Molecular Cloning and Sequence Analysis of GgUGE Gene in Glycyrrhiza glabra / 中国实验方剂学杂志

Objective::To clone the cDNA sequence of UDP-glucose 4-epimerase (UGE) in Glycyrrhiza glabra and analyze its sequence, so as to explore the potential relationship between the UGE gene and the molecular regulatory mechanisms of glycyrrhizic acid biosynthesis. Method::The cDNA sequence of UGE was cloned from the root of G. glabra by reverse transcription polymerase chain reaction (RT-PCR), then sequenced and analyzed by bioinformatics software. Results::A GgUGE cDNA sequence with the full length of 1 121 bp was obtained. The open reading fame (ORF) of GgUGE was 1 053 bp, encoding 350 amino acid residues. The GgUGE cDNA sequence was submitted to GenBank, and the accession No. was MK638908. Sequence analysis showed that GgUGE was an unstable hydrophilic protein, its average relative molecular weight was 39.02 kDa, and isoelectric point was 6.13. It contained no signal peptides or transmembrane domains. Its secondary structure mainly constituted of α-helix and had a conversed domain of UDP-glucose 4-epimerase superfamily. The homologoue analysis showed that the cDNA and amino acid sequences of GgUGE had the closest evolutionary relationship to Leguminosae and relatively distant evolutionary relationship to Salicaceae. Conclusion::In this study, GgUGE cDNA sequence is successfully cloned from G. glabra for the very first time, which will provide reference for studying the function of GgUGE and explaining the molecular regulatory mechanisms of glycyrrhizic acid biosynthesis in G. glabra.

Optimization of UDP-glucose supply module and production of ginsenoside F1 in Saccharomyces cerevisiae / 中国中药杂志

Ginsenoside F1 is a rare ginsenoside in medicinal plants such as Panax ginseng,P. notogingseng and P. quinquefolius. It has strong pharmacological activities of anti-tumor,anti-oxidation and anti-aging. In order to directly produce ginsenoside F1 by using inexpensive raw materials such as glucose,we integrated the codon-optimized P.ginseng dammarenediol-Ⅱ synthase(Syn Pg DDS),P.ginseng protopanaxadiol synthase(Syn Pg PPDS),P. ginseng protopanaxatriol synthase(Syn Pg PPTS) genes and Arabidopsis thaliana cytochrome P450 reductase(At CPR1) gene into triterpene chassis strain BY-T3. The transformant BY-PPT can produce protopanaxatriol. Then we integrated the Sacchromyces cerevisiae phosphoglucomutase 1(PGM1),phosphoglucomutase 2(PGM2) and UDP-glucose pyrophosphorylase 1(UGP1) genes into chassis strain BY-PPT. The UDP-glucose supply module increased UDP-glucose production by 8. 65 times and eventually reached to 44. 30 mg·L-1 while confirmed in the transformant BY-PPT-GM. Next,we integrated the UDPglucosyltransferase Pg3-29 gene which can catalyze protopanaxatriol to produce ginsenoside F1 into chassis strain BY-PPT-GM. The transformant BY-F1 produced a small amount of ginsenoside F1 which was measured as 0. 5 mg·L-1. After the fermentation process was optimized,the titer of ginsenoside F1 could be increased by 900 times to 450. 5 mg·L-1. The high-efficiency UDP-glucose supply module in this study can provide reference for the construction of cell factories for production of saponin,and provide an important basis for further obtaining high-yield ginsenoside yeast cells.

Effect of endophytic fungi on expression of key enzyme genes in polysaccharide and alkaloid synthesis from Dendrobium officinale / 中草药

Objective: To analyze the effect of endophytic fungi GXRz2, GXRz3 and GXRz10 on content of poly saccharide and alkaloid, and the expression of key enzyme genes UGPase, HMGR, and FPS in Dendrobium officinale. Methods: The endophytic fungi liquid were added to D. officinale seedlings. Polysaccharide and alkaloid content were measured by spectrophotometry method. With 18S rRNA as internal control gene, the expression of key enzyme genes was detected by real-time quantitative PCR method. Results: It was found that the content of polysaccharide in D. officinale was higher, mainly concentrated in stem, but the lowest in root. And the content of alkaloid in D. officinale was lower, mainly accumulated in leaf, but the lowest in root. In addition, the three endophytic fungi strains could promote accumulation of polysaccharide and alkaloid in D. officinale to a certain extent. The expression of UGPase, HMGR and FPS genes in D. officinale induced by different strains was detected by real-time quantitative PCR. The results showed that endophytic fungi GXRz3 and GXRz10 could significantly increase the expression of UGPase, HMGR and FPS genes in D. officinale. In the polysaccharide synthesis pathway, the UGPase gene had the highest relative expression in the stem, followed by the leaf, and the least in the root. In the alkaloid synthesis pathway, HMGR gene had the highest relative expression in the stem, followed by the leaf, and the least in the root. However, FPS gene had the highest relative expression in the leaf, followed by the stem, and the least in the root. Conclusion: Endophytic fungi may affect the synthesis of polysaccharide by regulating the expression of UGPase. Considering the accumulation of polysaccharide, it is speculated that UGPase may be a key enzyme in the polysaccharide synthesis pathway of D. officinale. Endophytic fungi may affect the synthesis of alkaloid by regulating the expression of HMGR and FPS. Considering the accumulation of alkaloid, it is speculated that FPS may be a key enzyme in the alkaloid synthesis pathway of D. officinale.

In vitro glycosylation of Vitamin C / Монголын Анагаах Ухаан

BACKGROUND: Glycosylation process helps in stabilization and solubilization natural of compounds. Glycosyltransferase (YjiC) provides for high efficient glycosylation product with an incredible variety of sugar moieties, typically from UDP-glucose. Vitamin C (L-ascorbic acid) is an essential nutrient for humans and certain other animal species. Vitamin C functions in many biological processes, such as collagen synthesis, antoxidation, intestinal absorption of iron. UDP-glucose acts as a starting material for glycosyltransferase (YjiC). In order to recycle UDP-glucose after glycosylation with glycosyltransferase (YjiC), sucrose synthase (AtSUS1) carry out than reversible conversion of sucrose and UDP to UDP-glucose and fructose. MATERIALS AND METHODS: DNA was extracted than E.coli BL 21 and E.coli JM 109 hosts were used for expression of proteins. The purified protein was then analyzed by 12% SDS-PAGE than used for enzymatic recycle system. TLC analyse of the products were carried out to the test glycosylation. RESULTS: In this study, we choose substrate vitamin C for the enhancement of enzymatic recycling system glycosylation. In this recycle system due to the high concentration of sucrose and vitamin C but low concentration of UDP-glucose with is relatively expensive made the system more economic. TLC analyses of the products were carried out to the recycled system worked and glycosylation product. CONCLUSION: Based on proved function an enzymatic recycling system with glycosyltransferse (YjiC) and sucrose synthesis (AtSUS1) to be applicable to enzymatic production of vitamin C glucoside and resveratrol glucoside. Further analysis by HPLC and MS will elucidate the products.

Effect of application of uridine diphosphate-glucose on self-repairment potentiality of immature cerebral white matter invivo / 临床儿科杂志

Objectives To explore the effect of application of uridine diphosphate-glucose (UDP-glucose) on self-repairment potentiality of immature white matter (WM) in vivo. Methods Five-day-old rats were randomly divided into sham, periventricular leukomalacia (PVL) and UDP-glucose groups. The PVL model was constructed in the PVL and UDP groups, and UDP-glucose (2000mg/kg) was induced by an intraperitoneal injection at once to the rats of UDP group. PVL in-duced proliferation and differentiation of WM-glial progenitor cells invivo were detected by using the three-label lfuorescent immunoanalysis, the apopotosis in WM cell was observed by TUNEL test, and the pathology of WM and myelination were evaluated by light and electron microscopy at day 7 and day 21 after PVL model construction. Results The numbers of new WM-progenitors (NG2+), oligodendrocytes (OLs) progenitor marker (O4+), OL precursors, cyclic nucleotide phosphodiesterase (CNPase+), immature OLs and myelin basic protein (MBP+), and mature OLs in the UDP-glucose group are signiifcantly grea-ter than those in the PVL group at each time interval after induction of PVL (P<0.05). The numbers of the apoptotic cells in UDP-glucose group are less than those in the PVL groups. Under light and electron microscopy, the white matter pathological changes and myelination were found to be better than those in the PVL group (P<0.05). Conclusion The application of UDP-glucose can induce the WM-progenitors to activate, proliferate and differentiate into immature and mature OLs. UDP-glucose can also signiifcantly reduce the apoptotic rate of the WM-new glia cells;improve the white matter pathological changes and the myelin formation.

Cloning and endogenous expression of a Eucalyptus grandis UDP-glucose dehydrogenase cDNA

UDP-glucose dehydrogenase (UGDH) catalyzes the oxidation of UDP-glucose (UDP-Glc) to UDP-glucuronate (UDP-GlcA), a key sugar nucleotide involved in the biosynthesis of plant cell wall polysaccharides. A full-length cDNA fragment coding for UGDH was cloned from the cambial region of 6-month-old E. grandis saplings by RT-PCR. The 1443-bp-ORF encodes a protein of 480 amino acids with a predicted molecular weight of 53 kDa. The recombinant protein expressed in Escherichia coli catalyzed the conversion of UDP-Glc to UDP-GlcA, confirming that the cloned cDNA encodes UGDH. The deduced amino acid sequence of the cDNA showed a high degree of identity with UGDH from several plant species. The Southern blot assay indicated that more than one copy of UGDH is present in Eucalyptus. These results were also confirmed by the proteomic analysis of the cambial region of 3- and 22-year-old E. grandis trees by 2-DE and LC-MS/MS, showing that at least two isoforms are present. The cloned gene is mainly expressed in roots, stem and bark of 6-month-old saplings, with a lower expression in leaves. High expression levels were also observed in the cambial region of 3- and 22-year-old trees. The results described in this paper provide a further view of the hemicellulose biosynthesis during wood formation in E. grandis.

Uridine 5'-diphosphate glucose 4-epimerase from Ehrlich ascites carcinoma cells.

Uridine 5'-diphosphate glucose 4-epimerase (EC 5.1.3.2) from Ehrlich ascites carcinoma cells was purified to apparent homogeneity using conventional procedures and NAD-hexane-agarose affinity chromatography. The protein had a molecular weight of 96,000. The ascites enzyme had an absolute requirement for exogenously added NAD (10 μM) for stability. This appears to be a unique feature of ascites epimerase since epimerase from other mammalian sources did not exhibit such a dependence. Exogenously added NAD was also needed for catalysis with an apparent Km value of 2·5 μM. NADH was a very potent competitive inhibitor (K i = 0·11 μM with respect to NAD) of the enzyme activity at pH values close to intracellular pH. The dependence of the enzyme on NAD for stability and its inhibition by NADH may have some potential significance in tumor metabolism.