| Polima cytoplasmic male sterility(pol CMS) is not only a major pattern for exploiting heterosis in Brassica napus, but also a model for the researches on nuclear-cytoplasmic interactions. In our study, the restorer gene of the pol CMS, Rfp, was cloned by map-based cloning approach. The cytology and molecular researches provided further clues for understanding the mechanism about the abortion of the pol CMS and the recovery function of Rfp gene in Brassica napus. The main findings were as follows:1. The restorer gene of pol CMS(Rfp) was mapping to a 32 kb regeion on A9 chromosome of Brassica napus by map-based cloning approach in the backcross populations from the crossing of the sterile line 6330 A and the restorer line Bing409. One orf in this region being a member of PPR family was regarded as the candidate gene. Genetic transformation of this orf confirmed that it was the restorer gene of the pol CMS.2. Sequencing of the full-length gene(including promoter region, CDS and 3,UTR) in the sterile line 1141 A and the restorer line Bing409 showed that the promoter and 3,UTR regions of Rfp and rfp were all the same but a SNP mutantion, respectively. However, 45 SNPs in CDS region were identified, which led to the variation of 30 amino acids, and 12 of them changed the polarity of the amino acids. The subcellular proteins of Rfp and rfp both localized in the mitochondria. This suggests that the functional differences in Rfp and rfp were owing to the variation of the CDS region, instead of the localization of these proteins. Based on the sequencing analysis, we developed a functional marker for.the Rfp gene, which could be used to efficiently select restorer line in backcrossed breeding.3. The semi-thin sections analysis showed that the anther of the sterile plants could not form a normal shape of four corners in the 5 stage which led to produce no pollen or shriveled pollen at laster development. TEM anlysis showed that the mitochondria in the anther cell of the sterile plant became abnormal or collapsed in anther development stage 3, which resulted in the archesporial cells processing PCD advanced. Additionally, the L2 layer cells in the sterile plants could not properly differentiate into endothecium, middle layer, tapetum and microspore mother cells.4. RT-PCR and q PCR analysis showed that Rfp gene was widely expressed in roots, stems, leaves, flowers and floral buds Brassica napus, with the highest expression in young bud. GUS analysis was consistent with the results of RT-PCR and q PCR analysis. These indicated that Rfp gene may play an important role in anther development. Moreover, GUS staining showed that Rfp gene was expressed in endothecium, middle layer, tapetum and pollen mother cells(microspores), which were differentiated from the L2 layer cells of the anther. This corresponds to the sterility phenotype.5. Northern blot detection was taken using the mitochondrial orfs different between the pol cytoplasm(CMS line) and the nap cytoplasm(maintainer line) as probes. Compared with the CMS line, the transcripts of orf224-atp6 were obviously reduced in the fertility restored line and transgenic restored line by using orf224 as a probe, while no band was detected in the maintainer. These indicated that the Rfp gene may be involved in the processing transcript orf224-atp6 through a post-transcriptional mechanism to suppress male sterility.6. There were 15 PPR domains in the Rfp gene, which were associated with RNA recognition. However there was no domain assoicated with RNA splicing or processing. It indicated that there may be some unknown proteins involved in the process of post-transcriptional mechanism. Four genes, possiblely interacted with the Rfp gene, were identified from the c DNA library of Brassica napus by yeast two-hybrid. These interactions were confirmed in the mitochondria of Brassica napus by bimolecular fluorescence complementation. These selected genes were interfered by genetic transformation.and the functions of these genes need further study. |
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