Cloning And Expression Analysis Of Anthocyanin Biosynthesis Genes In Potatoes

Potato (Solanum tuberosum) is the forth food crop following wheat, maize and rice inthe world. Potato tubers are multiplicative organs as well as product organs, which there aredifferent colors, e.g. white, red and purple. Anthocyanins are key factors that determinetuber color. As natural pigments, they have been used in foods, pharmaceuticals andcosmetics due to their function of anti-cancer, cardioprotection and antioxidization. So inrecent years, scientists have showed a great interest in biochemical and molecularbiological investigation of anthocyanin biosynthesis all over the world.The biosynthetic pathway of anthocyanins is one of the most extensively studiedpathways of plant secondary products, and it is clearly elucidated in some model organism.A few genes of anthocyanin synthesis have been cloned from potato cultivars (S. tuberosumL.), e.g., chalcone synthase (CHS), dihydroflavonol 4-reductase (DFR), flavanone3-hydroxylase (F3H), and flavonoid 3', 5'-hydroxylase (F3'5'H) genes. However, noreports in this aspect have been seen until now in wild potato species. In this research,cDNAs encoding CHS, F3H, DFR and 3GT were isolated from S. pinnatisectum, and theirexpression was analyzed. The function of 3GT was basely confirmed by transgenic plant.The expression of CHS, F3H, F3'5'H, DFR and 3GT genes from S. tuberosum cv. Chieftainwas investigated. The main results were showed as following:1. Preliminary studies on the categories, contents of skin color pigments and effectsof growth regulators, antibiotic and sucrose on anthocyanin synthesis in potatoes.The skin color pigments of potatoes were extracted in 1ï¼…HC1 (v/v) in methanol fromred and purple tubers, respectively. The extract was separated with hexane, purified by ThinLayer Chromatography (TLC). The Rf and UV-Vis spectra analysis indicated that the redpigments were primarily anthocyanin pelargonins and the purple pigments were primarilyanthocyanin petunins. The content of anthocyanins in purple tubers were 2.9 times of thatin red tubers. The stability of anthocyanins was significantly influenced by light, heat andpH value, but the anthocyanins from purple tubers were more stable than that from red tubers.The callus originated from the explants of S. tuberosum cv. Chieftain. The lowercontents of 2,4-D promote accumulation of anthocyanins in red callus, the higher contentsof 2,4-D stimulated callus growth and inhibited anthocyanin production in red callus. Thehigher contents of 6-BA promoted accumulation of anthocyanins in red callus, inducedanthocyanin production in white callus and inhibited callus growth. The white callus couldbe induced to turn red and accumulated anthocyanins by different concentrations ofkanamycin, while the higher contents of kanamycin inhibited callus growth and turnedbrown to lead callus dead eventually. The elevated contents of sucrose could stimulateanthocyanin production and inhibite callus growth.2. Cloning and sequence analysis of anthocyanin biosynthetic genes in wild potatospecies (S. pinnatisectum).Four complete-length cDNAs encoding CHS, F3H, DFR and 3GT were isolated fromthe sprouts by RT-PCR with the degenerated primers. The sequence analysis indicated thatCHS, F3H, DFR and 3GT cDNAs encoded the polypeptide of 389, 358, 382 and 448 aminoacid residues, respectively. Sequences of the encoded polypeptide comparison showed thatthey shared 76-96ï¼…identities with each corresponding solanaceous proteins reportedpreviously. The multiple alignment and phylogenetic analysis demonstrated that each genewas a member of a multigene family.3. The function of 3GT gene was confirmed by Agrobacterium-mediatedtransformation of arabidopsis thaliana.Arabidopsis thaliana was transformed by floral dip method with AgrobacteriumGV3101 carrying expression vector pG3GT. Four transformants were selected for theirgrowth ability on 1/2MS medium containing 50mg/L kanamycin. The frequency oftransformation was 0.13ï¼…. PCR analysis confirmed that three survival transformants werepositive. Southern blot confirmed that two transformants were positive, in which only onecopy of 3GT gene was detected. Among the transformants, there was one whose stem andleaves turn purple. The analysis showed that the content of anthocyanin was 7.01 times ofwild type. These results indicated that 3GT gene normally expressed in the transformant.4. Expression analysis of anthocyanin biosynthetic genes in potatoes.The spatial expression analysis of CHS, F3H, DFR and 3GT genes in wild potatospecies (S. pinnatisectum) indicated that these genes were preferentially expressed inflowers, stolons and terminal buds. In roots, and their transcripts could not detected except 3GT. The expression of CHS was not detected in tubers. In white tubers the genes wereexpressed a little or not, but after receiving light all the genes were induced to express intuber skins and the expression level increased greatly. The color of tubers changed fromwhite into purple with the prolongation of lighting.The spatial expression patterns of CHS, F3H, DFR, F3'5'H and 3GT genes in potatocultivar (S. tuberosum cv. Chieftain) were examined by RT-PCR. The expression analysisindicated that the expression of the genes was higher in stolons, and lower in tubers androots.The green, white and red callus were isolated from the explants of S. tuberosum cv.Chieftain on the MS medium containing 6-BA 2mg/L and 2,4-D 0.5 mg/L. The contents ofchlorophyll and anthocyanins were significantly different in the three different callus. Therewere few anthocyanins in green and white callus, but a great deal of anthocyanins in redcallus. The expression analysis of CHS, F3H, DFR, F3'5'H and 3GT genes in threedifferent callus were performed by RT-PCR. The results showed that no DFR transcriptswere detected in green and white callus to lead no anthocyanin accumulated in them.The effects of cerium on callus growth, anthocyanin content and expression ofanthocyanin biosynthetic genes in callus suspension cultures of S. tuberosum cv. Chieftainwere studied. The results indicated that 0.1 mmol L^-1 Ce⁴⁺ could promote callus growth,increase accumulation of anthocyanins, and enhance expression of five anthocyaninbiosynthetic genes (CHS, F3H, F3'5'H, DFR and 3GT) most efficiently. But higherconcentration (1mmol·L^-1) of Ce⁴⁺ inhibited partially callus growth and 2 mmol·L^-1 of Ce⁴⁺caused cell death eventually. The results revealed that Ce4^could induce the expression ofanthocyanin biosynthetic genes and accumulation of anthocyanins.