| Heterosis was the widespread natural phenomenon in biosphere, wheat as the largest food crop in the world, heterosis clear, using it's heterosis effectivly was an important way to improve the wheat yield. Approaches of wheat heterosis were mainly: (1) Chemical hybridizing, (2) Male sterile line. But it's male sterility mechanism research has became a scientific difficulty in the wheat heterosis utility, and still during the exploration. The analysis and the research were mainly concentrated in the fertility factors at cell nucleus and the mitochondria in recent years. In this study, 1376 wheat as the material to study the gametocide induced physiological male sterity ms(A)–Xi-nong 1376, genetic male sterity ms (S)–Xi-nong1376, and the corresponding nuclear fertile line MS(A)–Xi-nong1376, purified complete spikelet chloroplast with the combination of differential centrifugation and sucrose density gradient centrifugation, precipited protein with TCA-acetone, compared protein by IEF / SDS-PAGE two-dimensional electrophoresis, gel-digestion, digging point, biological mass spectrometry sequencing, searching bio-informatics database, studied chloroplast proteins of spikelet on the uninucleate anther stage of wheat. Concluded as follow:1. Extracted spikelet chloroplast with different methods on the uninucleate anther stage of wheat, starch KI staining, electron microscopy, showen that integrity chloroplast was rod-shaped and was coloured blue or olive, the broken chloroplasts were not stained, high impurity content.2. Precipitation protein with TCA-acetone, detected with 7cm, pH3～10 prefabricated dry linear strips for IEF/SDS-PAGE, found that chloroplast integrity and purity with three-layers combination of differential sucrose density gradient centrifugation were higher than the differential centrifugation techniques, as well as two sucrose density gradient centrifugation obtained from chloroplasts, mitochondrial and nuclear pollution were greatly reduced, make up drawbacks of simple differential centrifugation impurities, as well as two layers of sucrose density gradient centrifugation technology.3. Compared chloroplast proteins of spikelet on the uninucleate anther stage(the pollen germination to form a small single-core) by 7cm, pH4～7 linear strips of ms(A)–Xi-nong1376, (A)–Xi-nong1376, as well as ms(S)–Xi-nong1376, background removal by PDQuest software, spot detected, matching, obtained location of spots, counted protein spots, in the pH4～7, 14.4～94.0KD, may be 130～200 protein spots, most of the protein in the pH4.5～7 (strip alkaline side), 10-60KD. Found no obvious difference between gametocide induced physiological-type male sterity and the maintainer, only a few expression of quantity differences, up-regulated expressed of protein spots in ms(A)–Xi-nong1376 are 5, down-regulated expressed 7 compared (A)– Xi-nong1376, also some miny qualitative differences between them, there were 8 ms(A)– Xi-nong1376 expressed while (A)–Xi-nong1376 lost , the missing protein had 2, but not obvious. There were more than 150 spots matched between ms(S)-1376 and (A)–Xi-nong1376, up- regulated expressed 4, down-regulated 18 compared (A)–Xi-nong1376, almost no expressed in(A)–Xi-nong1376, clear-specific expressed in ms(S)-1376 were 15 points, instead14 points. The two male sterity both expressed while (A)–Xi-nong1376 lost were 6, (A)–Xi-nong1376 and ms(A)–Xi-nong1376 expressed while ms(S)-1376 lost were 6, (A)–Xi-nong1376 down-regulated expressed. However, two male sterity up-regulated expressed 4point.instead 2 point.4. Selected six points for mass spectrometry sequencing, got the PMF, searching in NCBInr database with profound and mascot software, identification results as follows: Spot 1 was acyl-coenzyme A dehydrogenase domain protein, spot3 was calmodulin-knot protein phosphatase, spot4 was multi-catalytic peptidase, spot5 was heat shock protein 60, spot 41 was unknown function protein in arabidopsis thaliana chloroplast gene discovered recently.5. Functional analysis known protein, speculated that male sterility of wheat may be related to energy metabolism disorder, signal transduction blocked, accumulation of reactive oxygen species, apoptosis and regulation of the role of developmental genes that control floral organ. These differentially expressed proteins were likely directly or indirectly involved in the test material for the normal development of fertility and abortion in certain key ways, while the three test materials were Isogenic lines, or Isophysiological system, the differencies of PMF were likely related to CMS .
|
PDF Full Text Request