Impact of mannitol and poly ethylene glycol 6000 induced water deficit on plant biomass and major secondary metabolites in Centella asiatica (L.) Urb. in vitr

Low water potential related stresses are regulated by modifying water uptake and loss to avoid low water potential, accumulating solutes which in turn enhance active principles and its gene expressions. Present study examined effect of in vitro induced absorption of mannitol and PEG (poly ethelene glycol) 6000 in Indian pennywort, Centella asiatica (L.) Urb., neutraceutical plant, evidenced by phenotypic, molecular and phytochemical analyses. Both mannitol and PEG 6000 induce water deficit conditions in plants and retarded normal plant biomass in terms of fresh and dry weights. These effects were significantly less severe in plants subjected to mannitol, compared to PEG. PEG and mannitol imposed water deficit, resulted in decline in major active compound, asiaticoside evidenced by HPTLC of asiaticoside content. Differential expression of some selected key genes in the asiaticoside pathway including squalene synthase and ? amyrin synthase by qPCR, confirmed decrease in transcript level expression of asiaticoside, whereas upregulated transcript level expression was observed in cycloartenol synthase for synthesis of phytosterols. Estimation of total flavonoids and phenolics under different water deficit conditions were found declined. In conclusion, water deficit by mannitol and PEG 6000 can significantly affects processes associated with biomass growth and ability to synthesize secondary metabolites in C. Asiatica.

Adaptive evolution analysis of squalene synthase in medicinal plants of Araliaceae / 中草药

Objective: To analyze the adaptive evolution of squalene synthase (SS) in the medicinal plants of Araliaceae. Methods: The adaptive evolutionary analysis of 23 SS genes of seven medicinal plants of Araliaceae was carried out by using the branch model, site model, branch-site model, MEC model, SLAC, FEL, and REL of PAML software. Results: In the analysis of PAML and MEC models, most of the branches and loci were found to be under strong negative selection, and no positive selection sites were found. The analysis by SLAC, FEL, and REL also showed that there were a large number of negative selection sites, only 412P, 413N, and 415K were positive selection sites. Conclusion: This indicates that negative selection plays a leading role in SS gene of Araliaceae. The 412P, 413N, and 415K sites found may be involved in the activity of SS.

Cloning and expression analysis of three critical triterpenoid pathway genes in Osmanthus fragrans

Background: Osmanthus fragrans is an important ornamental tree and has been widely planted in China because of its pleasant aroma, which is mainly due to terpenes. The monoterpenoid and sesquiterpenoid metabolic pathways of sweet osmanthus have been well studied. However, these studies were mainly focused on volatile small molecule compounds. The molecular regulation mechanism of synthesis of large molecule compounds (triterpenoids) remains unclear. Squalene synthase (SQS), squalene epoxidase (SQE), and beta-amyrin synthase (BETA-AS) are three critical enzymes of the triterpenoid biosynthesis pathway. Results: In this study, the full-length cDNA and gDNA sequences of OfSQS, OfSQE, and OfBETA-AS were isolated from sweet osmanthus. Phylogenetic analysis suggested that OfSQS and OfSQE had the closest relationship with Sesamum indicum, and OfBETA-AS sequence shared the highest similarity of 99% with that of Olea europaea. The qRT-PCR analysis revealed that the three genes were highly expressed in flowers, especially OfSQE and OfBETA-AS, which were predominantly expressed in the flowers of both "Boye" and "Rixiang" cultivars, suggesting that they might play important roles in the accumulation of triterpenoids in flowers of O. fragrans. Furthermore, the expression of OfBETA-AS in the two cultivars was significantly different during all the five flowering stages; this suggested that OfBETA-AS may be the critical gene for the differences in the accumulation of triterpenoids. Conclusion: The evidence indicates that OfBETA-AS could be the key gene in the triterpenoid synthesis pathway, and it could also be used as a critical gene resource in the synthesis of essential oils by using bioengineered bacteria.

Cloning and expression analysis of squalene synthase gene in Hedera helix / 中草药

Objective To obtain a full-length cDNA of squalene synthase gene from Hedera helix (HhSS) by cloning technique, and to carry out bioinformatics analysis and expression analysis. Methods Primers were designed based on H. helix transcriptome data, and HhSS was cloned by using RACE technologies. DNAMAN, PROTPARAM, TMHMM, PSORT, ScanProsite, SOPMA, and SWISS-MODEL were used for analysis of sequence and physical and chemical properties and domain of encoded protein. The relative expression of HhSS was detected by qRT-PCR. Results The cDNA sequence of HhSS (GenBank accession number: KX056078) was 1 889 bp which obtained by RACE cloning. It contained an ORF from 248 bp to 1 477 bp and a 5'UTR with 247 bp and a 3'UTR with 412 bp. It encoded a 409-amino-acid protein with a molecular weight of 46 800and an isoelectric point (pI) of 5.68. The HhSS protein had the characteristic domain and transmembrane region of plant SS protein and closer relationship with Eleutherococcus senticosus, Panax ginseng et al. The qRT-PCR results indicated that it had positive correlation between relative expressing level of HhSS gene and contents of saponins in H. helix leaves. Conclusion The cloning and expression analysis results of HhSS provide a theoretical and technical basis for elucidating the role of HhSS in saponins biosynthetic pathway and metabolic regulation.

Effect of light quality on total gypenosides accumulation and related key enzyme gene expression in Gynostemma pentaphyllum / 中草药·英文版

Objective: Light quality has effect on the accumulation of gypenosides in the medicinal plant Gynostemma pentaphyllum in the family Cucurbitaceae, while the squalene synthase (SS) and squalene epoxidase (SE) are the key enzymes for gypenoside biosynthesis. The objective of this study was to elucidate the relationship between light quality and biosynthesis key enzyme involving the regulation of gypenoside accumulation. Methods: The content of total gypenosides was measured by colorimetric method and the expression of SS and SE gene was determined by quantitative Real-time PCR in the seedlings of G. pentaphyllum which were grown with different light quality. Results: Light quality showed remarkable impacts on the accumulation of total gypenosides. The highest content of total gypenosides in the plant under red light condition was determined, followed by blue light and white light, while the lowest content was recorded under dark condition. qRT-PCR analysis proved that the expression levels of SS and SE genes were also affected by light quality. The high-level gene expressions of SS and SE were found in the plant under red light condition, followed by blue light, with the least content in darkness. The statistical analysis revealed that the total gypenosides were significantly different in different light treatment and the content of total gypenosides was positively related to the expression of SS and SE genes. Conclusions: Light quality regulates gypenoside accumulation via altering the expression of SS and SE in G. pentaphyllum.

Cloning and characterization of squalene synthase and cycloartenol synthase from

Mogrosides and steroid saponins are tetracyclic triterpenoids found in. Squalene synthase (SQS) and cycloartenol synthase (CAS) are key enzymes in triterpenoid and steroid biosynthesis. In this study, full-length cDNAs ofandwere cloned by a rapid amplification of cDNA-ends with polymerase chain reaction (RACE-PCR) approach. ThecDNA has a 1254 bp open reading frame (ORF) encoding 417 amino acids, and thecDNA contains a 2298 bp ORF encoding 765 amino acids. Bioinformatic analysis showed that the deduced SgSQS protein has two transmembrane regions in the C-terminal. Bothandhave significantly higher levels in fruits than in other tissues, suggesting that steroids and mogrosides are competitors for the same precursors in fruits. Combinedprediction and subcellular localization, experiments in tobacco indicated that SgSQS was probably in the cytoplasm or on the cytoskeleton, and SgCAS was likely located in the nucleus or cytosol. These results will provide a foundation for further study ofandgene functions in, and may facilitate improvements in mogroside content in fruit by regulating gene expression.

Prokaryotic expression, functional identification of squalene synthase in Alisma orientale and its immunoassay study / 中国中药杂志

Squalene synthase of Alisma orientale catalyzes farnesyl diphosphate (FPP) to form squalene, which is the key regulatory enzyme of the carbon source flow to protostane triterpenes biosynthesis. For further research on the function and expression of AoSS gene, the open reading frame (ORF) of squalene synthase gene (accession no. JX866770) from A. orientale was subcloned into a prokaryotic expression vector pCzn1 and induced the expression of AoSS gene in Escherichia coli BL21(Roseta). The fusion protein was mainly in the form of inclusion bodies and purified to obtain high purity protein. By verifying its functionality through vitro enzymatic reaction, the results showed that the catalytic protein had the catalytic activity of FPP into squalene. In order to research the expression of AoSS in A. orientale, the purified protein was used to immunized rabbits to prepare polyclonal antibody which was then purified, the titer of the antibody was greater than 1∶51 200 by ELISA detection, and displayed good specificity by Western blotting. The prepared antibody was used for immunoassay of AoSS in different organs of A. orientale, and the results showed that the AoSS expression level was the highest in tubers, followed by leaves, and lowest in root. Successful construction of prokaryotic expression vector, validation of gene functions and establishment of rapid immunoassay lay the foundation for further researches on the function and regulation of AoSS gene, and also provide scientific basis on the application of the protostane triterpenes of A. orientale in the field of synthetic biology.

Grey relational analysis of relationship between limonin compounds accumulation and functional genes expression of Citri Reticulatae Semen / 中草药

Objective: To study the biosynthesis mechanism and transshipment law of limonin compounds in Citri Reticulatae Semen, traditional medicinal variety Citrus reticulata "Dahongpao". Methods: In the process of seed growth and development, the contents of limonin, nomilin, and obacunone in stem, vane, peel, and seeds were determined by UPLC tangerine; Using grey relational analysis method, the correlation analysis between the contents and the cloning of squalene synthase gene (ss), squalene epoxidase gene (se), and glucose transferase (lgt) expression was performed. Results: Limonin compounds in different organs had the largest accumulation in seeds; The accumulation of limonin compounds in different organs was significantly correlated with ss, se, and lgt gene expression, but in the seeds the accumulation of limonin has the most closely correlation with ss, se, and lgt gene expression; The ss gene expression had the largest contribution on the accumulation of limonin, nomilin, and obacunone. Conclusion: The study provides reliable and scientific envidendce for the synthesis mechanism and transshipment law of limonin compounds.

Prediction of CpG islands and functional verification on promoters of FPS, SS, SE in Eleutherococcus senticosus / 中草药

Objective: To obtain the distribution and functional activity of CpG islands in promoters of FPS, SS, and SE from Eleutherococcus Senticosus. Methods: Based on the promoter sequence of FPS, SS and SE, CpG islands were predicted by using EMBOSS and Li Lab. The functional verification of transformed Arabidopsis thaliana was mediated by Agrobacterium tumefaciens by using GUS and pCAMBIA1301 plasmid as the reporter gene and expression vector respectively. Results: Two CpG islands were found in FPS and SS promoter with the lengths of 520 bp, 218 bp and 108 bp, 103 bp, and three CpG islands in SE promoter in 290 bp, 119 bp and 149bp. The promoters of FPS, SS and SE all had promoter activities at different level, in which SS promoter was the highest one. Conclusion: The functional verification and distributions of CpG islands in the promoters area of FPS, SS, and SE were reported in this research at first time, which established the foundation for the methylation analysis of FPS, SS, and SE and the further studying of the mechanism expression regulation in E. Senticosus.

Cloning and expression analysis of squalene synthase gene in Tripterygium wilfordii / 药学学报

In this paper, we cloned the full-length cDNA of TwSQS from Tripterygium wilfordii suspension cells (GenBank:KR401220) and performed the bioinformation and mRNA expression analysis. The expression after methyl jasmonate (MJ) treatment of the gene was detected by RT-PCR. The full-length cDNA of TwSQS was 1800 bp containing a 1242 bp open reading frame (ORF) encoding a polypeptide of 413 amino acids. The theoretical isoelectric point (pI) was 7.94 and the calculate molecular weight was about 47.20 kD. The relative expression level of TwSQS was deduced by MJ and reached the highest at 4 h after the treatment. The gene information we got in this study enriched the biosynthesis pathway of triterpenoids in Tripterygium wilfordii and laid foundation for further studies.

Study on mechanism of regulating synthesis of β-sitosterol by SiSQS1 and SiSQS2 in Saussurea involucrata cells / 中草药

Objective: To explore the synthetic mechanism of β-sitosterol by comparing the locus mutation, prokaryotic expression, expression level of SiSQS1 and SiSQS2, and the content of β-sitosterol in three color types of cells. Methods: Firstly, we preformed the cloning and bioinformatic analysis of SiSQS1 and SiSQS2 which were key enzymes involved in the biosynthesis of β-sitosterol in Saussurea involucrata cells. Secondly, we compared the differences of prokaryotic expression between SiSQS1 and SiSQS2, then optimized the expression conditions. Finally, we compared the expression levels of SiSQS1 and SiSQS2 by Real-time PCR and the content of β-sitosterol by GC-MS in three color types of cells, and made the correlative analysis on the expression level and the content of β-sitosterol. Results: There was a locus mutation of amino acid residues in 242E/D between SiSQS1 and SiSQS2. The results of prokaryotic expression analysis and conditions optimization showed that both target proteins had been expressed successfully, but the optimal prokaryotic expression system was different. The results of expression level and quantitative analysis showed a positive correlation to the expression levels of SiSQS1 and SiSQS2 and the content of β-sitosterol, the correlation coefficients were 0.92 and 0.89, respectively. Conclusion: A locus mutation of amino acid residues in 242E/D between SiSQS1 and SiSQS2 may influence the expression of SiSQS, and there may exist the functional differences in catalytic activity and the accumulation of β-sitosterol. The study will provide technical support and lay a theoretical foundation for studying the accumulated mechanism of β-sitosterol regulated by SQS in S. involucrata cells.

Cloning and sequence analysis of squalene synthase gene from Trichosanthes rubriflos / 中草药

Objective: To clone the full length cDNA encoding squalene synthase (SS) from Trichosanthes rubriflos and to carry on its sequence analysis, so as to lay the foundation for the further study on the positively selected sites and function correlation analysis of SS which is the key enzyme for triterpene synthesis pathway. Methods: According to the cDNA comparison on SS gene from Gynostemma pentaphyllum and Siraitia grosvenorii, 5'-upstream degenerate primers of the cDNA of SS gene from T. rubriflos were designed and the full length cDNA of SS gene from T. rubriflos was amplified by 3'RACE kit. Results: The full length cDNA of SS gene from T. rubriflos composed of 1 466 nucleotides was obtained. The open reading frame (ORF) of SS gene from T. rubriflos was 1 254 bp in length, corresponding to a predicted polypeptide of 417 amino acid residues. The results of homologous alignment analysis in GenBank demonstrated that the cDNA sequence of SS gene from T. rubriflos had 78%-94% similarity on the nucleotide sequence compared with SS from known plant and 74%-93% similarity on the deduced amino acid sequence compared with SS gene from other plants. Conclusion: The full length cDNA of SS gene from T. rubriflos has been cloned, which not only lays a foundation for the further study on the gene expression, gene structure, and gene mutation, but also provides the important data base for the association study between the positively selected sites and function correlation analysis of which is the key enzyme for triterpene synthesis pathway.

Cloning and identification of two squalene synthase genes from Salvia miltiorrhiza / 中草药

Objective: To clone and identify the squalene synthase genes in Salvia miltiorrhiza. Methods: The primers were designed based on the predication result of S. miltiorrhiza genome genes and two squalene synthase genes (SmSQS1 and SmSQS2) from S. miltiorrhiza were amplified via RT-PCR method. Some bioinformatic methods and softwares were used for the gene structure analysis, the analyses of sequence homology and protein conserved domains of the two gene encoding polypeptides were carried. Real-time quantitative PCR (RT-qPCR) method was used for the analysis of gene expression patterns. Results: The two squalene synthase genes of S. miltiorrhiza were obtained by RT-PCR method. The encoded polypeptide of the two genes has the related domains and a conserved motif for squalene synthases. The two genes had the different exon/intron characteristics and the different tissue-specific and time-specific expression patterns. Conclusion: There are two squalene synthase genes existing in the genome of S. miltiorrhiza, which have the different gene structures and expression patterns, and probably play the different roles in the biosynthesis process of steroids and triterpenoids in S. miltiorrhiza.

Gene cloning of squalene synthase in Alisma orientalis and its bioinformatic analysis / 中草药

Objective: To clone the full-length cDNA encoding squalene synthase (SS), a key enzyme of protostane type triterpenes biosynthesis, from Alisma orientalis and to perform bioinformatic analysis. Methods: With the total RNA as template, the full-length cDNA of SS in A. orientalis was cloned via homology-based cloning approach and rapid amplification of cDNA ends technique. The bioinformatics of the cloning SS gene was analyzed by DNAMAN and ExPASy online analysis. Results: The full-length cDNA (1 577 bp) of SS gene was obtained (GenBank accession number JX866770), with an open reading frame of 1 230 bp, encoding 409 amino acid polypeptides, which had higher homology with the known SS in other medicinal species. The calculated relative molecular mass was 4.68 × 104, the isoelectric point was 5.97, and there was no signal peptide in SS. The deduced protein sequence exhibited two conserved domains rich in Asp (DXXDD). Conclusion: The cDNA encoding SS from A. orientalis is cloned and reported for the first time. This work provides a foundation for exploring the biosynthetic pathway of protostane type triterpenes in A. orientalis and their applications in bioengineering.

Correlation between ginsenoside accumulation and SQS and SQE gene expression in different organs of Panax quinquefolius / 中草药

Objective: To examine the biological accumulation of total ginsenosides and their monomers, and determine their relationships with the expression of squalene synthase (SQS) and squalene epoxidase (SQE) genes that are involved in the ginsenoside biosynthetic pathway in different organs of Panax quinquefolius. Methods: Fourteen organs of four year-old P. quinquefolius were used as materials. Total ginsenosides were extracted using the Soxhlet ginsenoside extraction method, and the contents of total ginsenosides and their monomers Rg1, Re, Rb1, Rc Rb2 and Rd in the organs were determined by the Vanillin-sulfuric Colorimetry and HLPC methods, respectively. The expressions of the SQS and SQE genes in the organs were profiled by real-time quantitative PCR. Results: The biological accumulation of total ginsenosides and each of their monomers varied significantly (P<0.01) in different parts of P. quinquefolius.Except for ginsenoside monomer Rb 2, there were significantly positive correlations between total ginsenoside and monomers Re, Rg1, Rb1 and Rd (P<0.01). The expressions of both SQS and SQE genes were extremely significantly different among the 14 plant parts (P<0.01) and significantly positively correlated with the biological accumulation of total ginsenoside and monomers, Re, Rg 1, Rb1 and Rd (P<0.05). Conclusion: The results indicate that the SQS and SQE genes play the important roles in the biosynthesis of total gingenosides and their monomers.

Ergosterol biosynthesis and drug development for Chagas disease

This article presents an overview of the currently available drugs nifurtimox (NFX) and benznidazole (BZN) used against Trypanosoma cruzi, the aetiological agent of Chagas disease; herein we discuss their limitations along with potential alternatives with a focus on ergosterol biosynthesis inhibitors (EBI). These compounds are currently the most advanced candidates for new anti-T. cruzi agents given that they block de novo production of 24-alkyl-sterols, which are essential for parasite survival and cannot be replaced by a host's own cholesterol. Among these compounds, new triazole derivatives that inhibit the parasite's C14± sterol demethylase are the most promising, as they have been shown to have curative activity in murine models of acute and chronic Chagas disease and are active against NFX and BZN-resistant T. cruzi strains; among this class of compounds, posaconazole (Schering-Plough Research Institute) and ravuconazole (Eisai Company) are poised for clinical trials in Chagas disease patients in the short term. Other T. cruzi-specific EBI, with in vitro and in vivo potency, include squalene synthase, lanosterol synthase and squalene epoxidase-inhibitors as well as compounds with dual mechanisms of action (ergosterol biosynthesis inhibition and free radical generation), but they are less advanced in their development process. The main putative advantages of EBI over currently available therapies include their higher potency and selectivity in both acute and chronic infections, activity against NFX and BZN-resistant T. cruzi strains, and much better tolerability and safety profiles. Limitations may include complexity and cost of manufacture of the new compounds. As for any new drug, such compounds will require extensive clinical testing before being introduced for clinical use, and the complexity of such studies, particularly in chronic patients, will be compounded by the current limitations in the verification of true parasitological...

Cloning and Sequencing of Artemisia annua Squalene Synthase Gene and Its cDNA / 广州中医药大学学报

【Objective】 To clone the squalene synthase gene of Artemisia annua L. for improving the quality and production of Artemisia annua L. by genetic engineering. 【Methods】 PCR amplification, RT-PCR amplification, ligation of the target fragment with a T-vector and sequence analysis of the interested gene were performed. 【Results】 An expected 3590 bp fragment was amplified by PCR and an expected 1257 bp fragment was amplified by RT-PCR. The two cloned fragments were identified by PCR and restriction enzyme digestion respectively. The preliminary sequence data indicated that the results obtained were similar to that from GenBank, and the difference was only found in several base pairs. 【Conclusion】 The squalene synthase gene and cDNA of Artemisia annua L. were successfully cloned and sequenced.

Cloning of Squalene Synthase Gene from Saccharomyces cerevisiae and Construction of Yeast Expression Vector / 广州中医药大学学报

Objective To lay the foundation for studying the synthesis of artemisinin in microorganism,squalene synthase(SS) gene,a key enzyme gene from Saccharomyces cerevisiae,was cloned and a yeast expression vector was constructed.Methods After amplification of SS gene by polymerase chain reaction(PCR),ligation to T-vector and analysis of the cloned sequence,enzyme digestion and reconfirmation of the target gene,the antisense yeast expression vector was constructed by inverted insertion of the target gene into a yeast expression vector,pGAPZ?A,and digested with two restriction enzymes for vertifying the recombinant.Results The length of SS gene was 1335bp.The preliminary sequence data indicated that SS gene obtained from the experiment had a high sequence homology with that from GenBank,except for a few base pairs.The antisense yeast expression vector has been constructed and vertified by digesting with two enzymes.Conclusion SS gene from Saccharomyces cerevisiae has been successfully cloned and sequenced.An antisense yeast expression vector has been also constructed.

Gene targeting of squalene synthase in Artemisia annua / 中草药

Objective To increase artemisinin yield in transgenic Artemisia annua plants by regulating metabolic affluxion through metabolic pathway engineering. Methods The gene targeting vector was constructed by squalene synthase (SS) gene of A. annua, green fluorescent protein (GFP) gene, and cytosine deaminase (CodA) gene, and the vector was introduced into Agrobacterium tumefaciens by freeze-thawing procedure. A. annua was transformed through Leaf Disk method and regenerated transgenic plants were screened by the “Step-by-Step Selection”. Results Among the transgenic A. annua plants emitting green fluorescence after expression of GFP gene, the exogenous GFP gene rather than endogenous SS gene was detected in one transgenic plant by PCR as well as hybridization of PCR products. The preliminary data showed that the wild-type SS gene was replaced by mutated SS gene in the transgenic A. annua plant. Conclusion Gene targeting of squalene synthases of A. annua is successful.