Cloning and Expression Analysis of Squalene Epoxidase Gene in Poria cocos / 中国实验方剂学杂志

ObjectiveTo clone squalene epoxidase （SE）， a potential key rate-limiting enzyme involved in the synthesis pathway of Poria cocos triterpenes， from P. cocos and analyze for bioinformatics and expression. MethodThe total RNA was extracted by the kit and reverse-transcribed to cDNA. Specific primers were designed， and the cDNA was used as a template for cloning the SE gene， which was analyzed for bioinformatics. The expression of P. cocos qualene epoxidase（PcSE） was examined by Real-time polymerase chain reaction（Real-time PCR） in P. coco Shenzhou No. 10， Xiangjing 28， and 5.78 strains. ResultThe full length of PcSE is 1 571 bp， containing four exons and three introns. The obtained CDS sequence is 1 413 bp， encoding 470 amino acids. This protein is a hydrophobic protein with no signal peptide structure and has two transmembrane structural domains with a FAD/NAD （P） binding domain and SE structural domain localized to the mitochondrial membrane and the plasma membrane. The homologous sequence alignment with fungi of the Poriferae family is 80.92%， and the phylogenetic tree shows that PcSE protein is most closely related to P. cocos from the US. The results of Real-time PCR showed that the PcSE was expressed in all three strains， with the highest expression in 5.78 strain， and there was no significant difference in PcSE expression among the three strains. ConclusionFor the first time， the PcSE gene was cloned and analyzed from P. cocos， providing a basis for further research on the function of PcSE and the analysis of P. cocos triterpene biosynthesis pathway.

Screening novel inhibitors of squalene epoxidase and their effects on hepatocellular carcinoma / 药学学报

Squalene epoxidase (SQLE) is a potential target for the treatment of liver cancer. Bioinformatics analysis indicated that the high expression of SQLE was closely related to the clinical stage and poor prognosis of patients with liver cancer. However, the existing inhibitors against SQLE 195 tyrosine residue (Y195) cannot be used clinically due to severe side effects. In this study, 35 small-molecule compounds targeting SQLE 335 tyrosine residue (Y335) were selected by computer virtual screening. Combined with MTT assay, 3 candidate compounds (19#, 31# and 35#) with significant inhibitory effects on the proliferation of Huh7 cell line were obtained. Further studies showed that these 3 compounds could inhibit the migration of Huh7 cells, reduce the contents of total and free cholesterol, up-regulate the expression of tumor suppressor gene PTEN, and down-regulate the expression of PI3K and AKT proteins. The results showed that the novel inhibitors 19#, 31# and 35# targeting SQLE Y335 could reduce cholesterol content, inhibit the proliferation and migration of Huh7, thus playing an anti-liver cancer role.

Construction and optimization of squalene epoxide synthetic pathway in Escherichia coli / 生物工程学报

Triterpenoids are a class of natural products of great commercial value that are widely used in pharmaceutical, health care and cosmetic industries. The biosynthesis of triterpenoids relies on the efficient synthesis of squalene epoxide, which is synthesized from the NADPH dependent oxidation of squalene catalyzed by squalene epoxidase. We screened squalene epoxidases derived from different species, and found the truncated squalene epoxidase from Rattus norvegicus (RnSETC) showed the highest activity in engineered Escherichia coli. Further examination of the effect of endogenous cytochrome P450 reductase like (CPRL) proteins showed that overexpression of NADH: quinone oxidoreductase (WrbA) under Lac promoter in a medium-copy number plasmid increased the production of squalene epoxide by nearly 2.5 folds. These results demonstrated that the constructed pathway led to the production of squalene epoxide, an important precursor for the biosynthesis of triterpenoids.

Cloning and Prokaryotic Expression of Squalene Epoxidase Gene from Panax vietnamensis var. fuscidiscus / 中国实验方剂学杂志

Objective: To clone the squalene epoxidase genes of Panax vietnamensis var. fuscidiscus(PvfSE),and perform bioinformatics analysis and prokaryotic expression. Method: Total RNA was extracted from root of P. vietnamensis var. fuscidiscus by trizol method, and reverse-transcribed into first stand of cDNA. Specific primers for PvfSE cloning were designed according to the transcriptome data of P. vietnamensis var. fuscidiscus,and the cDNA sequence of PvfSE gene was isolated. Bioinformatics of PvfSE was analyzed by relevant software. The prokaryotic expression vector pMal-c2X-PvfSE was built to express recombinant protein in Escherichia coli cells. Result: The PvfSE gene contained a 1 887 bp open reading frame,encoding a predicted protein of 628 amino acids. The calculated molecular weight was 68.8 kDa,the theoretical isoelectric point was 9.28,the aliphatic index was 95.18,the grand average of hydropathicity was -0.060, and the instability index was 40.36. The protein was unstable. Bioinformatics analysis showed that PvfSE had two transmembrane domains and no signal peptide. PvfSE was most likely to be located in chloroplast or cytoplasmic membrane. PvfSE was a mixed protein with FAD/NAD(P) binding domain and squalene epoxidase domain. Sequence alignment and phylogenetic analysis demonstrated that PvfSE had a relatively close relationship with CpSE1,CpSE3,OsSE1 and OsSE2,which was involved in the biosynthesis of triterpene saponins in Cucurbita pepo and Ononis spinosa. In addition,PvfSE protein was expressed in E. coli. Conclusion: In this study,PvfSE gene was cloned and expressed in BL21(DE3),which lays a foundation for the further study on the gene functions of PvfSE and the biosynthetic pathway of triterpenoid saponins in P. vietnamensis var. fuscidiscus.

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, bioinformatics, and expression analysis of squalene epoxidase in Antrodia cinnamomea / 中草药

Objective To clone the squalene epoxidase gene of Antrodia cinnamomea (AcSE) and analyze the bioinformatics and expression of the gene. Methods AcSE was cloned by rapid-amplification of cDNA ends (RACE) from cDNA of A. cinnamomea. The physical and chemical properties of AcSE protein were analyzed, and its secondary structure, tertiary structure, and function were predicted by using bioinformatics analysis. The expression of AcSE in mycelium and fruit body of A. cinnamomea at different culture time was detected by using quantitative real-time PCR (qRT-PCR). Results The full-length cDNA sequence of AcSE were 1 446 bp (Genbank: KT070558), encoding a 481-amino-acid polypeptide. The molecular weight of AcSE was 53 300 and pI was 6.36. Domain analysis results showed that AcSE had three transmembrane domains without coiled-coil structure, and hydrophobic and hydrophilic regions existed alternately. The gDNA sequence of AcSE was 1 607 bp, contained four exons and three introns. A gene expression analysis by relative qRT-PCR showed that the highest expression level of AcSE was in mycelia incubated for 7 d of A. cinnamomea, and it was 7.89 times than that in fruiting body, and a gradual decline was observed with the extension of the culture time. Conclusion Gene AcSE was firstly cloned from A. cinnamomea, and it would lay a foundation for exploring the mechanism of terpenoid biosynthesis in A. cinnamomea.

Construction of prokaryotic expression and over-expression vectors of squalene epoxidase gene from Sanghuangporus baumii / 中草药

Objective To obtain prokaryotic expression and over-expression vectors of squalene epoxidase (SE) gene from Sanghuangporus baumii. Methods The entire protein-coding cDNA of SE was cloned into the expression vector pET-32a (+). Then the recombinant plasmid was transformed into Escherichia coli BL21 (DE3) cells. SDS-PAGE was used to investigate the situation of expression after IPTG induction for 2-10 h. Additionally, primers were designed according to the gpd promoter sequence of Lentinula edodes in GenBank, and the gpd promoter fragment was obtained by PCR. Subsequently, the plant binary expression vector pCAMBIA1301 was selected as the basic vector, and then the 35 S promoter replaced with L. edodes gpd promoter through enzyme digestion and connection. Finally, the coding region of SE was cloned to the downstream of the gpd promoter to construct over-expression vectors. Results The prokaryotic expression vector pET-32a-SE was successfully obtained. SDS-PAGE results showed a significant protein band was found in the vicinity of the relative molecular weight of approximately 55 000, consistent with molecular weight of the predicted protein. Moreover, the over-expression vector pCAMBIA1301-gpd-gpd-SE was constructed successfully through different detection ways. Conclusion These results lay the foundation for the further study of SE in triterpenoid biosynthesis pathway of S. baumii.

Cloning and expression analysis of squalene epoxidase genes from Siraitia grosvenorii / 中国中药杂志

Siraitia grosvenorii, vine plant of Cucurbitaceae family, has been used as natural sweetener and folk medicine. The major components and sweet substances are both known as mogrosides which are cucurbitane-type tetra-triterpenoids. Squalene epoxidase (SQE) has been generally recognized as the common rate-limiting enzyme in triterpenes and phytosterols, catalyzing into their common precursor 2,3-oxidosqualene (OS); however, in the biosynthesis of mogrosides, the precursor was 2,3,22,23-dioxidosqualene (DOS) instead of OS. To explore the specific SQE in S. grosvenorii, we cloned two full-length SQEs (SgSQE1, SgSQE2), performed bioinformatic analysis, analyzed the expression patterns in different periods of fruits by Real-time PCR, and induced the prokaryotic expressions. Finally, the interactive sites between SQE and substrate were predicted by docking, which would provide evidence for SQE gene function study of mogrosides and also lay foundation for triterpene biosynthesis in other plants. SgSQE1 and SgSQE2 both encoded predicted proteins of 524 amino acids, and shared 84% identity to each other at residues level, but had high specificity at N-terminal region. They both accumulated in fruits, but with different patterns, SgSQE1 increased rapidly and reached the highest level at 15 d, which had identical co-expression pattern with cucurbitadienol synthase (CS). SgSQE2 had a relatively constant level. The docking results showed that predicted proteins of SgSQE1 and SgSQE2 can interact with OS, with different contact sites (R348 for SgSQE1, H349 for SgSQE2). The recombinant proteins had no activities by prokaryotic expression, which were caused by transmembrane regions. However, all the results strongly suggested that SgSQEs were both involved in secondary metabolites biosynthesis in S. grosvenorii. SgSQE1 might be involved in mogrosides biosynthesis and SgSQE2 might participate in other cucurbitane-type triterpenes or phytosterols biosynthesis.

Antifungal Activity of Aspidin BB from Dryopteris fragrans against Trichophyton rubrum Involved Inhibition of Ergosterol Biosynthesis / 中草药·英文版

Objectives Aspidin BB, a typical phloroglucinol derivative from Dryopteris fragrans, possesses significant antifungal property. This study aimed to investigate potential mechanism of antifungal activity of Aspidin BB against Trichophyton rubrum which is the most common pathogens responsible for chronic dermatophytosis. Methods The minimum inhibitory concentration (MIC) of Aspidin BB against strains was determined by broth microdilution. The effects of Aspidin BB on ergosterol biosynthesis were investigated by content determination based on UPLC method. Besides, the effects of drugs on squalene epoxidase (SE) in T. rubrum cell membrane were analyzed. Results MIC value of Aspidin BB against T. rubrum was 25.0 μg/mL. Aspidin BB reduced ergosterol content significantly, but no notable effect on squalene epoxidase activity. Conclusion The results suggested that Aspidin BB inhibited ergosterol biosynthesis. However, it was not squalene epoxidase but other components may sever as possible targets in ergosterol biosynthesis pathway.

Cloning and expression of squalene epoxidase from Paris polyphylla var. yunnanensis / 中草药

Objective: To clone and express a full-length cDNA encoding squalene epoxidase which is key to the biosynthetic pathway of triterpenoid saponins in Paris polyphylla var. yunnanensis. Using real-time PCR method to detect the relative content of SE1 gene and SE2 gene from the cDNA sample. Methods: Total RNA was extracted from the roots of P. polyphylla var. yunnanensis and transcription was reversed. Using the reversed transcription of cDNA as template, specific primer was designed according to the transcriptome data about two groups of SE gene sequence from the HiSeq2500 sequencing platform, and then SE gene was cloned. The production was inserted to pEASY-T1 Simple Cloning Vector, and pEASY-E1-SE expression vector was built after sequencing appraisal right. The ArtMedia protein Expression/Amp+ medium was used to induce expression automatically. Using real-time PCR method to detect the relative contents of SE1 gene and SE2 gene from the cDNA sample. Results: Two SE genes of P. polyphylla var. yunnanensis were obtained, which were named as ppSE1 and ppSE2, repectively. cDNA was 1 598 bp of ppSE1 and 1 509 bp of ppSE2, respectively. The full length for ppSE1 was 1 932 bp, length of ORF was 1 578 bp, coding 525 AA; The full length for ppSE2 was 1 828 bp, length of ORF was 1 548 bp, coding 515 AA. Fluorescence quantitative PCR results showed that the expression of ppSE1 and ppSE2 genes had significant differences in stem and leaf, and the expression of ppSE1 was most pronounced in the leaf. Enzyme digestion and sequencing results showed that the prokaryotic expression vector pEASY-E1-SE was built successfully. SDS-PAGE analysis showed that two fusion proteins of SE genes were induced expression successfully in BL21 (DE3) express competent cells. Conclusion: The SE gene of P. polyphylla var. yunnanensis has been cloned, and the SE protein with biological activity in vitro has been obtained. The ppSE1 and ppSE2 genes have different expression patterns in P. polyphylla var. yunnanensis, and play a different role in the synthesis of secondary metabolites.

Cloning and ectopic expression analysis of squalene epoxidase 1 gene from Pseudostellaria heterophylla / 中草药

Objective: To clone the full-length cDNA encoding squalene epoxidase 1 (SQE1), a key enzyme of triterpenes biosynthesis, from Pseudostellaria heterophylla and to perform functional analysis. Methods: With the total RNA as template, the full-length cDNA of SQE1 in P. heterophylla was cloned via RT-PCR and rapid amplification of cDNA ends (RACE) techniques. The bioinformatics of the cloned SQE1 gene was performed. The target gene was transfered into tobacco by Agrobacterium-mediated transformation. Results: The full-length cDNA (2 038 bp) of SQE1 gene was obtained with an open reading frame of 1 554 bp, encoding 517 amino acid polypeptides, which had higher homology with the known SQEs in other medicinal species. The calculated relative molecular mass was 5.67 × 104, the isoelectric point was 8.8. The deduced protein sequence exhibited FAD-binding domains and four transmembrane regions. The content of total triterpenes was increased in transgenic tobacco plants. Conclusion: This is the first report that the full-length cDNA encoding SQE1 from P. heterophylla is cloned. The ectopic expression of SQE1 could promote to increase the content of total triterpenes in transgenic plant. This work provides a foundation for exploring the biosynthetic pathway of triterpenes in P.heterophylla and their applications in bioengineering.

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.

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.

Molecular Cloning and Expression of Squalene Epoxidase from a Medicinal Plant, Bupleurum chinense / 中草药·英文版

Objective: In plant, squalene epoxidase (SE) catalyzes the first oxygenation step in the biosynthetic pathway of triterpenoid and phytosterol, representing one of the rate-limiting enzymes in this pathway. Bupleurum chinense is an important medicinal herb with its major active constituents such as triterpenoid saponins and saikosaponins. In order to obtain the series of enzymatic genes involved in saikosaponin biosynthesis, a cDNA of SE, designated BcSE1, was cloned from B. chinense. Methods: The BcSE1 gene was cloned by homology-based PCR and 5'/3' RACE methods from the adventitious roots of B. chinense. The physical and chemical parameters of BcSE1 protein were predicted by protparam. In order to discover hints in amino acid sequences on the dominant functions in the biosynthesis of saponin or phytosterol, sequences of SE from other plants were downloaded from NCBI for sequences alignment and phylogenetic analysis. BcSE1 was cloned into a yeast mutant KLN1 (MATa, erg1::URA3, leu2, ura3, and trp1) to verify the enzyme activity of BcSE1. Additionally, the tissue-specific expression and methyl jasmonate (MeJA) inducibility of BcSE1 were investigated using quantitative real-time PCR. Results: The predicted protein of BcSE1 is highly similar to SEs from other plants sharing amino acid sequence identities of up to 88%. The BcSE1 can functionally complement with yeast SE gene (ERG1) when expressed in the KLN1 mutant (MATa, erg1::URA3, leu2, ura3, and trp1). Using as controls with β-amyrin synthase (β-AS) which is presumed to catalyze the first committed step in saikosaponin biosynthesis and a cycloartenol synthase (CAS) relating to the phytosterol biosynthesis, the transcript of BcSE1 was significantly elevated by MeJA in adventitious roots of B. chinense and the transcript of BcSE1 was most abundant in the fruits and flowers of plants, followed by that in the leaves and roots, and least in stems. Conclusion: It is the first time to illustrate the molecular information of SE in B. chinense and to clone the full-length SE gene in plants of genus Bupleurum L.

Cloning and sequence analysis of squalene epoxidase gene from Inonotus baumii / 中草药

Objective: To clone the full-length cDNA encoding squalene epoxidase (SE), which is the key enzyme involved in the triterpenoid biosynthesis pathway in Inonotus baumii, and analyze its bioinformates. Methods: Taking total RNA as template, the full length cDNA and DNA of SE in I. baumii was cloned through RT-PCR and the rapid amplification of cDNA ends (RACE) technique. The bioinformatics of SE gene were analyzed by ExPASy on line. Results: Sequence analysis showed that it consisted of 2145 bp with an open reading frame (ORF) of 1 452 bp, encoding 483 amino acid polypeptides. SE gene contained six exons and five introns. The relative molecular mass of SE calculated was 5.3 × 104, the isoelectric point (pI) was 8.41, and there was no signal peptide in SE. Conclusion: It is the first report that the cDNA encoding SE from I. baumii is cloned. This work provides a scientific basis for exploring the triterpenoid biosynthesis pathway of the medicinal ingredient and improving its quality in I. baumii.

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.