The Effects Of Two Types DFR Gene On Flower Colour Of Petunia

Anthocyanins together with carotenoids and alkaloids, are the majorpigments responsible for flower and fruit color. Three classes ofanthocyanidins, including cyanidin (red to pink), delphinidin (purple to blue)and pelargonidin (orange to brick red), are responsible for the primary shade ofthe flower color. It has been reported that Dihydroflavonol-4-reductase (DFR)is an important regulatory point in the anthocyanin pathway and is theupstream of both anthocyanin and proanthocyanidin production. DFR ofpetunia has the highest activity with dihydromyricetin and does not acceptdihydrokaempferol, so there is no pelargonidin derived pigments.Petunia hybrida (Vilm) is a popular garden plant, appreciated for itsflowers available colors from white via salmon, pink and red to violet.Calibrachoa hybrida is an ornamental plant of worldwide economicimportance. Flower of C. hybrida is similar with Petunia and they are relatives.The flower colour of P. hybrida integrated with DFR from Calibrachoa wasmore likely to change. In this study, based on the petunia DFR gene sequences published byNCBI, the full length cDNA of DFR was isolated from the petals of2petuniastrains. Both Pe1DFR and Pe2DFR were1473bp, bioinformatics analysisshowed that the open reading frame of these genes were1143bp, encoding380amino acids. The similarities to the DFR-A were93.8and99.6%respective, the putative amino acid similarities to the DFR-A were98.2%and100%respective. This result confirmed that DFR of petunia belonged to Asptype. Real-time quantitative PCR (qRT-PCR) was performed to determine theexpression pattern of DFR in different tissues of petunia. The expression ofDFR gene was detected in all kinds of tissues of petunia. But the expressionlevels were different; the expression level of this gene was highest in anther,medium in petal of blooming flower and flower just openned, lower in flowerbud, and the lowest expression level of DFR was in leaf. DFR activity analysisrevealed there was almost no DFR activity in leaf and anther, low activity inbuds, the highest activity was found in blooming flower. In9702·, there iscorrelation between DFR activity and DFR mRNA concentration except inanther. There was correlates positively with petal color and DFR enzymeactivity, the highest activity in purple petal while the lowest activity in W115·petal. The obsorption of cyanidins and delphindins were detected only in9702·.We obtained the full-length cDNA of DFR from Calibrachoa hybridathrough RACE-based technique. The cDNA of CaDFR is1351bp with anopen reading frame of1152bp, which encodes a putative protein of383amino acids. The CaDFR protein is predicted with42.74KD in molecularweight and a pI value of5.83. Sequence alignment showed that CaDFR had aclose relationship with DFRs from other species of the Solanaceae family, andit belonged to the Asp-type. On the other hand, the DFRs from Solanumlycopersicum, Gerbera jamesonii, Rosa chinensis were cloned according to thesequences gained from NCBI. We constructed these four DFR overexpressionvectors under the control of CaMV35S promoter and transformed them intofour strains of Petunia hybrida using the Agrobacterium-mediated method. Afragment corresponding to DFR was amplified in57%putative plantlets.Southern blot indicated that the copy number was from1to5. The phenotypesof T0plants were obvious. For example, overexpression of CaDFR changedthe white buds and white flowers of the strain ’2512’ into pink buds and whiteflowers with pink stream (’2H36’, it means36th transformant of ’2512’, and theother was similar). The strain ’9702’ produced red flowers with yellow anthers,whereas the transgenic plants displayed multiple phenotypes, including red flowers with pink spots (’9H30’,’9R8’), pink and red variegated flowers(’9T19’and ’9H31’), or deep red flowers with purple anthers (’9H33’,’9R9’).Most transformants of strain ’Lx’ had no phenotype, except one branch of atransformant (’LH26’) producing white flowers with purple spots, and thepurple spots were varied in different flowers and flower of ’LR25’ becamepurplish red. There were only three phenotypes of ’W115’ transformants, onewas just like their receptors, the other was pink flowers and pink buds (21transgenic plants integrated with RoDFR), the last was ’WR13’, a part of itspetals were white, and the other part were pink.Real time RT-PCR results showed that the concentration of exogenousDFR mRNA was higher than that of endogenous DFR, the biggest was up to64times. The time of highest concentration of DFR was different betweendifferent DFRs. The concentration of CaDFR mRNA was very low in leaf andanter, the highest in bud Ⅱ, and was lower in bud Ⅲ and budⅠ, it increasedin fully open flowers. The concentration of RoDFR mRNA was higher in leaf,and it was17.056in budⅠ, then it decreased in the followed stages (budⅡand bud Ⅲ), but the highest concentration was in petals starting to open.DFR activity analysis revealed that the intensity of red coloring in thetransgenic flowers was positively correlated with the level of DFR activity. DFR activity was low in leaf, anther and budⅠ, high activity was detected inblooming flower or petal of opened flower. DFR activity could be increased bythe integrated of exogenous DFR.The anthocyanin was extracted from various transformants. The resultsshowed that pelargonidin was emerging with the increasing of cyanidin in’9R9’,’9H33’ and ’9R12’. After integrated with CaDFR, the anthocyaninsconcentration in anthers and petals of ’9H33’ was increased significantly, andthere were44.23mg and99.61mg pelargonidin3-o-glucoside in per kilogramfresh weight respectively.The phenotypes of T1progenies were very obvious, in14progenies of’9H30’, there were4phenotypes, one was similar to ’9702’, second was similarto T0, third was red flower with pink spots, the last was pink flower. In ’9R9’offsprings,5plants were similar to ’9702’, and5plants were similar to T0. Afragment corresponding to CaMV35S was amplified in47.76%T1progenies.It can be drawn a conclusion that the exogenous DFR gene in Petunia hybridacan be inherited.