Effects Of Transcription Factor FtMYB1and FtMYB2from Fagopyrum Tatacuricum On Key Genes’ Expression Of Flavonoid Biosynthesis And Flavonoid Accumulation In Tobacco

Fagopyrum tataricum (L.) Gaertn., also known as the Tatary buckwheat, is an annual herb belongs to Polygonaceae genus. Tartary buckwheat is one of the cultivars, which not only has some advantages for cultivation, such as cold resistance, radiation resistance, and barren resistance, but also rich in flavonoids. The biosynthesis of flavonoids in tartary buckwheat is regulated by the key enzymes and the transcription factors. The former directly interrelates with each enzymatic reaction, while the latter regulates the expression intensity of the single or multiple key enzyme genes at the transcriptional level.Through the phenylpropanoid pathway, flavonoids are synthesized from several individual branches and can be classified into different groups of derivatives. In this study, we focused on four important enzymes, which involved in the flavonol biosynthetic branch, including phenylalanine ammonia-lyase (PAL), chalcone isomerase (CHI), flavanone3-hydroxy enzyme (F3H), and flavonol synthase (FLS) and the transcription factor FtMYB1and FtMYB2. By analyzing the relation ship of the key enzyme genes and the transcription factor in transgenic tobacco, our study will provide a stably fundament for understanding the flavonoids biosynthesis and the knowledge for creating high flavonoid Tartary buckwheat by metabolic engineering technology. The main findings are as follows:1. Two plant expression vectors, named pCAMBIA1301-FtMYBl and pCAMBIA1301-FtMYB2were constructed. Then, these vectors mentioned above were successfully transformed into the tobacco leaves by mediation of Agrobacterium tumefacients strain LBA4404, respectively.2. Using housekeeping gene N-Actin as internal control, the expression levels of Pal, Chi, F3h and Fls, in transgenic tobacco leaves, were detected by semi-quantitative PCR. The results showed that disseminated48h by Agrobacterium tumefaciens, the expression levels of four key enzyme genes in FtMYB1transgenic tobacco leaves were51.68%(Pal),70.02%(Chi),62.87(F3h), and0%(Fls), respectively, and in FtMYB2transgenic tobacco leaves were32.01%(Pal),38.30%(Chi),57.65%(F3h), and0%(Fls), respectively. In FtMYB1transgenic tobacco leaves, the expression levels of Pal, Chi and F3h were1.54,3.18, and1.46folds as much as that in the control groups, and in FtMYB2transgenic tobacco leaves, they were0.57,1.28, and1.26folds, respectively. In contrary, the expression levels of Fls in transgenic tobacco FtMYB1and FtMYB2were sharply decreased from19.51%to0%. Thus, the results suggested that FtMYB1and FtMYB2could significantly enhance the expression levels of Pal, Chi, and F3h genes and inhibite the expression of Fls gene completely in transgenic tobacco.3. The total flavonoids were prepared by ethanol extraction method, and their contents were determined by UV-Vis Spectrophotometry. The results revealed that the total flavonoids content in the leaves of transgenic tobacco FtMYBl and FtMYB2were2.81and1.29folds as much as that in the control groups after their transgenic tobacco leaves were cultivated for48h. Thus, the results suggested that the transcription factors FtMYB1and FtMYB2might significantly boost the biosynthesis of anthocyanidins and inhibit the production of flavonols in transgenic tobacco leaves.