And Evolution Analysis Of The Restriong Gene Of Brassica Napus Cenic Male Sterility7365a

The Brassica napus recessive genetic male sterility7365A was considered to be an efficient way in the heterosis utilization of rapeseed, because it can be generated a100%male sterile population by crossing with temporary maintain line and it has wide restores. The male sterility of7365A was controlled by two interacting genes (Bnms3and BnRf). In order to understand the sterile molecular mechanism of7365A, the restoring gene BnaC9. Tic40(BnMs3) was isolated by a map-based cloning approach. Then the evolution dynamics of Tic40in the Brassicaceae genomes was analyzed. Furthermore, the sterile molecular mechanism of7365A and the function of the restoring gene BnaC9.Tic40in anther development were studied by cytological and molecular biological methods. The results were described as follows:1. The phenotypic characterization of7365A by cytological analysesResults of transmission electron microscopy (TEM) suggested, at the meiosis stage, in the7365A mutant, the tapetal cells became abnormally enlarged. At the tetrad stage, the mutant tapetal cell walls were still intact and the tapetal cell seemed to be vacuolated. The organelles in the tapetum such as plastids, endoplasmic reticulum and vesicles were seldom. Subsequently, the tapetal cells in the7365A mutant did not synthesize and accumulate lipid compounds. The callose wall surrounding the tetrads was not dissolved. Finally, the microspores were degraded as tetrads. Lipid staining and TUNEL assay showed the tapetal cells in the7365A mutant could not synthesize and accumulate lipid compounds throughout anther development, and the retardation of tapetal PCD resulted in the failure of timely tapetal degradation in the7365A mutant. These results suggested the defects of the7365A mutant anther development were initiated at the meiosis stage. The transition of the tapetal cells to the secretory cells in7365A was defective, resulting in a loss of the secretory function of the tapetum, as suggested by abortive lipid accumulation, absent callose dissolution and retarded tapetal degradation.2. The cloning of the restoring gene BnaC9.Tic40The BnMs3gene was located between the molecar markers IP20and IP17by exploiting IP markers and scanning of two BnMs3/Bnms3near-isogenic line populations. Subsequently, the BAC overlapping clusters about the target region was constructed using the BAC libraries of B. oleracea and the B. napus cultivar Tapidor. Then the BnMs3gene was narrowed into a49.279kb region between T1and T4. By ORF finding, four candidate genes were identified in this region. Four resulting complementation constructs were introduced into the7365A line by Agrobacterium tumefaciens-mediated transformation. The genetic complementation suggested BnaC9.Tic40, the ortholog of atTic40, corresponds to the restoring gene BnMs3.3. The expression patterns and function study of BnaC9.Tic40RT-PCR analysis suggested that BnaC.Tic40was highly expressed in seedlings, anthers and20-dayold seeds. The expression of the fusion construct, Pro BnaC9.Tic40-GUS and RNA in situ hybridization of BnaC9. Tic40in Arabidopsis wild-type anthers, were used to determine precisely the spatial and temporal patterns of BnaC9. Tic40expression during anther development. At stage5, the BnaC.Tic40gene was first expressed in the tapetum and microspore mother cells. The expression level became higher at the tetrad stage, and was reduced in the tapetum and microspores along with microspore development and tapetal degradation. The expression was disappeared when pollen became mature. The subcelluar localization assay showed the BnaC.Tic40protein was localized in the chloroplast of the protoplast, which is consistent with its ortholog atTic40. Tic40is an inner membrane-anchored translocon in the chloroplast and functions during protein translocation across the inner membrane. It is proposed that BnaC.Tic40participates in protein translocation in the tapetal plastid.4. Duplication and deletion of Tic40in the diploid Brassica speciesThe genomic regions around Tic40in the Brassicaceae species were conserved and displayed high microsynteny. The Tic40genomic regions were triplicate duo to the whole-genome triplication of the tribe Brassiceae ancestry. Subsequently, in the diploidization process of the tribe Brassiceae ancestry, frequent gene loss was present in the triplicate Tic40genomic regions, resulting in the deletion of Tic40locus in the B. oleracea C3and B. rapa A3syntenic regions. Currently, the diploid Brassica species B. oleracea, B. rapa and B. nigra have two Tic40loci, located on B. oleracea C2and C9, B. rapa A2and A10, respectively.5. Homeologous chromosomal rearrangement between A10and C9caused different origins of BnaC9.Tic40(BnMs3) and bnac9.tic40(Bnms3), originating from BolC9.Tic40and BraA10.Tic40, respectivelyPhylogenetic tree constructed and sequence identity analysis revealed that BnaC9.Tic40originated from BolC9.Tic40. However, its allele bnac9.tic40was derived from BraA10.Tic40. Partial sequences of twenty-three flanking genes ofBnaC9.Tic40and bnac9.tic40were used to compare with their homologs from B. oleracea C9, and B. rapa A10. Nucleotide diversity demonstrated that chromosomal rearrangement around the Tic40in B. napus N19linkage was caused by homeologous recombination between A10and C9. Finally, about2Mb fragments around Tic40in the N19linkage of7365A was derived from B. rapa A10homologs, resulting in the evolutionarily close between bnac9.tic40and BraA10. Tic40.6. Function divergence of Tic40in the diploid Brassica species resulted by positive selection, caused a gain-of-function variation of BolC9.Tic40Genetic complementation assay of Tic40genes from three diploid Brassica species and A. thaliana indicated that in comparison with the Brassicaceae ancestral Tic40, BolC9.Tic40had a gain-of-function variation. The six possible variation sites related to function differences exits in the TPR and Hop domains, resulting in BolC9. Tic40and its derivative BnaC9.Tic40gain a new function which can restore the fertility of7365A duo to present of the dominant gene BnRf. And only B. oleracea and its derivatives displayed the possible gain-of-function variation sites. Subsequently, selective pressure analysis explored strong purifying selection of Tic40in the Brassicaceae before the Brassica genus divergence from other genus of the tribe Brassiceae. After divergence of the Brassica genus, the evolutionary clade of BolC9.Tic40displayed distinct positive selection. Furthermore, the six possible variation sites related to function differences had a Bayesian posterior probability greater than90%, and3of6with a probability more than...