Cloning And Expression Verification On MYB Genes Regulating Anthocyanin Synthesis Of Pansy

Viola×wittrockiana Gams. is an ornamental plants with typical spotted petal, which is a good material for flower blotch research due to its simple blotch inheritance, stable large area of spot and visible boundaries. It is very important to clone the MYB genes in pansy flower to find out the numbers and types of MYB transcription factors involved in anthocyanin synthesis, and to confirm their expression patterns for revealing the flower blotch forming mechanism determined by them. In this research, yellow pansy with black-blotched petal were used as materials, and the sequence informations of MYB genes were analyzed by transcriptome sequencing. Real-time PCR was applied to analyze the differential expression of MYB genes between the blotched and non-blotched area of pansy flowers, and the full length of MYB genes were cloned by RACE, then transient expression was adopted to confirm the function of MYB gene after constructing expression vector. The detailed results as follows:1. There were118747transcripts and43908unigenes through the transcriptome sequencing29MYB genes were obtained according to the CDS prediction, and the expression level of MYB29in the fifth stage was the highest in the29genes according to the analysis of gene differential expression. The expression differences of MYB29between the blotched and non-blotched area of pansy flowers were not obvious. The expression level of MYB8was higher than others, and the expression differences between the blotched and non-blotched area were not obvious either.2. The differential expression level of MYB8and MYB29in different stages according to Real-time PCR revealed that the expression trends variation of MYB8was similar to the structural genes F3H and DFR which was involved in the anthocyanin synthesis of pansy flower, except for the fourth stage, in which the expression level of the blotched area was higher than the non-blotched area. Above all, we can deduce that MYB8may corresponded with pigments biosynthesis of pansy flower. But the expression pattern of MYB29between blotched and non-blotched area of pansy flowers was not obviously different, and the variation trendency of its expression pattern in petals is basically the same situation. 3. RACE was used to obtain the full cDNA length of MYB8and MYB29, the length of the open read frame are849bp and600bp, encoding282and199amino acids, respectively.4. The bioinformatics analysis revealed that MYB8and MYB29were the non-transmembrane proteins without signal peptide. they were both R2R3-MYB transcription factors, might have regulatory functions.5. Transient expression was conducted by particle bombardment system. The result showed that there was a color deepened phenomenon in pansy. And red spots appeared in Rosa chinensis Jacq. It suggested that MYB29may be associated with pigments biosynthesis of pansy flower, but not with the formation of spot formation according to the results of Real-time PCR experiment.