| Auxin response factor（ARF）is a kind of transcription factor that can bind auxres elements in plants,and plays an important role in the regulation of plant growth and development.ARF plays an important role in the process of leaf,flower organ,root,fruit development and secondary growth.In this study,Pag ARF7 was cloned from Populus alba×P.glandurosa.The main results were as follows:（1）Bioinformatics analysis of ARF geneAccording to blast analysis of ARF gene sequence in Arabidopsis,it was found that there were 37 ARF genes in poplar,mainly distributed on 14 chromosomes.Phylogenetic evolution tree showed that 37 ARF genes of poplar could be divided into 6 groups,and Pt ARF7belonged to the fourth group,and had high homology with Pt ARF2.Using GSDS2.0（http://gsds.gao-lab.org/）,the results show that ARFs gene generally consists of three regions,namely,DNA binding domain（DBD）at N-terminal,core structure in the middle（MR）and protein dimerization domain（CTD）at C-terminal.DBD can be combined with auxres,which is a growth hormone responsive cis element.Mr binding domain mainly starts from transcription activation or inhibition The CTD structure can be divided into three domains,III and IV,which can bind to AUX/IAA protein,and then regulate the response of ARF gene to auxin.（2）Cloning and bioinformatics analysis of Pag ARF7 geneThe total length of Pag ARF7 gene was 3360 bp and the relative molecular weight was123736.46 Dalton,which was composed of 1120 amino acids,including 84 strongly basic（+）amino acids（K,R）.),91 strongly acidic（-）amino acids（D,E）,331 hydrophobic amino acids（A,I,L,F,W,V）,411 polar amino acids（N,C,Q,S,T,Y）.The phylogenetic tree analysis of the ARF gene of poplar and the ARF gene family of Arabidopsis showed that Pag ARF7 is divided into the same region as At ARF7 and At ARF19,and the nucleotide sequence homology of the three is 61.54%.The subcellular localization results showed that Pag ARF7 gene was located in the nucleus,indicating that Pag ARF7 gene of poplar is a kind of transcription factor,which can play a role of transcription activation or inhibition.（3）Expression pattern of Pag ARF7 geneUsing popgenie（http://popgenie.org/gene）The results showed that Pag ARF7 was mainly expressed in stem and root.Then,the expression patterns of Pag ARF7 gene in different tissues of Populus silver were analyzed by q RT-PCR.The results showed that Pag ARF7 was expressed in young leaves,mature leaves,internodes 1-3,4-6 and roots,and the highest expression was found in root.Pro ARF7::GUS staining showed that Pag ARF7 gene was expressed in roots,mature stems and mature leaves of Populus alba×P.glandulosa,mainly concentrated in roots.（4）Response of Pag ARF7 gene to auxinThe expression of Pag ARF7 gene was induced by IAA treatment with different IAA concentrations(0μmol·L-1,10μmol·L-1,20μmol·L-1,30μmol·L-1,50μmol·L-1,80μmol·L-1)in Populus alba×P.glandulosa tissue culture seedlings.The results showed that the expression of ARF7 gene was the highest when IAA was treated with different concentrations.Then,at the concentration of 30μmol·L-1IAA,the treatment time was 0 h,1h,2 h,3 h,4 h,6h,8h,10h respectively.The results of q RT-PCR showed that IAA could induce the expression of Pag ARF7 gene to increase first and then decrease,and the highest expression was observed at 3 hours of treatment.It indicated that Pag ARF7 could respond to exogenous hormone IAA,and then it might regulate the expression of downstream gene.（5）Phenotypic analysis of Pag ARF7 OEThe transgenic plants of Pag ARF7-OE were obtained by genetic transformation of poplar.Then the phenotype of Pag ARF7-OE was analyzed to explore the effect of ARF7 gene expression on the growth and development of poplar.The results showed that the rooting time of Pag ARF7-OE was significantly earlier than that of the control,and the number and length of roots were also significantly higher than that of the control.The number of common Populus alba×P.glandulosa roots is 3-5,and the number of Pag ARF7-OE transgenic plants increases,up to 6;the average length of common Populus alba×P.glandulosa roots is4.4cm,and the length of Pag ARF7-OE transgenic plants has been improved,and the longest is 5.8cm.The results show that Pag ARF7 can promote the root development of poplar.（6）RNA-seq analysisRNA from the roots of control and Pag ARF7-OE was extracted and analyzed by transcription group.The results of transcription group showed that 391 differential expression genes were found in the combination of control and OE-7,334 up regulated genes,57 down regulated genes,2501 differential expression genes in the combination of control and OE-14,including 1447 up regulated genes and 1054 down regulated genes.Wayne map analysis showed that 322 differentially expressed genes were found in OE-7 and OE-14.The results of the analysis of the enrichment of go function showed that the differential expression genes were concentrated in response to stimulus go:0050896,response to stress go:0006950,signal transduction go:0007165,etc.39 genes related to metabolic pathways were obtained by KEGG enrichment analysis,accounting for 51.32%,25 genes related to the biosynthesis of secondary metabolites,accounting for 32.89%,13 genes related to hormone signaling pathway,accounting for 17.11%.These results showed that ARF7 gene may be related to metabolism pathway and plant hormone signal transduction pathway,which provides a basis for further analysis of ARF7 gene regulation mechanism in the root of poplar.At the same time,q RT-PCR technology was used to quantitatively analyze and verify the 12 selected downstream target genes（including 7 up-regulated expression and 5 down-regulated expression）that may be Pag ARF7,and the results were consistent with the transcriptome data.This experiment is mainly about the role of Pag ARF7 in the growth and development of poplar.The results show that Pag ARF7 can regulate the growth and development of plants by regulating the number and length of rooting.These studies provide an important basis for revealing the regulation mechanism of ARF gene in poplar growth and development. |
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