Identification And Expression Characteristics Of Paeonia Ostii MirRNAs Response To Copper Stress

Paeonia ostii is a perennial ornamental plant which belongs to Paeonia family.It can not only be used in traditional Chinese medicine as well as processed to peony seed oil,but also absorb heavy metals strongly.Strong copper resistance capability makes it excellent candidate plant for phytoremediation of polluted soil.Currently,previous work demonstrated that P.ostii showed high heavy metal tolerance,but the molecular mechanism underlying it response to copper remains to be studied.In order to understand further Copper-responsive miRNAs in P.ostii,we observed physiological reactions and changes in gene expression under copper stress.In addition,we performed high-throughput sequencing by constructing cDNA libraries of control group and copper stressed group to identify differentially expressed miRNAs,and predicted its target genes through bioinformatics analysis.In this work,we identified differently regulated sRNAs in P.ostii under Copper stress,and predicted their target genes.Furthermore,we analyzed the spatial and terminal expression of miRNAs under copper stress.This study could enrich theoretical molecular mechanism of resistance to copper of P.ostii and lay a scientific foundation for applications of phytoremediation.The main findings are as follows:1 Physiological responses and differential expressions of important genes under copper stressIt was found that short-time stress treatment had no significant adverse effect on the growth of the P.ostii but long-time stress treatment does.In P.ostii,content of MDA,SOD,POD varied in root,stems and leaf.The most sensitive and vulnerable tissue appeared to be root tissue,followed by stem and leaf.To investigate the expression of stress response gene expression changes in P.ostii copper stress gene expression differentially.Expression patterns of 12 selected genes derived from previous researches were observed by qRT-PCR.One unigene encoded ABC membrane transporter,and two unigenes encoded POD and SOD,while another two unigenes encoded ACS and ETR which are related to ethylene biosynthesis and perception.Seven unigenes were putative stress-related transcription factor genes that,respectively,encoded ERF,DREB,CBF,WRKY,MYB,NAC,and TGA.Results showed that,the majorities of differentially expressed gene expression were up-regulated,respectively,unigenes encoded ABC membrane transporter,antioxidant enzymes POD,SOD,ACS,ERF,CBF,WRKY,DREB,MYB and NAC.Among them,some showed obviously upward trend,such as ERF,CBF,WRKY,DREB,NAC and MYB.Inversely,unigenes encoded ETR and TGA were down regulated.Expression levels of all the tested unigenes varied under copper stress,with ETR and TGA down-regulated and other genes up-regulated,which were similar to those obtained through high-throughput sequencing.This work verified the accuracy and reliability of high-throughput sequencing results and provided reference for the functional identification of miRNAs in adaptability of P.ostii under copper stress.2.Results of small RNA and mRNA-seq library high-throughput sequencingMiRNAs are a class of non-coding,small RNAs recognized as important regulators of gene expression.Although plant miRNAs have been extensively studied in model systems,less is known in other plants with limited genome sequence data,including P.ostii.We used high-throughput sequencing to identify conserved and nonconserved miRNAs and other short RNAs in P.ostii under control and copper stressed condition.102 previously known plant miRNAs were identified and classified into 89 families according to their gene sequence identity.Combined our transcriptome sequencing data of P.ostii under same conditions,34 novel potential miRNAs were identified.The potential targets of the identified known and novel miRNAs were also predicted based on sequence homology search.Comparing the two libraries,it was observed that 12 conserved miRNAs and 18 novel miRNAs showed significantly changes in response to copper stress.In this work,the reliability of high-throughput sequencing was validated by qRT-PCR.Some of the new identified potential miRNAs might be involved in P.ostii-specific regulating mechanisms under copper stress.These results provide a framework for further analysis of miRNAs and their role in regulating P.ostii response to copper stress.3.Spatial and temporal expression patterns of the copper-responsive miRNAs in P.ostii.To reveal spatial and temporal expression patterns of miRNAs and discuss their role in P.ostii response to Copper-stress,five differentially expressed miRNAs were selected from above miRNAs,the relative expression of the five miRNAs and their target genes were detected and validated by qRT-PCR in different tissues at Oh,6 h,12 h,24 h after copper treatment.It showed that the selected miRNAs such as miRNA902c-3p,novel_miR22,novel_miR27,novel_miR32,novel_miR₃ and novel_miR25 had stable expression in all tissues.MiRNA902c-3p,novel_miR22,novel_miR32,novel_miR3 had similar regulating pattern by targets in the above-mentioned tissues of P.ostii,whereas the other two miRNAs were down regulated.The expression patterns of corresponding target genes were opposite to the expression patterns of miRNAs.These results suggested that the data deprived from RNA-Seq assay of this study were reliable and met the general characteristics that miRNAs negatively regulates target gene expression level.Under copper stress,three miRNAs?miRNA902-3p,novel_miR₃,novel_miR₂2?in roots,stems and leaves had different degrees of reduction:the expression of miRNA902₃p always kept a slight downward trend in all tissues,showing no significant difference.Novel_miR₃ reached the peak at 6 h.The two miRNAs?novel_miR27,novel_miR32?were up-regulated.Novel_miR27 were up-regulated immediately after 1 h;novel_miR₃2 had low expression level in roots,but overall trend of the expression of novel miR 32 showed upward.Meanwhile,the results of target genes expressions validated by qRT-PCR were opposite to that of the corresponding miRNAs expression.CL939.Contig8_all,target gene of miRNA902-3p,showed down regulation in all tissue sample.CL1240.Contigl_all and Unigene26288_All,target genes of novel_miR₃ and novel_miR₂2 respectively,showed upward trend in root,stem and leaf.Under copper stress,CL1240.Contigl_all,target gene of novel_miR₃ mainly differential expressed in roots and leaves without significant.Unigene26288_All,target gene of novel_miR₂2,showed up regulation trend,mainly expressed in root and stem tissue.CL4800.Contig2_all and Unigene25888_All,target genes of novel_miR₂7 all and novel_miR₃2 respectively,showed overall downward trends in roots,stems and leaves.These results proved that the miRNAs and their target genes play an important role in the process of copper stress.The research provided important information for revealing the molecular mechanism of the response to copper stress.