-PAGE双向电泳技术进行差异蛋白比较;最后胶内酶切进行挖点、生物质谱测序技术以及生物信息学数据库检索技术,对小麦小花在花粉小孢子萌发的单核早期叶绿体蛋白质进行了研究,得出以下重要结果:1、利用不同方法提取了供试材料单核早期小麦小花叶绿体,经淀粉碘化钾染色压片并结合电子显微镜观察,结果表明完整叶绿体呈棒状或橄榄状并被染成蓝色;而破碎的叶绿体不被染色,杂质含量也很大。2、经TCA-丙酮沉淀蛋白,7cm,pH3～10的线性预制干胶条进行IEF/SDS-PAGE检测,发现三层蔗糖密度梯度结合差速离心技术所得叶绿体完整性和纯度都高于差速离心技术,以及两层蔗糖密度梯度离心所得的叶绿体;核污染以及线粒体污染大大降低,弥补了单纯差速离心杂质多,以及两层蔗糖密度梯度离心技术高丰度蛋白过多而低丰度蛋白损失严重的缺点。3、用17cm,pH4～7的线性胶条对小麦ms(A)-西农1376、(A)-西农1376以及ms (S)-西农1376单核早期(花粉小孢子萌发形成单核)小花叶绿体蛋白质组进行比较分析,利用PDQuest软件对图像进行背景消减,斑点检测、匹配,获取斑点位置坐标,并分析计数蛋白点,每个图谱在pH4～7,分子量14.4～94.0KD的范围内,均可以得到130～200个蛋白点,而且大多数蛋白在等电点4.5～7(胶条的碱性端),分子量10～60KD居多。发现杀雄剂诱导的生理型雄性不育系和对应可育系间叶绿体蛋白差异点不明显,仅有少数差异只是表达量上的差异,其中在生理型雄性不育系中上调表达的蛋白点有5个,下调表达的蛋白点有7个;还有一些不是很明显的微小的质的差异点,在生理性雄性不育系中表达而可育系中没有表达的有8个,丢失的蛋白有2个,但都是不很明显的点。遗传型雄性不育系ms (S)-1376叶绿体蛋白保持系相匹配的蛋白点有150多个,表达上调的蛋白点有4个,下调的蛋白点有18个,在对应可育系中几乎没有表达,而在遗传型雄性不育系中明显特异表达的有15个点,在对应可育系中表达而遗传型不育系中未表达的有14个。两不育系中都表达而在对应可育系中缺失的蛋白有6个,正常可育系和生理型雄性不育系材料中表达而遗传型不育系中未表达的6个点;可育系中下调表达但在两不育系中上调表达的4个点。两种不育系下调表达,可育系中上调表达的点有2个。4、选取具有代表性的6个差异蛋白点进行质谱测序,获得质量较好的肽指纹图谱(PMF),用Profound和Mascot软件搜索NCBInr数据库,鉴定结果,Spot1为酰基辅酶A脱氢酶结构域蛋白,Spot3钙调结蛋白磷酸酶,Spot4被鉴定为多催化功能肽酶,Spot5为热休克蛋白60,其中蛋白点41是在拟南芥叶绿体中新发现的未知功能基因表达的蛋白。5、对已知蛋白的功能进行分析,推测小麦雄性不育可能主要与能量代谢紊乱导致代谢供能不足,信号转导受阻导致细胞抗性减弱,活性氧积累导致细胞中毒,细胞凋亡导致花药发育异常及花粉败育,以及调节花器官发育基因作用失控导致的花丝不伸长或不产生有功能的花粉等有关。这些差异蛋白很可能直接或间接地参与了供试材料育性正常发育与败育的某些关键途径,而供试遗传型雄性不育系和杀雄剂SQ-1诱导的生理型雄性不育系和对应可育系互为等基因或者等生理差异系,其叶绿体蛋白图谱中表现出的差异蛋白很有可能与雄性不育或者SQ-1诱导小麦雄性不育有关。更多还原

【Abstract】 Heterosis was the widespread natural phenomenon in biosphere, wheat as the largest food crop in the world, heterosis clear, using it’s heterosis effectivly was an important way to improve the wheat yield. Approaches of wheat heterosis were mainly: (1) Chemical hybridizing, (2) Male sterile line. But it’s male sterility mechanism research has became a scientific difficulty in the wheat heterosis utility, and still during the exploration. The analysis and the research were mainly concentrated in the fertility factors at cell nucleus and the mitochondria in recent years. In this study, 1376 wheat as the material to study the gametocide induced physiological male sterity ms(A)–Xi-nong 1376, genetic male sterity ms (S)–Xi-nong1376, and the corresponding nuclear fertile line MS(A)–Xi-nong1376, purified complete spikelet chloroplast with the combination of differential centrifugation and sucrose density gradient centrifugation, precipited protein with TCA-acetone, compared protein by IEF / SDS-PAGE two-dimensional electrophoresis, gel-digestion, digging point, biological mass spectrometry sequencing, searching bio-informatics database, studied chloroplast proteins of spikelet on the uninucleate anther stage of wheat. Concluded as follow:1. Extracted spikelet chloroplast with different methods on the uninucleate anther stage of wheat, starch KI staining, electron microscopy, showen that integrity chloroplast was rod-shaped and was coloured blue or olive, the broken chloroplasts were not stained, high impurity content.2. Precipitation protein with TCA-acetone, detected with 7cm, pH3～10 prefabricated dry linear strips for IEF/SDS-PAGE, found that chloroplast integrity and purity with three-layers combination of differential sucrose density gradient centrifugation were higher than the differential centrifugation techniques, as well as two sucrose density gradient centrifugation obtained from chloroplasts, mitochondrial and nuclear pollution were greatly reduced, make up drawbacks of simple differential centrifugation impurities, as well as two layers of sucrose density gradient centrifugation technology.3. Compared chloroplast proteins of spikelet on the uninucleate anther stage(the pollen germination to form a small single-core) by 7cm, pH4～7 linear strips of ms(A)–Xi-nong1376, (A)–Xi-nong1376, as well as ms(S)–Xi-nong1376, background removal by PDQuest software, spot detected, matching, obtained location of spots, counted protein spots, in the pH4～7, 14.4～94.0KD, may be 130～200 protein spots, most of the protein in the pH4.5～7 (strip alkaline side), 10-60KD. Found no obvious difference between gametocide induced physiological-type male sterity and the maintainer, only a few expression of quantity differences, up-regulated expressed of protein spots in ms(A)–Xi-nong1376 are 5, down-regulated expressed 7 compared (A)– Xi-nong1376, also some miny qualitative differences between them, there were 8 ms(A)– Xi-nong1376 expressed while (A)–Xi-nong1376 lost , the missing protein had 2, but not obvious. There were more than 150 spots matched between ms(S)-1376 and (A)–Xi-nong1376, up- regulated expressed 4, down-regulated 18 compared (A)–Xi-nong1376, almost no expressed in(A)–Xi-nong1376, clear-specific expressed in ms(S)-1376 were 15 points, instead14 points. The two male sterity both expressed while (A)–Xi-nong1376 lost were 6, (A)–Xi-nong1376 and ms(A)–Xi-nong1376 expressed while ms(S)-1376 lost were 6, (A)–Xi-nong1376 down-regulated expressed. However, two male sterity up-regulated expressed 4point.instead 2 point.4. Selected six points for mass spectrometry sequencing, got the PMF, searching in NCBInr database with profound and mascot software, identification results as follows: Spot 1 was acyl-coenzyme A dehydrogenase domain protein, spot3 was calmodulin-knot protein phosphatase, spot4 was multi-catalytic peptidase, spot5 was heat shock protein 60, spot 41 was unknown function protein in arabidopsis thaliana chloroplast gene discovered recently.5. Functional analysis known protein, speculated that male sterility of wheat may be related to energy metabolism disorder, signal transduction blocked, accumulation of reactive oxygen species, apoptosis and regulation of the role of developmental genes that control floral organ. These differentially expressed proteins were likely directly or indirectly involved in the test material for the normal development of fertility and abortion in certain key ways, while the three test materials were Isogenic lines, or Isophysiological system, the differencies of PMF were likely related to CMS .更多还原