The Small RNA And Proteomic Studies On Brassica Polyploid Plants

Polyploidization plays an important role in promoting plant speciation and evolution. All most all the angiosperms have undergone at least one time whole genome duplication. Small RNAs play an essential role in genomic and transcriptomic changes in polyploidy through regulating gene expression and structure of chromatin. Based on the research that transcriptomes on the Brassica hexaploid and its paternal parent Brassica rapa, maternal parent Brassica carinata in our group, in this study, we used the same materials to characterize expression patterns of small RNAs and proteins in Brassica hexaploid.We used high-throughput sequencing to compare miRNA and siRNA expression profiles between Brassica hexaploid and its parents. As a result,981 known miRNAs (613,784, and 742) were identified in the B. rapa, B. carinata, and Brassica hexaploid, respectively. We detected 441 miRNAs present in all three libraries, which accounted for 44.95% of the 981 known miRNAs. We detected 618 miRNAs were differentially expressed between Brassica hexaploid and its parents, we associated GO terms with coherent target genes of miRNAs that exhibited differential expression between Brassica hexaploid and its parents to identify the significantly changed GO biological process terms. The results suggested that targets involved in response to stimulus are significantly enriched in coherent target genes of down-regulated miRNAs. More differentially expressed miRNAs with larger differences in expression exist between Brassica hexaploid and B. rapa. We detected 425 miRNAs were non-additively expressed in Brassica hexaploid, which suggest a trend of non-additive miRNA regulation following hybridization and polyploidization.We assigned target genes of additive and non-additive miRNAs to secondary classification GO terms. In the biological process category, seven functional groups:metabolic process, cellular process, death, localization, establishment of localization, response to stimulus, and multi-organism process were significantly enriched in coherent target genes of non-additive miRNAs. Remarkably,255 (60.00%) non-additively expressed miRNAs in the Brassica hexaploid are repressed, and there was a bias toward repression of B. rapa miRNAs, which is consistent with the progenitor-biased gene repression in the synthetic allopolyploids. In addition, we identified 653 novel mature miRNAs in Brassica hexaploid and its parents. Finally,204 siRNA clusters (100,73, and 84) in B. rapa, B. carinata, and Brassica hexaploid were identified, respectively. Among them, 6 siRNA clusters were present in all three RNA libraries which accounted for 0.61% of the 204 siRNA clusters. Among them,92.16% of the total identified siRNA clusters non-additively expressed. We found 90.91% of the non-additive expressed siRNA clusters exhibited a low-parent pattern in Brassica hexaploid. We examined genes that highly correlated with siRNA clusters that exhibited non-additive expression against the GO database to determine the GO biological process terms. The deviating clusters were categorized into a range of gene ontologies, including metabolic process, cellular process, and response to stimulus. Non-additive small RNA regulation is involved in a range of biological pathways, probably providing a driving force for variation and adaptation in allopolyploids.We reported quantitative analysis of proteomic changes in leaves of Brassica hexaploid and its parents using isobaric tags for relative and absolute quantitation (iTRAQ) coupled with mass spectrometry. A total of 2044 reproducible proteins were quantified by at least two unique peptides. We detected 452 (22.11%) proteins differentially expressed between Brassica hexaploid and its parents. Compared with all the proteins identified in the study, proteins related to functional classes of electron carrier activity, antioxidant activity, catalytic activity, response to stimulus, multi-organism process, and immune system process were significantly enriched in differentially expressed proteins. We found 100 proteins, approximately 4.89% of the total identified proteins, were non-additively expressed in Brassica hexaploid, which suggested a trend of non-additive protein regulation following genomic merger and doubling. Functional categories of cellular component biogenesis, immune system process, and response to stimulus, were significantly enriched in non-additive proteins which probably providing a driving force for variation and adaptation in allopolyploids. In particular,311 proteins,68.88% of the total 452 differentially expressed proteins, showed expression level dominance of one parental expression, and there was an expression level dominance bias toward the tetraploid progenitor. In addition, the percentage of differentially expressed proteins that matched previously reported differentially genes were relatively low.In conclusion, we characterized the expression patterns of small RNAs and proteins in Brassica hexaploid and its parents, and we found small RNAs and proteins showed non-additive and progenitor-biased expression patterns in Brassica hexaploid; the functional group of response to stimulus was significantly enriched in up-regulated proteins and coherent target genes of down-regulated miRNAs in Brassica hexaploid. These results increase our understanding of the gene expression regulation mechanism in Brassica polyploidy.