Appropriate light intensity is favorable for the photosynthesis, biomass accumulation, key enzyme activity, and secondary metabolite synthesis of medicinal plants. This study aims to explore the influence of light intensity on growth and quality of Panax quinquefolius. To be specific, sand culture experiment was carried out in a greenhouse under the light intensity of 40, 80, 120, and 160 μmol·m~(-2)·s~(-1), respectively. The growth indexes, photosynthetic characteristics, content of 6 ginsenosides of the 3-year-old P. quinquefolius were determined, and the expression of ginsenoside synthesis-related enzyme genes in leaves, main roots, and fibrous roots was determined. The results showed that the P. quinquefolius growing at 80 μmol·m~(-2)·s~(-1) light intensity had the most biomass and the highest net photosynthetic rate. The total biomass of P. quinquefolius treated with 120 μmol·m~(-2)·s~(-1) light intensity was slightly lower than that with 80 μmol·m~(-2)·s~(-1). The root-to-shoot ratio in the treatment with 120 μmol·m~(-2)·s~(-1) light intensity was up to 6.86, higher than those in other treatments(P<0.05),and the ginsenoside content in both aboveground and underground parts of P. quinquefolius in this treatment was the highest, which was possibly associated with the high expression of farnesylpyrophosphate synthase(FPS), squalene synthase(SQS), squalene epoxidase(SQE), oxidosqualene cyclase(OSC), dammarenediol-Ⅱ synthase(DS), and P450 genes in leaves and SQE and DS genes in main roots. In addition, light intensities of 120 and 160 μmol·m~(-2)·s~(-1) could promote PPD-type ginsenoside synthesis in leaves by triggering up-regulation of the expression of upstream ginsenoside synthesis genes. The decrease in underground biomass accumulation of the P. quinquefolius grown under weak light(40 μmol·m~(-2)·s~(-1)) and strong light(160 μmol·m~(-2)·s~(-1)) was possibly attributed to the low net photosynthetic rate, stomatal conductance, and transpiration rate in leaves. In the meantime, the low expression of SQS, SQE, OSC, and DS genes in the main roots might led to the decrease in ginsenoside content. However, there was no significant correlation between the ginsenoside content and the expression of synthesis-related genes in the fibrous roots of P. quinquefolius. Therefore, the light intensity of 80 and 120 μmol·m~(-2)·s~(-1) is beneficial to improving yield and quality of P. quinquefolius. The above findings contributed to a theoretical basis for reasonable shading in P. quinquefolius cultivation, which is of great significance for improving the yield and quality of P. quinquefolius through light regulation.
Farnesyl-Diphosphate Farnesyltransferase/metabolism* ; Ginsenosides ; Panax/metabolism* ; Plant Roots/metabolism* ; Sand ; Squalene Monooxygenase

Glycyrrhiza uralensis Fisch. ex DC is widely used in traditional Chinese medicine (TCM). Among its various active components, glycyrrhizic acid is believed to be the marker component. Squalene synthase (SQS) and beta-amyrin synthase (beta-AS) are key enzymes in the biosynthetic pathway of glycyrrhizic acid in G uralensis. To reveal the effects of co-expression of SQS1 and beta-AS genes on this pathway, 7 yeast expression vectors harboring different SQS1 variants and beta-AS were constructed and expressed in Saccharomyces cerevisiae as fusion proteins. TLC and GC-MS results showed that co-expression of SQS1 and beta-AS enhanced the accumulation of beta-amyrin. The effects of SQS12 were more obvious than the other two SQS1 variants. This study is significant for further investigations concerned with exploring the biosynthesis of glycyrrhizic acid in vitro and strengthening the efficacy of G. uralensis by means of increasing the content of glycyrrhizic acid.
Farnesyl-Diphosphate Farnesyltransferase ; genetics ; metabolism ; Glycyrrhiza uralensis ; genetics ; Intramolecular Transferases ; metabolism ; Oleanolic Acid ; analogs & derivatives ; metabolism ; Plant Proteins ; genetics ; Recombinant Proteins ; metabolism ; Saccharomyces cerevisiae ; metabolism

According to the designed specific primers of gene fragment based on the Salvia miltiorrhiza transcriptome data, a full-length cDNA sequence of SQS2 from S. miltiorrhiza f. alba was cloned by the method of reverse transcription polymerase chain reaction (RT-PCR). The SmSQS2 cDNA sequence was obtained, this sequence is named SmSQS2 and its GenBank registration number is KM244731. The full length of SmSQS2 cDNA was 1245 bp, encoding 414 amino acids including 5'UTR 115 bp and 3'UTR 237 bp. Sequence alignment and phylogenetic analysis demonstrated that SmSQS2 had relative close relationship to the SQS2 of S. miltiorrhiza. The induction of E. coli [pET28-SQS2] in different temperature, induction time, IPTG concentrations and density of inducing host bacterium (A600) were performed, Shaking the culture at 30 degrees C until the A600 is approximately 0.6 and add IPTG to final concentration of 0.2 mmol x L(-1), and then the optimal expression of SmSQS2 recombinant protein were accumulated after the induction time of 20 h. The research provided important base for the study of sterol and terpene biosynthesis of SQS2 in S. miltiorrhiza f. alba.
Cloning, Molecular ; Farnesyl-Diphosphate Farnesyltransferase ; chemistry ; genetics ; metabolism ; Models, Molecular ; Molecular Sequence Data ; Phylogeny ; Plant Proteins ; chemistry ; genetics ; metabolism ; Salvia miltiorrhiza ; chemistry ; classification ; enzymology ; genetics ; Sequence Alignment

A squalene synthase gene cloned (GuSQS1, accession number in GenBank database: AM182329) from Glycyrrhiza uralensis was transferred into G. uralensis via Agrobacterium rhizogenes A4 for investigating biosynthesis pathway and enhancing synthesis of glycyrrhizic acid (GA). Hypocotyl explants from G. uralensis were infected with A. rhizogenes A4 containing GuSQS1 gene to induce the hairy roots. The hairy root lines established were selected in medium containing 0.8 mg x L(-1) phosphinothricin (PPT) and analyzed by PCR and southern blotting. The transgenic hairy roots were cultured in liquid MS medium. GA contents in transgenic hairy roots were detected by HPLC. Results showed that maximal GA content in transgenic hairy root lines was 3.6 times as high as in wild type hairy roots.
Cells, Cultured ; Farnesyl-Diphosphate Farnesyltransferase ; genetics ; metabolism ; Glycyrrhiza uralensis ; enzymology ; genetics ; growth & development ; metabolism ; Glycyrrhizic Acid ; metabolism ; Molecular Sequence Data ; Plant Proteins ; genetics ; metabolism ; Plant Roots ; enzymology ; genetics ; growth & development ; metabolism ; Plants, Genetically Modified ; enzymology ; genetics ; growth & development ; metabolism

The present study was designed to determine the effects of copy number variations (CNVs) of squalene synthase 1(SQS1) gene on the mevalonate (MVA) pathway. SQS1 gene from G. uralensis (GuSQS1) was cloned and over-expressed in Pichia pastoris GS115. Six recombinant P. pastoris strains containing different copy number of GuSQS1 were constructed. HPLC was used to assay the level of ergosterol in all transgenic P. pastoris strains containing GuSQS1. HPLC analysis showed that the contents of ergosterol in all of the transgenic P. pastoris containing GuSQS1 were higher than that in the negative control. And with the increase of copy number of GuSQS1, the content of ergosterol showed an increasing-decreasing-increasing pattern. The contents of ergosterol in 10-copy-GuSQS1 P. pastoris and 47-copy-GuSQS1 P. pastoris were significantly higher than that in the rest recombinant P. pastoris strains. In conclusion, the CNVs of GuSQS1 influence the content of secondary metabolites in the MVA pathway. The present study provides a basis for over-expressing GuSQS1 and increasing the content of glycyrrhizin in G. uralensis cultivars.
Amino Acid Sequence ; genetics ; Chromatography, High Pressure Liquid ; DNA Copy Number Variations ; genetics ; Ergosterol ; biosynthesis ; Farnesyl-Diphosphate Farnesyltransferase ; genetics ; Glycyrrhiza uralensis ; genetics ; Mevalonic Acid ; metabolism ; Pichia ; metabolism ; Plasmids ; genetics ; Real-Time Polymerase Chain Reaction ; Recombinant Proteins ; metabolism

OBJECTIVETo analyze the effect of endophytic fungi on expression amount of key enzyme genes SS (squalene synthase gene), SE (squalene epoxidase gene) and bAS (beta-amyrin synthase gene) in saponin biosynthesis and saponins content in Eleutherococcus senticosus.

METHODWound method was used for back meeting the endophytic fungi to E. senticosus. With GAPDH as internal control gene, the expression of key enzyme genes was detected by real time PCR method. E. senticosus saponins content was measured by spectrophotometry method.

RESULTWhen wound method back meeting P116-1a and P116-1b after 30 d, the expression content of SS improved significantly (P < 0.05), however the back meeting of P109-4 and P312-1 didnt change the expression of SS. After that SS expression showed reduction-equality-reduction varying trend. Thirty days after back meeting P312-1, the expression content of SE improved significantly (P < 0.05). Ninty days after back meeting P116-1b and P312-1, the expression content of SE improved significantly to 130%,161%, respectively (P < 0.05). After 120 d, back meeting four endophytic fungi, the expression of SE were significantly higher than the control (P < 0.05). Back meeting four endophytic fungi form 60 d to 120 d, the expression of bAS was significantly higher than the control (P < 0.05). The back meeting four endophytic fungi improved E. senticosus saponins content significantly (P < 0.05).

CONCLUSIONEndophytic fungi P116-1a, P116-1b, P1094 and P312-1 significantly effected the expression of key enzyme genes SS, SE and bAS and then affected E. senticosus saponins content. Among the genes, bAS was key target gene.

Eleutherococcus ; chemistry ; metabolism ; microbiology ; Endophytes ; physiology ; Farnesyl-Diphosphate Farnesyltransferase ; genetics ; Fungi ; physiology ; Gene Expression Regulation, Enzymologic ; Gene Expression Regulation, Plant ; Intramolecular Transferases ; genetics ; Saponins ; analysis ; biosynthesis ; Squalene Monooxygenase ; genetics

OBJECTIVETo analyse the polymorphism of squalene synthase gene and reveal the influence of squalene synthase (SQS) gene polymorphism on the catalytic efficiency of its encode enzyme in Glycyrrhiza uralensi.

METHODThe total RNA was extracted. PCR was used to amplify the coding sequences of squalene synthase gene, which were sequenced and analysed. The expression vectors containing different SQS gene sequences, including SQS1C, SQS1F, SQS2A, SQS2B, were constructed and transformed into Escherichia coli BL21. The fusion protein was induced to express by IPTG, then was isolated, purified and used to carry out the enzymatic reaction in vitro. GC-MS was used to analyse the production.

RESULTThere were three kinds of gene polymorphism existing in SQS1 gene of G. uralensis, including single nucleotide polymorphism (SNPs), insertion/deletion length polymorphism (InDels) and level of amino acid, the proportion of conservative replace of SQS1 was 53.94%, and there were 2 mutational sites in structural domains. The proportion of conservative replace of SQS2 was 60%, and there was 1 mutational site in structural domains. The production squalene could be detected by GC-MS in all the 4 kinds of enzymatic reactions. The capacity of accumulating squalene of SQS1F was higher than other SQS genes.

CONCLUSIONThe polymorphism of SQS gene was quite abundant in G. uralensis, which maybe the molecular foundation of the formation of high-quality liquorice.

Amino Acid Substitution ; Biocatalysis ; Cloning, Molecular ; DNA, Complementary ; chemistry ; genetics ; Electrophoresis, Polyacrylamide Gel ; Escherichia coli ; genetics ; Farnesyl-Diphosphate Farnesyltransferase ; genetics ; metabolism ; Gas Chromatography-Mass Spectrometry ; Glycyrrhiza uralensis ; enzymology ; genetics ; INDEL Mutation ; Isoenzymes ; genetics ; metabolism ; Molecular Sequence Data ; Plant Proteins ; genetics ; metabolism ; Polymorphism, Genetic ; Polymorphism, Single Nucleotide ; Recombinant Proteins ; metabolism ; Sequence Analysis, DNA ; Squalene ; metabolism

OBJECTIVETo study the gene expression profile of liver of young apoE(-/-)/LDLR(-/-)/Lepr(db/db) treble genes mutant mice and disclose its relationship to hyperlipidemia and the following atherosclerotic lesion.

METHODSThe gene expression profile was investigated using cDNA microarray technique; the plasma total cholesterol(TC) and triglyceride(TG) levels were analyzed by COD-PAP and GPO-PAP method. And morphological observations of the aorta were made.

RESULTSAmong the 4000 target genes, 92 genes were up-regulated and 105 genes were down-regulated in the treble genes mutants, compared with wild type control. Among the differentially expressed lipid metabolism related genes, cholesterol synthesis gene coding for farnesyl diphosphate farnesyl transferase was down-regulated, while triglyceride metabolism gene e.g. pancreatic lipase related protein 1 gene (Pnliprp1) was up-regulated. Expression profile of carbohydrate, cell skeleton and immune related genes were also altered. On the other hand, in the plasma from the treble genes mutant mice at 5 weeks of age, hyperlipidemia was found to be combined with atheroslerotic lesion. All these biochemical and pathological changes were aggravated following aging.

CONCLUSIONThe data suggested that the multiple genes mutations, especially those involved in lipid metabolism, were contributing to the alteration of liver gene expression profile that might lead to hyperlipidemia and atherosclerotic lesion in the young apoE(-/-)/LDLR(-/-)/Lepr(db/db) mutants.

Animals ; Apolipoproteins E ; genetics ; Cholesterol ; blood ; Farnesyl-Diphosphate Farnesyltransferase ; genetics ; metabolism ; Female ; Gene Expression Profiling ; methods ; Hyperlipidemias ; blood ; genetics ; metabolism ; Lipase ; genetics ; metabolism ; Lipid Metabolism ; Male ; Mice ; Mice, Knockout ; Oligonucleotide Array Sequence Analysis ; methods ; Receptors, LDL ; genetics ; Receptors, Leptin ; genetics ; Triglycerides ; blood

The total triterpene saponins of Psammosilene tunicoides have significant pharmacologic activity. Psammosilene tunicoides squalene synthase (PSS) is a gateway enzyme to regulate the biosynthesis of total triterpene saponins extracted from the root of Psammosilene tunicoides which is an endangered species. In this paper, cDNA encoding of PSS was cloned by the degenerate primer PCR and rapid-amplification of cDNA ends (RACE). The full-length of cDNA of PSS is 1663 bp, with an open reading frame (ORF) of 1 245 bp, encoding 414 amino acid polypeptide (calculated molecular mass, 47.69 kDa), 5'UTR (untranslated region) and 3'UTR are 260 bp and 158 bp, respectively. The deduced amino acid sequence of PSS has higher homology with the known squalene synthases of several species such as Panax notoginseng (83%), Panax ginseng (82%) and Glycyrrhiza glabra (82%) than that with Schizosacharomyces pombe (35%), Candida albicans (39%) and Homo sapiens (47%). The characterization of PSS was done by a series of methods, such as prokaryotic expression, the activity of enzyme in vitro, capillary gas chromatography (GC) and capillary gas chromatography mass spectrometry (GC-MS). The results showed that the cell-free extract of E. coli transformed with the recombinant plasmid can effectively convert farnesyl diphosphate into squalene in vitro. GenBank accession number is EF585250. Our research provided important base for the study of Psammosilene tunicoides secondary metabolism and metabolic engineering.
Caryophyllaceae ; enzymology ; genetics ; Cloning, Molecular ; DNA, Complementary ; genetics ; Endangered Species ; Escherichia coli ; genetics ; metabolism ; Farnesyl-Diphosphate Farnesyltransferase ; genetics ; metabolism ; Gas Chromatography-Mass Spectrometry ; Open Reading Frames ; Phylogeny ; Plant Proteins ; genetics ; metabolism ; Plants, Medicinal ; chemistry ; genetics ; Plasmids ; Polymerase Chain Reaction ; Recombinant Proteins ; metabolism ; Sequence Homology, Amino Acid ; Transformation, Genetic

PNS (Panax notoginseng saponins) is the main medical bioactive component in Panax notoginseng. The medical value of PNS cannot be extended because of its low production. With the deep study of saponins biosynthetic pathway, the control of PNS biosynthesis through metabolic engineering has gradually become possible. In this study, the Squalene synthase (SS) over-expression vector was established. By the way of agrobacterium-mediated method, the vector was transfered and integrated into the Panax notoginseng genome. The result of the PCR detection and the saponin content detection shows that over-expression SS is able to produce high level of Panax notoginseng saponins, and confirms the regulatory function of SS in the biosynthesis of ginsenosides in Panax notoginseng. It provides a theoretical basis and technical basis for the construction of PNS homologous or heterologous efficient expression system in the future.
Agrobacterium tumefaciens ; Amino Acid Sequence ; Cell Line ; Cloning, Molecular ; DNA, Complementary ; genetics ; Farnesyl-Diphosphate Farnesyltransferase ; genetics ; metabolism ; Gene Transfer Techniques ; Genetic Vectors ; genetics ; Panax notoginseng ; chemistry ; cytology ; genetics ; microbiology ; Plants, Genetically Modified ; chemistry ; cytology ; genetics ; microbiology ; Plants, Medicinal ; chemistry ; cytology ; genetics ; microbiology ; Saponins ; metabolism ; Transformation, Genetic