| Maize is one of the plants which utilized heterosis the earliest in the world. As to make full use of maize cytoplasmic male-sterile lines in heterosis, it is very important to elucidate the mechanism of cytoplasmic male-sterility theoretically. A great deal of research about abortion mechanism and fertility restoration has been reported. As an altered pattern of epigenetic modifications, DNA methylation plays a critical role in regulating gene expression and maintaining genomic stability. Understanding the dynamics of DNA methylation in the whole process of growth and development is vital for illustrating the roles played by epigenetic paradigms during plant development and genome evolution. But up to date the reports associated cytoplasmic male-sterility with epigenetic modification are still relatively rare. Maize materials with homoplasm but heteronuclei and matreials with homonucleus but alloplasms were used in this experiment, the mitochondrial DNA and nuclei DNA of different development tassel were isolated, and DNA methylation were detected by HPLC(High Performance Liquid Chromatography). The object was to investigate the changing rule of DNA methylation levels in the materials with homoplasm but heteronuclei and materials with homonucleus but alloplasms as well as the changing regularity at different stages across development, also to offer further basic data on epigenetic regulation in the process of cytoplasmic male-sterility. The main results are as follows:1. HPLC methodologies have been optimised for mitochondrial DNA and nuclei DNA in this study, the methodological study showed that this method had a good linear relationship, accuracy and repeatability, and can be used to detect the DNA methylation level of mitochondrial DNA and nuclei DNA. The procedure was as follows:After hydrolysis with Benzonase, alkaline phosphatase and phosphodiesterase I, the purified DNA were hydrolyzed to a single deoxynucleoside mixture, and separated by HPLC, the absorption peak area was determined and quantified by HPLC with a certain wavelength, finally the methylation level of each sample was calculated according to DNA methylation=5mC/(5mC+dC). The optimized chromatographic conditions are as follows:C18 column(4.6×250mm,5μm), flow rate of 0.8 mL/min, injection volumn of 20μL, mobile phase consist of methanol, sulfonic acid sodium salt and triethylamine(10:90:0.2, V/V), the optimized pH of mobile phase used for detecting mitochondrial DNA and nuclear DNA is 3.42 and 3.0, respectively. Detection was performed with SPD-20A detector at 287nm, column temperature was 35℃, all the samples were separated completely in 20min, the results showed that this method was simply and reliably.2. Mitochondrial DNA methylation of maize materials (CMol7, C698-3, C48-2, C478) with homoplasm but heteronuclei was performed by high performance liquid chromatography (HPLC), the results showed that, during four periods of microspore development, mitochondrial DNA methylation levels of the above materials were different, which indicated that the mitochondrial DNA methylation degree was influenced partly by nuclear background, and this influence appearred development-specific and material-specific:mitochondrial DNA methylation levels of the same material in different periods and different materials in the same period were both different; Our study also showed that except for individual period, with Mo 17 for nuclear background, the C-CMS mitochondrial DNA methylation level was the highest, while with 48-2 for nuclear background, the C-CMS mitochondrial DNA methylation level was the lowest.3. Nuclear DNA methylation of maize materials (TMo17, CMo17, SMo17, Mo17; C698-3, 698-3; C48-2,48-2; CHuangzaosi, Huangzaosi) with homonucleus but alloplasms was carried out by high performance liquid chromatography (HPLC), the results showed that, during four periods of microspore development, nuclear DNA methylation levels were different, which proved that there was a certain influence of cytoplasm on the nucleus. The article also focused on comparison of the C-CMS and their maintainer lines which were under the same nuclear background, the results demonstrated that apart from individual periods, the nuclear DNA methylation degree of C-CMS material was higher than their maintainer lines, especially at tetrad and mononuclear periods. The same tendency was found in material SMo17 and it was concluded that more or less it might be involved in abortion.4. At four periods of microspore development, we explored the changing regularity of CMS mitochondrial DNA and nuclear DNA methylation, the results showed that dynamic changes did occur during the growth development and interestingly different cytoplasm type had its specific changing way. C-CMS, T-CMS, S-CMS mitochondrial DNA and nuclear DNA methylation level generally reached its maximum in respective period of abortion, which probably be a proof that CMS is the result of nuclear and cytoplasm interaction. It is speculated that abortion may have a certain relationship with elevated DNA methylation levels, which then led to abnormal expression of some key enzymes or fertility genes in the anther metabolic process, further contributed to the material of anthers and energy metabolism disorders, finally resulted in the abnormal pollen growth. |
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