Tartary Buckwheat Light-induced Transcription Factor FtMYB5 Involved In Flavonoid Biosynthesis Regualtion

Tartary buckwheat(Fagopyrum tataricum Gaertn.),a member of the Polygonaceae family,is a traditional medicinal and edible cereal crop.Tartary buckwheat contains high flavonoid contents,which exhibit antioxidant activity,antiproliferative capacities,anti-inflammatory and antitumor activity in pharmacological investigations.Tartary buckwheat is recognized as a nutraceutical and functional food in Asian countries.Because rutin is the main quality character and economic indicator of tartary buckwheat,to understand the regulatory mechanism of rutin biosysthesis becomes the hot research points in tartary buckwheat.Flavonoids are synthesized from the phenylalanine by the phenylpropanoid metabolic pathway.The whole system is under the tight control of several transcription factors,especially the R2R3-MYB proteins.In this study,FtMYB5,an R2R3-MYB transcription factor was isolated from tartary buckwheat cultivar 'Xiqiao No.2'.Firstly,its molecular characterization was identified.Then the tissue-specific expression patterns and photoresponse characteristics were analyzed.Finally,the effect of FtMYB5 on flavonoids biosynthesis was explained through its overexpression in transgenic tobacco.This study initially revealed the regulatory mechanism of FtMYB5 in flavoniods biosynthesis patheway,and builds a good theoretical foundation for improving the flavonoids contents in buckwheat.The main results are as follows:1.According to the tartary buckwheat flower transcriptome database,I identified one gene,named FtMYB5,encoding a putative R2R3 MYB protein that exhibited high similarities with Arabidopsis AtMYB111.The recombinant plasmid pBridge-FtMYB5 was constructed and transformed into yeast AH 109.Based on the ?-galactosidase specific activity analysis,the FtMYB5 showed transcriptional activation activity in yeast cell.The yellow fluorescent protein expression vector PA7-FtMYB5-YFP was constructed and transformed into Arabidopsis protoplast by PEG-Ca mediated method.Results showed that the FtMYB5 is localized in cell nucleus.2.The expression levels of FtMYB5 in tartary buckwheat different tissues at flowering stage were determined by qPCR.FtMYB5 expressions were detected in all tissues with the highest levels in flower and stem which coincides with expression of flavonoid pathway genes in different tissues.Tartary buckwheat seedlings were treated by dark and light treatments,and then the expression levels of FtMYB5 and flavonoid contents were analysed.The results showed that the early induction of FtMYB5 gene expression upon light exposure in tartary buckwheat seedlings correlates well with the expression pattern of flavonoid pathway genes including FtC4H,FtCHS1,FtF3H,FtF3'H and FtFLS1.The subsequent increase in flavonoid levels in tartary buckwheat seedlings confirms the inducibility of flavonoid biosynthesis,providing evidence that this response is likely to be mediated by FtMYB5 in response to light.3.The promoter sequences of FtMYB5 contain several putative light regulatory elements,such as Box 4(ATTAAT),GATA box(GATA),GT1CONSENSUS(GRWAAW),GT1CORE(GATAA),IBOXCORE(GATAA),INRNTPSADB(YTCANTYY),SORLREP3AT(TGTATATAT).By GUS dyeing analysis of transgenic plants,the results suggested the 5' terminal deletion segments were able to drive the expression of GUS gene.Under the light treatment,the plants showed a deeper blue compare to those under dark.These results showed that the FtMYB5 promoter can be induced by light.4.In transgenic plants,the amounts of total flavonoid were increased by 4.6 to 5.9-fold in the leaves of FtMYB5 overexpressing tobacco plants,and were inversely correlated with the accumulation levels of anthocyanins.Expression of NtPAL,NtCHS,NtCHI,NtF3'H and NtFLS involved in the flavonol biosynthesis was significantly up-regulated in leaf tissues of FtMYB5 overepressing lines in comparison to the wild type plants.However,no change was detected in the NtDFR gene,which is involved in late flvonoid(anthocyanins)biosynthesis.These results suggest that FtMYB5 activates the expression of genes involved specifically in the flavonol branch of phenylpropanoid pathway and thereby acts as flavonol specific regulator in plant.