Identification And Interaction Study Of Key MicroRNAs And Their Target Genes In The Physiological Differentiation Stage Of Longan Flower Buds

Longa(Dimocarpus longan Lour.)is an important tropical/subtropical fruit tree of the Sapindaceae family.Longan flower bud differentiation directly affects fruit yield and quality and determines the success of commercial orchards.MicroRNA(miRNA)plays an important role in regulating flower bud differentiation.However,limited reports on key miRNAs and their interactions with target genes during longan flower bud differentiation are available.In this study,small RNA(sRNA)and degradome libraries were constructed using the apical buds of fruiting branches at four stages of longan flower bud differentiation(October 30,T1;November 30,T2;December 30,T3;January 30,T4).Degradome analysis was performed to identify and analyse differentially expressed miRNAs(DEmiRNAs)and their target genes.The lncRNA-miRNAmRNA competitive endogenous RNA(ceRNA)network was constructed by combining miRNAome and transcriptome data,and the interactions of three important miRNAs(dlo-miR164a,dlo-miR171f,and dlo-miR319)with their target genes and lncRNAs during longan flower bud differentiation were investigated in tobacco.The results revealed the molecular mechanisms by which dlo-miR1 64a and dlo-miR171f regulated longan flower bud differentiation by repressing their target genes that interact with auxin signaling and photoperiod pathways,and increased the understanding of the post-transcriptional regulatory mechanisms of flower bud differentiation in longan and other woody fruit trees.The main research results are as follows:1 Histological observation and determination of endogenous hormones in the apical buds of longan fruiting branches.The morphological appearance of the apical buds of longan fruit branches was observed,and paraffin sections were used to understand the changes in the internal morphology of the apical buds during flower bud differentiation.It was found that the leaf bud primordia at the T3 stage(physiological differentiation stage)appeared conical-tipped,while the inflorescence primordia at the T4 stage(morphological differentiation stage)transformed into flattened semicircular,revealing the internal morphological histological differences between the leaf bud primordia and inflorescence primordia of longan and confirming the reliability and accuracy of the sampling material.The levels of endogenous hormones showed that the increase of IAA was beneficial for the morphological transformation in the physiological differentiation stage of longan flower bud differentiation.At the same time,the high content of ETH was involved in regulating the physiological differentiation stage.2 Identification and expression analysis of miRNA and its target genes during longan flower bud differentiation.Small RNA(sRNA)sequencing technology was used to identify the miRNAs in the apical buds of longan fruiting branches at four stages(T1,T2,T3,T4).A total of 429 miRNAs(109 known and 320 novel miRNAs),including 104 DEmiRNAs,were identified by sRNA sequencing.dlo-miR164a,dlo-miR160a,dlo-miR171f were expressed down-regulated at the T3 stage.In contrast,dlo-miR294,dlo-miR318 and dlo-miR319 were expressed up-regulated at the T3 stage,suggesting that these miRNAs may be involved in post-transcriptional regulation during the physiological differentiation stage of longan flower bud differentiation.A total of 193 target genes of 52 DEmiRNAs were identified by degradome.These DEmiRNAs targets were significantly enriched in MAPK signaling pathway-plant,photosynthesis-antenna protein,and phytohormone signaling pathways during flower bud differentiation.Based on this,a regulatory network of 38 DEmiRNA-mRNA pairs related to flower bud differentiation was constructed.3 Combined analysis of miRNAs and transcriptome during longan flower bud differentiation.The expression correlation of 38 pairs of DEmiRNA-mRNAs was analyzed in combination with transcriptomic data,among which 11 pairs of DEmiRNA-mRNAs showed high negative correlation in expression levels(Pearson correlation coefficient<-0.6),including dlo-miR164aDlNAC100,dlo-miR171f-DlSCL15,dlo-miR319-DlTCP4,dlo-miR160dDlARF18 and other modules.Weighted gene co-expression network analysis(WGCNA)revealed that two target genes,DlNAC100 and DlSCL15,were significantly up-regulated in expression at the T3 stage,and may be involved in plant hormone signal transduction and photoperiod regulation pathways.In conjunction with the analysis of lncRNA data,five functional signature modules conforming to the lncRNA-miRNA-mRNA competitive endogenous network(ceRNA)were mined,including five DEmiRNAs and five lncRNAs.These lncRNAs as endogenous target mimics(eTMs)positively affect DEmiRNAs target genes involved in flower development,auxin signal transduction,and cell proliferation.These results provide new insights into a molecular regulatory mechanism of lncRNA-miRNA-mRNA cascad regulation mechanism centered on miRNAs that may contribute to longan flower bud differentiation.4.Validation analysis of key miRNA-mRNA and miRNA-lncRNA interactions.The interactions between miRNAs and mRNAs of three pairs,including dlo-MIR164a-DlNAC100,dlo-MIR171f-DlSCL15 and dloMIR319-DlTCP4 and dlo-MIR164a-DllncRNA42466,were investigated by GUS tissue staining and dual luciferase in tobacco leaves.The results of the GUS histochemical staining assay showed that the blue area of tobacco leaves was significantly reduced after transient co-transformation of all three pre-miRNAs with their target genes,indicating the presence of a targeted cutting effect.The dual luciferase assay further verified the targeted cleavage effect of the three pre-miRNAs with their target genes.In addition,the effect of dlo-miR164a on DlNAC100 was blocked by the overexpression of DllncRNA42466,indicating that DllncRNA42466 could act as an eTM for dlo-MIR164a.5 Interaction and validation analysis of the target genes DlNAC100,DlSCL15,DlTCP4 and co-expressed genes.Subcellular localization and yeast self-activation assays in tobacco leaves confirmed that DlNAC100 and DlSCL15 have transcription factor nuclear localization properties and transcriptional activation ability.In contrast,DlTCP4,which has nuclear localization properties,has no transcriptional activation ability.The expression patterns in different tissues of longan suggest that DlNAC100 and DlSCL15 play an important role in the physiological differentiation stage of longan flower bud differentiation.At the same time,DlTCP4 may integrate the flowering signal from leaves to induce flower bud differentiation.Dual luciferase assay confirmed that DlNAC100 and DlSCL15 significantly activated the promoter activity of auxin response factor DlARF18 and photoperiod gene DlCOL4,respectively.In addition,DlNAC100 significantly activated the promoter activity of the rhythm gene DlFKF1,while DlSCL15 significantly activated the promoter activity of the rhythm gene DIGI,suggesting that DlNAC100 and DlSCL15 may be involved in regulating the physiological differentiation phase of longan flower bud differentiation through auxin signaling as well as rhythm and photoperiod pathway genes.The exogenous auxin inhibitor TIBA was sprayed in field longan plants during the physiological differentiation stage.The quantitative PCR results showed that the expression levels of DlARF18,DlNAC100,DlSCL15,DlFKF1,DIGI,DlCOL4,and DlSOC1 were highly significantly downregulated compared with the control,further indicating that the growth hormone pathway,photoperiod pathway and DlNAC100,DlSCL15 interacted during longan flower bud differentiation.