- characterization of long non-coding RNAs involved in the early somatic. - KEGG analysis revealed that most of the differentially expressed target genes (mRNAs) of the lncRNAs were enriched in the “ plant-pathogen interaction ” and “ plant hormone signaling ” pathways during early longan SE. - lncRNAs come from the intergenic, intronic or coding gene regions in the sense and antisense directions. - and miR160 [19] in the regulation of the auxin response factors were demonstrated in longan SE. - Now, however, with the release of longan genome data from our laboratory [24], investigation of the regulatory roles of lncRNAs in longan SE has become possible. - We explored the functions of the lncRNAs and their target genes in the three samples and speculated that lncRNAs might participate in the developmental process, and then predicted the regula- tion of lncRNAs, miRNAs and lncRNAs during early longan SE. - The results enhance our understanding of the regulation of lncRNAs and provide valuable information for re- search on lncRNAs during early longan SE.. - Notably, 74.39% of lncRNAs in the early longan SE samples contained only one or two exons, whereas the number of exons in 82.41% of the mRNAs ranged. - RNA-Seq expression analysis revealed that among the 7643 lncRNAs and 47,169 mRNAs identified, 6005 novel lncRNAs, 15,233 novel mRNAs, and 23,886 known mRNAs were expressed in the three samples. - Of the 6005 expressed lncRNAs, 5254 were detected in the EC stage, 5320 in the ICpEC stage, and 5568 in the GE stage. - According to the locations of the nearest protein coding genes, 3422 of the 6005 expressed lncRNAs were. - c Venn diagram showing the specifically expressed lncRNAs in the EC, ICpEC and GE samples. - In the significant difference analysis of the 6005 lncRNAs, we found that the number of significantly differentially expressed lncRNAs in EC vs. - Compared with ICpEC, 604 of the 1028 lncRNAs were up-regulated and the other 424 lncRNAs were down-regulated in GE.. - 2c, the expression trends of the 3680 lncRNAs in the three samples were divided into nine major categories. - The expression levels of 273 lncRNAs, which accounted for 7.42% of the total lncRNAs, from the first category were lower in the ICpEC stage than in the EC and GE stages. - The lncRNAs in the fifth (386 lncRNAs) and seventh (361 lncRNAs) categories were expressed at higher levels in the ICpEC stage than in the other stages. - The lncRNAs in the second and fourth categories showed stable expression from the EC to ICpEC stage. - The second category included 369 lncRNAs (9.89% of the. - total) whose expression decreased in the GE stage.. - of the total) whose expression increased in the GE stage. - of the total) classified into the third category exhib- ited higher expression levels in the GE stage, whereas the 340 lncRNAs (9.24% of the total) classified into the sixth category showed higher expression levels in the EC stage. - The lncRNAs in the eighth and ninth categories showed stable expression from the ICpEC to the GE stage, but the 417 lncRNAs (11.33% of the total) classified into the eighth category exhibited lower expression levels in the EC stage, while the 373 lncRNAs (10.14% of the total) classified into the ninth category exhibited higher expression levels in the EC stage. - The lncRNAs in the second, fourth, and eighth categories were accumulated from the EC to the GE stage, indicating that the related lncRNAs in these three categories contributed to the formation of the GE stage. - The expression decrease from the EC to GE stage in the sixth and ninth categories indicated that the lncRNAs in these two categories were mainly involved in the maintenance of the EC stage. - The lncRNAs in the second, fifth and seventh categories were highly expressed in the ICpEC stage, and these lncRNAs were possibly involved in ICpEC morpho- genesis. - Longan lncRNA target prediction and functional annotation The potential target genes of the lncRNAs were pre- dicted according to their regulatory methods, which were divided into cis- and trans-regulation. - Of the 6005 lncRNAs, only 4682 lncRNAs were predicted to target protein-coding genes, including 5051 cis-regulated target genes (5712 pairs) and 1605 trans-regulated target genes (3618 pairs) (Additional file 3).. - Among the potential target genes (mRNAs) of the lncRNAs, 852 genes were deferentially expressed in EC vs. - Gene Ontology (GO) analysis was performed to investigate the differentially expressed target genes in the EC, ICpEC and GE stages (Fig. - GE, genes in the biological process, cellular component, and molecular function categories were also enriched. - In the molecular function category, kinase activity, transferase activity/. - To confirm the significant differences in the expression of lncRNAs and their predicted target genes in early longan SE, 20 lncRNAs with significant differences in expression were selected from among 641 up-regulated and 316 down-regulated lncRNAs in the RNA-Seq data.. - Additionally, 11 potential target genes (mRNAs) and five lncRNAs related to auxin response factors were verified by qPCR in the three stages of early longan SE.. - Twenty-four of the lncRNAs were detected in EC, ICpEC and GE (excluding LTCONS-00027337).. - Cluster analysis of the expression patterns of the 24 lncRNAs in qPCR (Fig. - The expression patterns of the 24 lncRNAs were divided into three categories according to the timing of their highest expression level. - In category I, eight lncRNAs exhibited high levels in the EC stage, but low levels in the other two stages. - In category II, four lncRNAs had the highest expression in the ICpEC stage. - LTCONS-00045469 in the EC and GE stages were basically unchanged. - The expression levels of LTCONS-00029834 and LTCONS-00055024 in the EC stage were higher than in the GE stage. - In category III, the expression of the 12 lncRNAs was highest in the GE stage. - These re- sults indicated that different lncRNAs might be involved in the maintenance of different stages of longan SE, i.e. - Comparing the expression trends of the lncRNAs in qPCR (Fig. - 4b) showed that the expression trends of 16 of the 24 lncRNAs were the same. - The inconsistencies of the other eight lncRNAs were as follows: LTCONS-00053938 showed highest expression in the GE stage and the lowest expression in the EC stage according to qPCR, but its expression was lowest in the GE stage and highest in the EC stage in RNA-Seq. - The expression of LTCONS-00031272 in the GE stage was higher in qPCR than in RNA-Seq. - For LTCONS-00030223, LTCONS- 00006334 and LTCONS-00045113, the expression in qPCR was highest in the EC stage and in RNA-Seq was highest in the GE stage. - The expression of LTCONS-00055024 and LTCONS-00045469 was highest in the ICpEC stage in qPCR, but highest in the GE stage in RNA-Seq.. - In the qPCR analysis of 11 lncRNAs and their poten- tial target genes (mRNAs), except for LTCONS- 00013909 and the target gene MTCONS-00013943 of LTCONS-00027337, all of the lncRNAs and their target genes were detected (Fig. - According to the expres- sion patterns of the lncRNAs and their target genes mRNAs, LTCONS-00022307, LTCONS-00045469 and LTCONS-00010999 were found to target cis mRNAs 20 kbp downstream (cis mRNA dw20k), while LTCONS LTCONS-00022673, LTCONS-00037848 and LTCONS-00038201 were trans-acting, and LTCONS- 00050060 and LTCONS-00031543 targeted cis mRNAs 10 kbp upstream (cis mRNAs up10k).. - 4 Analyses of differentially expressed lncRNAs and their targets in the RNA-Seq libraries and qPCR. - Heat maps were constructed for (a) relative expression levels in qPCR (b) and log2 (FPKM) values in the RNA-Seq library. - Functions of lncRNAs in the lncRNA-miRNA expression network in longan early SE. - As a new type of regulatory factor, eTMs play an important regulatory role in the process of plant cell differentiation and development [25, 35]. - In the present work, 40 lncRNAs were predicted to function as eTMs for 15 miRNAs (Fig. - Some of the lncRNAs responding to early longan SE might be regulated as targets by miRNAs. - In KEGG enrichment analysis of the 39,119 differentially expressed potential target genes during the early development of longan SE, 30 differ- entially expressed genes (5.33% of genes annotated to the pathway) in EC vs. - GE were enriched in the phytohormone signaling pathway. - Five target genes in the auxin signaling pathway were identified by lncRNA target gene prediction, including ARF4 targeted by LTCONS-00025525, IAA6 and AUX22 targeted by LTCONS-00008111, ABF targeted by LTCONS-00030223 and LTCONS-00055024, and AUX2 targeted by LTCONS-00006334 (Fig. - To profile the expression patterns of the lncRNAs associated with auxin response factors, five lncRNAs, their target genes (auxin response factors) and the corre- sponding miRNAs were analyzed by qPCR in EC, ICpEC and GE (Fig. - Thus, lncRNAs with different modes of action were involved in the auxin signaling pathway, with different potential target genes and corresponding differential expression. - patterns in different stages of longan SE, which indicated that lncRNAs were involved in the auxin signaling pathway through complex regulatory mechanisms.. - In summary, the lncRNA-miRNA-mRNA regulatory network in the auxin signaling pathway was constructed from lncRNAs, miRNAs and mRNAs, and verified by qPCR of five lncRNAs and related miRNAs (Additional file 7). - In the miR172a net- work, LTCONS-00042843 targeted Dlo-018542.1 and acted on miR172a as an eTM (Fig. - In the miR159a.1 network (Additional file 8), LTCONS-00046326 was predicted to be a Dlo-miR159a.1 target gene and to act as an eTM for Dlo-miR529d, and both Dlo-017525.1 and Dlo-028266.1 were predicted to be target genes of LTCONS-00046326. - In the miR398a network (Additional file 9), Dlo-miR398a acted on the target genes LTCONS- 00032074 and LTCONS-00039356, which in turn acted as eTMs for Dlo-miR157d and Dlo-miR406, respectively.. - Most of the five genes showed the same trend. - with overexpression and inhibition of Dlo-miR398a in longan EC, but Dlo-miR157d and Dlo-miR406 showed no significant response trends in the same material. - The above results shows that lncRNAs bound to miRNAs at different binding sites and had different expression in the same material.. - 9880 lncRNAs), the number of lncRNAs obtained from early longan SE was moderate, the poor conservation of lncRNAs among different spe- cies and the different tissues might be an important rea- son for the significant differences in the number of lncRNAs identified during early SE. - In the three stages of early longan SE, 375 lncRNAs were specifically expressed in the GE stage, which was much more than in the EC stage (159) and the ICpEC stage (153). - Among the three stages of the longan SE, the number of differ- entially expressed lncRNAs was highest in the EC vs.. - 7 qPCR validation of the Dlo-miR172a lncRNA-miRNA-mRNA network. - c Dlo-miR172a overexpression inhibited lncRNA-miRNA-mRNA expression in the sample material. - In total, 74.39% of the lncRNAs involved in the early stages of longan SE had only one or two exons, which might account for their low expression levels. - Therefore, differential enrichment analysis of the target genes (protein-coding genes) of lncRNAs during early longan SE provided valuable information. - Of the top five most enriched pathways, three were com- monly enriched in EC vs. - These results indicate that similar molecular mechanisms are active between the pre-embryonic EC and the embryogenic ICpEC, and the main change leading to GE formation occurs in the ICpEC to GE stages. - Previous studies reported that the plant-pathogen interaction pathway was enriched in lncRNAs [46], miR- NAs [47, 48] and gene transcripts [23], which suggested the importance of plant-pathogen interactions in the embryonic developmental network in longan and other plants. - In the KEGG enrichment results, it seemed that longan had begun to establish disease resist- ance mechanisms and other metabolic components based on hormones in the GE stage. - This change not only involved a large number of mRNAs, but also a large number of lncRNAs that participate in the molecular regulation and morphogenesis of the GE stage by regulating target genes (mRNA).. - The target genes of the five lncRNAs associated with auxin response factors were cis-regulated, which implied that the lncRNAs associated with auxin response factors mainly function as cis-acting regulators during early longan SE.. - Our study is the first to discover that lncRNAs act as eTMs of miR172a and miR529d in the EC stage in longan.. - In the present work, based on miRNA target predic- tion, 1765 lncRNAs were predicted to be targets of 85 miRNAs of 74 families. - Five lncRNAs related to the auxin response factors selected for miRNA target prediction, and seven miR- NAs (lncRNAs as miRNA target genes) were predicted to be involved in the action of lncRNAs. - Through qPCR analysis of the EC. - The number of lncRNAs specifically expressed in the GE stage was much greater than that in the EC stage and the ICpEC stage. - qPCR verification of five ARF- related lncRNAs and their target genes (mRNA) sug- gested that lncRNAs tend to positively regulate target genes in the early stages of longan SE. - 50% of the. - The expression of the three samples was analyzed as a time series according to previous studies [72, 73].. - The potential target genes of the lncRNAs were pre- dicted according to their regulatory methods, which were divided into cis- and trans-acting. - Prediction of the relationships between lncRNAs, miRNAs and mRNAs. - Functional categorization of potential target genes of lncRNAs based on the biological process category of the Kyoto Encyclopedia of Genes and Genomes (KEGG). - Additional file 8: qPCR validation of the lncRNA-miRNA-mRNA relationships of the miR159a.1 and miR398a networks. - All data presented in this study are provided either in the manuscript or additional files.. - Study of the progress on chemical constituents and pharmacological activities of Longan. - Analysis of the global transcriptome of longan (Dimocarpus longan Lour.) embryogenic callus using Illumina paired-end sequencing.. - LIT1, an imprinted antisense RNA in the human KvLQT1 locus identified by screening for differentially expressed transcripts using monochromosomal hybrids. - Overlapping and non-redundant functions of the Arabidopsis auxin response factors MONOPTEROS and
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