Xueyi Han

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.

Improving Phytase Enzyme Activity in a Recombinant phyA Mutant Phytase from Aspergillus niger N25 by Error-Prone PCR

The mutant acid phytase (phyAm) gene was modified by random mutagenesis to improve enzymatic activity by using an error-prone PCR (ep-PCR) strategy. The mutated gene was linearized and inserted into plasmid vector pPIC9K and transformed by electroporation into Pichia pastoris GS115. A single transformant, PP-NPep-6A, showing the strongest phytase activity from among the 5,500 transformants, was selected for detailed analyses. Southern blot analysis of the mutant yeast transformant showed that phyAep gene was integrated into the chromosome genome through single crossover with one copy of phyA. The kinetic parameters indicated that the mutant one showed 61% higher specific activity and 53% lower km value than that of PP-NPm-8 (P < 0.05). In addition, the overall catalytic efficiency (kcat/km) of the mutant one was 84% higher (P < 0.05) than that of PP-NPm-8. Nine bases were altered in the mutant sequences, which resulted in three amino acid changes, namely, Glu156Gly, Thr236Ala, and Gln396Arg. The structural predictions indicated that the mutations generated by ep-PCR somehow reorganized or remodeled the active site, which could lead to increasing catalytic efficiency.

An antifungal peptide from Fagopyrum tataricum seeds.

A major trypsin inhibitor was isolated and characterized from the seeds of the tartary buckwheat (Fagopyrum tataricum) (FtTI) by ammonium sulfate precipitation, ion exchange chromatography and centrifugal ultrafiltration. SDS-PAGE analysis under reducing condition showed that FtTI is a single polypeptide chain with a molecular mass of approximately 14kDa. The complete amino acid sequence of FtTI was established by automatic Edman degradation and mass spectrometry. It was found that the trypsin inhibitor molecule consists of 86 amino acid residues containing two disulfide bonds which connect Cys(8) to Cys(65) and Cys(49) to Cys(58). The active site of the inhibitor was found to contain an Asp(66)-Arg(67) bond. MALDI-TOF analysis showed that FtTI has two isoforms (Mr: 11.487 and 13.838kDa). Dixon plots revealed a competitive inhibition of trypsin with inhibition constants (Ki) of 1.6nM. Analysis of the amino acid sequence suggests that FtTI is a member of the protease inhibitor I family. What is more, FtTI exhibited strong inhibitory activity against phytopathogenic fungi.

Comparative Proteomic Analysis of Differentially Expressed Proteins in Amaranthus hybridus L. Roots Under Cadmium Stress

Amaranthus hybridus L. has great potential for use in phytoremediation of soils contaminated with cadmium (Cd). In this study, we found higher absorption of Cd by the roots of A. hybridus than by its other organs. To understand the mechanism of Cd accumulation in A. hybridus roots, a comparative proteomic approach was used to differentiate the two-dimensional electrophoretic profiles of root proteins in Cd-free and Cd-treated plants. Twenty-eight differentially expressed proteins were successfully identified using matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry. Of these, 10 were specifically expressed under Cd stress, and another 11 were upregulated and 7 downregulated by >2.5-fold under Cd stress. We observed increased expression of proteins involved in energy metabolism, protein metabolism, stress and defense, and signal transduction. These changes likely enhanced Cd tolerance and enrichment in A. hybridus. The downregulated proteins were mainly involved in the synthesis of microRNAs, cell walls, and other structural components. These observations were further confirmed by quantitative fluorescence PCR. The resulting differences in protein expression patterns suggest that redirection of root cell metabolism might be an important survival mechanism for A. hybridus under Cd stress.

Genetic relationships among buckwheat (Fagopyrum) species from southwest China based on chloroplast and nuclear SSR markers

Buckwheat (Fagopyrum, family Polygonaceae), originates from China (Linnaeus 1753), has great value as the green food material and medicinal plant. The genus Fagopyrum includes 23 species, three variations and two subspecies (Yang and Xia 2008), which can be divided into smallachene and big-achene buckwheat groups. Phylogenetic relationships and classification of Fagopyrum species have been debated ever since Gross (1913) first classified them in China. The origin and classification of buckwheat gradually became the worldwide concern. Studies have provided strong support for the hypothesis that the Jinsha river basin (Chinese western region) is the distribution centre or centre of origin for buckwheat (Ohnishi and Yasui 1998; Wang et al. 2008). Buckwheat resources in Sichuan Pan’xi regions are quite rich (Wang et al. 2006) and new wild Fagopyrum species are continuously being discovered. We investigated the genuslevel phylogenetic relationship and genetic backgrounds of 12 wild and two cultivated accessions representing the main distribution of buckwheat in western Sichuan by using chloroplast simple sequence repeat (cpSSR) and nuclear (nSSR) markers.