Cloning And Functional Analysis Of FvMYB6-like Gene In Strawberry

Strawberry,a vital fresh and processed fruit,is welcomed by consumers due to its unique aroma and nutritional value.Skin color,which is one of the most significant criteria for evaluating the appearance of strawberries,is mainly due to the accumulation of anthocyanins.The synthesis of anthocyanin is regulated by many transcription factors.R2R3-MYB transcription factors(TFs)is a key factor in the regulation of anthocyanin synthesis.In this study,FvMYB6-like gene was isolated from woodland strawberry ‘Ruegen'.We were constructed the overexpression vector of FvMYB6-like.Overexpression vector were transformed into Arabidopsis.It was revealed that FvMYB6-like gene is not only involved in anthocyanin accumulation but SAM differentiation.The main results are revealed as follows:(1)FvMYB6-like gene was isolated from woodland strawberry ‘Ruegen' by RT-PCR.The CDS of FvMYB6-like gene is 768 bp,the amino acids of FvMYB6-like is 256,with an isoelectric point of 8.60 and a reckoned molecular weight of 29.22 kD.(2)DNAMAN software was performed to analyze nucleotide sequence alignments between FvMYB6-like and different species.This result indicated that a highly conserved R2R3-MYB domain was contained in the N-terminal and a bHLH motif was also observed in R3 domain of FvMYB6-like.In addition,two conserved regions exist in the C-terminus of FvMYB6-like named as C1-motif and C2-motif.Thus it belongs to R2R3-MYB subgroup 4.(3)The full-length Fv MYB6-like without the TAA was fused in a vector which containing GFP gene and the CaMV 35 S promoter.We performed this experiment by transient transformation the FvMYB6-like-GFP recombinant plasmids in Arabidopsis protoplast.Confocal microscopy analysis indicated that the FvMYB6-like fusion protein was merely localized in the nucleus.So this gene encodes a nuclear protein.Transactivation analysis assay indicated that FvMYB6-like is no transcriptional activation activity in yeast(4)The promoter sequence of FvMYB6-like gene was obtained in the GDR database and analyzed using PlantCARE online software.There is a MYB binding site involved in flavonoids biosynthesis genes regulation(MBSIIG)contains in this sequence.Real-time PCR showed that the expression level of FvMYB6-like gene was significantly up-regulated in FvMYB10 overexpression strawberry fruit.The results of tissue-special showed that the expression of FvMYB6-like gene was relatively high in leaves,stems and flowers,and relatively low in roots and fruits.Real-time PCR was used to detect the expression levels of FvMYB6-like in different stage of strawberry fruit.The results showed that the expression of FvMYB6-like performed a decreasing trend with the maturity of strawberry fruit.(5)Overexpression of FvMYB6-like in Arabidopsis showed dark-green colour in leaves and multiple shoot apical meristems(SAMs).The expression levels of some key anthocyaninrelated and SAM-related genes were significantly higher in Arabidopsis plants overexpressing FvMYB6-like compared with control plants.