Molecular Cloning Flavoniod-related Genes From Tartary Buckwheat And Their Responses Under UV-B, Cold, And Drought Stresses In Sprouts, And Enzymatic Activity Analysis Of The Recombinant FtPAL And FtFLS Proteins

Tartary buckwheat (Fagopyrum tataricum Garetn) is an excellent medicinal and nutrient-rich crop containing abundance of rutin, which is a kind of flavonol glycoside, and it has become one of the hottest research points for its health benefits of lipid-lowering, hypoglycemic and antioxidant activity. Nowadays, researchers have concentrated on three aspects about Tartary buckwheat:the development of flavonoids-rich products, the identification of flavonoids types, and the enzymes of flavonoids biosynthesis. However, the flavonoids biosynthetic pathway in Tartary buckwheat is more complex, and most of the flavonoids-related genes have not been cloned, biological activity has not been verified, and the stress research is relatively weak.Due to the pivotal role of enzyme genes in flavonoids biosynthesis, our study focused on the cloning and molecular characterization of functional flavonoids-related genes from Tartary buckwheat. Thus,11ORF sequences of flavonoids-related genes were cloned from Tartary buckwheat. By analyzing the relationships between total flavonoids content and expression level of genes under different stress treatments, the key enzyme genes were found. Two key enzyme genes were expressed by prokaryotic expression, and their enzymatic characteristics were identified.1. Using the cDNA from flowers as template,11ORF sequences and a conserved fragment of flavonoid-related genes were isolated from Tartary buckwheat, including phenylalanine ammonia-lyase (FtPAL), cinnamate-4-hydroxylase (FtC4H),4-coumarate coenzyme A ligase (Ft4CL), chalcone synthase (FtCHS), chalcone isomerase (FtCHI), flavanone3-hydroxylase (FtF3H), flavonoid3’-hydroxylase (FtF3’H), flavonol synthase (FtFLS), dihydroflavonol-4-reductase (FtDFR), anthocyanidin synthase (FtANS), and glutathione S-transferases (FtGST), and a fragment sequence was flavonoid3-glucosyl transferase(3GT).2. Sequence analysis showed that the ORF sequences of11flavonoids-related genes encoded complete enzyme proteins, and the fragment of3GT might be a pseudogene, which appeared early termination in translation. The deduced amino acid sequence of11flavonoids-related genes showed a higher level of homology (more than90%) with the corresponded genes in Fagopyrum dibotrys and Fagopyrum esculentum, and a moderate level with other plants’genes, ranging from38-95%. Signal peptides and subcellular localization prediction showed that all the11sequences had no signal peptide existed; FtCHI located in the mitochondria, FtC4H located in the endoplasmic reticulum, and the other9genes located in the cytoplasm. Senior Structural analysis showed that the each flavonoids-related gene had similar secondary and tertiary structure with its own protein family.3. Three different stress treatments, including UV-B, cold and drought stress, were treated with the12days old sprouts for8hours. The expression levels of FtPAL, FtCHS, FtCHI, FtF3H, FtFLS, FtDFR, and FtANS were tested by semi-quantitative RT-PCR. Results showed that FtPAL, FtCHS, FtCHI, FtF3H, FtFLS, FtDFR and FtANS could be examined in the cotyledons before and after different stress treatments. FtPAL, FtCHS, FtCHI, FtF3H, FtDFR and FtANS could be examined in the hypocotyls before and after different stress treatments. However, the expression level of FtFLS could not be detected in the hypocotyls before and after stresses. The statistical analysis results showed that, in UV-B stress condition, the expression levels of FtF3H in cotyledons and FtCHI in hypocotyls showed no significant difference with control (P>0.05). In cold stress condition, the expression levels of FtCHS in cotyledons and FtF3H in hypocotyls showed no significant difference with control (P>0.05). The expression levels of other genes in3different stress conditions showed significant differences with control (P<0.05). Thus, our results suggested that each gene expression patterns vary in different stress conditions, with a certain degree of tissue specificity, and the cold stress could up-regulate most of the genes both in cotyledons and hypocotyls.4. Total flavonoids in Tartary buckwheat were mainly distributed in the cotyledons. The contents and change trends of total flavonoids in Tartary buckwheat showed different degrees, which were accorded to different stress treatments. Statistical analysis results show that, with in a short period of treatment’s time (2h), the total flavonoids in cotyledons and hypocotyls could be induced and significantly increased by UV-B stress (P <0.05), and the significance were gradually increased along with the induction time. In cold stress, the total flavonoids in cotyledons were significantly increased after8hours’ induction (P<0.05), and it was significantly increased in hypocotyls after2hours’ induction (P<0.05). In drought stress condition, the total flavonoids in cotyledons could not show a certain significance after8hours’ treatment (P>0.05), and it had a short response time (2h) in hypocotyls and showed a maximum after4hours’induction (P<0.05). In addition, the maximum of total flavonoids were detected in cotyledons by8hours’cold treatment and in hypocotyls by4hours’drought treatment.5. By analyzing the relationships among total flavonoids content and expression level of genes under different stress treatments, we speculated that:1) In UV-B and cold stresses, the increase of total flavonoids might due to the rutin biosynthesis in cotyledons, and in3different stresses, the increase of total flavonoids might contribute to the accumulation of anthocyanins in hypocotyls.2) FtPAL might be controlled by some separate adjustment factors in Tartary Buckwheat and be a primary switch in flavonoids biosynthetic pathway. Its expression level of response to different stress condition might control the starting of flavonoids biosynthesis and affect the accumulation patterns of total flavonoids both in cotyledons and hypocotyls. The expression level of FtFLS might correspond with the accumulation and increase of total flavonoids in cotyledons, and the potential co-expression of FtDFR and FtANS might control the biosynthesis of anthocyanins in hypocotyls and cause the rising trend of total flavonoids in hypocotyls.6. Southern blot analysis indicated that FtPAL was a multi-copy gene in Tartary buckwheat. A non-fusion expression vector (named pET-30b(+)-FtPAL) was successfully constructed to prokaryotic express the recombinant FtPAL protein in Escherichia coli BL21(DE3). Induced by IPTG for7hours, the SDS-PAGE result showed that the recombinant FtPAL was about77kDa. The enzymatic activity of recombinant FtPAL was tested and results showed that FtPAL reached maximum yield at5hours’cultivation and its specific activity up to35.7IU/g by using L-Phe as substrate. TLC results suggested that the recombinant FtPAL could conversion L-Phe to trans-cinnamate in vitro.7. Southern blot analysis indicated that FtFLS was a multi-copy gene in Tartary buckwheat. A fusion expression vector (named pET-30b(+)-FtFLS) was successfully constructed to prokaryotic express the recombinant FtFLS protein in Escherichia coli BL21(DE3). After10hours of induction, the SDS-PAGE result showed that the recombinant FtFLS was about40kDa. His affinity chromatography results showed that the concentration of purified FtFLS was1.93g/L. Enzymatic activity of FtFLS showed that the total and specific activities of dihydroquercetin were36.55×10-3IU and18.94×10-3IU/mg, respectively, and the total and specific activities of dihydrokaempferol were10.19×10-3IU and5.28×10-3IU/mg. Thus, our study suggested that the recombinant FtFLS had a higher catalytic efficiency of dihydroquercetin than dihydrokaempferol in vitro.