Differential Proteomics Analysis Of Thermo-Sensitive Male Sterile Lines And Conversion Lines In BNS Wheat

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.