Study On The Function Of PlGIDI And PlDELLA Genes Involved In The Endodormancy Release Of Paeonia Lactiflora

Peony（Paeonia lactiflora Pall.）,which belongs to the family Paeonaceae,is a well-known ornamental perennial in the world.It has strong adaptability,high ornamental value and ecological value.It is widely used in landscaping and cut flower culture.However,the long dormancy time of its underground buds greatly limits its flowering regulation and landscape utilization.At present,researches on dormancy release of underground buds of peony are mostly focused on the chilling requirement and the use of growth regulators,and researches on molecular biology are relatively scarce.In our previous transcriptome study,abundant transcripts of peony buds were found change significantly during peony bud endodormancy release by artificial chilling treatment,and the differentially expressed genes（DEGs）were significantly enriched in plant hormone signal transduction pathways.Among them,in gibberellin signal transduction pathway,which is closely related to plant dormancy release and germination,two key genes,GID1 and DELLA,changed significantly in the process of dormancy release of peony.Existing research shown that GID1 and DELLA genes are related to plant dormancy,germination and growth regulation.In this study,GID1 and DELLA will be used as entry points to study their functions in the dormancy release of peony buds.Therefore,in this study,based on the transcriptome data of early stage in our laboratory,ORF full-length sequences of PlGID1 and PlDELLA genes were isolated from the buds of‘Da Fuigui’,and the basic sequence,structure,physical and chemical properties,homology and other aspects of PlGID1 and PlDELLA genes were analyzed by bioinformatics analysis and phylogenetic tree construction.High performance liquid chromatography tandem mass spectrometry（HPLC-MS/MS）was used to determine the content of endogenous activity GA1,3,4,7 and ABA hormone in the buds of peony in the process of dormancy release.On this basis,qRT-PCR was used to detect the response of PlGID1 and PlDELLA genes to external GA₃ and PAC（GA synthesis inhibitor）in the process,and the differential expression analysis in different tissues and organs was carried out at the same time.An overexpressed plant system was established to transform into Arabidopsis.The function of the two genes was preliminarily identified.The virus-induced gene silencing（VIGS）technology was used to establish the PlDELLA gene silencing system to infect peony,and the function of the homologous transformation of PlDELLA gene in peony was further identified through the positive and negative directions.The yeast two hybrid technology was used to construct the PlDELLA bait protein to verify the protein interaction relationship with the peony related PlWRKYs.The main conclusions of this experiment are as follows:1.The ORF sequence of two key genes from peony was cloned,PlGID1（Gen Bank accession number:MG720011）,was 1035 bp in length,encoding 344 amino acids.PlDELLA（Gen Bank accession number:MG720010）,was 1866 bp in length,encoding 621 amino acids.2.PlGID1 has typical domains of HGG and GXSXG of HSL gene family,which encodes unstable non secretory protein,partial hydrophilicity,no signal peptide and transmembrane region.The N-terminal of PlDELLA has two very conservative acid domains,DELLA and VHYNP.The C-terminal of PlDELLA is one of the eight subfamilies of GRAS gene family,which has a conserved domain of RVER and SAW.3.During chilling process to release dormancy,the content of endogenous activity GA1,3,4 and ABA hormone showed a downward trend.The results showed that chilling treatment did not induce the increase of endogenous GAs content in peony,but the release of peony dormancy was positively related to the decrease of GAs and ABA content,and may also be related to GAs/ABA.In this process,the expression of PlGID1 was strongly induced and rapidly increased.After chilling plus with exogenous GA₃ and PAC treatment,the expression of PlGID1 was still in an upward trend,but the trend was relatively gentle.The expression level of PlDELLA decreased rapidly.After chilling plus with exogenous GA₃ and PAC,the expression level of PlDELLA still decreased.The results showed that the response of PlGID1and PlDELLA to chilling was more obvious than that of exogenous GA₃ and PAC.4.Overexpression of PlGID1 gene in Arabidopsis showed that the overexpression strains（OE_S）significantly accelerated the germination and bolting of Arabidopsis.The height,pods number,dry and fresh weight of the above ground parts of OE_S were significantly higher than those of WT.It is suggested that PlGID1 plays a key and positive role in Arabidopsis growth and development.The expression levels of five DELLA genes were further detected and analyzed in OE_S plants.It was found that the expression levels of DELLA genes were significantly inhibited.After overexpression of PlDELLA gene in Arabidopsis,the plant phenotype was significantly different,which was manifested as seed germination and bolting were significantly delayed,leaf development was small,the growth of main root was inhibited,the plant was shorter,and the character of pod was also affected to varying degrees.The expression levels of three GID1 genes were further detected and analyzed in OE_S plants.It was found that the expression levels of GID1 genes were up-regulated in varying degrees.5.qRT-PCR was used to detect the expression of PlDELLA gene in the VIGS test group and the control group.It was found that the expression of PlDELLA gene in the test group decreased significantly,the highest silencing efficiency was 67%,and the lowest was 34%,while the expression of PlDELLA gene in the two control groups was basically the same,indicating that the virus-induced gene silencing significantly reduced the expression of PlDELLA gene in peony.After the PlDELLA gene was silenced,the peony plants in the experimental group germinated early,while the germinating time of the empty control group and CK control group was the same.Compared with the two control groups,the germination of the experimental group was 8～10 days earlier,and the nutritional growth of the experimental group was significantly accelerated,especially in the early growth stage.It is proved that PlDELLA plays a key role in the dormancy release,bud differentiation and nutritional growth of peony.6.When the concentration of Ab A was 125 ng·ml^-1,the PlDELLA bait protein could completely inhibit its autoactivation and non-toxic.The results of yeast two hybrid showed that there was no interaction between the PlDELLA with the four screened PlWRKYs,PlWRKY13,PlWRKY18,PlWRKY40 or PlWRKY50.