Cloning And Functional Analysis Of Transcription Factors Related To Biosynthesis Of Anthocyanins From Purple-fleshed Sweet Potato

Purple-fleshed sweet potato is a kind of potato with purplish black skin, purple red flesh and a creeping habit, which belongs to the Convolvulaceae Ipomoea. The skin and flesh of Purple-fleshed sweet potato can be edible with a sweet taste and less fiber. It has high starch content, contain a variety of vitamins, amino acids and other nutrients as common sweet potato. The extract of purple-fleshed sweet potato has many pharmacological effects including ascorbic acid, anticancer, anti high blood sugar, atherosclerosis, antimutagenic, antibacterial etc. Most of these pharmacological effects are related to anthocyanin of purple-fleshed sweet potato. The content of anthocyanin is20-180mg in100g fresh purple-fleshed sweet potato. The extracted anthocyanin from purple-fleshed sweet potato is high-quality, which could replace the artificial pigment in food, medicine, cosmetics application. Anthocyanins synthesized from the flower pigments and various monosaccharides are the most important water-soluble flavonoid pigments which belong to plant secondary metabolites. The biosynthetic pathway of anthocyanin, which consist of phenylalanine synthesis pathway and flavonoids biosynthetic pathway, require a series of enzyme catalysis and the regulation of transcription factors.The anthocyanin synthesis pathway is predominantly regulated by class R2R3-MYB, class bHLH and class WD40transcription factor. A protein complex conformed from MYB, bHLH and WD40protein, directly regulate structure gene transcription. Five genes belong to MYB, bHLH and WD40transcription factor family are firstly cloned from YuZi263with RACE technology, in order to illuminate the regulation mechanism of anthocyanin biosynthesis pathway and provide a new target for anthocyanin secondary metabolic engineering. In addition, the anthocyanin content in various tissue is analyzed with HPLC and colorimetry.The regulative activity of MYB transcription factor family is the main reason of coloration patterns of plant in nature. The R2R3MYB family and flavonoids are closely related to anthocyanin biosynthetic pathway regulation. A full-length cDNA of IbMYB1(GenBank accession number: JQ337861) is cloned via RACE technology from YuZi263. Bioinformatics analysis showed that full-length cDNA of IbMYBl was1198bp, containing a750bp CDS,184bp5’-UTR and164bp3’-UTR. Besides, the ORF encodes249amino acids with a calculated molecular mass of28.6kDa and an isoelectric point of8.57. The putative protein is an alkaline protein containing36.55%alpha helix,8.03%extended chain,4.42%turn and51%irregular coiled. Amino acid sequence multiple alignment shows that IbMYBl exists mainly the N end of the R2R3DNA binding domain compared with other species in R2R3MYB transcription factor sequence, but the similarity of C-terminal is very low in each species. Tertiary structure model is successfully established in the R2R3region, which is formed respectively by2and3alpha helix. The phylogenetic analysis indicated that IbMYB1belonged to plant MYB family relative to anthocyanins transcription factor. Quantitative RT-PCR showed that expression level of IbMYBl was highest in tuberous roots, followed by diameter of0.5cm tuberous roots, periderms, stems, knots of stems, petioles, fibrous roots, leaves and shootapexes.bHLH transcription factor can be identifid by E-BOX (CANNTG), which is a promoter region of key enzyme in anthocyanin synthesis pathway. When the transcription factor combined with DNA, it requires two different bHLH transcription factors to form dimmer. The full-length cDNA of bHLH was isolated from YuZi263using Rapid Amplification of cDNA Ends (RACE) technique and characterized. The new cDNAs were designated as IbbHLHl and IbbHLH2and submitted to GenBank(?) to be assigned with an accession number:JQ337862and JQ337863. The full-length IbbHLH1was2467bp containing a427bp5’-UTR and150bp3’-UTR and encoding a629amino acids peptide with a calculated molecular mass of69.5kDa and an isoelectric point of5.10. Secondary structure prediction revealed that IbbHLH1consists of52.94%random coil,9.38%extended strand,2.86%p-turns and34.82%alpha helix. The full-length IbbHLH2was2467bp containing a2025bp CDS,427bp5’-UTR and150bp3’-UTR. The gene of IbbHLH2encodes a674amino acids peptide with a calculated molecular mass of75.1kDa and an isoelectric point of5.07. Secondary structure prediction revealed that IbbHLH2consists of39.17%random coil,10.83%extended strand,4.60%β-turns and45.4%alpha helix. Bioinformatic analysis revealed that IbbHLH land IbbHLHl grouped into different branch, but they are in greatly similar in N-terminal part of HLH domain. Tertiary structure model analysis indicated that both/IbbHLH1and IbbHLH2 combined with N-terminus of protein were made up of two alpha helix and random coil. Quantitative RT-PCR showed that expression level of IbbHLH1was highest in fibrous roots, followed by petioles, shootapexes, knots of stems, tuberous roots, diameter of0.5cm tuberous roots, stems, leaves and periderms. However, expression level of IbbHLH2was highest in diameter of0.5cm tuberous roots。WD40transcription factor is necessary for activating anthocyanin transcriptional complex in many plants. The new cDNAs were designated as IbWDR1and IbWDR2and submitted to GenBank(?) to be assigned with an accession number:JQ340206and JQ337864. The full-length IbWDRl was1239bp containing a64bp5’-UTR and143bp3’-UTR and encoding a343amino acids peptide with a calculated molecular mass of38.09kDa and an isoelectric point of4.97. Secondary structure prediction revealed that IbWDR1consists of51.31%random coil,34.99%extended strand,3.50%β-turns and10.20%alpha helix. The full-length IbWDR2was1299bp containing a136bp5’-UTR and121bp3’-UTR and encoding a346amino acids peptide with a calculated molecular mass of39.09kDa and an isoelectric point of4.71. Secondary structure prediction revealed that Ib6WDR2consists of52.31%random coil,32.37%extended strand,4.91%(3-turns and10.40%alpha helix. Tertiary structure model analysis indicated that both IbWDR1and Ib&WDR2were combined with seven regions surrounded by a ring-shaped structure and each region formed with β-extended strand. Quantitative RT-PCR showed that expression level of IbWDR1in petioles, shootapexes, diameter of0.5cm tuberous roots and periderms is a few higher than in stems, fibrous roots, tuberous roots and leaves. Expression level of IbWDR2in diameter of0.5cm tuberous roots, tuberous roots and shootapexes is much higher than in knots of stems, fibrous roots, petioles, periderms, leaves and knots of stems.In the research, anthocyanin was extracted with10mL1%formic acid methanol as extracting solution. In order to establish YuZi263tissue content spectrum, HPLC and UV spectrophotometer methods were adopted. The result shows that the content of anthocyanin was highest in periderms, followed by tuberous roots, basic stems, and lowest in leaves and petioles.The gene cloning and functional analysis is helpful to illuminate the biosynthesis pathway of three types of transcription factors in anthocyanin, and provides a new target for anthocyanin biosynthesis. Analysis compared with content of spectrum shows that the gene expression level of IbMYB1and IbbHLH2is related to anthocyanin biosynthesis, which provides an introduction for metabolic engineering.