Cloning And Transcriptional Activation Characterization Of MYB Transcription Factor Genes From Fagopyrum Tataricum

Fagopyrum tataricum, also called tartary buckwheat, is an annual herbaceous plant subordinated to family Polygonaceae, genus Fagopyrum, dicotyledon. As one of the main buckwheat cultivar widely distributing in southwest China's remote areas, such as cold mountainous regions where precipitation is also scarce. Tartary buckwheat is not only cultivated as one kind of food resources, but also as a sort of officinal plants resulting from its remarkable content of flavonoids (rutin in specific). Both key enzymes and transcription factors participate the regulation of flavonoids'biosynthesis in F. tataricum. MYB, a member of the biggest transcription factor family in plants, possesses various functions, such as fundamental units involving in response mechanisms regarding to environmental stress and hormones, cell cycle, cell differentiation, morphogenesis, etc. As part of stress-resistance mechanisms, biosynthesis of flavonoids in F. tataricum is also believed to be regulated by MYB.In this paper, novel genes in F. tataricum encoding MYB are cloned and characterized. This would be helpful for further understanding of networks involving in F. tataricum's flavonoids biosynthesis and quotable for creating high flavonoid-content tartary buckwheat by molecular breeding technology. Main results of this research are as follows.1. A pair of degenerated primers was designed based on plant MYB's conserved domains and applied to amplificate three independent conserved fragments from cDNA of F. tataricum's flower bud using PCR. Then RACE (Rapid Amplification of cDNA Ends) was performed to obtain three MYB cDNA sequences in full-length, respectively designated as FtMYBl (JF313345), FtMYB2 (JF313347), and FtMYB3 (JF313349). A forth MYB cDNA sequence FtMYB-P(HQ214132) was obtained by primers designed based on Fagopyrum dibotrys'FdMYBP1(EF522147). In all four cloned MYB cDNA sequences, two MYB domains were detected in N-terminal by sequence analysis, demonstrating their categories as type R2R3 MYBs; a putative acidic activation domain was also found in C-terminal of every sequence. Phylogenetic analysis showed that FtMYB1, FtMYB2, and FtMYB-P were related to regulation of flavonoids metabolism.2. DNA sequences of FtMYB1, FtMYB2, Ft MYB3, and FdMYBP1 were obtained by PCR. Results of sequence analysis showed that FtMYBl, FtMYB2, and FtMYB3 all contained 2 introns, whereas FtMYB-P possessed only one. Splittings of all introns abide to the "GU-AG law".3. Using Yeast one-hybrid technique, all four MYB transcriptional factors were tested for their transcriptional activation activities. Recombinant plasmids, pBridge-FtMYB1, pBridge-FtMYB2,pBridge-FtMYB3, and pBridge-FtMYB-P, were constructed and transformed into AH 109 yeast strain. After screening by SD (Synthetic Dropout) auxotrophic media, Whatman filter paper color reaction andβ-galactosidase activity test were performed. Results of color reaction presented to be positive for FtMYB1, FtMYB2, and FtMYB-P, while negative for FtMYB3. Results ofβ-galactosidase activity assessment for FtMYB1, FtMYB2, FtMYB-P, and FtMYB3 were 29.37U,11.80U,28.10U, and 2.24U, respectively, indicating their differences in transcriptional activation activities.