| The cloning genes associated with cytoplasmic male sterility is a key for explorating of the molecular mechanism of cytoplasmic male sterility. Studies on the molecular mechanism of cytoplasmic male sterility can provide research techniques for creating new CMS system and a theoretical basis for the heterosis utilization in kenaf. The mtDNA were isolated from cytoplasmic male sterility (CMS) line P3A, maintainer P3B and Fi hybrid of kenaf. Then the full-length of atp9, atp6, atpA genes were obtained by using homology cloning and hiTAIL-PCR methods. Subsquently, the expression level of atp9, atp6, atpA in the CMS line P3A, maintainer P3B and F1hybrid were analysed by real-time quantitative method. However, when compared the full-length sequences of atp9, a47bp deletion was characterized at the3’flanking of atp9in CMS line. In order to confirm whether the47bp deletion located on3’flanking of atp9were related to male sterility, the plant expression vectors containing the different fragment of3’flanking of atp9were constructed. Subsquently, the function of these target genes were analysed by transforming into tobacco plants. Meanwhile, based on the differences of atp9and atp6in CMS line, two molecular markers distinctively to male sterile cytoplasm were development in kenaf. Other104varieties were used for further analysis. The results showed that these different fragments were specifically to male sterile cytoplasm (MSC) of kenaf. In addition to these different fragment related to male sterility, the RNA editing also were related to male sterility. Some editing sites were found when the RNA editing of the CDS of atp9, atp6, atpA were analysed.The main results obtained of the dissertation were as follows:1. The full-length of atp9gene in the CMS line and its maintainer were obtained by using homology cloning and hiTAIL-PCR methods. When comparing their sequences, As a result, a1215bp fragment was amplified from CMS line P3A, and a1262bp fragment could be obtained from the maintainer P3B, and a47bp deletion was characterized at the3’-flanking of atp9in CMS line. Meanwhile, the expression level of atp9was quantified at the dual-core period of microspore mother cell in CMS line, its maintainer and F1hybrid. The results indicated that the expression level of atp9in CMS line was0.937folds, and F1was1.59folds compared to that of its maintainer.2. The full-length of atp6gene in the CMS line and its maintainer were obtained by using homology cloning and hiTAIL-PCR methods. When comparing their sequences, As a result, a4892bp fragment was amplified from CMS line P3A, and a4922bp fragment could be obtained from the maintainer P3B, and a33bp deletion and3bp insertion were characterized at the CDS of atp6in CMS line. Meanwhile, the expression level of atp6was quantified at the dual-core period of microspore mother cell of CMS line, its maintainer and F1hybrid. The results indicated that the expression level of atp6in CMS line was0.963folds, and F1was0.987folds compared to that of its maintainer.3. The full-length atpA gene in the CMS line and its maintainer were obtained by using homology cloning and hiTAIL-PCR methods. When comparing their sequences, As a result, a3194bp fragment was amplified from CMS line P3A and its maintainer P3B. There was not obvious differenct fragment, just several bases differential between the two. The expression level of atpA was quantified at the dual-core period of microspore mother cell of CMS line, its maintainer and F1hybrid. The results indicated that the expression level of atpA in CMS line was0.760folds, and F1was1.87folds compared to that of its maintainer.4. RNA editing pattern of the CDS of atp9, atp6, atpARNA editing of atp9CDS region was analyzed using five CMS lines K03A,722A,917A, F3A, P3A with homoplasmic but heteronucleic and the CMS line and its maintainer which were homonucleic but alloplasmic. The results showed that seven of the same editing sites were found in all materials. All bases were C-to-U transformation. The editing frequency was61.5%-100%. The stop codons were created in262th site that produced a specific restrict enzyme site PvuI among all the materials, the map of Pvul-digested showed that the262th site was fully edited site. These materials with bomoplasmic but heteronucleic or homonucleic but alloplasmic had not only the same editing sites but also specific fully editing sites. There were no significant differences among these same editing sites, but there were significant differences among these specific editing sites which were more in maintainer722B,917B, F3B, P3B than in the CMS lines. It was good explanation for the materials with normal fertility leading to fully editing.Additionally, RNA editing of atp6, atpA CDS region were analyzed using the CMS line P3A, its maintainer P3B and F1. The results showed that43editing sites were found in the CDS of atp6containing18cytidine (C)-to-uridine (U) conversions, the rest were A-G, T-C, A-U, G-A transformation.37amino acids were altered because of37editing action generally in the first or second position of codon. And there were three co-editing sites. Similarly,36editing sites were found in the CDS of atpA containing10cytidine (C)-to-uridine (U) conversions, the rest were A-G, T-C, A-U, G-A, G-U, A-C, G-C transformation.24amino acids were altered because of28editing action generally in the first or second position of codon. Some amino acids trans formated were analyzed, the results showed that the hydrophilic amino acids (S) was easy to be edited which transfered to hydrophobic amino acids (L or F). Moreover, new termination codon was created during the the proceeding of RNA editing. Besides, the number and frequency of editing sites were increasing in hybrid F1being the restoring gene introduced into F1progeny.5. Four different plant expression vectors were constructed based on the differences in3’-flanking region of atp9in the CMS line. These plant expression vectors pBI121containing chimeric genes were then introduced to tobacco plants by Agrobacterium-mediated T-DNA transformation. The results showed that certain transgenic plants containing MTS-HM184-GFP and MTS-HM184were male sterility. During all the positive transgenic plants, some were male sterility or semi-sterility, while some were not. The expression analysis further demonstrated that higher level of expression were showed in the sterility plants, while no expression or less expression in fertility plants, the levels of expression of semi-sterility were in between. And the sterile plant (containing MTS-HM184-GFP) had abnormal anther produced malformed/shriveled pollen grains stained negative that failed to germinate (0%), the corresponding fruits was shrunken, the semi-sterile plants having normal anther shape produced about10%-50%normal pollen grains, the corresponding fruits were not full, and the germination rate was58%. Meanwhile these transgenic plants which altered on fertility were further analyzed in phenotype. As a result, the metamorphosis leaves were observed in the seedling stage, the plant height of transgenetic plants were shorter than wild type. The growth duration of transgenic tobacco was delayed30-45days compared to the wild type. The copies number of target genes of transgenic tobacco were analyzed using the real-time quantitative method. The results showed that these transgenic plants targeting-expression in mitochondrial containing MTS-HM184-GFP had1copies and2copies, the other two plants containing MTS-HM184both had3copies, but0copies in wild type. In summary, the chimeric genes HM184might be related to male sterility in kenaf.6. Two specific molecular markers distinctively to male sterile cytoplasm based on the differences regions of atp9and atp6in the CMS line had been developed. In order to screen male sterile cytoplasm from the germplasm using the two molecular markers, other104varieties were used for further analysis. The results showed that these varieties were divided into two types of cytoplasm, one type was male sterile cytoplasm (MSC) and the other was male fertile cytoplasm (MFC) by using the two molecular markers. Subsquently, ten varieties containing male sterile cytoplasm of UG93variety were identified and characterized from104germplasm resources of kenaf. These ten varieties were indigenous to Africa. |
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