| Cytoplasmic male sterility is a widespread phenomenon in plant kingdom, and attracts the interest of breeders mainly for facilitating the utilization of hysteresis to produce vigorous F1 hybirds. Thus, research on the molecular mechanism of rice CMS and the fertility restore is important for the use of hybrid rice in breeding The main characters of CMS were the vegetative development is normal, while the microspores development is defect. Previous researcher have shown that:the failure of pollen development is suggested to be associated with chimeric open reading frames(ORFs) that have arisen from unusual recombination events in mitochondria and dysfunction of the mitochondria in CMS plant. Until now most research on CMS mainly focus on the development of pollen, little attention was concerned on vegetative development. So in this work, we investigated the mitochondria function in the seedlings of HL-CMS rice(YtA) and its maintainer line (YtB), the major result are shown as following:1. Previous studies suggested that pollen abortion of the sterile plants characterized in a special programmed cell death (PCD) program caused by ROS accumulation. To investigate the potential relationship between the CMS and the ROS, we treated the rice seedlings with 50mM H2O2. Biochemical and molecular analysis showed that ATP content and mitochondrial membrane potential in the HL CMS line YtA declined significantly compare with those of the maintainer line YtB. At the same time it was observed that ROS content swiftly rose in the CMS line, but remained stable in the maintainer line. These biochemical changes were closely accompanied by nuclear DNA fragmentation and mitochondrial cytochrome c release in the root cells of the CMS lines. Expression analysis revealed that the antioxidation enzyme and mtETC complex activity were also significantly down-regulated. These biochemical index disturbances indicated that the CMS line was more susceptible to H2O2 stress than the maintainer line, which might be the result from co-damage between the CMS associated ORFH79 peptide and the outside H2O2 stress.2、Flow cytometric analysis on the mitochongdria of HL-CMS rice seedling revealed that both linescontained two distinct mitochondria fluorescence populations:high (HFP) and light (LFP) fluorescence populations, and a somewhat lower LFP/HFP ratio was detected in conjunction with the lesser ATP content in YtA. In addition, use of the specific effector H2O2 demonstrated a correlation between ATP levels and the LFP/HFP ratio in both lines. Higher ROS content caused a more rapid ATP decline in YtA than in YtB, and lower ATP levels resulted in a decline in the LFP/HFP ratio in both lines. Furthermore, a mitochondrial genomic DNA smear was detected by pulsed-field gel electrophoresis (PFGE). Taken together, different fluorescence intensity mitochondria with different size of mitochondrial genomic particles might be coexsited in YtA and YtB, furthermore, they could carry out different physiological functions by LFP/HFP ratio variation triggered by different ATP contents in the two lines.3、For further study the molecular mechanism of CMS in rice,we extracted RNA from anthers of F1 hybrid rice of HL CMS line, then constrcted the phage display library in accordance with protocol for operation, and succeed in buiding an library whose titer was 1.03×106pfu/ml, and its recombination-rate was 100%,97% of inserts were larger than 300 bp, without any rRNA and genomic DNA contamination.4、The mammalian BAG (Bcl-2-associ-atedathanogene) protein shared a family of chaperone regulators that modulated an number of diverse processe ranging from proliferation to growth arrest andcell death.Here,we present the results of using human BAG conservative sequence blast for uncover rice family of BAG proteins. In fact we found ten rice BAG proteins. And we select one homozygous gene for further research. The RNAi rice was much more shorte than wide type. And the mutant which was bought from Korea was still dwarf all the time of growth, as well as its seed was much shorter than wide type. Moreover the yeast which contian this gene can hamper the cell death induced by H2O2, and that the function of this protein may realize by cooperation of the two conserved structures. Western results indicated that this gene was highly expressed in the root and callus, which indicated that this protein might participate in cell growth which may result in PCD regulating induced by environment stress. In addition, we also attempted to find its interaction protein using the T7-phage library, but evern more work are needed now. |
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