Cloning And Functional Analysls Of PlARF2 Gene From Paeonia Lactiflora

Paeonia lactiflora was one of the traditional famous flowers in China.It had rich flower types,colorful flowers and ornamental characteristics,which can be comparable to the peony with national beauty and natural fragrance.However,there was a serious cross incompatibility phenomenon in the distant hybridization of Paeonia,and the abnormal accumulation of callose is one of the important reasons.The research team had successfully cloned and analyzed the key domain of callose synthase gene Cal S in the early stage,and preliminarily explored the regulation network of callose.Related studies had shown that At ARF17 in Arabidopsis can directly bind to the At Cal S5 promoter to regulate the formation of callose.In order to explore whether there is a similar regulatory relationship between ARF gene and Cal S gene in paeonia lactiflora,this study screened a PlARF2 gene from the transcriptome database,and carried out cloning,bioinformatics analysis and spatial expression analysis.A c DNA library for yeast two hybrid screening was constructed using 36 h hybridized stigma as material,and the protein interacting with PlARF2 was screened.The main research results were as follows:(1)Based on transcriptome data,an ARF transcription factor was screened and named PlARF2.The CDS of the gene was 2391 bp,encoding 797 amino acids,containing B3-DNA binging domain,Auxin response factor auxin element,Aux/IAA element of plant Auxin primary response gene.The relative molecular weight of PlARF2 was about 88.6k D,the total number of atoms was 12281,the predicted molecular formula was C3897 H6045 N1113 O1197 S29,and the theoretical isoelectric point(p I)was 6.40,which was a hydrophilic protein.Phylogenetic tree analysis showed that PlARF2 was closely related to Vv ARF2 protein of grape.Fluorescence quantitative analysis showed that PlARF2 gene had the characteristics of constitutive expression,and its expression in self pollinated stigma was higher than that in hybrid stigma,indicating that the gene expression was conducive to cross compatibility.Subcellular localization showed that PlARF2 was located in the nucleus.(2)Auxin response factors ARFs,as important transcription factors in response to auxin signals in plants,play a very important role in plant growth and development.To investigate the response of PlARF2 gene to IAA and ABA hormones,different concentrations of IAA and ABA were used to treat roots and leaves,respectively.The expression of PlARF2 gene in applied IAA and ABA: After ABA treatment of 50μmol·L-1,the expression of PlARF2 gene was slightly higher than that of water treatment,but with the extension of treatment time,the expression of PlARF2 gene was inhibited,which was lower than that of water treatment;However,the expression of PlARF2 gene in leaves was higher than that in water treatment all the time during the treatment period,and was 2.78 times higher than that in water at 4h,and significantly increased compared with that in roots.As the treatment concentration reaches 100μmol·L-1,PlARF2 gene expression levels in roots and leaves were lower than 50μmol·L-1 treatment.Different from ABA treatment,with the above two concentrations of IAA,the expression of PlARF2 gene in roots and leaves was higher than that in water treatment,and at the concentration of 50μmol·L-1,the expression was slightly higher than 50μmol·L-1 treatment suggested that the PlARF2 gene table was positively regulated by IAA.The expression of PlARF2 gene increased under low concentrations of IAA and ABA,suggesting that it may be involved in the regulation of peony’s response to IAA and ABA regulation.(3)Plant gene promoters contain a variety of important homeopathic elements that play an important role in responding to external stimuli and regulating downstream responsive gene expression.The PlARF2 promoter was successfully cloned by chromosome walking.It was found that the PlARF2 promoter contained hormone response elements,light response elements,endosperm expression elements and light sensitive elements.GUS staining showed that the activity of PlARF2 promoter was strong;The hormone treatment experiment showed that IAA and ABA treatment could improve the activity of PlARF2 promoter,which was more significantly affected by IAA.(4)To further explore the effect of PlARF2 gene on plant growth,Arabidopsis thaliana was infected by inflorescence transfection,and the positive plant OE-PlARF2-2 was successfully obtained after screening.Phenotypic observation showed that OE-PlARF2-2 had earlier flowering time and bearing time,faster plant growth,larger rosette leaf,delayed rosette leaf senescence,and larger seeds than WT plants,and its pods were more plump.It was peculated that PlARF2 plays an important role in promoting fruit enlargement,delaying senescence,and enlarging leaves.(5)Yeast two-hybrid technology can be used to verify whether there was interaction between proteins,which plays an important role in screening proteins that interact with target proteins.In order to screen related proteins that interact with PlARF2 protein,the research team successfully constructed a Paeonia lactiflora hybrid stigma c DNA library.The detection of the primary library and the secondary library capacity showed that the total clone number of the library was about 1.4x107,which fully met the standard of high-quality library and could be used to screen yeast interacting proteins in the next step.The PlARF2 gene was successfully recombined with p GBKT7 plasmid to form bait plasmid.The toxicity test showed that the bait plasmid was non-toxic to yeast.The self activation test showed that the bait plasmid had self activation,but the self activation was weak.It could be completely inhibited on the SD/-Trp Lcu His Ade plate with10 m M 3-AT,which could be used for the next screening test.Using Mating,14 positive proteins interacting with PlARF2 were successfully screened.Analysis showed that these 14 proteins played an important role in plant growth and development,secondary metabolite synthesis,plant gamete growth,hormone response and stress resistance.It is speculated that PlARF2 interacts with these proteins to affect the production of secondary metabolism and the development of gametes,thereby affecting the cross compatibility of Paeonia.