Cloning And Analysis Of Structural Genes FpDFD And FpANS And Their Promoters For Anthocyanin Synthesis In Red-flowered Strawberry

Red-flowered strawberry is an intrageneric hybridization of white flowered cultivated strawberry(Fragaria × ananassa)and red-flowered Potentilla palustris.It is a new member of strawberry family.It has high ornamental value,edible fruit and good development space and prospect.In recent years,with the development of high-throughput sequencing technology,it can provide a new idea for the research mechanism of petal color of red-flowered strawberry.The petals of hybrid progenies of red-flowered strawberry had significant character separation,and the color of the progenies changed from white to red.In this paper,the red petals and white petals of hybrid progenies were used as materials by means of transcriptome sequencing analysis,hoping to analyze the difference of flower color from transcriptional level.Based on the previous research,the key structural genes and promoter sequences in anthocyanin synthesis pathway of red flower strawberry petals were cloned,and the structural characteristics of different flower colors were analyzed,which is of great significance to further understand the flower color regulation mechanism of red-flowered strawberry.The main results are as follows:‘Sijihong' was used as the material to select the petal samples of three development stages with obvious color change in the petal development process,namely,bud stage(PF_L),color transition stage(PF_Z),and large bud stage(PF_D)for high-throughput sequencing.According to FPKM value and log2(foldchange),three members of DFR gene family Fp DFR1,Fp DFR2,Fp DFR3 and one member of ANS gene family Fp ANS1 were preliminarily selected.According to TPM value,it can be seen that the expression of these four genes increases gradually with the development of flower bud.Four structural genes,Fp DFR1,Fp DFR2,Fp DFR3 and Fp ANS1,were cloned from the petals of red-flowered strawberry cultivar "Sijihong" and hybrid white-flowered "B1".The ORF length of open reading frame was 1077 bp,1050bp,1116 bp and 1152 bp respectively,and 358,349,371 and 383 amino acids were encoded.The similarity of the cloned sequence of the four structural genes is over 97%,and each of them has a special conserved domain.The cloned genes Fp DFR1,Fp DFR2 and Fp DFR3 have two special domains: a conservative NADPH binding site and a substrate specific binding site;all three DFR genes have NADB Rossmann conservative domain and unique FR-SDR-e element;Fp ANS1 gene belongs to PLN03178 superfamily and has [2OG Fe(II)] dioxygenase family gene conservative domain There are active sites of Fe2+ binding site and ?-ketoglutarate.According to amino acid sequence alignment,the 134 th amino acid of Fp DFR2 is asparagine(n),belonging to Asn type DFR;the specific binding site of Fp DFR1 gene is alanine(a),and the 134 th amino acid of Fp DFR3 is threonine(T),and the latter two DFR types belong to non Asn / Asp type.Subcellular proteins are located in the nucleus and other locations.Compared with other species,almost all of them have the highest homology with Rosaceae.The real-time fluorescence quantitative experiments were carried out on the petals and different tissue parts of different flower colors and different developmental stages of hybrid progenies of red-flowered strawberry.The results showed that among petals,fruits,leaves,petioles and stolons,DFR1 and DFR2 genes were the most expressed in the fruit,DFR3 and ANS1 genes were the most expressed in the petals,and in the gene expression analysis of five colors in the petals are red,deep pink,pink,light pink and white,the gene expression was the highest in safflower,The expression of Fp DFR1 and Fp DFR3 was the lowest in the light pollen,the lowest in the deep pollen,and the lowest in the white petal;in the expression analysis of three different stages of red strawberry,such as L(bud stage),Z(color transition stage),D(big bud stage),the expression of all genes was the highest in the big bud stage(PF_D),the lowest in the bud stage(PF_L),and the lowest in the gene D FR3 is special,and its expression level is the lowest in the color changing period.Four promoter sequences,Fp DFR1 Q,Fp DFR2 Q,Fp DFR3 Q and Fp ANS1 Q,were extracted and cloned from the petals of red-flowered strawberry cultivar ‘Sijihong' and white-flowered ‘B1' of hybrid progeny.The results of promoter cloning showed that except for Fp ANS1 Q,the DFR promoter sequences of red flower and white flower were the same.the length of the three promoters except Fp DFR1 Q was more than 2000 bp.Besides TATA box and CAAT box,there were more Box4,G-box,are and abre in the promoter region.Through the analysis of the myb binding sites of cloned promoters,it was found that there was no difference in the number of MYB binding sites between the red and white promoters of the same gene,but there was a significant difference in the number of MYB binding sites between different promoters.Fp DFR2 Q had the largest number of MYB binding sites and the highest expression.