Studies On Relationships Among Brassica Species And Comparative Proteomics In The Early Generations Of Newly Synthesized Brassica Napus Allotetraploid

Brassica is the third largest oilseed crop in the world, providing approximately 13% of the world's supply of vegetable oil. The relationship between three diploids and three allotetraploids was demonstrated in a classical cytogenetic study by U; this relationship is commonly referred to as the"U triangle". On our research, cytological, molecular biological, and comparative proteome analyses were tentatively applied to study the relationship between them. This studies have provided important basis for Brassica evolution. Main results were described as follows:1. Mitotic and meiotic FISH analysis of 45S rDNA in Brassica diploidsThe position and number of 45S rDNA on mitotic and meiotic chromosomes of the three basic diploid species (Brassica rapa, B. oleracea and B. nigra) were detected by FISH analysis using 45S rDNA as probe. Ten out of 20 chromosomes can be easily identified in B. rapa, six out of 16 in B. nigra, and four out of 18 in B. oleracea. Among them, meiotic FISH analysis of 45S rDNA of B. rapa and B. nigra was reported for the first time. These investigations are useful for identifying the chromosomes of genome A, B and C and studying the evolution and phylogeny of the Brassica genomes.2. Relationships among genome A, B and C revealed by centromere repetitive sequence FISH and CAPS analysisPhysical locatization of repetitive DNA sequence from genome A (151bp) was carried out on the chromosome of the selected Brassica species by FISH (fluorescence in situ hybridization). The signals distributed on all the chromosomes of A (Brassica.rapa, 2n=20) or C genome (B. oleracea, 2n=18). For B. Juncea (AABB,2n=36), the signals were found on all the chromosome of genome A and the strength of signal varied among different chromosomes, while the chromosomes of genome B showed weak or no signals. Application of this method to 151bp centromere satellite repeats distinguished the 10 chromosome of AA from B. juncea (AABB). FAE1 gene is a related-limiting gene for erucic acid synthesis in Brassica. The genes from six Brassica species of U-triangule were cloned by PCR. These PCR products were digested with different restriction endonucleases. MboI and MspI were found to produce informative CAPS patterns of FAE1 gene. Three diploids display different patterns, the pattern of genome A was very similar to that of genome C, while the pattern of genome B was the most diverged out of the patterns of the A and C genomes. Three amphidiploids generally exhibited additive patterns of the progenitors, but not strictly in all cases, indicating that rearrangements and recombinations did occur in the formation and evolution of amphidiploids. Genetic relationships among Brassica species could be demonstrated through CAPS analysis of FAE1 gene and FISH method when repetitive DNA sequence (not ribosomal RNA genes) was used as a probe.3. The analysis of constitute of protein subunits and clusteringSeed storage proteins extracted from six species of Brassica were analysed with SDS-PAGE technique. The constituents of seed storage proteins contained 24 subunits, and polymorphisms and specific protein bands among different species were observed. Prominent bands were characterized at all the diploid species. The result of cluster analysis indicates that 6 species are classified into two types. First type includes B. rapa, B. oleracea and B. napus. The second type includes B. nigra, B. juncea and B. carinata.4. Characterization of seedling proteomes and development of markers to distinguish the Brassica A and C genomesThe diploid species Brassica rapa (genome AA) and B. oleracea (genome CC) were compared by full-scale proteome analyses of developing seedling. A total of 95 and 111 protein spots were detected in Brassica rapa and B. oleracea, correspondingly. Among these were 29 mutual spots, accounting for 28.2 % of the total spots, indicating the existence of a basal or ubiquitous proteome. while 66 spots were specific to the A genome and 82 to the C genome (32.0 % and 39.8 %, respectively). Nine A genome specific spots and eleven C genome specific spots were excised for subsequent MALDI-TOF-MS analyses. Six of the excised nine spots from the A genome and four of the eleven C genome spots could be reliably identified by MALDI-TOF-MS and protein database searching, the identification rate was 50%. Based on the sequences of deduced contrasting AA and CC protein spots, a number of PCR-based markers to distinguish B. rapa (genome AA) and B. oleracea (genome CC) species were developed as well. Ten primer pairs were contrived and tested for their efficiency in detecting polymorphisms between the A and C genomes.5. Comparative proteomics in the early generations of newly synthesized Brassica napus allotetraploidPolyploidy is a prominent process in higher plants and is often described as a genomic shock that may induce stress and defense responses. Recent studies indicate that extensive genetic and epigenetic changes often occur at the early stage of allopolyploid's genome formation, these changes have played a significant role in stabilization of newly formed genome of species. Synthetic Brassica napus provides a model system to study early events in the evolution of ploidy genomes, because the exact progenitors (B. rapa and B. oleracea) for a synthetic polyploid are known. Large-scale analysis of proteome from leaf organ can be studied accurately compared to their homozygous diploid progenitors B. rapa and B. oleracea, and gene expression changes of F1~F4 generations, which might help enriching the mechanisms of genetic and epigenetic changes. The detailed results are as follows:1) Comparative proteomics between newly synthesized B. napus and its progenitorsLeaf proteins of B. napus and its pprogenitors (B. rapa and B. oleracea) were separated with 2-DE. It was remarkable that the abundance of all these differentially expressed proteins in the hybrid was different from that of corresponding proteins expressed in its progenitors, some of them deviated relatively mid-parent predictions, exhibiting somewhat non-additive expression repatterning. Seven of the differentially expressed proteins were identified by MALDI-TOF-MS and database searching. Biological functional analysis showed these identified proteins were related with substance and energy metabolism: ATP synthase beta subunit was manifested involving in energy metabolism; ADP-glucose pyrophosphorylase small subunit may be an enzyme in substance metabolism; sedoheptulose-bisphosphatase precursor was involving in photosynthesis; DNA-directed RNA polymerase beta-2 chain was related to transcriptional regulation; Serine/Threonine protein kinase, possible related to disease resistance; and a hypothetical unknown protein. The function of these proteins was showed to be related with heterosis, and our explanation of heterosis mechanism was carried out at the protein level. In addition, our analysis of leaf proteome indicated that genome restructuring by recombination occurred in B. napus synthetics the first generation. This process created novel plant phenotypes and should be addressed in terms of evolutionary consequences.2) Comparative proteomics in early generations of newly synthesized B. napus allotetraploidWe analyzed genetic and epigenetic changes in resynthesized Brassica napus from the first to forth generations using comparative proteomics. The differentially expressed proteins were sorted into five categories after statistical analyses of the 4 generations: 1) polypeptide specific in F2; 2) polypeptide only occurred in F3; 3) polypeptide loss in F1 and reoccurred in F2~F4; 4) polypeptide loss in F1~F2 and reoccurred in F3~F4; 5) gradually up-regulated polypeptide from F1 to F4. The results indicated that gene silencing was not permanent phenomenon and could be reactivated at any moment. Biological function showed these identified proteins related with substance and energy metabolism, Signal transduction, detoxification enzymes, protein degradation, retrotransposon protein and other unknown proteins. Although leaf proteins were extensively modified in synthetic B. napus, however, the distribution of the"housekeeping"proteins into metabolic pathways, functional categories and signal transduction were not disturbed. Moreover, there is no evidence for chaos or a large disorder following the merging of two genomes. Instead, a novel order established quickly and may evolve in further generations of synthetic B. napus.