Studies On The Molecular Regulation Of The Anthocyanin Biosynthesis Of Purple-fleshed Sweetpotato (Ipomoea Batatas (L) Lam.)

Sweetpotato(Ipomoea batatas (L) Lam.) belongs to the family of convolvulaceae, genus Ipomoea and speciesⅠ. batatas, is a kind of herbaceous annual or perennial crop, which is an important source for food, feed and industrial material being widely cultivated in tropical, subtropical and warm regions. Anthocyanin, a kind of important soluble flavonoid compound including glycosylation derivates of anthocyanidin through combining different monosaccharide with glucosidic bond, can be produced by a wide range of flowering plants. Purple-fleshed sweetpotato means sweetpotato with purple tuber flesh, it is deemed as a special variety due to its high anthocyanin content. Accordingly, purple-fleshed sweetpotato enjoys high edible and medicinal value since natural anthocyanin contains a variety of biological activities including antioxidant, antidiabetics and curbing gene mutation etc. Although its health function begins to be acknowledged, molecular mechanism of anthocyanin biosynthesis in purple-fleshed sweetpotato is extremely limited, thus it is difficult to breed varieties containing high anthocyanin content. This thesis aims to elucidate biosynthetic pathway of anthocyanin through cloning and characterizing key genes, thus provides theory foundation for breeding varieties producing high level of anthocyanin as well as producing anthocyanin with purple-fleshed sweetpotato as plant metabolism bioreactor. The main results in our research are as follows:1. Cloning and functional analysis of dihydroflavonol 4-reductase (IbDFR) gene from purple-fleshed sweetpotatoBased on homogenous clone strategy and RACE technique, the full length cDNA of dihydroflavonol 4-reductase designed as IbDFR was cloned from purple-fleshed sweetpotatao variety 263 (Genbank accession number:HQ441167). The sequence analysis reveales that the full-length cDNA of IbDFR is 1392-bp long fragment gcontaining a 1182-bp open reading frame (ORF) encoding a polypeptide of 394-amino acids. Blastn and blastp analysis on NCBI showes that nucleotide sequence of IbDFR shares a high similarity with DFRs from other plants, especially with Ipomoea nil (96% identity), likewise, amino acid sequence of IbDFR shares a high similarity with other reported DFR proteins, especially withⅠ. nil (94% identity). The phylogenetic analysis indicates that IbDFR and DFR from Ⅰ. nil develop into one small group. Analysis of conserved,domain indicates that IbDFR protein contains highly conserved NADPH combining and substrate specific motifs. In addition, IbDFR is similar with known DFR proteins in active spots and advanced structure. Thus it is predicted that IbDFR protein possesses biological function.Expression plasmid of IbDFR was constructed and was used for transformation to tobacco(Nicotiana tabacum) W38. Transgenic tobacco plants were obtained and expression patten of IbDFR in transgenic plants was analysed. Transgenic plants grew and developed normally after transplanted to field. Observation during growth period suggested that transgenic plants did not show obvious difference in growth trend and plant type compared to wild type plants. During flowering period, it is observed that the flower color in transgenic plant number 9 was sharply deepened, anthocyanin content in flowering organ was improved dramatically (P<0.01), as much as 50% higher to control plants. Above results demonstrated that the expression of IbDFR driven by a strong promoter can up-regulated the anthocyanin content in tobacco. In subsequent molecular detection by real-time quantitative PCR(qPCR), the high expression level of IbDFR in transgenic line number 9 proved that improved anthocyanin content was positively co-related with the expression of IbDFR. However, positive transgenic line number 1 and number 7 did not show IbDFR's function, which may be resulted from the gene silence of introduced IbDFR. Q-PCR results also suggested that expression level of IbDFR was relatively low in both line number 1 and number 7.The prokaryotic expression vector of IbDFR gene was constructed and transformed into Escherichia coli BL21(DE3), also the IbDFR protein expressed and purified through Ni column ion exchange chromatography. After that, used circular dichroism spectrum(CD) to analyze the secondary structure component of the recombinant protein as follows:13.9%α-helix,41.2%β-sheet,10.1% turn,34.7% random coil, and it was easy come to thatβ-sheet and random coil take the dominance. However, amino acid sequence on the site expasy with SOPMA analysis to predict the secondary structure of the recombinant protein had a different show: with 153α-helix,49β-sheet,25 turn,167 random coil, that was 38.83%,12.44%,6.35% and 42.39%, respectively, with a-helix and random coil take the most. Took the (±)taxifolin as substrates to do experiments on enzyme activity, and the enzyme activity determination of the recombinant protein showed that the prokaryotic expressed recombinant protein of IbDFR could catalyze (±)taxifolin to change into downstream substances, which demonstrated that the recombinant protein had enzymatic activity. The circular dichroism spectrum(CD) determination result was not consistent with the predicted result on expasy, which indicated that the fold mode of prokaryotic expressed recombinant protein may be not consistent with the protein expressed in plant. At the same time, it showed that the prokaryotic expressed protein could form a conserved domain which combined to NADPH and substrate when folding. Finally, the enzymatic activity results also fully illustrated the key role of that enzyme in anthocyanin biosynthesis pathway.2. Cloning and characterization of IbF3H and IbANS fromⅠ. batatas (L) Lam. Yuzi263 Based on homogenous clone strategy and RACE techniques, it was cloned that the full-length cDNA of Flavanone 3-hydroxylase (IbF3H) and Anthocyanidin synthase(IbANS) of Yuzi263 and the sequences of full-length cDNA were submitted to GenBank and got the Accession Number: HQ441168 and GU598212, respectively. The physical full-length cDNA of IbF3H gene was 1280bp long, including 5'terminal UTR,3'terminal UTR, a polyA tail and a coding region which encoded 368 amino acid residues. Additionally, IbF3H protein had conserved amino acids H219,D22 and H277 which could combine ferrous ion(Fe2+), and a RXS sequence R287-S289 which could combine Oxoglutarates. What's more, the domain located in the centre of the protein, that was to say that Fe2+ was surrounded in the centre of the enzyme. The physical full-length cDNA of IbANS gene was 1375bp long, including 5'terminal UTR,3'terminal UTR, a polyA tail and a coding region which encoded 362 amino acid residues. Conservation search on NCBI showed that IbANS protein also had conserved amino acids H242,D244,H298 which could combine ferrous ion(Fe2+) and a RXS sequence R308-S310 which could combine Oxoglutarates. IbANS together with IbF3H are both 2-ODD belong to the enzyme family on the biosynthesis pathway of flavanoid. Compared IbF3H and IbANS with other species' IbF3H and IbANS in GenBank, it came out that IbF3H and IbANS both had high sequence similarity with these two genes in other species. Protein homology alignment and Phylogenetic analysis showed that they had high amino acid similarity with submarginal species and also can get together into a branch with F3H or ANS in the Convolvulaceae plants. Bioinformatical analysis results indicated that the IbF3H and IbANS cDNAs cloned fromⅠ. batatas (L) Lam.were the right genes in plants, with biologic activity.3. The biosynthesis of anthocyanin in purple-fleshed sweetpotato [Ⅰ. Batatas (L.) Lam] and tissue expression analysis of IbF3H, IbDFR and IbANS We analysed the characters of the biosynthesis of anthocyanin in purple-fleshed sweetpotato by grafting method and the expression character of purple-fleshed sweetpotato 263 tissues (fibrous root, 0.5 cm tuberous root,3.0 cm tuberous root, external periderm, stem joint, middle stem, petiole, leaf and tenderstem tip) were studied by Real-time quantitative Polymerase Chain Reaction(qPCR) based on the three enzyme gene (IbF3H, IbDFR and IbANS) which we cloned, and the results showed that: (1) The anthocyanin relative contents in the tissues including fibrous root(FR) and tuberous root(TR) of ps53 and ps53+263 are relatively low; while the anthocyanin relative contents in TR and FR of Yuzi263 and 263+ps53 are obviously higher than other tissue; the contents in TR and FR of 263+ps53 engrafting plants are higher than Yuzi 263's TR and FR, and the content accumulated after engrafted. It can be concluded that the biosynthesis position of anthocyanin in purple-fleshed sweetpotato is mainly in tuberous root and then transport to the fibrous root, stem, leaf and other organs. (2) The anthocyanin relative contents and the tissue expression character of Yuzi263 by qPCR analysis results showed:the IbF3H,IbDFR,IbANS genes expressed obviously higher in 0.5 cm tuberous root,3.0 tuberous root, external periderm than in stem joint, middle stem, petiole, leaf and tender stem tip; the IbF3H,IbDFR,IbANS gene' high expression resulted in the high contents of anthocyanin. The content of anthocyanin is closely related to the structure gene expression in the biosynthesis pathway of anthocyanin. The gene mainly expression in tuberous roots of sweetpotato is the main cause of the high content of sweetpotato tuberous root, which also explained that the formation of purple-fleshed sweetpotato is only for the underground parts but not for the upper-ground parts of the sweetpotato plant in our engrafting experiment.