Analysis Of Cytology Research And Gene Expression Profiles Of Fertile And Sterile Anthers Of Thermo-Stnsitive Genic Male Sterile Wheat BNS

To reveal the mechanism of the pollen abortion to the thermo-sensitive male sterile lines BNS, this study aimed at turning the fertile conversion and suitable sowing by the method of interval sowing trial; Staining the Microspores with DAPI、 aceto-carmine and KI-I2; The method of electron microscopy and histological staining techniques was conducted in observeing the properties of pollen grains, identifying the changes in tapeta and microspores, and analyzing the nutrient substances in microspores; we used Affymetrix gene chip technology of wheat to analyze the differential expression genes with anthers at the tetrad stage and at bicellular of the sterile and fertile lines of BNS. The semi-quantitative PCR was utilized to characterize some sterile candidate genes. The key findings of this study are as follows:Interval sowing results showed:wheat BNS fertility gradually restored with the delayed sowing, in November 1, the percentage setting of wheat BNS is 61.4%, and be the fertile lines; while when the sowing data delayed to November 15, the setting rate presents slight decline. Among this four sowing stages, when the sowing stage was November 1, The wheat of BNS had lowest tillering tillers and highest yield, and it consumed relatively little demand for soil nutrients and moisture, so this time would be the most suitable stage to breeding BNS sterile line in yang ling district of shanxi province.Semi-thin sections stained histological results showed:compared to (F)-BNS, The tapetum of (S)-BNS anther eventuated an earlier degradation in early uninucleate, There are less tapetum structure in late uninucleate, the structure of tapetum disappeared at the bicellular. The microspores of (S)-BNS exhibited Significant differences in bicellular, a majority of microspores wall Started retracted, and could not developed to bicellular pollens. At tricellular stage, The microspores of (S)-BNS exacerbated the vacuolization, the pollens could not be dyed, pollens wall developed to Crescent-type, T-type, L-type and other irregular shapes. This implied the bicellular is the key stage which made the wheat BNS pollens abortion.Electron microscopy sections statistical results showed:From late uninucleate stage to bicellular stage, the trends is consistent for the area of tapeta and microscopes between the sterile and fertile lines, while the area of tapeta reduction is faster than fertile lines, the area of microscopes increase is rather slower than fertile lines. Semi-thin sections stained statistic results showed that all of the Anther walls and microscopes and fertile lines and sterile lines obtained polysaccharide, lipid, protein and other nutrients, after this developed stage, microscopes gradually accumulate lots of nutrients. From late uninucleate stage to tricellular stage, the sterile lines of nutrient content which accumulated in the tapeta and microscopes were significantly less than the fertile lines. This suggested that metabolism disorder was associated with the pollens abortion.Expression profiling results showed that there are a large number of differentially expressed genes between the sterile and fertile lines. Functional classification indicated that these genes participated a series of significant life processes like metabolism process, cytological process, biological regulation and irritative reaction etc. Moreover, to check chip data, we used the cDNA semi-quantitative PCR to verify 10 genes (Ta.17228.1, Ta.21809.1, Ta.22570.1, Ta.37113.2, Ta.19531.1, Ta.18368.1, Ta.9239.1, Ta.37113.1, Ta.21983.1, Ta.18097.1)that demonstrated a significant expression differences as well. Result showed that the expressing models of genes were concordant with the result of gene chip detection. Conservative structure domain analysis showed that Ta.18091.1, Ta.21809.1 belong to ATP energy metabolism-related genes and Ta.19531.1 is Lipid metabolism genes. The three genes demonstrated a significant expression differences and played an important role in the microspores material and energy metabolism, It could be the key genes which made the pollens abortion in wheat BNS.Conclusion:In tetrad period and early uninucleate, (S)-BNS ATP energy metabolism related genes Ta.18091.1、Ta.21809.1 are down regulated in anther, which means the energy metabolism process abnormal, then (S)-BNS tapetum of (S)-BNS anther eventuated an earlier degradation in early uninucleate, which made the nutrient substances could not continuing to meet the the normal growth and development of microspores required. At binucleate stage the microspores Started the process of programmed cell death (PCD), At the trinucleate stage, cytoplasmic inclusions could not continue to increase, The accumulation of polysaccharides, lipids, proteins and other substances terminated and consumed lots of it, which eventually the microspores without the substances, and become the empty spores. This implied, It could be the energy metabolism process abnormal made the (S)-BNS tapetum eventuated an earlier degradation, and caused the abnormal metabolism, which eventually leaded to the pollens abortion.