Cloning And Expression Analysis Of DOPA 4,5-dioxygenase Extradiol In Viola Wittrockiana Gams.

Anthocyanin and Betalain are two of the most important water-soluble pigments in plants,but so far there is no coexistence of these two pigments found in one single plant,and the molecular mechanisms of this mutual exclusion is still not clear.Pansy(Viola wittrockiana Gams.)is an excellent ornamental flower in cold seasons,which belongs to Viola,Violaceae,and of which the petals contains anthocyanin.Our research has found a key enzyme gene of betalain synthesis,DOPA 4,5-dioxygenase extradiol(DODA),showing a high expression level in pansy petals.This research tried to study the function of VwDODA in pansy,and to provide a new idea of the mutual exclusion of anthocyanin and betalain using petals of pansy as researching material.Real-time PCR was applied to analyze the different expression levels of VwDODA in different organs of pansy,then the cDNA full-length of VwDODA was obtained by RACE.The bioinformatic analysis was conducted on the cDNA sequence,and the expression vector containing VwDODA was constructed and transferred to Arabidopsis thaliana to confirm the function of VwDODA gene.In addition,the transformation ability of the gene for DOPA was researched by infiltration of pansy seedlings with L-DOPA.The main results were as follow:(1)Differential expression levels of VwDODA gene were showed in pansy flower,stem,leaf and root were analyzed by Real-time PCR.The VwDODA gene expression was observed in each organ of pansy,but the lowest level was in blotched area of petal with the highest level in the non-blotched area of petal.(2)The cDNA full length sequence of VwDODA by RACE was 1055bp,with 157bp of 3'UTR sequence,28bp of 5'UTR sequence,and 795bp open reading frame sequence encoding 264 amino acids.(3)The VwDODA-pOE1 expression vector was constructed successfully,then transferred to Arabidopsis thaliana by Agrobacterium tumefaciens.Seeds of the transgenic Arabidopsis thaliana were collected,and the phenotype of progeny was observed to analyze the gene function of VwDODA.(4)No obvious color change was observed on pansy seedlings cultivated with L-DOPA.Pansy seedlings all died when concentration of L-DOPA solution was higher than 0.1mM.The differential expression levels of VwDODA in pansy seedlings under different concentrations of L-DOPA were analyzed.The result showed that VwDODA expressed in each concentrations of L-DOPA treatments,with the lowest level in 0.05mM,conversely,the highest level in 0.1 mM.