Bioinformatics Analysisof Auxin Response Factor (ARF) Gene Family And Functional Research Of Mir167 And Its Target ARF6 In Strawberry

Auxin is involved in the regulation of many stages of plant growth and development,and it is an important plant hormone.At the same time,the ARF family regulates auxin signaling and it is a signification transcription factor.Up to now,there are arabidopsis,grape,papaya,apple and so on have been reported,but little research about strawberry ARF family.Therefore,bioinformatics analysis of auxin response factor(ARF)gene family was expansion in the study.MIR167 and ARF6 overexpression vectors and ARF6 interference vectors were constructed and transgenic strawberry plants were obtained.The phenotypic observation of transgenic plants revealed the functions of strawberry MIR167 and ARF6 genes.The main results are as follows:(1)Twelve Fv ARFs were obtained from the NCBI database,namely Fv ARF1?Fv ARF2?Fv ARF3?Fv ARF4?Fv ARF5?Fv ARF6?Fv ARF8?Fv ARF9?Fv ARF19 and Fv ARF23.Phylogenetic analysis of family genes by using bioinformatics analysis methods,12 genes of the ARF family were divided into four branches in the phylogenetic tree.Fv ARF6 and At ARF6,Fv ARF3 and Md ARF3,Fv ARF23 and Md ARF23,Fv ARF1 and Md ARF1 were sister gene pairs of the same branch among the phylogenetic.There are close evolutionary relationship to each.Fv ARFs are unevenly distributed in five chromosomes.Fv ARFs contain three conserved domains,and only Fv ARF3?Fv ARF17 and Fv ARF18 have truncated CTD domains.All genes contain a lot of exons and introns,and the number of introns varies from 1 to 16.Conserved motifs of family genes are analysised by MEME database,family genes have conserved motifs distribution structure except Fv ARF17.(2)The ‘Yanli' strawberry was used as a test material,the q RT-PCR method was used to analysis of relative expression about the different tissues organs and developmental stages of the‘Yanli' strawberry.Quantitative results in different tissues of the same period,Fv ARFs were expressed in all tissues;the relative expression levels of Fv ARFs in different fruit developmental stages Fv ARFs expression decreased gradually with the ripening of the fruit.(3)Treatment of tissue culture of 'Ruegen' strawberries with 10 ?mol·L-1 for 0 h,6 h,12 h,24 h,48 h and different IAA concentrations(0 ?mol·L-1,0.1 ?mol·L-1,1 ?mol·L-1,10 ?mol·L-1,25 ?mol·L-1)for 12 h.q RT-PCR results showed that ARF family gene expression was highest at treatment with 12 h IAA(10 ?mol·L-1).(4)The MIR167 and its target gene ARF6 were cloned from 'Yanli' strawberry by RT-PCR.Obtaining 2646 bp CDS sequence,it encodes 882 amino acids.The sequence is the similar with the diploid ARF6 sequence in the NCBI database,and the Similarity is 98.68%.The length of the MIR167 gene was 305 bp.(5)The overexpression vector of mutational ARF6 gene,overexpression vector of MIR167 gene and ARF6 interference vector were constructed.Theywere transferred into 'Ruegen' tissue culture seedling by agrobacterium-mediated genetic transformation.Finally,four strains of MIR167 overexpressing plants and three strains of ARF6 interfering plants were obtained.Phenotypic observation showed that the number of branches and lateral roots is increased in MIR167 overexpressing plants and ARF6 interfering plants.ARF6 interfered plants with the dark green leaves,and leaves showed an inward curling and shrinking phenomenon.(6)Recombinant plasmid of p GBKT7-ARF6 and empty vector p GBKT7 transformed into yeast cell.The growth analysis of a deficiency and three-deficient medium identified ARF6 is a transcriptional activator.