Inhibition Of Anthocyanin Synthesis By Transcription Factor FtMYB3 From F. Tataricum

Flavonoids are a large class of natural secondary metabolites which exist widely in terrestrial plants. Flavonoids not only play an important role not in the basal metabolism of plants but also show a positive impact on human health as the ingredients of food and medicine. Tartary buckwheat (Fagopyrum tataricum) is an excellent medicinal and health-care coarse cereal due to its high rutin content. For rutin is the main quality character and economic indicator for tartary buckwheat, to understand the regulatory mechanism of rutin biosysthesis becomes the theoretical fundament of molecular breeding. Nowadays, researchers concentrate on the flavoniods-related stress, key enzymes involved in flavoniods accumulation and transcriptome in tartary buckwheat. However, the knowledge about its MYBs transcription factors is not systematically reported yet. In this study, FtMYB3, a transcription factor isolated from tartary buckwheat cultivar’Xiqiao No.2’,was focused on its function identification. Firstly, FtMYB3’s important molecular characteristics, as well as its temporal and spatial expression profile, were analyzed. Then, the effects of FtMYB3 on the key enzymes genes and the accumulation of metabolites were evaluated in transgenic tobacco. Finally, the cis-acting elements of FtMYB3 were screened and identified. In brief, this study reveals the regulatory mechanism of FtMYB3 in flavoniods mechanism, and builds a good foundation for improving the flavonoids content in buckwheat. The main results are as follows:1. The yellow fluorescent protein expression vector FtMYB3-PA7-YFP was constructed and transformed into the epidermal cells of onion via particle bombardmental method. Seen from its instantaneous expression image, FtMYB3 is localized in cell nucleus. Meanwhile, southern blot analysis showed that FtM YB3 is a multi-copies gene in tartary buckwheat.2. The relative expression levels of seven structure genes on flavonoids biosynthesis pathway and FtMYB3 were analyzed from flower stages to seed maturing stages in tartary buckwheat. The data reveal that FtPAL, FtANS and FtF3’H display the simlar expression patterns during these stages, which hints the three genes are coordinately regulated by transcription level. However, FtDFR and FtFLS are likely to be regulated by mutl-factors at transcription level.3. FtMYB3 gene was transformed into tobacoo for over-expression. In positive transgentic tobacoo plants, the relative expression levels of six genes on flavonoids biosynthesis pathway indicate that NtCHI, NtDFR and NtANS are decreased distinctly (P< 0.01), and NtPAL and NtCHS have no significant changes (P> 0.05). Moreover, the anthocyanin contents in transgentic tobacoo plants T-1 and T-2 are severely lessened (P< 0.01) to 38.75% and 21.25% of the control at average, respectively. Therefore, the results imply FtMYB3 plays an importance role in the inhibition of anthocyanin biosynthesis.4. Bacterial one hybrid system and electrophoretic mobility shift assay (EMSA) was used to screen and identify FtMYB3’s DNA binding sequence. The results reveal that FtMYB3 has the ability to read and bond B4 (CCAAAAAAAGAAACAAGA) and A7 (AAACACCAACACAGAAAA) motifs strongly, and their interactions lead to an obvious electrophoretic mobility effect. Furthermore, FtMYB3’s brachytmema protein without activation domain shows the same results as the protein in full length, which proves the activate domain is not necessary for FtMYB3 combination with cis-acting element.