| Populus is a crucial global afforestation and energy fast-growing tree species,which is not only of important economic value,but also significant impact on ecological balance and environmental protection.The research on molecular regulation mechanism of poplar growth and development is of great significance for selection of good characters and breeding of superior varieties.Recent studies have shown that microRNA plays an important regulatory role in many biological processes such as plant organ development,stress response,and plant signal transduction.Secondary growth is one of the vital life activities of woody plants,affecting the formation of wood and the accumulation of biomass.Pto-miR257 is a newly discovered microRNA that may be related to secondary growth in Populus tomentosa,but its biological function and regulatory role still remains unknown.N~6adenylate methylation(m~6A)could be involved in the regulation of RNA transcription,processing,cleavage and multiple life processes.It has become the latest research focus in the field of epigenetics,but detailed role of m~6A in plants is still poorly understood.In this paper,microRNA257 was successfully cloned and genetic transformed into 84K poplar.Based on the comparison of the phenotypes between transgenic plants and wild-type plants,the role of microRNA257 in the secondary growth and development of poplar was preliminarily revealed.Using Me RIP-seq sequencing,the effects of m~6A methylation modification on the heterophylly growth of lanceolate and serrated ovate leaves in Populus euphratica were uncovered,which provided scientific and theoretical basis for revealing the role of m~6A in epigenetic regulation of woody plants.The main results are as follows:1.Cloning of Pag-miR257 gene and construction of overexpression vector.The precursor sequence of Pto-miR257 gene was obtained in the NCBI database and the Pag-miR257 gene in 84K poplar was cloned.p CAMBIA2300-35S-miR257-GFP overexpression vector was successfully constructed by using molecular biology experiments.2.Acquisition and phenotypic analysis of Pag-miR257 overexpressing plants.Agrobacterium-mediated leaf disc method was used for genetic transformation of poplar,and Pag-miR257 overexpression positive plants were obtained by screening and identification.The results of real-time fluorescence quantitative PCR showed that the expression of miR257 was significantly increased in the transgenic plants.During the growth process,the average plant height of Pag-miR257 overexpressing poplar was higher than that of wild type,as well as the diameter and fresh weight of internodes increased compared with that of wild type.The results of photosynthetic rate measurement showed that the photosynthetic rate of Pag-miR257 overexpressing poplar was significantly enhanced than that of wild type.3.Microscopic observation of secondary structure of Pag-miR257 overexpressing plants and analysis of Fourier infrared spectroscopy.The results of cell wall autofluorescence showed that the xylem area and the number of ducts in Pag-miR257overexpressing poplar were significantly increased.The cell wall of the overexpressing plants was also thickened by examine of transmission electron microscopy.Furthermore,the FTIR results showed that the lignin content was significantly increased in Pag-miR257overexpressing poplar.These results indicated that microRNA257 could be involved in the regulation of the secondary growth of poplar stem.4.Construction of the m~6A methylation modification transcriptome map of Populus euphratica heterophylly.Through statistical analysis of Me-RIP sequencing data,it was found that there were 0.385 m~6A peaks in each transcript of JC＿leaf and 0.390 m~6A peaks in each transcript of PZ＿leaf.The ratio of m~6A/A in m~6A modified transcripts is 0.76%-0.82%in the serrated ovate leaf and lanceolate leaf tissue,respectively.Additionally,the methylation rates were 33.84%and 34.21%in the serrated ovate leaf(JC＿leaf)and lanceolate leaf(PZ＿leaf),respectively.5.Distribution characteristics and conservative sequence identification of m~6A peaks of Populus euphratica.The distribution of m~6A peak on the heterotypic leaves of Populus euphratica on the m RNA was enriched at specific sites.The m~6A peaks in JC＿leaf and PZ＿leaf were mainly concentrated near the CDS region and the starting position of 3’UTR.The proportion of genes containing a single m~6A modification site accounted for more than70%,while the proportion of genes containing multiple m~6A modification sites was low.The identified m~6A conserved motif UGUAYY sequence of plant was significantly enriched in the lanceolate and serrated ovate leaves of Populus euphratica.6.Differential methylation genes and potential functions analysis of m~6A methylation in the two kinds of leaves in Populus euphratica.The m~6A methylation-modified gene showed functional differences between the heterophylly of Populus euphratica.The results showed that 1962 and 1652 leaf shape differential m~6A peaks were identified in the serrated ovate leaves(JC＿leaf)and lanceolate leaves(PZ＿leaf).After correlation with genes,895genes with down-regulated differential methylation genes and 422 up-regulated genes were identified.It is found that differential genes(HSMGs)between these two leaves were mainly involved in biological processes such as plant-pathogen interaction,photosynthesis and plant hormone signal transduction.Taken together,by using molecular biology,poplar genetic transformation,cell biology analysis and other methods,it is found that Pag-miR257 could play a role in increase of the lignin deposition,and affect the morphological formation of secondary xylem.These findings preliminarily confirmed that Pag-miR257 could be involved in the regulation of secondary growth and development of poplar.This study laid foundation for further exploring the network relationship between miR257 and other genes and the regulatory mechanism of wood formation.Furthermore,sequencing analysis of RNA m~6A methylation in Populus euphratica lanceolate and serrated ovate leaves was carried out,revealing the potential transcriptional regulatory function of m~6A methylation modification among Populus euphratica heteromorphic leaves.The results of this study laid a foundation for further revealing the mechanism of m~6A methylation modification involved in regulating heteromorphic leaves formation of Populus euphratica,and provide new scientific evidence for epigenetic influence of RNA on the development of heteromorphic leaves in plants. |
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