| Flower color is one of the important ornamental characters of red-flower strawberry.In the early studies,the genes regulating anthocyanin synthesis in petals were studied and explored.Among them,MYB gene can indirectly regulate anthocyanin accumulation by regulating the expression of structural genes.However,the regulatory mechanism of MYB transcription factor on anthocyanin synthesis in petals of red strawberry is still unclear.Red-flower strawberry is a new member of ornamental flower with bright color,long flowering period and edible fruit,which is obtained by crossing(Fragaria × Potentilla).The different from the previous cultivated strawberry with white flowers,there are red,deep pink,pink and other flower colors.Early reports indicated that pink is a dominant trait,while recessive gene regulates white.After distant hybridization with Potentilla palustris(L.)Scop.,the hybrid offspring have rich flower color,and both ornamental and edible value.In plants,MYB transcription factor belongs to one of the largest transcriptional regulatory factor families.In this study,five MYB genes related to anthocyanin synthesis in petals of red-flower Strawberry ’Sijihong’ and other hybrids with different flower colors were screened,and two MYB genes were cloned and verified.In the later stage,MYB gene with high expression in petals was selected for protein expression in vitro to explore the regulatory binding sites of transcription factors.The main results are as follows:1.Based on the transcriptome data of red-flower strawberry,the phylogenetic tree of MYB gene in L,Z and D stages and Arabidopsis thaliana was constructed by MEGA.Furthermore,the FpMYB genes that regulate anthocyanin biosynthesis were screened by using Z-score standardized TPM value to draw thermal map for cluster analysis and qRT-PCR for temporal and spatial expression pattern analysis.According to the known species functions,they were divided into two categories: positive regulatory factors and negative regulatory factors.2.There are two FpMYB genes were amplified and cloned by PCR,named of FpMYB90 and FpMYB308.The results of preliminary bioinformatics analysis showed that the two had typical MYB domain at N-terminal,and had high homology with the MYB transcription factor domain reported in other species.The results showed that the two were R2R3-MYB transcription factors,and subcellular localization prediction showed that the main role of these was in the nucleus.3.The subcellular localization vector and overexpression vector of FpMYB90 and FpMYB308 were constructed.The function of the vector was identified.The fusion expression vector of FpMYB90-eGFP and FpMYB308-eGFP was constructed.After the tobacco leaves were infected momentarily,the main location was located in the nucleus,which was consistent with the predicted results of the software.The expression vector was constructed by fusion of pRI-101-AN-FpMYB90 and pRI-101-AN-FpMYB308,and infected tobacco,but the phenotype was not obvious.RT-PCR was used to determine the completion of infection.4.The Dap-seq of FpMYB90 and identification of downstream target genes were carried out.The DNA fragment of protein binding was studied in vitro through protein expression in vitro,and transcription factor binding sites were studied.The possible interaction genes were PRDM1 and AT1G69570;The qRT-PCR was used to predict the target genes of anthocyanin synthesis pathway.It was found that the downstream F3 H,DFR,ANS and UFGT genes might be regulated by FpMYB90. |
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