| Maize (Zea mays L.) is one of the original crops which breed hybrids by using male sterile lines in the world, and CMS (cytoplasmic male sterility) is the most important type of male sterility. As the CMS-T can be specific infected by Helminthosporium maydis Nisik, CMS-S belongs to gametophytic sterility which is instability and sensitive to the environment, so CMS-C is the main and very important type in maize breeding. Many studies on the abortion mechanism of CMS-C have shown the abortion time, the cytologic features when abortion occur, the discovery of chimeric genes in mitochondrial genome, the RNA editing, the differentially expressed proteins, but a relatively clear and consistent explanation for CMS-C abortion molecular mechanism is still not formed.Proteins are the end-products of genes expression and real supporters of life activities. So based on the previous research of CMS-C, mitochondrial proteomes from different cytoplasm are chosen in my study to do further research on mechanism of CMS. In this study, three sterile lines (C-Huangzaosi, C48-2, C478), their maintainer lines (N-Huangzaosi, N48-2, N478) and Fl (C-Huangzaosi×068, C48-2×18bai, C478×068)that were fertility restorations of three sterile lines were selected as our study materials, from which mitochondrial proteins were extracted in three times:etiolated seedling, tassels of tetrad stage and uninucleate stage of microspore development. Mitochondrial proteins of each material were separated by two-dimensional electrophoresis and PDQuest software was used to analyse the 2-D maps. Finally,28 differential spots were chosen to do mass spectrometry. The main results obtained are as follows:1.2-D maps with good reproducibility of each material were obtained, after PDQuest software analysis,400 average spots were found in 2-D maps of etiolated seedling with Huangzaosi nuclear background, and 340 average spots with 48-2 nuclear background; 370 average spots were found in 2-D maps of tassels of tetrad stage of microspore development with 48-2 nuclear background, and 350 average spots with 478 nuclear background, the spot amounts are close between the two times. In tassels of uninucleate stage of microspore development with 48-2 nuclear background, only 242 average spots were found in 2-D maps, the amounts are much lower than etiolated seedling and tetrad stage of microspore development, the abortion and disintegration of microspore may be the main reason.2. Under the differential spots choice standard that spots had the same expression in maintainer lines and F1, but special expression, loss expression, down or up-regulated expression in sterile lines,16 differential spots were found between N478 and C478 in etiolated seedling, after comparison with F1, only 10 spots left. Likewise,14 of 32 spots between N-Huangzaosi and C-Huangzaosi in etiolated seedling, six of 12 spots between N48-2 and C48-2 in tetrad stage,30 of 65 spots between N478 and C478 in tetrad stage,11 of 17 spots between N48-2 and C 48-2 in uninucleate stage. The influence of comparison with F1 is significant, the left differential proteins are more closer to abortion.28 of differential spots were selected to do mass spectrometry (MALDI-TOF/MS) as their good repeatability, two from etiolated seedling,21 from tetrad stage of microspore and five from uninucleate stage of microspore.3. By mass spectrometry,27 spots acquired peptide mass fingerprintings, and eight spots were identified by the Mascot software in the NCBI protein database, respectively:loss expression of malate dehydrogenase,down-regulated expression of stomatinl,up-regulated expression of succinate dehydrogenase flavoprotein subunit (mitochondrial precursor) and low molecular weight heat shock protein precursor from tetrad stage of microspore in C478, loss expression of NADH-ubiquinone oxidoreductase 51 kD subunit from tetrad stage of microspore and loss expression of NADH-ubiquinone oxidoreductase B17.2 down-regulated expression of Rieske [2Fe-2S] domain loss expression of actin from uninucleate stage of microspore in C48-2. Among these eight proteins, five are involved in energy metabolism in mitochondria, including glucose metabolism and electron transfer chain, and others are associated with ion transportation in membrane, molecular chaperone and apoptosis, cytoskeleton and so on.The abortion of CMS-C has close relationship with mitochondrial abnormal, in this study, many differentially mitochondrial proteins were found, they are closely related to mitochondrial energy metabolism, which further reveals that the shortage of energy supply when microspore is developing is the main reason of pollen abortion, while the process of abortion may also be involved with apoptosis, cell structure destruction. This study provided a new basis and reference for further explanation of molecular mechanism of CMS-C.
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