Molecular Basis Of Polima Cytoplasmic Male Sterility And Fertility Restoration In Brassica Rapa L.ssp. Pekinensis

Cytoplasmic male sterility(CMS)have been widely used to produce hybrid seeds in a variety of valuable crop species due to strong heterosis.The Polima CMS of rapeseed and Chinese cabbage was closely associated with the co-transcription and processing of mitochondrial dicistronic orf24-atp6.Although several restorer genes of rapeseed and Chinese cabbage have been identified,but the mechanism of fertility restoration remains elusive.Transgenic Polima male sterile lines with these restoring genes can restore the pollen fertility.In mustard type rape of Polima CMS,the longer transcripts of the co-transcription of orf224-atp6 are 2.0,2.2 kb in male sterile lines(without restoring gene),but degraded to 1.3,1.4 kb in F1(with restoring gene).Only one 1.1 kb atp6 transcript exist in restorer and maintainer lines(with normal cytoplasm).In this paper,we studied the integrity and toxicity of orf224,especially there translation-inhibiting effect to atp6.Through constructing eukaryotic expression vectors with full length and 3'-region of orf224(inserted between 35S promoter and Gus gene),transformation of Arabidopsis thaliana(Col-0),many transgenic plants had been obtained.After histochemical GUS staining of seedling and flower,we can easily visualize translation inhabitation by these constructs.The results show that both full length and truncated orf224 greatly inhibit the translation of GUS,but 3'-region show less extent.Over expression of the full-length orf224 in Arabidopsis thaliana(Col-0)cause no male sterility,but the 3'-region do.In order to understand the mechanism of Polima CMS,we studied the morphology,physiology and gene activity of different development stage of flower,both transgenic Arabidopsis and Chinese cabbage male sterile lines.The result of the DAB and NBT staining show that the concentration of H2O2 and reactive oxygen in sterile line is much higher than that of maintainer line at the beginning of the flowering,but the AIP content were significantly lower in sterile line than that in maintainer.These results also proved the early hypothesis that lack of ATP in sterile line at the early flowering stage cause Polima CMS.By comparing of the PPR gene sequence cloned from our sterile line and restorer line,sterile plants and fertile plants of F2 generation,we obtain the restoring genes of Polima CMS and confirmed its function by transformed Chinese cabbage.Polima sterile line transformed with this gene fully restoring fertility.Comparing other Polima CMS restoring genes of rapeseed and Chinese cabbage,our restoring gene PPR has little mutations and more conservative.