The Research On MiRNA Evolution And Target Genes In Poplar

Pupulus plants are fast-growing commercial trees. Owing to the availability of the Populus trichocarpa genome sequence and plenty of Populus-specific genetic databases, Populus is known as a model for research of woody plants. Populus serves as a reference for many fundamental scientific investigations of wood formation, secondary cell wall formation, perennial and seasonal growth, flowering, evolution and resistance physiology. MicroRNAs （miRNAs） are a class of short non-coding RNAs found widely in eukaryotic organisms. MiRNAs function as regulators of development and stress responses in plants. Study of miRNA will contribute to reveal molecular mechanisms of many phenomenon of life, and may be applied to genetic improvement of crops and forest trees. To understand the evolutionary mechanisms of miRNAs in poplar, the evolution and functional diversity of3more conserved and4less conserved poplar miRNA gene families was comparatively studied in this dissertation, and the poplar miRNA targets were investigated by degradome sequencing and gene ontology enrichment analysis, and then two miRNA target genes were cloned and their functions were analyzed. The main research contents and results are as fellows:1. Molecular evolution of the poplar MIR169gene family. Tandem duplications and chromosome segmental duplications, which happened16.6-83.1million years ago, contributed equally to the expanding of ptc-MIR169gene family, Several members of this family has disappeared during evolution. Before the differentiation of monocots and dicots, the plant MIR169family had been differentiated into two families, MIR169₁and MIR169₂, then MIR169₄and MIR169₃family differentiated from MIR169₁and MIR169₂respectively in poplar. The expression patterns diversified obviously among the gene family. MIR169u has highest expression activity in many tissues. The predicted target genes of poplar MIR169include transcription factors, protein kinases, etc. The NF-YAs are major targets of poplar MIR169. These findings suggest that the ptc-MIR169gene family is involved in complex regulatory networks, and plays significant roles in development and stress response in poplar.2. Evolutionary of the poplar MIR171genes. The poplar MIR171gene family expanded mainly through large-scale duplication48-54million years ago and the expression patterns and functions have diversified. MIR171l/m/n have the highest expression activity in many tissues. Poplar MIR171gene family involves in complex regulation of development, light responsiveness and photomorphogenesis by targeting GRAS transcription factors and signal transduction proteins.3. Molecular evolution of the poplar MIR156gene family. The ptc-MIR156gene family expanded mainly via large-scale duplications instead of tandem duplications. The expression patterns and cis-acting elements have diversified among the members in this family and the MIR156g/h/i/j have the highest level of expression. Owing to the variance in the mature sequences, the target genes, including29SBP domain proteins, also differ among these members. These findings suggest that ptc-MIRl56gene family have diversified in functions and formed complex regulatory networks in poplar.4. Evolution of4new miRNA gene families in poplar. The more conserved gene family, MIR1446, expanded mainly through chromosome large-scale duplications, and MIR1444expanded through chromosome large-scale duplications as well as tandem duplications, and the less conserved gene families, MIR6425and MIR6462expanded mainly via tandem duplications; the expression patterns of the4gene families have all diversified, the MIR1444/1446gene families expressed higher in multiple tissues than MIR6425/6462families; the mature sequences of different members are same in MIR1446/6425/6462families, therefore their target genes are same; on the other hand, the mature sequences of different members are different in MIR1444families, so their target genes are different. Comparative analysis of the above-mentioned seven miRNA gene families shows that the older and more conserved miRNA gene families generally contain more members, higher expression levels, higher rates of functional diversification, and their target genes include mainly transcription factors; whereas the younger and less conserved miRNA gene families usually have fewer members, lower expression levels, lower rates of functional diversification, and their target genes are mainly functional protein （e.g., enzymes）.5. Degradome sequencing and gene ontology enrichment analysis of miRNAs target genes in polar. A leaf degradome library of Populus euphratica was constructed and sequenced by Illumina HighSeqTM platform, yielding42,659,177row reads. After removing the adaptors sequences, pollution sequences and low-quality sequences,34,657,804clean reads were obtained. A total of23miRNA targets were identified by analysis of the degradome sequences. All targets identified by degradome analysis were subjected to AgriGO to investigate gene ontology. The miRNA targets were significantly enriched in oxidoreductase activity, antioxidant activity and cell redox homeostasis. These results suggested that miRNAs play important roles in eliminating of ROS and maintaining of redox homeostasis under salt stress. The targets of highly conserved and lowly conserved miRNAs from poplar, Arabidopsis and rice were analyzed by singular enrichment analysis and the realist were cross compared. The results showed that the targets of more conserved miRNAs from above3species were enriched significantly in transcriptional factors, and the targets of more conserved miRNA from poplar were also enriched significantly in oxidation reduction process. In contrast, the enrichment significance of the targets of less conserved miRNAs was lower than more conserved miRNAs。5. Cloning and functional analysis of miRNA target genes in polar. The full length cDNA of one of the MIR171targets, Pescll, was cloned from Populus euphratica. The Pescll belongs to the GRAS transcription factor family and has a miR171a-3p/b target site which was validated by RNA ligase mediated5’RACE. Under salt stress conditions, the expression of Pescll rose in leaves, suggesting that the Pescll participates in response to salt stress in polar. The full length cDNA of Pecngcl, which belongs to the GRAS transcription factor family, was cloned from Populus euphratica. The PeCNGCl located in the cell membrane, which was validated by transient expression in tobacco epidermal cell. The Arabidopsis plants transferred by Pecngcl showed higher salt tolerance than wild type. Under salt stress conditions, the expression of Pecngcl rose in leaves, suggesting that the Pecngcl involved in maintaining of Na+/K+homeostasis in leaves under salt stress in polar.