| CMS (cytoplasmic male sterility, CMS) is a common biological phenomena in higher plants. Because the male reproductive system cannot produce a function of pollen, and the female reproductive system development and vegetative growth is fully functional, so the solution of the tedious step "emasculation" in hybrid seed production is an important way of crop heterosis utilization. The inner mechanism of CMS needs further investigation. So far, the research of cytoplasmic male sterility (CMS) has been carried out from ecology, genetics, molecular biology, but the protein expression of the research is less.From the developmental genetics angle,the protein comparisons from different developing stages of different organs of K-cytoplasmic male sterile(CMS)line and maintainer line in common wheat were carried on by means of two-dimesional gel electrophoresis(2-DE). The results are as follows:1.The effects of different protein extraction methods (TCA/acetone precipitation method,urea/thiourea extraction method and KCl-methanol/ammonium-acetate method) on two-dimensional electrophoresis (2-DE) gels were compared, and different lysis buffer and loading quantity of sample were also optimized for the analysis of 2-DE system on the binucleus-trinucleate stage anthers of wheat. The results showed that, more stable repetitiveness, higher clarity and more regular shape protein spots had observed in the samples prepared by TCA/acetone precipitation than by phenol extraction method, and clearer background and protein spots were obtained in the 2-DE map. The sample dissolved in the lysis bufferⅡwhich contained thiourea, could enhanced the protein solubility. Extracted proteins by the TCA/acetone method, dissolved proteins with the lysis bufferⅡ,with pH 3-7 17cm IPG scip , 1000μg loading quantity and 12% concentrations of the SDS-PAGE gel , the proteins were well separated .2.Two-dimensional electrophoresis(2-DE)was performed to generate the images of proteins of leaves and flag leaf of K-cytoplasmic male sterile(CMS) line and maintainer line at seeding and binucleus-trinucleate stages, respectively. The results showed that, almost no difference protein spots between K-cytoplasmic male sterile(CMS)line and maintainer line in the 2-DE map. Only Cytoplasmic Male Sterile(CMS)line and maintainer line of yunong 43 had slight differences in the 2-DE map.3.Two-dimensional electrophoresis was performed to generate the images of proteins of anthers at binucleus-trinucleate stage. The results indicated that most of the wheat proteins were acid proteins and laid in pH4-7 position of the gel. The protein expression pattern showed that 12 protein spots with differential expression were found.There were 5 five different spots between CMS line and maintainer line of yunong 43 ,namely A4,A5,A6,A7 and A8.Among them, A5 appeared only in anthers of the maintainer line, and A4, A6 and A8 only in those of the CMS line. A7 had high expression in anthers of the maintainer line.There were 7 five different spots between CMS line and maintainer line of yumai 21, namely B1,B2,B3,B4,B5,B6,B7.Among them, B1and B7 had high expression in anthers of the CMS line, and B2, B3, B4, B5and B6 had lower expression.4.According to LC/ESI-MS/MS analysis and protein database searching, 15 different protein spots were identified from the 2D protein maps, and 10 protein spots among them were identified succesfully.They represented 6 types of proteins: cytosolic malate dehydrogenase, triosephosphat-isomerase, S-adenosylmethionine synthetase, wall-associated kinase-like 1, glutamine synthetase isoform and OEC33kDa. This study speculated that these differentially expressed proteins were connected intensivly with plant physiological and bio-chemical activity metabolism and energy metabolism, which were key proteins of CMS.
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