Chenglei Li

Cloning, characterization, and activity analysis of a flavonol synthase gene FtFLS1 and its association with flavonoid content in tartary buckwheat.

Evidence from in vitro and in vivo studies indicates that rutin, the main flavonoid in tartary buckwheat ( Fagopyrum tataricum ), may have high value for medicine and health. This paper reports the finding of a flavonol synthase (FLS) gene, cloned and characterized from F. tataricum and designated FtFLS1, that is involved in rutin biosynthesis. The FtFLS1 gene was expressed in Escherichia coli BL21(DE3), and the recombinant soluble FtFLS1 protein had a relative molecular mass of 40 kDa. The purified recombinant protein showed, with dihydroquercetin as substrate, total and specific activities of 36.55 × 10(-3) IU and 18.94 × 10(-3) IU/mg, respectively, whereas the total and specific activities were 10.19 × 10(-3) IU and 5.28 × 10(-3) IU/mg, respectively, with dihydrokaempferol. RT-PCR revealed that during F. tataricum florescence there was an organ-specific expression pattern by the FtFLS1 gene, with similar trends in flavonoid content. These observations suggest that FtFLS1 in F. tataricum encodes a functional protein, which might play a key role in rutin biosynthesis.

Application of magnetic hydroxyapatite nanoparticles for solid phase extraction of plasmid DNA.

We developed a facile method for plasmid DNA (pDNA) extraction from crude Escherichia coli lysate using magnetic hydroxyapatite nanoparticles (MHapNPs) in the presence of polyethylene glycol (PEG)/NaCl. DNA condensation induced by PEG/NaCl is a prerequisite for achieving pronounced DNA recovery. The quality and quantity of MHapNP-purified pDNA under optimal binding buffer conditions (0.5 volume of 20% PEG 8000/2M NaCl) were comparable to those obtained using organic solvents or commercial kits. This MHapNP technique is rapid, simple, cost-effective, and environmentally friendly and has the potential to extract DNA from other cell lysates.

Temperature-dependent selective purification of plasmid DNA using magnetic nanoparticles in an RNase-free process

Carboxyl group-functionalized magnetic nanoparticles were used to develop an RNase-free method for plasmid DNA (pDNA) purification directly from RNA-containing crude Escherichia coli lysates. This method takes advantage of differing adsorption behaviors of pDNA and RNA onto magnetic nanoparticle surfaces at different temperatures. Pure pDNA can be isolated between 70 and 80°C without sacrificing DNA quality and quantity, as evidenced by comparison with that obtained using organic solvents or commercial kits. This RNase-free method is rapid, simple, cost-effective, and environmentally friendly, and it can be easily scaled up for the production of pharmacological-grade pDNA.

Identification, isolation and expression analysis of eight stress-related R2R3-MYB genes in tartary buckwheat (Fagopyrum tataricum)

Key messageEightR2R3-MYBgenes in tartary buckwheat were identified, and their expression patterns were comprehensively analyzed, which reveals role in plant response to abiotic stresses.AbstractThe proteins of the R2R3-MYB superfamily play key roles in the growth and development processes as well as defense responses in plants. However, their characteristics and functions have not been fully investigated in tartary buckwheat (Fagopyrum tataricum), a strongly abiotic resistant coarse cereal. In this article, eight tartary buckwheat R2R3-MYB genes were isolated with full-length cDNA and DNA sequences. Phylogenetic analysis of the members of the R2R3-MYB superfamily between Arabidopsis and tartary buckwheat revealed that the assumed functions of the eight tartary buckwheat R2R3-MYB proteins are divided into five Arabidopsis functional subgroups that are involved in abiotic stress. Expression analysis during abiotic stress and exogenous phytohormone treatments identified that the eight R2R3-MYB genes responded to one or more treatments. This study is the first comprehensive analysis of the R2R3-MYB gene family in tartary buckwheat under abiotic stress.

Anthocyanins accumulate in tartary buckwheat (Fagopyrum tataricum) sprout in response to cold stress

Tartary buckwheat (Fagopyrum tataricum) is rich in flavonoids. Anthocyanins, a special class of flavonoids, offer significant nutrients and provide the red pigment found in sprouts. The anthocyanins biosynthesis has been shown influenced by environmental stress. In this study, we investigate the effects of cold stress on anthocyanins biosynthesis of tartary buckwheat sprouts. On cellular level, cross-sectional observations were performed and result indicated that anthocyanins accumulated primarily in the epidermal and cortex cells of hypocotyls. Biochemical analysis, including anthocyanin quantification, thin-layer chromatography and radical scavenging assay, showed that cold stress significantly increased the synthesis of anthocyanins and antioxidant activity of tartary buckwheat sprouts. At the molecular level, semi-RT-PCR was used to investigate 14 anthocyanin-related genes in tartary buckwheat sprouts treated with cold stress. All the selected genes were upregulated in cold-stressed sprouts, except for FtGST, FtAHA, and FtMYB. More importantly, the expression level of three late biosynthesis genes, FtF3′H, FtDFR, and FtANS, was highest in hypocotyl tissue. Our results suggest that anthocyanins play a considerable role in the cold stress tolerance of tartary buckwheat sprouts.

Overexpression of a tartary buckwheat R2R3-MYB transcription factor gene, FtMYB9, enhances tolerance to drought and salt stresses in transgenic Arabidopsis.

Tartary buckwheat (Fagopyrum tataricum) is a traditional coarse cereal that exhibits strong plasticity in its adaptation to harsh and complicated environmental stresses. In an attempt to study the strong tolerance of tartary buckwheat, the FtMYB9 gene, which encodes an R2R3-MYB transcription factor protein, was functionally investigated. FtMYB9 expression was rapidly and strongly induced by ABA, cold, salt, and drought treatments in the seedling stage. A yeast one-hybrid system assay indicated that FtMYB9 is an activator of transcriptional activity, consistent with its roles as a transcription factor. Its overexpression in plants resulted in increased sensitivity to ABA at the germination and seedling stages compared to wild type. The overexpression of FtMYB9 increased tolerance to drought and salt stresses by the activation of some stress-related genes from both ABA-independent and ABA-dependent pathways in transgenic Arabidopsis. Furthermore, enhanced proline content and the activation of the P5CS1 gene implied that FtMYB9 may be involved in proline synthesis in plants. Collectively, these results suggest that FtMYB9 functions as a novel R2R3-MYB TF which plays positive roles in salt and drought tolerance by regulating different stress-responsive signaling pathways.

Overexpression of a Tartary Buckwheat Gene, FtbHLH3, Enhances Drought/Oxidative Stress Tolerance in Transgenic Arabidopsis

bHLH (basic helix-loop-helix) transcription factors play important roles in the abiotic stress response in plants, but their characteristics and functions in tartary buckwheat (Fagopyrum tataricum), a flavonoid-rich cereal crop with a strong stress tolerance, have not been fully investigated. Here, a novel bHLH gene, designated FtbHLH3, was isolated and characterized. Expression analysis in tartary buckwheat revealed that FtbHLH3 was mainly induced by polyethylene glycol 6000 (PEG6000) and abscisic acid (ABA) treatments. Subcellular localization and a yeast one-hybrid assay indicated that FtbHLH3 has transcriptional activation activities. Overexpression of FtbHLH3 in Arabidopsis resulted in increased drought/oxidative tolerance, which was attributed to not only lower malondialdehyde (MDA), ion leakage (IL), and reactive oxygen species (ROS) but also higher proline (Pro) content, activities of antioxidant enzymes, and photosynthetic efficiency in transgenic lines compared to wild type (WT). Moreover, qRT-PCR analysis indicated that the expression of multiple stress-responsive genes in the transgenic lines was significantly higher than in WT under drought stress. In particular, the expression of AtNCED, a rate-limiting enzyme gene in ABA biosynthesis, was increased significantly under both normal and stress conditions. Additionally, an ABA-response-element (ABRE) was also found in the promoter regions. Furthermore, the transgenic Arabidopsis lines of the FtbHLH3 promoter had higher GUS activity after drought stress. In summary, our results indicated that FtbHLH3 may function as a positive regulator of drought/oxidative stress tolerance in transgenic Arabidopsis through an ABA-dependent pathway.

Tartary buckwheat FtMYB10 encodes an R2R3-MYB transcription factor that acts as a novel negative regulator of salt and drought response in transgenic Arabidopsis

Tartary buckwheat is a strongly abiotic, resistant coarse cereal, but its tolerance mechanisms for stress are largely unknown. MYB transcription factors play key roles in various physiological, biochemical and molecular responses, which can both positively and negatively regulate the stress tolerance of plants. In this study, we report that the expression of FtMYB10, a R2R3-MYB gene from Tartary buckwheat, was induced significantly by ABA and drought treatments. A seed germination test under ABA treatment indicated that transgenic lines were less sensitive to ABA. The overexpression of FtMYB10 in Arabidopsis reduced drought and salt tolerance. Further studies showed that the proline contents in the transgenic plants are markedly decreased associated with reduced expression of the P5CS1 gene under both normal and stress conditions. Furthermore, the expression of some stress-responsive genes, including DREB1/CBFs, RD29B, RD22, and several genes of the DRE/CRT class, decreased in response to FtMYB10 overexpression in Arabidopsis. These results suggest that FtMYB10 may play a key role in ABA signaling feedback regulation and act as a novel negative regulator of salt and drought stress tolerance in plants.

Fagopyrum tataricum FtWD40 Functions as a Positive Regulator of Anthocyanin Biosynthesis in Transgenic Tobacco

Tartary buckwheat (Fagopyrum tataricum Gaertn.) contains high concentration of flavonoids, which are mainly represented by rutin, anthocyanins, and proanthocyanidins. WD40 transcription factors (TFs) play significant roles in the transcriptional regulation of the anthocyanin biosynthetic pathway. In this study, a WD40-repeat protein gene (designated as FtWD40) was identified and characterized from tartary buckwheat. The bioinformatics analyses showed that the putative FtWD40 shared a high level of similarity with MtWD40-1, which is a positive regulator in anthocyanin biosynthesis of Medicago truncatula. The yeast one-hybrid assay indicated that FtWD40 had transcriptional activation activities. During florescence, FtWD40 was highly expressed in flowers compared to other organs. Furthermore, its overexpression in tobacco resulted in a remarkable deepening of petal pigmentation in flowers due to a significant increase in anthocyanins accumulation. Meanwhile, the expression of dihydroflavonol-4-reductase (DFR) and anthocyanin synthase (ANS) was upregulated 1.95- and 1.56-fold, respectively. In contrast, the expression level was lower for flavonol synthase (FLS) in the transgenic lines. To the best of our knowledge, this is the first functional characterization of a WD40 transcription factor (FtWD40) from tartary buckwheat that controls the anthocyanin pathway.

β‐Amyrin synthase from Conyza blinii expressed in Saccharomyces cerevisiae

Conyza blinii H.Lév. is a widely used medicinal herb in southwestern China. The main pharmacological components of C. blinii are a class of oleanane‐type pentacyclic triterpene glycosides known as conyzasaponins, which are thought to be synthesized from β‐amyrin. However, no genes involved in the conyzasaponin pathway have previously been identified. Here, we identify an oxidosqualene cyclase (OSC), a β‐amyrin synthase, which mediates cyclization of 2,3‐oxidosqualene to yield β‐amyrin. Ten OSC sequences were isolated from C. blinii transcript tags. Phylogenetic analysis was used to select the tag Cb18076 as the putative β‐amyrin synthase, named CbβAS. The open reading frame of CbβAS is 2286 bp and encodes 761 amino acids. Its mature protein contains the highly conserved motifs (QXXXGXW/DCTAE) of OSCs and (MWCYCR) of β‐amyrin synthases. Transcription of CbβAS was upregulated 4–24 h after treatment of the seedlings of the C. blinii cultivar with methyl jasmonate. Furthermore, expression of CbβAS in Saccharomyces cerevisiae successfully yielded β‐amyrin. The chemical structures and concentrations of β‐amyrin were confirmed by GC‐MS/MS. The target yeast ultimately produced 4.432 mg·L−1 β‐amyrin. Thus, CbβAS is an OSC involved in conyzasaponin biosynthesis.