Identification Of Anthocyanin Transport Related GST Gene Family And Molecular Simulation Analysis In Red-Flowered Strawberry

The red-flowered strawberry is an interspecific cultivar,and the characteristics of them are introduced by distant hybridization from red-flowered Potentilla pulsetris to strawberries(Fragalia × ananassa)with white flowers.Since the role of Glutathione S-transferase(Glutathione S-Transferase,GST)in the accumulation of anthocyanin in the petals of red-flowered strawberry is still unclear.In this study,functional genes were screened by transcriptome data and bioinformatics analysis of Honghua-strawberry ’Sijihong’,a self-bred variety of red-flowered strawberry,and other hybrid offspring of different colors.We used gene cloning,subcellular mapping,molecular docking and promoter cloning to analyze the correlation between anthocyanin transport and GST gene,and discussed the mechanism of anthocyanin accumulation in flower petals.The main results are as follows:(1)In this study,a total of 66 GST gene members were selected from the Fragaria vesca strawberry genome and compared with the GST gene sequence of Arabidopsis.Phylogenetic tree was constructed by combining with GSTs with known functions of Arabidopsis.The 66 FvGST genes were divided into 7 subfamilies,among which the Tau subfamily had 42 genes and had the largest number of members.Phi contains 7 genes that may be involved in the transport of anthocyanin.The FvGST gene family was unevenly distributed on seven chromosomes,with a large number of tandem repeated events.The members of each family showed certain similarities in the species,number and structure form of conserved motifs and domains.Based on transcriptome data of red-flowered strawberry,differential gene expression analysis showed that only 3 of the 7 Phi genes were differentially expressed in the petals of strawberry,which were named FpGST1,FpGST2 and FpGST3,respectively.(2)The three genes were screened by real-time fluorescence quantitative assay,and the results showed that FpGST1,FpGST2 and FpGST3 were all specifically expressed in petals.The expression levels of the three genes were higher in pink and red petals than in white flowers.In different stages of flower development(from bud stage,beginning coloration stage to big bud stage),with the deepening of petal color,the expression level showed an increasing trend.Therefore,it is speculated that FpGST1,FpGST2 and FpGST3 genes may all be involved in anthocyanin transport.(3)Complete coding region sequences were cloned from flowering petals of ’Sijihong’and flowering petals of B1.Amino acid sequence alignment between red and white flower colors revealed that there was no difference between FpGST1 and FpGST2,and there was a codon mismatch in FpGST3.The results of subcellular localization showed that all three genes could be localized to the endoplasmic omentum and nucleus where GSH binds to anthocyanin.(4)Regarding the cloning of the promoters of the three genes Q-FpGST1 and Q-FpGST3 obtained by screening,the cloning results are basically the same for red flowers and white flowers,and there is only one difference in the base of Q-FpGST1.The three genes are regulated by various cis and trans-acting factors,and Q-FpGST2 has different colors.Of the three genes,TATA-box has the highest number of core promoter elements to initiate transcription at around-30,and CAAT-box as a common cis-acting element has more in the promoter and enhancer regions.All three genes contain basic functional elements,and each has a binding site for flavonoids such as MYB transcription factors.(5)The three-dimensional structure of the three genes is similar,and the modeling results are similar.All of them were similar to the glutathione transferase crystal of Populus tomentosa,with a coverage rate of over 50%.The three genes were molecularly docked with cornulin and geranium,respectively,and the optimal ligands were selected for the three protein structures by molecular dynamics simulation.Molecular docking data showed that the three genes had high homology and could covalently bind to anthocyanins.FpGST2 had similar binding ability to both anthocyanins,while FpGST1 had better binding ability to the main anthocyanin in petals,and FpGST3 had better binding ability to the main anthocyanin in fruits.