Flavonols are produced by the desaturation of dihydroflavanols, which is catalyzed by flavonol synthase (FLS). FLS belongs to the 2-oxoglutarate iron-dependent oxygenase family. The full-length cDNA and genomic DNA sequences of the FLS gene (designated as GbFLS) were isolated from Ginkgo biloba. The full-length cDNA of GbFLS contained a 1023-bp open reading frame encoding a 340-amino-acid protein. The GbFLS genomic DNA had three exons and two introns. The deduced GbFLS protein showed high identities with other plant FLSs. The conserved amino acids (H-X-D) ligating ferrous iron and residues (R-X-S) participating in 2-oxoglutarate binding were found in GbFLS at similar positions like other FLSs. GbFLS was found to be expressed in all tested tissues including roots, stems, leaves, and fruits. Expression profiling analyses revealed that GbFLS expression was induced by all of the six tested abiotic stresses, namely, UV-B, abscisic acid, cold, sucrose, salicylic acid, and ethephon, consistent with the in silico analysis results of the promoter region. The recombinant protein was successfully expressed in the E. coli strain BL21 (DE3) with a pET-28a vector. The in vitro enzyme activity assay by high performance liquid chromatography indicated that recombinant GbFLS protein could catalyze the formation of dihydrokaempferol to kaempferol and the conversion of kaempferol from naringenin, suggesting that GbFLS is a bifunctional enzyme within the flavonol biosynthetic pathway.

Phenylalanine ammonia-lyase (PAL) is the first key enzyme of the phenypropanoid pathway. A full-length cDNA of PAL gene was isolated from Juglans regia for the first time, and designated as JrPAL. The full-length cDNA of the JrPAL gene contained a 1935bp open reading frame encoding a 645-amino-acid protein with a calculated molecular weight of about 70.4 kD and isoelectric point (pI) of 6.7. The deduced JrPAL protein showed high identities with other plant PALs. Molecular modeling of JrPAL showed that the 3D model of JrPAL was similar to that of PAL protein from Petroselinum crispum (PcPAL), implying that JrPAL may have similar functions with PcPAL. Phylogenetic tree analysis revealed that JrPAL shared the same evolutionary ancestor of other PALs and had a closer relationship with other angiosperm species. Transcription analysis revealed that JrPAL was expressed in all tested tissues including roots, stems, and leaves, with the highest transcription level being found in roots. Expression profiling analyses by real-time PCR revealed that JrPAL expression was induced by a variety of abiotic and biotic stresses, including UV-B, wounding, cold, abscisic acid and salicylic acid.

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Flavonoids are secondary metabolites that contribute substantially to the quality of Ginkgo biloba. Plant flavonoid accumulation is controlled by transcriptional regulation of the genes that encode the biosynthetic enzymes, in which the R2R3-MYB transcription factor is a key factor. In this study, we describe the cloning and functional characterization of a R2R3-MYB transcription factor gene, GbMYBF2, isolated from G. biloba. GbMYBF2 encodes a protein belonging to a small subfamily of R2R3-MYB transcription factors. Comparative and bioinformatics analyses showed that GbMYBF2 is more closely related to the repressor R2R3-MYB subfamily involved in flavonoid biosynthesis. Tissue expression pattern analysis showed that GbMYBF2 was constitutively expressed in leaves, fruits, stems, and roots, wherein the level of transcription in the roots is significantly higher than that in the stems, leaves, and fruits. During G. biloba leaf growth, the transcription of GbMYBF2 is negatively correlated with the flavonoid content, suggesting that the GbMYBF2 gene is responsible for the repressed flavonoid biosynthesis. Transgenic Arabidopsis plants that overexpress GbMYBF2 exhibit an inhibition of flavonoid and anthocyanin biosynthesis compared with the untransformed Arabidopsis plants. In addition, the overexpression of GbMYBF2 in Arabidopsis clearly downregulates the expression of the structural genes that control the synthesis of flavonoids and anthocyanins. These findings suggest that GbMYBF2 may have a key role in repressing transcription in regulating the biosynthesis of flavonoids in G. biloba.

Lactobacillus plantarum ZDY 2013, a novel strain isolated from Chinese traditional fermented acid beans, was systematically evaluated for its survival capacity under stress conditions (pH, bile salt, simulated gastrointestinal tract, and antibiotics), production of exopolysaccharide and antagonism against 8 pathogens. Its effect on mice gut microbiota was also investigated by quantitative PCR and PCR-denaturing gradient gel electrophoresis. The results showed that ZDY 2013 can grow at pH 3.5 and survive at pH 2.0 for 6 h and at 0.45% bile salt for 3 h. The exopolysaccharide yield was up to 204±7.68 mg/L. The survival rate of ZDY 2013 in a simulated gastrointestinal tract was as high as 65.84%. Antagonism test with a supernatant of ZDY 2013 showed maximum halo of 28 mm against Listeria monocytogenes. The inhibition order was as follows: Listeria monocytogenes, Salmonella typhimurium, Escherichia coli, Pseudomonas aeruginosa, Shigella sonnei, Enterobacter sakazakii, and Staphylococcus aureus. Lactobacillus plantarum ZDY 2013 was sensitive to some antibiotics (e.g., macrolide, sulfonamides, aminoglycoside, tetracyclines and β-lactams), whereas it was resistant to glycopeptides, quinolones, and cephalosporins antibiotics. Denaturing gradient gel electrophoresis profile demonstrated that ZDY 2013 administration altered the composition of the microbiota at various intestinal loci of the mice. Moreover, the quantitative PCR test showed that the administration of ZDY 2013 enhanced the populations of Bifidobacterium and Lactobacillus in either the colon or cecum, and reduced the potential enteropathogenic bacteria (e.g., Enterococcus, Enterobacterium, and Clostridium perfringens). Lactobacillus plantarum ZDY 2013 exhibited high resistance against low pH, bile salt, and gastrointestinal fluid, and possessed antibacterial and gut microbiota modulation properties with a potential application in the development of dairy food and nutraceuticals.

Gaotian, one typical conservative village in rural area of South China, is differentiated from other adjacent village for its longevity and health situation of residents. To ascertain the difference of intestinal microbial community between Gaotian and other region, high-throughput sequencing and systematical bioinformation analyses was adopted to compare 21 samples in long life group with 28 in control group. The α diversity showed that the diversity of species of intestinal flora of Gaotian villagers was higher than that of control group, while the β diversity showed that the similarity of intestinal flora for Gaotian residents was also much higher than that of control group. OTU cluster analysis and Venn diagram showed that the intestinal microbial community of Gaotian villagers is different from that of control group. To quantitatively compare the main flora constitution in all samples, real-time PCR was performed, and the results showed that the biomass of Enterococcus, Lactobacillus, Enterobacteriaceae, Clostridium perfringens, and Bacteroides of Gaotian villages is generally significantly higher than that of control group. Remarkably, some special species, i.e., Methanobacterium, Butyricimonas, Deinococcus, and Streptococcaceae, have been found in Gaotian villagers. Overall, this study lays a preparatory basis for exploration of the resources of special species from healthy and long-living elderly Gaotian villagers and for proposal of a hypothesis, namely, the diversity in intestinal flora of Gaotian might contribute to the longevity and health of local residents. Further study should be focused on screening and functional evaluation of the special species in the long-life residents.

Dihydroflavonol-4-reductase (DFR, EC1.1.1.219) catalyzes a key step late in the biosynthesis of anthocyanins, condensed tannins (proanthocyanidins), and other flavonoids important to plant survival and human nutrition. Three DFR cDNA clones (designated GbDFRs) were isolated from the gymnosperm Ginkgo biloba. The deduced GbDFR proteins showed high identities to other plant DFRs, which form three distinct DFR families. Southern blot analysis showed that the three GbDFRs each belong to a different DFR family. Phylogenetic tree analysis revealed that the GbDFRs share the same ancestor as other DFRs. The expression of the three recombinant GbDFRs in Escherichia coli showed that their actual protein sizes were in agreement with predictions from the cDNA sequences. The recombinant proteins were purified and their activity was analyzed; both GbDFR1 and GbDFR3 could catalyze dihydroquercetin conversion to leucocyanidin, while GbDFR2 catalyzed dihydrokaempferol conversion to leucopelargonidin. qRT-PCR showed that the GbDFRs were expressed in a tissue-specific manner, and transcript accumulation for the three genes was highest in young leaves and stamens. These transcription patterns were in good agreement with the pattern of anthocyanin accumulation in G.biloba. The expression profiles suggested that GbDFR1 and GbDFR2 are mainly involved in responses to plant hormones, environmental stress and damage. During the annual growth cycle, the GbDFRs were significantly correlated with anthocyanin accumulation in leaves. A fitted linear curve showed the best model for relating GbDFR2 and GbDFR3 with anthocyanin accumulation in leaves. GbDFR1 appears to be involved in environmental stress response, while GbDFR3 likely has primary functions in the synthesis of anthocyanins. These data revealed unexpected properties and differences in three DFR proteins from a single species.

Decreased serum omentin-1 levels could be considered as an independent predictive marker of the presence and severity of OSAS.