Mechanism Of VwNEGAN And VwRTO Genes Regulating The Pigmentation Patterning Of Floral Blotches In Viola × Wittrockiana

Pansy(Viola × wittrockiana)are wonderful biennial flowers of Violaceae for their abundant cultivars and various colors.They are also is the ideal materials for studying the formation and inheritance of flower blotches for their variegated flowers and stable genetic traits.In this study,VwMYB transcription factor genes in Viola×wittrikianan ‘Meng Die’ were identified using full-length transcriptome data.We also found that a large number of MYB genes had significantly differential expression by the next-generation transcriptome sequencing of Pansy.According to the sequence alignment,two MYB transcription factors,VwRTO and VwNEGAN,might act as the repressor and activator to regulate anthocyanin spot patterning in pansy.VwRTO and VwNEGAN were cloned and qPCR validated,and were transformed into tobacco.The subcellular localization and virus-mediated gene silencing(VIGS)validation experiments were performed then.The results were as follows:(1)A total of 121 MYB genes were identified in pansy,of which 61 belonged to1R-MYB,57 belonged to R2R3-MYB and 3 belonged to 3R-MYB.About twenty motifs were obtained,in which motif2 had the highest distribution frequency(15.2%),and motif20 was the lowest one(0.3%).Web Logo analysis showed that R2R3-MYB transcription factors have typical W type R2R3 conservative motifs.The gene functions of MYB factors were predicted based on the homology and evolutionary relationship between pansy and Arabidopsis thaliana.A total of 23 subgroups were obtained through clustering analysis with Arabidopsis thaliana,of which 7 subgroups could be clustered in Arabidopsis subgroups,suggesting these VwMYBs might have similar biological functions.(2)Sequence alignment of the third-generation and second-generation transcriptome differential expression data of pansy with two MYB genes in Mimulus,Mg RTO and Mg NEGAN,showed the VwRTO and VwNEGAN in pansy were similar to Mg RTO and Mg NEGAN,We speculated that VwRTO might be a negative regulator of anthocyanin spots,and VwNEGAN might be an anthocyanin-activating R2R3-MYB transcription factor.The qPCR was used to check the expression of the VwRTO and VwNEGAN genes.The results showed that VwRTO had higher expression levels in the blotch areas in the Ⅰ-Ⅳ periods.When the petals senesced and the spots color faded,VwRTO increased in the non-blotch areas,and decreased in the blotch areas.VwNEGAN gene in the blotch areas showed the higher expression than that in the non-blotch areas,and the expression levels of the blotch areas gradually increased during the formation of blotches,and the highest expression levels occurred when the blotches formation was stable.The expression levels of the two genes in the non-blotch areas were much lower than that in the blotch areas.The expression level of VwRTO gene in the blotch areas gradually decreased with the formation of blotches,while VwNEGAN gene in the blotch areas gradually increased with the formation of blotch areas of pansy.Bioinformatics analysis showed that two genes were hydrophilic proteins and didn’t contain transmembrane structures.The protein tertiary structures of the two genes were consistent with the structure of the R2R3-MYB transcription factors.(3)The open reading frame(ORF)of VwRTO and VwNEGAN genes were cloned to use for subcellular localization,and the VwRTO gene length was 633 bp and the VwNEGAN gene length was 735 bp.pNC-Green-Sub N-VwRTO and pNC-Green-Sub N-VwNEGAN vectors were constructed.Subcellular localization recombinant vectors were introduced into agrobacterium,and the tobacco was infected.The localization of the protein was observed by laser confocal microscope,showing that the VwRTO and VwNEGA genes were expressed in the cytoplasm,nucleus and cell membrane.(4)The open reading frame were recombined with the pCAMBIA2300 vector to obtain pCAMBIA2300-VwRTO and pCAMBIA2300-VwNEGAN recombinant vectors.The recombinant vectors were introduced into agrobacterium for genetic transformation of tobacco.VwNEGAN gene significantly promoted the anthocyanin accumulation in some tobacco callus and tobacco plants.VwRTO gene was observed to inhibite the growth of callus in tobacco,and no tobacco plants were obtained.(5)The cDNA fragments of VwRTO and VwNEGAN genes for the VIGS were cloned,and the VwRTO fragment length was 380 bp and the VwNEGAN gene fragment length was 331 bp.The pTRV2-VwRTO and pTRV2-VwNEGAN recombinant vectors were constructed.The recombinant vector was introduced into agrobacterium,and introduced into pansy plants.It was found that the petals of pansy that the VwNEGAN gene was silenced became smaller,while the area of blotches were significantly smaller and the color became lighter.This result indicated that VwNEGAN gene can promote the formation of blotches.The pansy petals infected by VwRTO gene did not show significant changes.