| Expression and regulation of numerous genes involved in stamen sterility. The correlation of pollen development process and sterility gene expression at different levels was the important approaches to elaborate molecular mechanism. In order to understand the regulatory mechanism of male sterility expression, the various expression of transcriptome and proteome in the male sterile (Ms-4) and fertile (MF) plant of buds with alfalfa were studied and combined the physiological and biochemical indexes in the process of metabolism. The main findings were summarized as follows:(1) The key factor of cDNA-AFLP technique were optimized by using of Ms-4and MF buds, and to establish and perfect the cDNA-AFLP technique system that attained to fitting the flower buds of alfalfa. It was obtained that the pair of69primer from all of256primer according to the selection standard, which the69primer had clear amplification band and reasonable dispersibility.(2) The difference of Ms-4and MF gene expression of alfalfa was compared by using cDNA-AFLP technique. The103various differential expression fragments were obtained by cloning and sequencing, in comparison to Blast,34differential expression fragments were relatively lower or no homology that we presumably them could be the new gene. However, the69differential expression fragments have good homology and73.9%of fragments are highly consistent with Medicago truncatula. Function analysis showed that differential expression fragments were involving in process of substance metabolism and synthesis(28.36%), signal transduction and transcriptional regulation(20.90%), energy metabolism(8.96%) and so on. The quantitative analysis of six differentially expressed fragments showed that the process of buds development between Ms-4and MF significantly had difference. The results implied gene expression level and specificity could be related to plant sterility.(3) The differential proteomics of Ms-4and MF bud with alfalfa were analyzed by using two-dimensional gel electrophoresis (2-DE). The results showed:6000expressed protein points were found from silver color spectrum of Ms-4and MF bud with alfalfa,98spots were differential expressed,35.7%of protein points was up-regulated and64.3%of protein points was down-regulated in Ms-4.2600expressed protein points were found from coomassie brilliant blue G-250spectrum,54spots were differential expressed,61.1%of protein points was up-regulated and38.9%of protein points was down-regulated in Ms-4. Meanwhile,22differentially expressed proteins spots were successfully identified by using mass spectrometry technology. The differentially expressed proteins major participate in the process of metabolism (27.08%), cell processing (18.75%), stimulus response (18.75%), development (8.33%), and so on, which played important molecular function on catalysis, binding and so on. Combined with the transcriptome differences analysis, different segments which affect the level of metabolic was related to the formation of fertility in the process of pollen development was found.(4) In order to further understanding the formation mechanism of male sterility of alfalfa, metaboilic level of Ms-4and MF were studied. The nutrients accumulation of Ms-4and MF stems and leaves in reproductive stage was higher than vegetative period, especially proline content and starch content. The content of proline, soluble sugar and starch showed the deficit the process of bud development on Ms-4, and soluble protein was relatively high. The content of chlorophyll, the enzymatic activity of POD and CAT were lower than MF and the content of MDA was higher than MF. The expression of sterility related gene and protein regulated antioxidant enzyme, chlorophyll, MDA,.nutrients to some extent, so they affected the formation of matter and energy and then affected pollen fertility in the process of pollen development. |
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