Identification Of ABC Gene Family And Cloning And Expression Of FpABC Gene In Red-flowered Strawberry

The red-flowered strawberry is a new member of the strawberry family.It is an intergenus hybrid(Fragaria × Potentilla)and has high ornamental and edible value.The transport of anthocyanins plays an important role in flower color changes.In the transport of various substances of red-flowered strawberry,the ABC gene family has an important role,but its transport mechanism is not yet very clear.In this study,131 red-flowered strawberry ABC genes were screened from red-flowered strawberry transcriptome data.Three genes related to anthocyanin were cloned.At the same time,combined with the real-time fluorescence quantification of ABC genes related to anthocyanin,the expression characteristics of ABC at different developmental stages,different tissue parts and different flower colors were analyzed.The main results were as follows:(1)From the red-flowered strawberry transcriptome data,131 ABC genes were identified and named Fp ABC1-131 respectively.Through analysis of its physical and chemical properties,it is found that the proteins encoded by these Fp ABC genes contain 68-4352,the protein molecular weight is about 6775 to 485455 Da,and the isoelectric point is 4.25-6.44.According to phylogenetic analysis,131 red-flowered strawberry Fp ABC genes were assigned to 8 subfamilies.Among them,ABCB,ABCC,and ABCG subfamilies have the most members,while other subfamilies have relatively few members.Members of the same subfamily show certain similarities in the types,numbers and structural forms of conserved motifs and domains.The ABCG and ABCB subfamilies contain multiple conserved domains.The number of conserved motifs contained in ABC family members is between 1 and 36,and the number of exons is between 1 and 36,indicating that it is a huge gene family,and the function of each subfamily is very different.Chromosome distribution analysis showed that the Fp ABC family of red-flowered strawberry was unevenly distributed on 7 chromosomes,mainly on the 6th chromosome,and there were tandem repeats.From the gene expression analysis of three stages of petals of red-flowered strawberry transcriptome,all genes will be expressed in at least one stage,and the gene expression period of the same subfamily is almost the same.In addition,common cis-elements involved in transcriptional regulation were identified in the promoter region of all Fp ABC genes of red-flowered strawberry.(2)Amplified clones in the petals of red-flowered strawberry 'Sijihong' yielded the complete CDS sequence of genes related to anthocyanin transport.Fp ABC55 had differences in the alignment of clones of red and white flowers,while Fp ABC33 and Fp ABC34 had the opposite.The sequence lengths of Fp ABC33,Fp ABC34,Fp ABC55-H and Fp ABC55-B are 2250 bp,2238bp,2169 bp and 2169 bp,encoding 750,736,723 and 723 amino acids respectively,and the gene accession numbers are MT362908,MT362909,MT362910 and MT362911 respectively.Through multi-sequence comparison analysis,it was found that Fp ABC33 and Fp ABC34 respectively have more than 80% identity with homologous protein sequences of various other species.The evolutionary relationship between these three genes and other species shows that the rose has the closest relationship with safflower strawberry.The secondary structures of these three transporters have great similarity.The heaviest part is ?-helix,followed by random coil.Fp ABC33 and Fp ABC34 have the same PLN03140 superfamily domain,while Fp ABC55 has the 3a01204 superfamily domain.(3)Through the ABCC gene screened by transcriptome,RNA was extracted from the petals of red-flowered strawberry in different tissue parts,different colors and different periods,and then real-time fluorescence quantitative experiments were performed.The results showed that the expression levels of Fp ABC33,Fp ABC34,and Fp ABC55 were highest in red-flowered,and almost the lowest in light pink flowers,with significant differences.The expression level of red-flowered is about twice that of white flower;in the expression analysis of bud stage(L),color transformation stage(Z)and large bud stage(D)in different periods,the results show that Fp ABC33,Fp ABC34,Fp ABC55 are all in The expression level in the big bud phase is the highest,the expression level in the bud phase is low,and the expression level in the relative bud phase is about 4-8 times;while in other genes,the expression levels of Fp ABC108 and Fp ABC69 show a downward trend,and the expression level in the big bud phase is the lowest;Tissue parts Petal,Fruit,Leaf,Petiole,and Runner are all significantly different.Fp ABC33,Fp ABC34,and Fp ABC55 are the highest expressed in petals.The remaining expression levels are highest in the leaves.