CDNA Cloning And Characterization Of UDP-glucose: Anthocyanin 3-O-glucosyltransferase In Freesia Hybrida

Freesia hybrida, a kind of perennial herbaceous plant, belongs to fressia genus of Iridaceae family. It originated from Southern Africa, and was a new brand of cut flowers in the international flower market. Freesias are excellent cut flowers and work well in corsages, which have a wide range of flower colors including white, yellow, red, blue, lavender and purple. Therefore they are good plant materials to study the flower color.Glycosylation is one of the major modification reactions that often occur in the final step of the natural compound biosynthesis. Glucosyltransferases catalyze the transfer of the glucosyl moiety from UDP-glucose to the 3-hydroxyl group of anthocyanidins. UF3GTs play important roles not only in modifying the flower color but also in increasing the solubility and stability of hydrophobic flavonoids. In this thesis, UF3GT in Freesia hybrida is the key enzyme of flavonoid biosynthetic pathway, which was chosen for gene cloning and function analysis. We also preliminary discussed the relationship between the expression of Fh3GT1 gene and the formation of flower color. The cloning and the relevance of the Fh3GT1 in this study paves a way which can be applied in changing flower pigments based on metabolic engineering. The main results were as follows:1. A cDNA clone encoding UF3GT was isolated by screening from a cDNA library of red petals from Fressia hybrida and 3’ rapid amplification of cDNA end (RACE) techniques. Sequence analysis showed that the cDNA is 1614 bp long and has a putative open reading frame of 1338 bp encoding 445 amino acid residues with a calculated molecular mass of 48,043 Da and an isoelectric point of 6.02, named Fh3GT1 (Genbank accession number was HM590645). A molecular phylogenetic analysis of the deduced amino acid sequence between Fh3GT1 and other plant glycosyltransferases showed that it is a new member of UF3GT group.2. The spatial and temporal expression pattern of Fh3GT1 was studied with semiquantitative RT-PCR (sqRT-PCR). The result indicated that the expression of Fh3GT1 in fully opened petals reached to the maximum and was more than the unpigmented buds. Besides, expression levels in all the pigmented petals were higher than stems and leaves. The Fh3GT1 transcripts in red and pink petals exhibited the same expression level and it was higher than in yellow petals, whereas in white petals, the expression of Fh3GT1 was rarely detected. 3. The open reading frame of Fh3GT1 was cloned into the pET-28a (+) vector for heterologous protein expression in E.Coli strain BL21 (DE3). The presence of target protein was confirmed by SDS-PAGE. The molecular mass of recombinant protein was 48 kDa which is consistent with the results of the calculated molecular mass of Fh3GT1. Characterization of the enzymatic assays in vitro using recombinant Fh3GT1 showed that it had the activity of UF3GT.4. Fh3GT1 was fused with GFP, and then the reconstruction vector was transformed into Agrobacterium tumefaciens strain EHA105 and transiently expressed after transformed into onion epidermal cells. Confocal laser microscopy was used to examine onion epidermal cells and revealed that the expression of Fh3GT1::GFP was mainly in cytoplasm, which suggested that Fh3GT1 gene encoded a cytoplasm protein.5. To elucidate the function of Fh3GT1 in plants, we constructed overexpression vector (pBI-Fh3GT1), and then transformed petunia. The results showed that 7 independent transgenic plants of petunia were obtained for pBI-Fh3GT1. PCR and Southern blot were performed to confirm the situation of genomic integration. RT-PCR was used to identify the level of Fh3GT1 transcription in transgenic petunia plants. All transgenic petunia plants showed the color change compared to the wild type petunia. For the single copy overexpression transgenic plant, the edge of corolla appeared white patches in different degrees, and some with color missing in petal. The average content of total anthocyanins was reduced to 40%. While in two copies overexpression transgenic plants, the color changed from deep red into pink in petals, and the average content of total anthocyanins was even reduced to 80%. The feasible reason for this phenomenon is sense cosuppression of homologous genes, which is a post-transcriptional gene silencing (PTGS) event.6. Constructed RNA interference vector (pART-Fh3GT1i). The target DNA was 347bp conserved domains of plant UF3GT family. Recombinant RNA interference vector encoded intron-spliced RNA with a hairpin structure, which could cause post-transcriptional gene silencing event in plant. The results showed that 5 independent transgenic plants of petunia were obtained for pART-Fh3GT1i. For the transgenic petunia plants with RNAi vector, all the flower colors were changed from red into pink, the average content of total anthocyanins was reduced to 85% in comparison with the wild type petunia, and the transgenic petunia plants have dwarf plant and flower type.