Gene Cloning And Biological Function Identification On Dihydroflavonol 4-reductase Of Fagopyrum Tataricum

Tartary buckwheat(Fagopyrum tataricum)is a kind of coarse grains with high nutritional value in Polygonaceae.Because it is rich in bioactive substances,which are rich in flavonols and anthocyanins,it has become an important functional food raw material.Anthocyanin play animportant role in the anti-reverse growth of buckwheat,and it widely involved in the physiological process of resisting cold,ultraviolet and drought stress.Furthermore,it is an important evaluation index for high quality tartary buckwheat with the bright red endowed with higher economic value added to buckwheat,and has many physiological functions,such as antioxidation,anticancer,prevention of cardiovascular disease and so on.Dihydroflavonol 4-reductase(DFR)is the first key enzyme of the anthocyanin synthesis and metabolic pathway.Its expression pattern and enzymology properties play a decisive role in the flow and flow of the branch.It is the bottleneck for the synthesis and accumulation of anthocyanins.In this study,a high flavonoid buckwheat cultivar(“Xi Qiao No.2”)was used as materials,the FtDFR1、2gene of tartary buckwheat was cloned successfully;The analysis of correlation was made between anthocyanin content and the expression pattern of the obtained genes during the growth and development of buckwheat germination from 4 d to 35 d;The expression model of FtDFR1、2 gene and tartary buckwheat bud anthocyanin synthesis was investigated under cold stress;Further,transgenic technology was used to identify its biological activity and its effect on anthocyanin biosynthesis and metabolism.This study can clarify the regulatory factors of the biosynthesis pathway of tartary buckwheat anthocyanins,and provide important material for understanding the metabolic network of flavonoids in tartary buckwheat.It lays the foundation for the further use of gene tic engineering to create high anthocyanin tartary buckwheat,improve the nutrition and economic value of tartary buckwheat.The main results are as follows:1.According to the transcriptome data of tartary buckwheat,FtDFR1、2 gene cDNA and DNA sequences were obtained by PCR technology,which were FtDFR1 and FtDFR2,respectively.Sequence analysis showed that the DN A sequence of FtDFR1 was2187 bp,it contains five introns and six exons;The ORF sequence was 1026 bp,encoding 341 amino acid residues.The DN A sequence of FtDFR2 was 1684 bp,it contains four introns and five exons;The ORF sequence was 1002 bp,encoding 333 amino acid residues.The sequence alignment results showed that the amino acid sequences encoded by FtDFR1 and FtDFR2 were 60.84% to 99.12% of the DFRhomology in Gen Bank,and FtDFR1、2 homology was 76.02%.The obtained sequence has the characteristic sequence “VTGASGYVGSWLVMKLLQYGY”(NADPH binding site)and “TVNVQEHQMSEYDESSWSDDDFCRRV”(substrate binding site)of the NADPH dependent reductase family,which is the dihydroflavonol4-reductase homologous gene of tartary buckwheat.Phylogenetic analysis showed that FtDFR1 and FtDFR2 had a close evolutionary relationship with the DFR protein of Fagopyrum esculentum,Fagopyrum cymosum and Dianthus caryophyllus.2.The determination of anthoc yanin content in 4 growth periods(4 d～35 d)of tartary buckwheat showed that anthocyanin were accumulated mainly in stems(2.329mg/g,1.306 mg/g,1.518 mg/g,FW)and the tatal content of stage2 reached the highest(3.579 mg/g,FW);The content of roots and leaves was relatively stable(0.534～0.68mg/g,FW).q RT-PCR analysis showed that the expression patterns of FtDFR1 and FtDFR2 were spatio-temporal and tissue-specific,and it were expressed in all tissues at different growth stage.In addition,the expression level of FtDFR1 gene in different tissues at different growth stages was much higher than that of FtDFR2 gene.It may be a major gene of transforming flavanonol to leucocyanidin.3.The buckwheat sprouting at under cold stress 8 d was used to analyze the correlation between the anthocyanin content and the expression model of FtDFR1、2gene.The results showed that after 0～12 h cold stress,the anthocyanin content of tartary buckwheat bud increased significantly(p< 0.05)and reached the peak after 12 h(1.46 times more than before treatment).After 24 h,the content of anthocyanin wa s reduced to89% before treatment.Although the expression of FtDFR1 was much greater than that of FtDFR2 under cold stress,the change trend is the same,and is similar to anthocyanin content;It reached the highest(p< 0.05)at 12 h and decreased after 24 h.The correlation analysis showed that FtDFR1 and FtDFR2 were positively correlated with anthocyanin content,which further indicated that FtDFR1 played a leading role in the synthesis of anthocyanin in Tartary buckwheat bud.4.Plant expression vectors pCHF3-Flag-FtDFR1 and pCHF3-Flag-FtDFR2 were constructed respectively,The Arabidopsis thaliana was transformed by flowering method by Agrobacterium mediated GV3101.The T1 generation of transgenic Arabidopsis thaliana was obtained by screening and identification of Kan resistant plates and PCR.The content of anthocyanin in T1 generation transgenic Arabidopsis thaliana and the expression of genes related to the flavonoid synthesis pathway were analyzed.The results showed that: The anthocyanin content of FtDFR1 transgenic Arabidopsis thaliana was slightly higher than that of FtDFR2 transgenic Arabidopsis,and both were significantly higher than that of wild Arabidopsis(p< 0.05);The flavonoids synthesis pathway related enzyme gene AtCHS,AtCHI,AtF3 H,AtFLS and AtANS genes expression all increased significantly(p< 0.05),and the expression of AtF3’H gene was not significantly changed,but the expression of endogenous AtDFR gene was significantly inhibited(p< 0.05);The expression of gene AtCHS,AtCHI,AtF3 H,AtF3’H,AtFLS and AtANS gene in FtDFR2 transgenic Arabidopsis thaliana was up-regulated,and the expression of AtDFR gene was also inhibited(p< 0.05).The results showed that the functional products encoded by FtDFR1 and FtDFR2 were involved in the biosynthesis of anthocyanins in Arabidopsis thaliana.