Molecular Mechanism Analysis Of Tyrosine Promoting The Formation Of Viola × Wittrockiana Floral Blotches

Viola × wittrockiana(pansy)is a biennial plant of Violaceae,which is a common landscaping plant in winter.It is also an ideal material for studying the inheritance of flower color and the formation mechanism of flower blotches.In previous studies,our team found that tyrosine decarboxylase(VwTYDC)expressed differently in blotches and non-blotches regions.It is speculated that tyrosine may be a potential factor in the formation of anthocyanins in pansy,which makes tyrosine become a new research direction in the mechanism of flower spot.In order to reveal the molecular mechanism and transcriptome characteristics of Viola×wittrockiana floral blotches effected by tyrosine and to explore the effect of tyrosine on anthocyanins biosynthesis,the species of Viola × wittrockiana,’Mengdei’,was used as the experimental material in this study.Transcriptome sequencing were performed on non-blotch areas with treatments of H2O or 3 mM tyrosine.The qPCR and VIGS were used to identify the expression of the key genes.The results display possible existing pathways of tyrosine and anthocyanin biosynthesis in Viola × wittrockiana,which provides a new idea and theoretical basis for further color breeding and genetic information in the future.The specific research contents are as follows:(1)19 438 different expressing genes were obtained by de novo assembly method.13037 unigenes were up-regulated and 6401 unigenes were down-regulated.9 genes differentially expressed in anthocyanin biosynthesis;(2)Cluster analysis was taken out on transcription factors from Viola × wittrockiana and Arahidopsis thaliana.Two differently expressed transcription factors were related to flower color,1 anthocyanin-related MYB transcription factor and I anthocvanin-related bZIP transcription factor;(3)Transcriptomic data analysis showed that exogenous application of tyrosine could promote abscisic acid formation,and abscisic acid could promote anthocyanin biosynthesis mechanism.(4)qPCR was used to analyze and validate the structural genes in nine anthocyanin biosynthesis pathways.It was confirmed that exogenous application of tyrosine could promote the up-regulation of structural gene expression in the anthocyanin biosynthesis pathway in Viola × wittrockiana:(5)qPCR was used to validate anthocyanin synthesis genes and key transcription factors under ABA treatment.The expression of VwCHS,VwANS,VwUGT,MYB transcription factors(Unigene0005403)and bZIP transcription factors(Unigene0087548)were up-regulated under ABA treatment.(6)VIGS expression vector pTRV2-VwCHS,pTRV2-VwTYDC,pTRV2-VwANS were conducted,mediated and transferred to Viola × wittrockiana though Agrobacterium tumefaciens.We found that the VwCH.S-silenced and VwANS-silenced flowers changed from patchy patches to striped patches.After VwTYDC-shenced,the accumulation of anthocyanins increased obviously,which proved that tyrosine was closely related to the anthocyanins biosynthesis in Viola × wittrockiana.(7)Putting the possible regulatory network of tyrosine-promoted anthocyanin biosynthesis in Viola × wittrockiana.The results showed that exogenous addition of tyrosine could promote the accumulation of anthocyanin content and improve the expression of anthocyanin structural genes and related transcription factors in Viola × wittrockiana.It could also promote anthocyanin biosynthesis and the formation of color spots by increasing ABA content in other ways.