Characterization Of Tartary Buckwheat WD40 Transcription Factor And Its Regulation Of Anthocyanin Biosynthesis

Tartary buckwheat (Fagopyrum tataricum) is a dicotyledonous cereal crop of the Polygonaceae family. As an edible and medicinal plant, flavonoids are the most important bioactive components in this species, in which flavonols are the main storage form. However, anthocyanin, another flavonoid form, plays an important role to maintain the physiological stability when tartary buckwheat suffers from cold or ultraviolet. At the transcriptional level, the anthocyanin biosynthesis is regulated by one protein of transcription factors R2R3-MYB, bHLH and WD40, or their ternary complex (MBW). Furthermore, WD40 protein provides a scaffold to facilitate the interactions between MYB and bHLH. In this study, cDNA sequence of FtWD40 related with anthocyanin biosynthesis was cloned from tartary buckwheat "Xiqiao No.2" firstly. Then, its transcriptional activation activity was identificated further. After FtWD40 expression and anthocyanin content was determinated, both of their tissues specifity and correlation were analyzed in tartary buckwheat under cold and UV-B stresses. Finally, the effect of FtWD40 on anthocyanin biosynthesis was explained through its over expression in transgenic tobacco. The main idea of this research is to reveal FtWD40's response genes on the anthocyanin biosynthesis patheway. In addition, it is helpful to understand more about the anthocyanin biosynthesis regulation in tartary buckwheat under stresses. The main results are as follows:1. Based on homology cloning and RACE technology, a WD40 gene cDNA named as FtWD40 was cloned from tartary buckwheat. FtWD40 ORF was 1035 bp in length, and encoded 344 amino acid residues. Phylogenetic tree analysis showed that the FtWD40 was highly homologous with AtTTG1 and VvWD involved in regulating anthocyanins biosynthesis. After FtWD40 gene expression and anthocyanin content were determined in roots, stems, leaves and flowers at the flowering phase of buckwheat, the results showed they presented the same tissues specifity as flowers> roots> leaves> stems, and their correlation coefficient was r=0.875 (P<0.05). Furthermore, their correlation coefficients under cold and UV-B stresses were r=0.785 (P<0.05) and r=0.891 (P<0.05), respectively. Seen from the above results, FtWD40 may be involved in tartary buckwheat's anthocyanins biosynthesis regulation.2. The recombinant plasmid pBridge-FtWD40 was constructed and transformed into yeast AH 109. Based on the selection on SD/-Trp/-His nutrient deficiencies medium, positive transformants were screened. The ?-galactosidase specific activity analysis in yeast one-hybrid system showed FtWD40 displayed the transcriptional activation activity.3. The plant expression vector pECMBIA1301-FtWD40 was constructed and introduced into leaf disks derived from tobacco using Agrobacterium-mediated transformation. The relative expression levels of six genes on anthocyanin biosynthesis pathway as well as the anthocyanin contents were determined in positive transgenic tobacco plants. The anthocyanin contents in transgenic tobacco plants T-1, T-2 and T-3 were severely improved to 3.45-,2.81-,3.24 times of the control at average, respectively (P<0.01). Moreover, qRT-PCR results indicated that NtPAL, NtCHS and NtF3'H had no significant changes (P>0.05), NtDFR and NtANS were increased by 1.95-,1.56 times of the control group respectively (P<0.01), and NtANS are reduced to 0.79 times of the control (P<0.05). Therefore, the results implied FtWD40 would play an importance role in enhance the anthocyanin biosynthesis in transgenic tobacco.