Screening Of The Interaction Genes Of ARRO-1 Based On Yeast Two-hybrid And Transcriptome Analysis In Tree Peony During Adventitious Rooting

Tree peony(Paeonia suffruticosa)has beautiful flowers,rich patterns,diverse colors and excellent ornamental and medicinal value.Tree peony is a perennial deciduous shrub,but the problems of poor rooting quality and non-connection of adventitious roots and vascular bundles lead to low survival rate in vitro,which seriously limits the rapid reproduction of tree peony.A total length of adventitious root occurrence related oxygenase(adventitious rooting related oxygenase,ARRO-1)gene was obtained by cloning in our group.The subcellular localization results showed that the gene was located in the plasma membrane and expressed higher in the early stage of adventitious root formation.PsARRO-1 gene may be the key gene in the development of adventitious root of tree peony.On the basis of previous research,the cDNA library of adventitious roots of tree peony was constructed,and the protein of PsARRO-1 interaction related to adventitious roots of tree peony was screened by yeast two-hybrid technique.Combined with transcriptome analysis of the key period of adventitious root,the expression and function of PsARRO-1 interacting genes were analyzed,which provided technical support for the construction of gene regulation network of adventitious roots,and provided theoretical support for the study of molecular mechanism of root occurrence and the solution of rooting problem of tree peony group.The main results are as follows:1.Construction of the yeast two-hybrid library of adventitious roots in tree peonyThe adventitious roots in different developmental stages of P.ostii were collected,the total RNA was extracted and purified by modified CTAB method,mRNA was isolated and purified,and then the double-stranded cDNA was synthesized.The adventitious root yeast two-hybrid library of P.ostii was constructed using the CloneMinerTM Ⅱ cDNA Library Construction Kit.The study found that the library capacity of pPR3-N membrane yeast two-hybrid system was 6.5×106 cfu/mL,the total clones was 1.3×107 cfu,insertion fragments were mainly focus on 500-3000 bp,the recombination rate was 100%,indicated that this library could be used for the screening of interaction proteins.2.Screening of proteins which interact with PsARRO-1 in tree peonyOn the basis of the cDNA sequence of PsARRO-1 gene in tree peony,we designed specific primers,pBT3STE-ARRO and pBT3SUC-ARRO bait vectors were constructed by Sfi I enzyme digestion.Self-activation and functional testing in yeast cells,neither the pBT3STE-ARRO or pBT3SUC-ARRO bait plasmids had self-activation capabilities,the function of pBT3SUC-ARRO was stronger.Through the screening of pPR3-N membrane yeast two-hybrid system by pBT3SUC-ARRO.we got 36 positive clones,and all the 36 positive clones could activate the HIS3、ADE2 and MEL1 reporter genes.Colony PCR,sequencing and gyration validation showed that 18 clones may be interacted with PsARRO-1.3.Transcriptome analysis of the critical period of root initiation in tree peonyTranscriptome sequencing of four growth stages of root(Gmfq,Gmf,Gtq,Gzc)was performed to obtain 141.77 Gb of sequencing data,50.44%unigenes were annotated,and 128571,163869,168379,170004 high-quality reads were identified,respectively.Gmf compared with Gmfq,48031 genes were up-regulated and 23687 genes were down-regulated;Gtq compared with Gmf,21165 genes were up-regulated and 8639 genes were down-regulated;Gzc compared with Gtq,13556 genes were up-regulated and 11156 genes were down-regulated.A large number of genes were involved in adventitious rooting.Through the analysis of plant hormone,especially the gene expression of auxin pathway,we found that a large number of hormone related genes was specifically highly expressed in the early stage of root germination,such as auxin key gene ARF6,ARF8,ARF2B,IAA9,IAA11,IAA16,GH3.5,GH3.6,LAX5,SAUR71,TIR1,ethylene related gene ERF113,ERF020,ERF071,ERF4,TOE038,and brassinolide related gene BSK3,BEH4,CYCD3-1,CYCD3-3,and so on,these genes would be specifically expressed in the early stage of root germination.Cytokinin,abscisic acid,gibberellin,salicylic acid and jasmonic acid were negative regulators in the formation of adventitious root.Moreover,a large number of oxidase activity related genes were highly expressed in the development of adventitious roots of tree peony,and the expression of PER21,PER70,PER52,PNC1,SODCC,SOD1 was higher in the early stage of germination of adventitious roots,which may be important oxidase activity gene in the development of adventitious roots.4.Bioinformatics analysis of the PsARRO-1 genes based on yeast two-hybrid and transcriptomeAlignment of 18 genes interacting with PsARRO-1 with sequences in the transcriptome,a total of 15 genes would be annotated into the transcriptome database.The protein stability prediction results showed that,The Unigene899_All,CL5321.Contigl_All,CL1669.Contig2_All,Unigene43742_All and CL683.Contig2_All of 15 proteins was stable proteins,the remaining 10 were all unstable proteins.Projections of protein hydrophilicity indicate that,only CL5321.Contigl_All,CL4478.Contigl_All,CL12001.Contig3_All and Unigene43742_All were hydrophobic proteins,The rest were hydrophilic proteins.Signal peptide prediction results showed that only CL18524.Contig2_All proteins may have signal peptides.Unigene43426_All,CL4478.Contig1_All,CL8996.Contigl_All and Unigene43742_All proteins had transmembrane domains,could be a transmembrane protein.The results of conservative domain analysis and prediction showed that 15 protein conserved domains were different,among them Unigene43426_All the conservative domain structure is not predicted.The results of transcriptome annotation showed that HMGB3(CL5321.Contigl_All)were mainly role in Gzc;POT2(CL4478.Contigl_All)was expressed substantially in Gtq;Cyb5(CL8996.Contigl_All)was expressed highly in Gmfq.Therefore,PsARRO-1 may interact with HMGB3,POT2,and Cyb5 to regulate the growth and development of adventitious roots of tree peony.5.Cloning and functional analysis of PsARRO-1 interacting genes in tree peonyBased on the transcriptome data,gene-specific primers were designed to amplify the cDNA of adventitious roots of P.ostii.The coding sequences of PsHMGB3,PsPOT2 and PsCyb5 genes were obtained,and the amino acid sequence alignment and evolutionary analysis were performed.Both direct Y2H and bimolecular fluorescence complementation assays confirmed that PsARRO-1 interacted with PsHMGB3 in the nucleus,and PsARRO-1 may not interact with PsPOT2 and PsCyb5.Therefore,the expression analysis and functional study of PsHMGB3 gene were carried out.It was found that PsHMGB3 gene was expressed in different developmental stages of adventitious roots of tree peony,mainly expressed highly in Gzc,and the expression level was up-regulated with the development of adventitious roots.PsHMGB3 gene was subcellular localized in the nucleus.The overexpression of PsHMGB3 gene in Arabidopsis resulted in the lengthened primary roots,more lateral roots,indicating that PsHMGB3 gene involved in root development.