摘要
杂种优势是生物界存在的一种普遍现象,利用这种现象可以有效地提高作物产量和品质。自花授粉作物利用杂种优势需要雄性不育为材料,小麦光温敏雄性不育系是这种材料中的重要一类。在小麦温敏雄性不育中,近年来新发现一种类型叫BNS型温敏雄性不育系,其不育性和恢复性良好,符合杂交小麦制种要求,是一个有重要利用价值的新材料。本论文在系统观察BNS性状特征的基础上,以不育条件和转化条件下产生的不育系和转换系的减数分裂期的幼穗和花药为材料,利用单向、双向电泳技术和基质辅助激光解吸电离飞行时间质谱技术,在差异蛋白水平探讨BNS的不育和转化的机制,为该系的有效利用提供遗传和生理的理论。论文获得如下结果:
1.初步探讨了小麦花药蛋白质组学的研究方法。由于取材花药难度较大,因此“TCA-丙酮沉淀法”是花药蛋白提取的适宜方法。蛋白质浓度测定可采用Bradford法;2-DE第一向IPG胶条适宜pH为4-7 ;第二向凝胶电泳分离胶适宜浓度为13%T。得到的结果用一级质谱与蛋白质数据库联合检索效果良好。
2.单向SDS-PAGE电泳发现,转换系中在分子量35KD与45KD之间有4条带的蛋白表达量明显高于不育系的蛋白表达量。
3.双向电泳在不育系材料中分离出223个蛋白质点,转换系中分离出284个蛋白点。拟合结果是,不育系中有而转换系无的蛋白点为8个,不育系中无而转换系中有的蛋白点10个,转换系中上调的且2倍以上差异蛋白点7个,不育系中上调且2倍以上差异蛋白点6个,共31个差异表达蛋白。
4.选取不育系和转换系互为有无的18个蛋白点,以及转换系中上调的7个点,不育系中1个10倍上调的点进行了“基质辅助激光解吸电离飞行时间质谱”(MALDI- TOF-MS)鉴定分析,得到了13个阳性结果。其中与质谱库中匹配上的蛋白有11个,未知蛋白2个。在匹配的11个蛋白点中,9个显示出实质表达差异,2个是不育和转换中都存在的假阳性。
5.表达差异的9个蛋白质点分别为:3个在不育花药中表达(点122,叶绿体放氧增强蛋白1;点18,Harpin-induced ADH1A 1;点129,山梨醇脱氢酶);3个在可育花药中表达(点15,推定的Rubisco亚基结合蛋白α亚基前体;点183,乙醇脱氢酶ADH1A;点237,烯醇化酶);3个在转换系中上调(点31,组蛋白H2B.2;点35,延伸因子TU;点89,23.5 kDa热激蛋白)。
通过对差异蛋白的功能分析,可把这些蛋白分为四类:第一类是对温度敏感的调节蛋白,如,23.5 kDa热激蛋白;第二类是参与能量代谢的功能蛋白,如,烯醇化酶、乙醇脱氢酶ADH1A;第三类是与光合作用有关的功能蛋白,如,推定的Rubisco亚基结合蛋白α亚基前体、叶绿体放氧增强蛋白1 ;第四类是其它类功能蛋白,如,组蛋白H2B.2、延伸因子TU、山梨醇脱氢酶。推测引起BNS不育的机制可能主要有两个方面,一是对温度敏感的热激蛋白表达异常,二是糖代谢紊乱、能量代谢失调。这些失调使四分体后的花药能量供应受阻,花粉发育败育。
Heterosis is a universal phenomenon in biological kingdom. There are many more records to show that by use of this phenomenon the production and quality of crops were increased and improved enormously. Heterosis utilization in self-fertilization crops need male sterility characteristics for the female parent. And the Photo-thermoperiod-sensitive male sterility is just the important sterility material. Recent years ,the photo-thermoperiod-sensitive male sterility in wheat, a new material, named BNS,was found and developed successfully, whose sterility and fertility restoration expreesed excellently, and was the male sterility that have important value in wheat Heterosis utilization. On the basis of systematic observation to biological chracteristic of BNS , this paper focused on the mechanism of sterility and conversion of sterility of BNS in the level of difference proteins that extrcted from both sterility anthers and fertile anthers during the period of meiosis by means of two-dimensional electrophoresis and MALDAI-TOF-MS. The results are as follows .
1. The proteomics methods of wheat anthers was studyed: The method "TCA-acetone precipitation "was the appropriate one for the extract of anther protein in the case that anthers materials were difficult to get. The method of modified Bradford could be used as determination of protein concentration; IPG strips of pH 4-7 for the first dimensional electrophoresis was better than others; And the appropriate of separating gel concentration for the second dimensional electrophoresis was at 13% T.
2. It was found that there were four difference proteins bands in the gel of SDS-PAGE.whose molecular weight was at between 35KD and 45KD. The expression in the conversion lines were significantly higher than the sterility lines. The protein extracted from young ears of two lines.
3. Proteins were separated by two-dimensional electrophoresis , the results showed that about 223 protein spots were tiled on the gel of sterile anthers, and about 284 protein spots were tiled on the gel of fertile anthers. By fitting that there were total of 31 differentially expressed proteins was identified. They were: 6 proteins up-regulated in the sterile line , 7 proteins up-regulated in the fertile line , 8 proteins existed only in the sterile line , 10 proteins existed only in the fertile line .
4. MALDAI-TOF- MS was adopted to identify the difference proteins . 8 proteins existed only in the sterile anthers , 10 proteins existed only in the fertile anthers , 7 proteins up-regulated in the fertile line, 1 up-regulated 10 times protein in the sterile line were analysised , and 13 positive results were obtained . but 2 proteins were unknown , only 11 proteins matched with the Protein database . In those matched 11 proteins , 9 proteins showed substantially differential expression , 2 proteins were false positive .
5. There were 9 differeniially expressed protein spots that were identified . spot 18 was Harpin- induced ADH1A 1 , spot 122 was chloroplast oxygen-evolving enhancer protein 1 , spot 129 was Sorbitol dehydrogenase , those 3 proteins were expressed only in the sterile anthers. Spot 15 was putative rubisco subunit binding protein alpha subunit precursor , spot 183 was alcohol dehydrogenase ADH1A , spot 237 was Enolase , those 3 proteins were expressed only in the fertile anthers . Spot 31 was histone H2B.2 , spot 35 was elongation factor TU , spot 89 was 23.5 kDa heat-shock protein , those 3 proteins were up-regulated proteins in the fertile line.
According to analysising those 9 differential proteins , those proteins could be divided into four categories. The first category was thermo-sensitive regulatory protein , for example , 23.5 kDa heat-shock protein . The second category was energy metabolism proteins , for example , Enolase , alcohol dehydrogenase ADH1A . The third category was photosynthesis proteins , for example , putative rubisco subunit binding protein alpha subunit precursor , chloroplast oxygen- evolving enhancer protein 1. The fourth category was the other functional proteins , for example , histone H2B.2 , elongation factor TU , Sorbitol dehydrogenase . In a word , two reasons may lead to BNS infertility . Firstly , thermo-sensitive heat shock proteins expressed abnormally.Secondly, glucose metabolism disorders and energy metabolism disorders . Those disorders made the energy supply disruptions after the tetrad and then the pollen abortion.
引文
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