- Results: In this study, we used high-throughput sequencing technology to analyze the expression profiles of miRNAs in leaf and flower tissues of O. - In the end, flavonoid content was measured in flower and leaf tissues and potential role of miR858 in regulating flavonoid synthesis was illustrated in O. - Conclusions: This study not only provided the genome-wide miRNA profiles in the flower and leaf tissue of O.. - For instance, miRNAs participate in the regulation of numerous bio- logical processes, such as cell proliferation, leaf and root development, phase transition [4–6].. - The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. - If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. - fragrans is one of the most known medicinal plants in China, typically used in folk medicine as expec- torant and anti-cough agent for thousand years. - fragrans has a mildly sedative effect on controlling the energy balance of the body in terms of the prevention of over-eating and gaining weight [18–20]. - fragrans development and application have been re- ported in the recent years there is no study reported to address the genome-wide identifica- tion of the microRNAs in O. - fragrans, which impeded the comprehensive understanding of the regulation net- works during O. - fragrans can further provide important additional information about the regulatory mechanisms in the biological processes of O. - In addition, miR858 was reported to putatively regulate MYB transcription factors in A.thaliana [34], and MYB family transcription factors MYB11, MYB12, as well as MYB111 were found to regu- late flavonol biosynthesis by targeting CHI, CHS and F3H [36]. - understand the functions of the identified conserved and novel miRNAs, as well as the ways that they play regula- tory functions in O. - fragrans, GO analysis and KEGG analysis of the target genes were also performed. - To obtain the RNA sequencing data for identification of the conserved and novel miRNAs in O. - In the end, 22.83 and 23.13 million clean reads were acquired from flower and leaf groups (Additional file 1: Table S1), re- spectively. - In the flower tissue of O. - fragrans, 83.9% of small RNAs were 20-24 nt, while 81.2% in the tissue. - Moreover, regardless of the samples source, most small RNAs were characterized with 24 nt in length, the percentages of 24 nt small RNAs were 44.2% in flowers and 41.3% in leaf. - 1 MiRNA sequencing analysis of flower and leaf tissues in O.. - Length distribution and frequency analysis of miRNAs in O. - In the following data analysis, the rRNAs, tRNAs, snRNAs and snoRNAs were not included. - To further address the tissue specificity of the small RNAs in O. - fragrans, the comparative analysis of the small RNAs reads between flower and leaf tissue was per- formed. - To identify the conserved miRNAs in O. - The read numbers of the conserved miRNAs in miRNA families varied dramatic- ally from 1to Additional file 2: Table S2).. - It was reported that miR-535 was induced in leaves under low ambient temperature treatment [39], which is consistent with the analyzing result in this study since the sampling time was in October and the temperature was comparatively low in the middle areas of China. - As mentioned before, TPIS_PETHY is one of the target genes of miR5538. - Taken together, these results had shown that the expression levels of conserved miRNAs highly varied, which could be because of the tissue-specific or stage-specific expres- sion patterns of conserved miRNAs in O. - To predict novel miRNAs in the obtained libraries, first of all, the hairpin structure of miRNA precursors is characterized. - We performed the sequences folding ana- lysis of potential miRNA precursors and identified the novel miRNAs in the libraries. - In the end, 45 novel miRNAs in the flower li- brary and 58 novel miRNAs in the leaf library were suc- cessfully detected (Additional file 3: Table S3). - In the flower library, the lengths of mature novel miRNAs were from 18 to 24 nt, while the lengths ranged from 18 to 25 nt in the leaf library. - Besides, the lengths of the novel miRNA precursors were also measured. - It ranged from 59 to 226 nt in the flower library and from 58 to 226 nt in the leaf library, with the average lengths 92 nt and 87 nt, respectively. - The MFEI of the precursor sequences ranges from − 0.67 to − 1.46 with an average of − 1.05 in flower and − 0.59 to − 1.80 with an average of − 1.03 in leaf, respectively. - Compared with the conserved miRNAs, the novel miRNAs in O. - Meanwhile, eight novel miRNAs were most abundant in the leaf tis- sue, including ofr-Novel_14, ofr-Novel_46, ofr-Novel_64, ofr-Novel_67, ofr-Novel_75, ofr-Novel_79, ofr-Novel_84 and ofr-Novel_85. - Given the sig- nificant difference of the read numbers between the novel and conserved miRNAs in O. - Differential expression analysis and target gene prediction of miRNAs in O. - MiRNAs play versatile roles in post-transcription regula- tion of the target genes expression, which are essential for plant development. - The reliability of the differentially expressed miR- NAs was further confirmed by qRT-PCR experiments in which twelve randomly selected differentially expressed miRNAs were tested (Fig. - Interestingly, some of the potential target genes of identified miRNAs belong to transcription fac- tors, such as the well-known WRKY family protein and GRAS family protein. - It is well known that GRAS gene family comprises several transcriptional regulators, and participates in the regulation of plant growth and devel- opment [43]. - For example, in the biological process category, most GO terms were distrib- uted in “organic cyclic compound metabolic process”,. - To better understand the biological functions and underlying mechanisms of the potential target genes, we took advantage of the KEGG database to explore the biochemical pathways in which the target genes partici- pate. - Interestingly, several target genes of the novel miRNAs identified in O. - Heatmap analysis of the differentially expressed miRNAs of flower and leaf tissues in O. - fragrans, which is useful to illustrate the biological functions of the miRNAs. - In this study, there were 133 identified miRNAs differently expressed in flower and leaf tissues. - In addition, the miRNA-mRNA networks of the DE- miRNAs were analyzed and some interesting mRNA. - 3 GO (Gene Ontology) analysis of the mRNA targets of differentially expressed miRNAs between flower and leaf tissues in O. - putatively regulate MYB transcription factors in A.thali- ana [34], while MYB transcription factors were found to regulate flavonol biosynthesis by interacting with CHI, CHS and FLS genes [35]. - MiRNAs are important non-coding small RNA mole- cules which participate in the regulation of numerous physiological processes in plant [1]. - Based on the advan- tages of high-throughput sequencing technology, the capacity in large-scale miRNAs detection and high sensi- tivity in the measurement of minimally expressed miR- NAs, it has been widely used to powerfully identify conserved miRNAs and species-specific miRNAs during. - fragrans, one of the widely cul- tivated perennial, evergreen broad leaved trees in Asia, is not reported yet. - Via bioinformatics analysis, there are 47 conserved miRNA families and 88 novel miRNAs identified from flower and leaf samples.. - The read numbers of the 47 conserved miRNA fam- ilies varied from 11 (miR5538) to miR166), which implies the expression patterns of different miRNA families dramatically differ. - They expressed in the flower and leaf tissues with at least ten thousand reads, which is in line with the previous study about the correlation between plant evolutionary conser- vation and expression abundance [39, 40]. - Moreover, unlike other miRNAs, the expression level of miR5538 was very low and only 11 reads de- tected in the flower tissue. - However, even though the ex- pression level is not abundant, the lowly conserved miRNAs may participate in the regulation of plant devel- opmental and cellular processes such as flower develop- ment and abiotic stress responses [14].. - Interestingly, most of the predicted potential targets of novel miRNAs in O. - It is worth to mention, some of the predicted target genes were important transcription factors in plant including WRKY and GRAS. - Flavonoid content analysis between flower and leaf tissues in O. - In this study, high-throughput sequencing technology is used to identify miRNAs in leaf and flower tissues of O.. - In the end, flavonoid content between flower and leaf tissues was measured and potential role of miR858 in regulating flavonoid biosynthesis was ex- plored in O. - Taken together, this study not only provided the genome-wide miRNA expression pro- files in the flower and leaf tissue, but also addressed the possible regulatory role of miR858 in flavonoid synthesis in O. - The flower and leaf samples were collected from O. - To check the A260/A280 and A260/A230 values, the purity of the RNA was con- firmed. - In the end, the RNA samples were stored in − 80 °C freezer for further use.. - Briefly, electrophoresis of the total RNAs isolated from O. - The criteria used to define the low-quality reads are listed as below: 1) The reads in which quality scores of more than 15% of the bases are less than 19. - The conserved and novel miRNAs identification. - The length distribution of the clean reads was summa- rized and the mapping of the clean reads to the O. - Finally, the secondary structure, the cleavage sites of Dicer com- plex and the minimum negative folding free energy of the unannotated small RNAs were measured via the RNAfold (http://www.tbi.univie.ac.at/~ivo/RNA/. - 3) The precursor should not contain the mature miRNA in the hairpin structures ’ terminal loop. - Analysis of the differentially expressed miRNAs. - To better understand the biological function of the identified miRNAs, it is necessary to analyze the ex- pression levels of miRNAs in leaf and flower tissue and screen the differentially expressed miRNAs. - In order to identify the differentially expressed miRNAs between flower and leaf tissues, we set the expression level of the miRNAs in leaf as a control and detected the up- or down-regulated miRNAs in flower. - To indirectly certify the sequencing data quality of small RNAs samples and directly confirm the differential ex- pression patterns of the identified miRNAs in the flower and leaf tissues of O. - fragrans, we randomly selected some of the miRNAs to perform qRT-PCR experiment.. - The qRT-PCR experiments were inde- pendently repeated three times to measure the expres- sion levels of the selected miRNAs in leaf and flower tissues. - All the qRT-PCR experiments were performed in the ABI StepOne Plus PCR instrument using miRNA qRT-PCR kit (#AMPR-0200, GeneCopoeia). - U6 was used as the internal control in these qRT-PCR experi- ments and the relative expression levels of the selected miRNAs were calculated using the standard comparative Ct method. - For each miRNA, the reactions were tripli- cate via the sample addition into three wells of the PCR plate. - The primer sequences used in this study were listed in the Additional file 8: Table S7.. - Prediction and annotation of the miRNAs target genes To further illustrate the biological functions of the iden- tified miRNAs, the software psRobot was employed to predict the potential target genes of the identified miR- NAs [42]. - Then, the functions of the potential target genes were annotated with the Gene Ontology (http://. - The flavonoid content of the acetonic extract in O. - Small RNA sequencing analysis for flower and leaf in O. - Additional file 3: Table S3: The identified novel miRNAs in O. - Additional file 4: Table S4: The differentially expressed miRNAs between flower and leaf tissues in O. - The funders had no role in study design, data collection, analysis and interpretation, decision to public, or writing of the manuscript.. - Identification and characterization of microRNAs and their targets in the bioenergy plant switchgrass (Panicum virgatum).. - Identification and quantitation of the bioactive components in Osmanthus fragrans fruits by HPLC-ESI-MS/MS. - Transcriptomic analysis of the candidate genes related to aroma formation in Osmanthus fragrans.. - The chromosome-level quality genome provides insights into the evolution of the biosynthesis genes for aroma compounds of Osmanthus fragrans. - Differential regulation of closely related R2R3-MYB transcription factors controls flavonol accumulation in different parts of the Arabidopsis thaliana seedling. - Genome-wide identification and evolutionary analysis of the plant specific SBP-box transcription factor family. - Small RNA discovery in the interaction between barley and the powdery mildew pathogen
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