Correlation study between accumulation of triterpenoids and expression of relative genes in Alisma orientale / 中国中药杂志

To research the correlation between accumulation of triterpenoids and expression of key enzymes genes in triterpenoid biosynthesis of Alisma orientale,the study utilized UPLC-MS/MS method to detect eight triterpenoids content in the tuber of A. orientale from different growth stages,including alisol A,alisol A 24 acetate,alisol B,alisol B 23 acetate,alisol C 23 acetate,alisol F,alisol F 24 acetate and alisol G,and then the Real time quantitative PCR was used to analyze the expression of key enzymes genes HMGR and FPPS in triterpenoid biosynthesis. Correlation analysis showed that there was a significant positive relation between the total growth of these eight triterpenoids and the average relative expression of HMGR and FPPS(HMGR: r = 0. 998,P<0. 01; FPPS: r = 0. 957,P<0. 05),respectively. Therefore,the study preliminarily determined that HMGR and FPPS genes could regulate the biosynthesis of triterpenoids in A. orientale,which laid a foundation for further research on the biosynthesis and regulation mechanism of triterpenoids in A. orientale.

Active expression and core sequnce analysis of HMGR gene promoter from Artemisia annua / 中草药

Objective: To analyze the expression character, screen the core sequence of 3-hydroxy-3-methyl glutaryl coenzyme A (HMGR) promoter from Artemisia annua, and provide a theoretical basis for the efficient biosynthesis of artemisinin. Methods: In this study, β-glucoside acid enzyme (GUS) gene was used as the report gene and constructed five different length expression vectors, including the whole length of HMGR promoter, based on the cloned HMGR promoter sequences, and successfully obtained transgenic plants by the method of agrobacterium mediation transform tobacco, then the tobacco was stained by GUS. Results: The GUS staining showed that the blue color could detect in the roots, stems, leaves, flowers, and fruit pods. And the leaves in the lower part could easily get the blue color than the leaves in the top part, this may due to that the accumulation of HMGR metabolite product is more and the permeability in leaves is better in the mature leaves. The results also showed that the color showed shallow in heat and drought stresses, and with no significant difference in cold stress, the tobacco leaves with paragraph of P-HMGR-1, P-HMGR-2, and P-HMGR-3 showed blue color but did not find blue in leaves with paragraph of P-HMGR-4, P-HMGR-5. Conclusion: HMGR promoter has a stable expression during the whole growth period of tobacco. HMGR promoter was not sensitive with cold environment, but sensitive with heat and draught resistance. The key area of HMGR promoter was in P-HMGR-3. And there are some regulatory elements but no necessary part of HMGR promoter between the non overlap area of P-HMGR-3 and P-HMGR-2.

Cloning and Bioinformatic Analysis of HMGS and HMGR Genes from Panax notoginseng / 中草药·英文版

Objective To clone and analyze 3-hydroxy-3-methylglutaryl coenzyme-A synthase (HMGS) and 3-hydroxy-3-methylglutaryl coenzyme-A reductase (HMGR) genes from Panax notoginseng of four-year old during the flowering period, the key genes involved in the mevalonic acid pathway for saponin biosynthesis. Methods The cDNA sequences of PnHMGS1 and PnHMGR2 were obtained by reverse transcription PCR (RT-PCR) and rapid amplification of cDNA ends (RACE) methods and were analyzed in their secondary structures, subcellular localizations, domains, and the three-dimensional structures of putative proteins by the bioinformatics tools. Fusion genes were constructed by the prokaryotic expression system. Results The two genes were cloned, named as PnHMGS1 and PnHMGR2, respectively, and were both predicted to be located in the chloroplast. PnHMGS1 (1410 bp) encoded a predictive unstable protein with 469 amino acids and covered hydroxymethylglutaryl-CoA synthase domain. PnHMGR2 (1690 bp) also encoded an unstable protein with 589 amino acids and possessed a hydroxymethylglutaryl-coenzyme A reductase domain and two transmembrane regions. Both of the genes were expressed most in flowers followed by roots, stems, and least in leaves. Conclusion PnHMGS1 and PnHMGR2 are firstly cloned from P. notoginseng as the new member of the HMGR family, and they show the same expression profile as P. ginseng and P. quinquefolius.

HMGR, SQS, β-AS, and Cytochrome P450 Monooxygenase Genes in Glycyrrhiza uralensis / 中草药·英文版

Glycyrrhiza uralensis is frequently used in traditional Chinese medicine. This plant contains a large amount of effective constituents, including triterpenoids and flavonoids. Among them, glycyrrhizin is believed to be the marker compound to evaluate the quality of G. uralensis based on Chinese Pharmacopoeia. Many studies showed that glycyrrhizin possesses various pharmacological activities, such as antibacterial, antiviral, antitumor, anti-inflammatory, and immune-stimulating activities. In this paper, we summarized the cloning, characterization, expression, and polymorphism analysis of several functional genes involved in glycyrrhizin biosynthesis in G. uralensis.

Cloning and characterization of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) gene from Paris fargesii Franch.

3-Hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR) plays an important role in catalyzing the first committed step of isoprenoids biosynthesis in mevalonic acid (MVA) pathway. Here, we cloned a full-length transcript of Paris fargesii Franch. The full-length cDNA of P. fargesii HMGR (Pf-HMGR, GenBank accession no. JX508638) was 1,973 bp and contained a 1,728 bp ORF encoding 576 amino acids. Sequence analysis revealed that the deduced Pf-HMGR had high similarity with HMGRs from other plants, including Ricinus communis (77%), Litchi chinensis (76%), Michelia chapensis (75%) and Panax quinquefolius (72%). It had a calculated molecular mass of about 62.13 kDa and an isoelectric point (pI) of 8.47. It contained two transmembrane domains, two putative HMGR binding sites and two NADP(H)-binding sites. The predicted 3-D structure revealed that Pf-HMGR had a similar spatial structure with other plant HMGRs. Three catalytic regions, including L-domain, N-domain and S-domain were detected by structural modeling of HMGR. Tissue expression analysis revealed that Pf-HMGR was strongly expressed in roots and stems than in leaves. Taken together, our data laid a foundation for further investigation of HMGR's functions and regulatory mechanisms in plants.

Overexpression of HMGR and DXR from Amomum villosum Lour . Affects the Biosynthesis of Terpenoids in Tobacco / 世界科学技术-中医药现代化

HMGR and DXR are key enzymes of terpenoids biosynthesis pathway. This study was aimed to discuss the effects of overexpression of HMGR and DXR from A momum villosum Lour. on the biosynthesis of terpenoids in transgenic tobacco. The real-time fluorescence quantitative PCR (RT-qPCR) was used to analyze the expression level of AvHMGR and AvDXR. Then, enzyme activities of HMGR and DXR were determined by spectrometer using the substrate-specific method. Different terpenoids were detected by GC-MS. The results showed that individual overex-pression of HMGR/DXR can inhibit the enzyme activities of HMGR and DXR but promote the biosynthesis of men-thene, neophytediene, cembrenene and sterol. The co-overexpression of HMGR and DXR had different enzyme activ-ities and can promote the biosynthesis of sterol and phytol, but inhibit the biosynthesis of neophytadiene. It was con-cluded that the overexpression of HMGR and DXR had diverse effects when regulating the biosynthesis of different terpenoids. This study provided the basis for using A vHMGR and A vDXR to regulate the metabolism of terpenoids.

Cloning and bioinformatic analysis of 3-hydroxy-3-methylglutaryl coenzyme-A reductase gene from Panax notoginseng / 中草药

Objective: Panax notoginseng is an important medicinal plant and its secondary metabolites, P. notoginseng saponins (PNS), synthesized by the mevalonate pathway are the active ingredients. The study on the gene of the key enzyme 3-hydroxy-3- methylglutaryl-coenzyme-A reductases (HMGR) in the mevalonate pathway is helpful for the regulation of PNS syntheses. Methods: The primers were designed according to P. ginseng HMGR (accession number: GU565097.1) from NCBI. Total RNA was extracted from the callus of P. notoginseng. The fragment of HMGR gene was amplified by reverse transcription PCR technology and analyzed. Results: Sequence analysis showed that the cDNA sequence of obtained fragment (PnHMGR) was 1 893 bp, containing an ORF spaning 1 725 bp, and exhibited 98%, 93%, and 80% sequence identity with HMGR in P. ginseng, Eleutherococcus senticosus, and Eucommia ulmoides. The cDNA was a new one as searching in the Genbank. The bioinformatic analysis showed that PnHMGR-encoding protein contained two transmembrane regions and the HMGR catalytic domain, without signal peptide. The expression level of PnHMGR was the highest when the callus of P. notoginseng has grown for 30 d. Conclusion: It is the first time to report HMGR gene isolated from P. notoginseng. The results will provide a groundwork for exploring the molecular function of PnHMGR involved in PNS biosynthesis based on the synthetic biology of P. notoginseng.

Promotion of HMGR activation and β-eudesmol biosynthesis in Atractylodes lancea suspension cell culture by hydrogen peroxide-mediated endopytic fungal elicitor / 中草药

Objective: To investigate the effect of endophytic fungal elicitor on key enzyme activity, inducing pathway and mechanism involved in the secondary metabolites of Atractylodes lancea. Methods: NADPH oxidase, HMGR activities, the concentration of hydrogen peroxide (H2O2) and β-eudesmol were determined by the co-culture of endophytic fungal elicitor and A. lancea suspension cell. Results: NADPH oxidase activity was notably enhanced by Fusarium sp5 elicitor which could induce oxidative burst, significantly promote H2O2 accumulation, and activate HMGR in the sesquiterpenoids metabolic pathway. Compared with the control, the yield of β-eudesmol increased 257.6% and reached 66.59 μg/g. CAT and DPI could inhibit the HMGR activity and β-eudesmol biosynthesis in A. lancea cell induced by Fusarium sp5 elicitor. Exogenous H2O2 also induced HMGR and promoted the β-eudesmol biosynthesis. Conclusion: H2O2 is necessary to induce β-eudesmol synthetic signal molecule by activating the HMGR.

Cloning and analysis of HMGR gene conserved fragments in Paris fargesii / 中草药

Objective: Paris fargesii is an important medicinal plant and its secondary metabolites, steroidal saponins, synthesized by the mevalonate pathway are the active ingredients. The study on the gene of the key enzyme 3-hydroxy-3-methyl- glutaryl-coenzyme A reductases (HMGR) in the mevalonate pathway is helpful for the regulation of steroidal saponins syntheses. Methods: In the basis of degenerate primers designed according to mRNA homologous conserved regions of 11 HMGRs from 11 plant species published on NCBI and total RNA extracted from the seedling of P. fargesii, a 404 bp fragment of HMGR was amplified by reverse transcription PCR technology, which is a new cDNA as searching in the Genbank. Results: Sequence analysis showed that the obtained fragment had 76%-81% identification compared with other seven plant species. The deduced amino acid seqence was predicted to have a wider and higher homology with other plants. Conclusion: Protein characteristic zones, conserved regions, and phylogenetic analysis by Prosite and ClustalX could give preliminary evidence for the existence of HMGR in P. fargesi, which is the report of HMGR gene isolated from the plants in Liliaceae for the first time.

Effect of Genistein and Soy Protein on Lipids Metabolism in Ovariectomized Rats

Postmenopausal women or ovariectomized rats are associated with increased cholesterol levels, which are risk factors of metabolic syndrome and cardiovascular diseases. Increased prevalence of metabolic syndrome after menopause might be associated with estradiol deficiency. Harmful effect of estradiol hampers the casual usage of hormone to prevent the metabolic syndrome. Soy protein has been reported to show several beneficial effects on health, however it is unclear which components of soy protein is responsible for anti-obesity and hypocholesterolemic effects. Soy isoflavones, genistein and daizein, are suggested to have anti-obesity and hypocholesterolemic effects but with inconsistency. The present study investigated the effect of supplementation of genistein (experiment I) and soy protein containing isoflavones (experiment II) to high fat diet on body weight gain, food intake, liver and fat tissue weight and the lipid levels in ovariectomized rats. Plasma and hepatic lipid contents and the mRNA levels of genes encoding lipid metabolism related proteins, such as CPT1 and HMGR were measured. Ovariectomy increased body weight, fat tissue weight and plasma and hepatic lipid levels which increase the risk of metabolic syndrome. Soy protein could improve plasma and hepatic lipids levels. Soy protein also increased hepatic CPT1 and HMGR mRNA levels. Plasma and hepatic lipids levels could not be decreased by dietary genistein alone. In contrast, lipids levels could be decreased by isoflavone-fortified soy protein, suggesting that the ingestion of soy protein enriched with isoflavone gives more benefit for protecting postmenopausal women from metabolic syndrome.

Cloning and analysis of HMGR gene conserved fragments in Atractylodes lancea / 中草药

Objective To clone and sequence cDNA encoding 3-hydroxy-3-methylglutaryl-coenzyme A reductase(HMGR) from Atractylodes lancea.Methods The cDNA,encoding HMGR in A.lancea,was amplified by RACE strategy with the cDNA of the total RNA of young leaves as the template.The partial fragments of HMGR were cloned and sequenced.Results The analysis results revealed that the conserved fragments were 458 bp.At the same time,the two fragments had been obtained 84.28% identification in nucleotide acid and 92.11% identification in corresponding amino acid,named as HMGRcr1 and HMGRcr2,respectively.It was deduced that they may be members of the HMGR gene family in A.lancea.Sequencing analysis showed that HMGRcr1 and HMGRcr2 had high identity with HMGR from other plants.Conclusion The cDNA encoding HMGR from A.lancea is cloned and reported for the first time.The work will provided a foundation for exploring the mechanism of terpenes biosynthesis and application to the other medicinal plants.