Identification And Functional Analysis Of MiRNAs Related To Activity Cycle Of Paulownia Tomentosa Vascular Cambium

The vascular cambium of trees exhibits periodicity in perennial plants.The growth and development process of vascular cambium is related to the regulation of a large number of genes.The regulation mechanism of miRNAs(microRNAs)on the activity cycle of vascular cambium has been widely concerned by researchers in recent years.Identifying the miRNAs associated with the activity cycle of the Paulownia tomentosa vascular cambium can better understand the molecular mechanism of vascular cambium.In this experiment,three small RNA(sRNA)libraries in the dormant,reactive and active cambium were constructed and sequenced by Solexa.The sRNA was isolated and identified by bioinformatics to identify the activity cycle of vascular cambium.The miRNAs were analyzed by real-time quantitative PCR(qRT-PCR).The expression levels of some miRNAs and their target genes in the dormant cambium(DC),reactive cambial(RC)and active cambium(AC)of Paulownia tomentosa were analyzed.The specific experimental results are as follows:1.Through the Solexa sequencing of the dormant cambium(DC),reactive cambium(RC)and active cambium(AC)of the Paulownia tomentosa,7722583,7938631,and 7972651 raw reads sequences were obtained in three samples.The low-quality sequence,the joint contamination,the sequence of N ratio greater than 10%,and the polyA/T/G/C sequence were removed,and 7522995,7803216,and 7821819 clean reads were obtained.2.Using the Paulownia tomentosa transcriptome as a reference sequence,a total of 49 known miRNA families were identified(the abundance of miRNA families varies widely,and the expression levels of different members of the same miRNA family are also significantly different)and 33 new miRNAs.3.Based on two criteria |log ?fold change?| ? 1 and P ? 0.05 comparing the expression levels of miRNA between AC vs DC,AC vs RC,and RC vs DC,360 differentially expressed miRNAs were found.Among them,107 differentially expressed miRNAs were found in the comparison between AC and DC;132 differentially expressed miRNAs were found in the comparison between AC and RC;121 differentially expressed miRNAs were found in the comparison of RC and DC.4.Using the psRNATarget web server to predict target genes for known miRNA families and new miRNAs,a total of 528 target genes were predicted.Of these,26 of the 49 known miRNA families predicted 457 target genes,and 25 of the 33 new miRNAs predicted 71 target genes.5.Perform GO functional analysis on target genes.In the functional classification of biological processes,biological processes,metabolic processes,and cellular process functional groups receive the most annotations;in the functional classification of cellular components,cellular components,The number of annotations for the functional group of cells and cells is the highest;in the classification of molecular function,the combination of binding,catalytic activity and heterocyclic compound is the most annotated.KEGG analysis was performed on target genes differentially expressing miRNAs,which were classified into 20 different pathways,such as starch and sucrose metabolism,RNA degradation,plant-pathogen interactions,etc.,to significant enrichment.It indicates that miRNA plays an important regulatory role in the growth and metabolism of plants.6.Six known miRNAs with known precursor sequences(pto-miR156 b,pto-miR164 a,pto-miR171 d,pto-miR172 c,pto-miR319 d,pto-miR399a)and three new miRNAs(pto-miR02,pto-miR16 and pto-miR21)were subjected to the real-time quantitative PCR analysis during the dormant cambium(DC),reactive cambium(RC),and active cambium,and the results show that the expression levels of these miRNA precursors determined by qRT-PCR have similar expression trends to those of mature miRNAs from high throughput sequencing.7.The qRT-PCR experiments on the selected 9 miRNA target genes showed that the expression of pto-miR160 a and pto-miR21 did not show a good correlation with the expression of potential target genes.However,there is an inverse relationship between the expression of the other seven miRNAs(pto-miR156 b,pto-miR171 d,pto-miR172 c,pto-miR319 d,pto-miR398 b,pto-miR02,and pto-miR16)and their potential target genes(SPL,SCL transcription factors,APETALA2,Laccase-14-like,Cu-Zn SOD,NBS resistance protein and receptor-like protein kinase),indicating that these miRNAs may be involved in the regulation of the cyclaline vascular cambium cycle.