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1.
Chinese Journal of Biotechnology ; (12): 443-459, 2022.
Article in Chinese | WPRIM | ID: wpr-927721

ABSTRACT

Triterpenoids are one of the most diverse compounds in plant metabolites, and they have a wide variety of physiological activities and are of important economic value. Oxidosqualene cyclases catalyze the cyclization of 2, 3-oxidosqualene to generate different types of sterols and plant triterpenoids, which is of great significance to the structural diversity of natural products. However, the mechanism of the diversified cyclization of 2, 3-oxidosqualene catalyzed by oxidosqualene cyclases remains unclear. This review summarized the research progress of oxidosqualene cyclases from the aspects of catalytic function, molecular evolutionary relationship between genes and proteins, protein structure, molecular simulation and molecular calculations, which may provide a reference for protein engineering and metabolic engineering of triterpene cyclase.


Subject(s)
Intramolecular Transferases/metabolism , Metabolic Engineering , Plants/genetics , Squalene/chemistry , Triterpenes
2.
China Journal of Chinese Materia Medica ; (24): 94-102, 2021.
Article in Chinese | WPRIM | ID: wpr-878916

ABSTRACT

This study cloned the transcription factor gene PnbHLH which held an open reading frame of 966 bp encoding 321 amino acids. This study constructed the overexpression vector of transcription factor PnbHLH of Panax notoginseng. The combination of PnbHLH overexpression and RNAi of the key enzyme gene PnCAS involved in the phytosterol biosynthesis was achieved in P. notoginseng cells, thus exploring the biosynthetic regulation of P. notoginseng saponins(PNS) by the synergistic effect of PnbHLH overexpression and PnCAS RNAi. The results showed that the PnbHLH transcription factor interacted with the promoters of key enzyme genes PnDS, PnSS and PnSE in the biosynthetic pathway of PNS, and then regulated the expression levels of key enzyme genes and affected the biosynthesis of saponins indirectly. Further study indicated that the synergistic effect of PnbHLH overexpression and PnCAS RNAi was a more effective approach to regulate the biosynthesis of saponins. Compared with the wild type and PnCAS RNAi cells of P. notoginseng, the contents of total saponins and monomeric saponins(Rd, Rb_1, Re, Rg_1 and R_1) were increased to some extent in the cell lines of PnbHLH overexpression and PnCAS RNAi. This indicated that the two ways of forward regulation and reverse regulation of saponin biosynthesis showed superposition effect. This study explored a more rational and efficient regulation strategy of PNS biosynthesis based on the advantages of multi-point regulation of transcription factors as well as the down-regulation of by-product synthesis of saponins.


Subject(s)
Intramolecular Transferases , Panax notoginseng , RNA Interference , Saponins , Transcription Factors/genetics
3.
China Journal of Chinese Materia Medica ; (24): 3967-3973, 2019.
Article in Chinese | WPRIM | ID: wpr-1008313

ABSTRACT

Lanosterol synthase( LS) is a key enzyme involving in the mevalonate pathway( MVA pathway) to produce lanosterol,which is a precursor of ganoderma triterpenoid. And the transcriptional regulation of LS gene directly affects the content of triterpenes in Ganoderma lucidum. In order to study the transcriptional regulation mechanism of LS gene,yeast one-hybrid technique was used to screen the transcription regulators which interact withthe promoter of LS. The bait vector was constructed by LS promoter,then the vector was transformed yeast cells to construct bait yeast strain. One-hybrid c DNA library was constructed via SMART technology. Then the c DNA and p GADT7-Rec vector were co-transformed into the bait yeast strain to screen the upstream regulatory factors of the promoter region of LS by homologous recombination. Total of 23 positive clones were screened. After sequencing,blast was performed against the whole-genome sequence of G. lucidum. As a result,8 regulatory factors were screened out including the transcription initiation TFIIB,the alpha/beta hydrolase super family,ALDH-SF superfamily,60 S ribosomal protein L21,ATP synthase β-subunit,microtubule associated protein Cript,prote asome subunit β-1,and transaldolase. Until now,the regulation effect of these 8 regulatory factors in G.lucidum has not been reported. This study provides candidate proteins for in-depth study on the expression regulation of LS.


Subject(s)
Gene Library , Intramolecular Transferases/metabolism , Reishi/genetics , Saccharomyces cerevisiae , Transcription Factors/metabolism
4.
China Journal of Chinese Materia Medica ; (24): 1341-1349, 2019.
Article in Chinese | WPRIM | ID: wpr-774550

ABSTRACT

In this study, the synthetic pathway of β-amyrin was constructed in the pre-constructed Saccharomyces cerevisiae chassis strain Y0 by introducing β-amyrin synthase from Glycyrrhiza uralensis, resulting strain Y1-C20-6, which successfully produced β-amyrin up to 5.97 mg·L~(-1). Then, the mevalonate pyrophosphate decarboxylase gene(ERG19), mevalonate kinase gene(ERG12), 3-hydroxy-3-methylglutaryl-CoA synthase gene(ERG13), phosphomevalonate kinase gene(ERG8) and IPP isomerase gene(IDI1)were overexpressed to promoted the metabolic fluxto the direction of β-amyrin synthesis for further improving β-amyrin production, resulting the strain Y2-C2-4 which produced β-amyrin of 10.3 mg·L~(-1)under the shake flask fermentation condition. This is 100% higher than that of strain Y1-C20-6, illustrating the positive effect of the metabolic engineering strategy applied in this study. The titer of β-amyrin was further improved up to 157.4 mg·L~(-1) in the fed-batch fermentation, which was almost 26 fold of that produced by strain Y1-C20-6. This study not only laid the foundation for the biosynthesis of β-amyrin but also provided a favorable chassis strain for elucidation of cytochrome oxidases and glycosyltransferases of β-amyrin-based triterpenoids.


Subject(s)
Fermentation , Glycyrrhiza uralensis , Genetics , Industrial Microbiology , Intramolecular Transferases , Genetics , Metabolic Engineering , Oleanolic Acid , Saccharomyces cerevisiae , Metabolism
5.
Acta Pharmaceutica Sinica ; (12): 118-122, 2015.
Article in Chinese | WPRIM | ID: wpr-251807

ABSTRACT

The cyclization of 2,3-oxidosqualene is the key branch point of ergosterol and triterpenoid biosynthesis. Downregulation of 2,3-oxidosqualene metabolic flux to ergosterol in Saccharomyces cerevisiae may redirect the metabolic flux toward the triterpenoid synthetic pathway. In our study, primers were designed according to erg7 gene sequence of S. cerevisiae. Three fragments including 5' long fragment, 5' short fragment and erg7 coding region fragment were amplified by PCR. 5' long fragment consists of the promoter and a part of erg7 coding region sequence. 5' short fragment consists of a part of promoter and a part of erg7 coding region sequence. These fragments were inserted reversely into pESC-URA to construct antisense expression plasmids. The recombinant plasmids were transformed into S. cerevisiae INVSc1 and recombinant strains were screened on the nutritional deficient medium SD-URA. The erg7 expression level of recombinant strains, which harbored antisense expression plasmid of erg7 coding region, was similar to that of INVScl by semi-quantitative PCR detection. But erg7 expression level of recombinant strains, which harbored 5' long antisense fragment and 5' short antisense fragment, was significantly lower than that of the control. The results of TLC and HPLC showed that the ergosterol content of recombinant strains, which harbored 5' long antisense fragment, decreased obviously. The ergosterol contents of the others were almost equal to that of INVSc1. Lanosterol synthase gene expression was downregulated by antisense RNA technology in S. cerevisiae, which lays a foundation for reconstructing triterpenoid metabolic pathway in S. cerevisiae by synthetic biology technology.


Subject(s)
DNA Primers , Down-Regulation , Gene Expression , Intramolecular Transferases , Genetics , Metabolism , Plasmids , Polymerase Chain Reaction , RNA, Antisense , Saccharomyces cerevisiae , Genetics , Squalene , Metabolism , Transformation, Genetic
6.
Acta Pharmaceutica Sinica ; (12): 906-909, 2015.
Article in Chinese | WPRIM | ID: wpr-257048

ABSTRACT

β-Amyrin synthase (β-AS) genes of Glycyrrhiza uralensis from 6 different regions were analyzed by PCR-SSCP and sequenced, then the correlationship between β-AS SNP and regions of Glycyrrhiza uralensis were determined. According to the 1 coding single nucleotide polymorphism on the first exon of β-AS gene at 94 bp site, Glycyrrhiza uralensis could be divided into 3 genotypes. In these genotypes, the percentage of 94A type in genuine regions was much higher, and it had significant differences with the percentage in non-genuine regions (P < 0.001). The results of the experiment proved that different β-AS genotypes at 94 bp site from different regions may be one of the important reasons to result in the genuineness of Glycyrrhiza uralensis.


Subject(s)
Exons , Genotype , Glycyrrhiza uralensis , Classification , Genetics , Intramolecular Transferases , Genetics , Plant Proteins , Genetics , Polymorphism, Single Nucleotide , Polymorphism, Single-Stranded Conformational
7.
Chinese Journal of Biotechnology ; (12): 1690-1699, 2015.
Article in Chinese | WPRIM | ID: wpr-337466

ABSTRACT

Penicillin expandase, also known as deacetoxycephalosporin C synthase (DAOCS), is an essential enzyme involved in cephalosporin C biosynthesis. To evaluate the catalytic behaviors of penicillin expandase under high penicillin G concentration and to identify mutants suitable for industrial applications, the specific activities of wild-type DAOCS and several mutants with increased activities toward penicillin G were determined by HPLC under high penicillin G concentrations. Their specific activity profiles were compared with theoretical predictions by different catalytic dynamics models. We evaluated the specific activities of wild-type DAOCS and previous reported high-activity mutants H4, H5, H6 and H7 at concentrations ranging from 5.6 to 500 mmol/L penicillin G. The specific activities of wild-type DAOCS and mutant H4 increased as penicillin G concentration increased, but decreased when concentrations of substrate go above 200 mmol/L. Other mutants H5, H6 and H7 showed more complex behaviors under high concentration of penicillin G. Among all tested enzymes, mutant H6 showed the highest activity when concentration of penicillin G is above 100 mmol/L. Our results revealed that the substrate inhibition to wild-type DAOCS' by penicillin G is noncompetitive. Other DAOCS mutants showed more complex trends in their specific activities at high concentration of penicillin G (>100 mmol/L), indicating more complex substrate inhibition mechanism might exist. The substrate inhibition and activity of DAOCS mutants at high penicillin G concentration provide important insight to help select proper mutants for industrial application.


Subject(s)
Catalysis , Intramolecular Transferases , Genetics , Mutation , Penicillin G , Pharmacology , Penicillin-Binding Proteins , Genetics , Streptomyces , Genetics
8.
Acta Pharmaceutica Sinica ; (12): 734-741, 2014.
Article in English | WPRIM | ID: wpr-245020

ABSTRACT

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.


Subject(s)
Farnesyl-Diphosphate Farnesyltransferase , Genetics , Metabolism , Glycyrrhiza uralensis , Genetics , Intramolecular Transferases , Metabolism , Oleanolic Acid , Metabolism , Plant Proteins , Genetics , Recombinant Proteins , Metabolism , Saccharomyces cerevisiae , Metabolism
9.
Acta Pharmaceutica Sinica ; (12): 742-746, 2014.
Article in Chinese | WPRIM | ID: wpr-245019

ABSTRACT

Lanosterol synthase is encoded by the erg7 gene and catalyzes the cyclization of 2, 3-oxidosqualene, which is a rate-limiting step of the inherent mevalonate (MVA)/ergosterol metabolic pathway in Saccharomyces cerevisiae. The intermediate 2, 3-oxidosqualene is also the precursor of triterpenoids. Therefore, the cyclization of 2, 3-oxidosqualene is the key branch point of ergosterol and triterpenoids biosynthesis. Down-regulation of 2, 3-oxidosqualene metabolic flux to ergosterol in S. cerevisiae may redirect the metabolic flux toward the triterpenoid synthetic pathway reconstructed by the synthetic biology approach. To construct erg7 knockout cassette harboring the loxP-Marker-loxP element, long primers were designed, which were homologous to the sequences of both erg7 ORF and plasmid pUG66. The cassette was transformed into diploid wild strain INVSc1 by LiAc/SS Carrier DNA/PEG method and then erg7 gene haploid deficient mutant was obtained by homologous recombination. The results of semiquantitative PCR and real-time quantitative PCR revealed that erg7 expression level in erg7 gene haploid deficient mutant is one time lower than that in wild strain. The results of TLC and HPLC showed that the ergosterol content in deficient mutant decreased to 42% of that in wild strain.


Subject(s)
Chromatography, High Pressure Liquid , DNA Primers , Down-Regulation , Ergosterol , Metabolism , Haploidy , Intramolecular Transferases , Genetics , Polymerase Chain Reaction , Saccharomyces cerevisiae , Genetics , Squalene , Metabolism
10.
Electron. j. biotechnol ; 16(5): 6-6, Sept. 2013. ilus, tab
Article in English | LILACS | ID: lil-690466

ABSTRACT

Background: The perennial medicinal herb Dioscorea zingiberensis is a very important plant used for steroid drug manufacturing for its high level of diosgenin in rhizome. Although the stimulation of diosgenin accumulation by ethylene has been reported in a few of plant species, its regulation is not yet characterized at the molecular level, the underlying molecular mechanism remains elusive. Results: In this study, the effects of ethylene on diosgenin biosynthesis in in vitro cultures of D. zingiberensis were described. The results showed that, in samples treated with ethylene at concentration E3 (10(4) dilution of 40% ethephon), the diosgenin biosynthesis was significantly promoted in comparison with the control samples. Treatment with high concentrations of ethylene had inhibitory effect, whereas with low concentration of the gas elicitor brought about no detectable deleterious effect on the growth rate and diosgenin content of the cultures. The considerable increase of diosgenin level in in vitro cultured Dioscorea zingiberensis by ethylene application is accompanied by the concomitant increase of soluble proteins and chlorophyll content. The gene expressions of cycloartenol synthase and 3-hydroxy-3-methylglutaryl-CoA reductase but not of squalene synthase or farnesyl pyrophosphate synthase were up-regulated by applied ethylene. Conclusions: Our results suggest that ethylene treatment enhanced diosgenin accumulation via up-regulation of the gene expressions of cycloartenol synthase and 3-hydroxy-3-methylglutaryl-CoA reductase.


Subject(s)
Intramolecular Transferases/genetics , Intramolecular Transferases/metabolism , Dioscorea/metabolism , Hydroxymethylglutaryl CoA Reductases/genetics , Hydroxymethylglutaryl CoA Reductases/metabolism , In Vitro Techniques , RNA/isolation & purification , Gene Expression , Up-Regulation , Reverse Transcriptase Polymerase Chain Reaction , Dioscorea/growth & development , Dioscorea/genetics , Diosgenin/analysis , Ethylenes
11.
China Journal of Chinese Materia Medica ; (24): 1715-1719, 2012.
Article in Chinese | WPRIM | ID: wpr-338775

ABSTRACT

<p><b>OBJECTIVE</b>To clone and sequence the open reading frame of cycloartenol synthase (CAS) from Huperzia carinata.</p><p><b>METHOD</b>After searching the transcriptome dataset of H. carinata, one unique sequence containing oxide squalene cyclases domain was discovered. The primers were designed according to the cDNA sequence of CAS from the dataset. And then, the open reading frame of CAS was cloned by RT-PCR strategy with the template of mixed RNA extracted from root, stem and leaf of H. carinata. The bioinformatic analysis of this gene and its corresponding protein was performed.</p><p><b>RESULT</b>One unique sequence of CAS, named as HcCAS1 (GenBank accession number JN790125) , was cloned from H. carinata. The open reading frame of HcCAS1 consists of 2 474 bp, encoding one polypeptide with 757 amino acids.</p><p><b>CONCLUSION</b>This study cloned and analyzed CAS from H. carinata for the first time. The result will provide a foundation for exploring the mechanism of sterol biosynthesis in Huperziaceae plants.</p>


Subject(s)
Amino Acid Sequence , Cloning, Molecular , Computational Biology , Evolution, Molecular , Huperzia , Genetics , Intramolecular Transferases , Chemistry , Genetics , Metabolism , Models, Molecular , Molecular Sequence Data , Phylogeny , Protein Structure, Secondary
12.
China Journal of Chinese Materia Medica ; (24): 2041-2045, 2012.
Article in Chinese | WPRIM | ID: wpr-338708

ABSTRACT

<p><b>OBJECTIVE</b>To 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.</p><p><b>METHOD</b>Wound 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.</p><p><b>RESULT</b>When 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).</p><p><b>CONCLUSION</b>Endophytic 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.</p>


Subject(s)
Eleutherococcus , Chemistry , Metabolism , Microbiology , Endophytes , Physiology , Farnesyl-Diphosphate Farnesyltransferase , Genetics , Fungi , Physiology , Gene Expression Regulation, Enzymologic , Gene Expression Regulation, Plant , Intramolecular Transferases , Genetics , Saponins , Squalene Monooxygenase , Genetics
13.
Acta Pharmaceutica Sinica ; (12): 250-255, 2012.
Article in Chinese | WPRIM | ID: wpr-323049

ABSTRACT

This study is to reveal the correlation between CNVs of HMGR, SQS1, beta-AS gene and genuineness of liquorice. Real-time PCR was used to detect the copy number of HMGR, SQS1, beta-AS gene of liquorice. According to the results, the range of the copy number variation of HMGR gene was between 1 and 3, the copy number of SQS1 gene was 1 or 2, and the copy number of beta-AS gene was only 1. On the basis of the copy number of HMGR, SQS1 and beta-AS gene, there were five groups, type A (2 + 1 + 1), type B (1 + 1 + 1), type C (3 + 2 + 1), type D (2 + 2 + 1) and type E (3 + 1 + 1). There were two types, type A and type B, in Hangjinqi of Inner Mongolia, and the ratio of A to B was 1:1.3. There were also two types, type A and type B, in Chifeng of Inner Mongolia, and the ratio of A to B was 3:1. There were four types, type A, type B, type C and type D, in Yanchi of Ningxia province, and the ratio of A to B was 1:5.1. There were three types, type A, type B and type E, in Minqin of Gansu province, and the ratio of A to B was 2:1. So CNVs mainly existed in the liquorice from Ningxia and Gansu provinces. While the genetic background of liquorice from Hangjinqi of Inner Mongolia was stabilized. The results of the experiment proved that the correlation between CNVs and origins was one of the reasons of genuineness of liquorice.


Subject(s)
China , DNA Copy Number Variations , DNA, Plant , Genetics , Farnesyl-Diphosphate Farnesyltransferase , Genetics , Glycyrrhiza uralensis , Genetics , Hydroxymethylglutaryl CoA Reductases , Genetics , Intramolecular Transferases , Genetics , Real-Time Polymerase Chain Reaction
14.
Chinese Journal of Biotechnology ; (12): 1717-1723, 2010.
Article in Chinese | WPRIM | ID: wpr-351543

ABSTRACT

Glutamate-1-semiadhyde aminotransferase (GSAT) is an enzyme in the upstream biosynthetic pathway of uroporphyrinogen III that is the substrate of uroporphyrinogen III methyltransferase (UPMT), a novel red fluorescent protein. In order to detect the effect of overexpression of GSAT with UPMT on the fluorescent intensity in Escherichia coli, we amplified maize upmt gene by PCR and inserted into the first cistron of pET Duet-1 plasmid to create the vector pETU. The expressed UPMT was fused histidine tag at N terminus. We also amplified E. coli hemL gene encoding GSAT by PCR reaction, eliminated Nco I site within the hemL gene by site-directed mutagenesis and subcloned into pET-51b plasmid. The resultant hemL gene was inserted the second cistron of pETU plasmid to produce the vector pETeGU. The expressed GSAT has the extra Strep-TagII at N terminus. Compared to overexpression upmt gene alone, coexpression both genes did not resulted in the remarkable change in either the amount of the UPMT, as estimated by western blot analysis, or the constitution of red fluorescent materials, as shown by UV/visible light scanning analysis, but increased cellular level of the fluorescent material trimethylpyrrocorphin with the specific absorption at 354 nm. The red fluorescence emitted by the colonies cooverexpressing both enzymes completely disappeared after treated by 2 mmol/L gabaculine, the GSAT inhibitor, suggested that the recombinant GSAT may increase the cellular level of uroporphyrinogen III, and thus enhanced the red fluorescence of the E. coli cells conferred by the recombinant UPMT.


Subject(s)
Escherichia coli , Genetics , Metabolism , Genes, Plant , Genetic Vectors , Genetics , Intramolecular Transferases , Genetics , Luminescent Proteins , Genetics , Methyltransferases , Genetics , Mutagenesis, Site-Directed , Recombinant Proteins , Genetics , Zea mays , Genetics
15.
China Journal of Chinese Materia Medica ; (24): 813-816, 2010.
Article in Chinese | WPRIM | ID: wpr-281711

ABSTRACT

<p><b>OBJECTIVE</b>To analyze the correlation between content of glycyrrhizic acid and the single nucleotide polymorphism of beta-amyrin synthase (bAS) in Glycyrrhiza uralensis.</p><p><b>METHOD</b>glycyrrhizic acid content in 80 samples of the cultivated G. uralensis were determined by HPLC; According to the very significant level (P < 0.000 1), 80 samples in accordance with glycyrrhizic acid will be grouped by SAS 9.0; Using RT-PCR strategy to amplification the Open Reading Frame of beta-amyrin synthase with the template of total RNA extracted from roots of G. uralensis and then using DNAman to analyze the relationship between glycyrrhizic acid content and the single nucleotide polymorphism of beta-amyrin synthase (bAS).</p><p><b>RESULT</b>There exited two mutation sites 94 bp and 254 bp, G/A conversion occurred at 94 bp site, which belonged to a missense mutation. G/A conversion led to the corresponding amino acid conversion (Gly --> Asp); C/T conversion occurred at 254 bp site, which belonged to a synonymous mutation. According to sequence variation, the samples were divided into four genotypes: G-T genotype, A-T genotype, G/A-C genotype and G-T genotype.</p><p><b>CONCLUSION</b>A-T genotype, G/A-C genotype and G-T genotype are correlated with the high content of glycyrrhizic acid.</p>


Subject(s)
Genotype , Glycyrrhiza uralensis , Genetics , Metabolism , Glycyrrhizic Acid , Metabolism , Intramolecular Transferases , Genetics , Polymorphism, Single Nucleotide , Reproducibility of Results
16.
China Journal of Chinese Materia Medica ; (24): 2941-2944, 2010.
Article in Chinese | WPRIM | ID: wpr-260714

ABSTRACT

<p><b>OBJECTIVE</b>To analyze heterologous expression in Saccharomyces cerevisiae of two genotypes: beta-AS (A-T) genotype which is related to high content of glycyrrhizic acid and beta-AS(G-C) genotype which is related to low content of glycyrrhizic acid, and compare two different genotypes on the impact of beta-amyrin production in order to provide a foundation for licorice molecular breeding.</p><p><b>METHOD</b>The 2 289 bp fragment in plasmid pMD-19T encoding beta-amyrin synthase was subcloned into the yeast-Escherichia coli shuttle vector pY26, thus an expression recombinant plasmid PY-beta-AS containing target gene was constructed. The PY-beta-AS was introduced into defective mutant INVSc1 of S. cerevisiae by LiAc method, after induced by IPTG, the content of beta-amyrin was determined by GC-MS.</p><p><b>RESULT</b>GC-MS analysis demonstrates that the an occurring peak corresponding to beta-amyrin standards was detected with the same retention time, which is absent in the cell transform with empty vector. Results showed the peak was beta-amyrin and the percentage of beta-amyrin in two genotypes: beta-AS (A-T) genotype and beta-AS (G-C) genotype were 19.08% and 1.40%, respectively. Thus the beta-amyrin synthase exhibited the activity of catalyzing 2, 3-oxidosqualene to beta-amyrin.</p><p><b>CONCLUSION</b>The catalytic efficiency of beta-AS(A-T) genotype is higher than that of beta-AS(G-C) genotype, which can lay the foundation for licorice molecular breeding.</p>


Subject(s)
Catalysis , Cloning, Molecular , Genotype , Glycyrrhiza uralensis , Chemistry , Genetics , Intramolecular Transferases , Chemistry , Genetics , Metabolism , Plant Proteins , Chemistry , Genetics , Metabolism , Polymorphism, Genetic , Recombinant Proteins , Chemistry , Genetics , Metabolism , Saccharomyces cerevisiae , Genetics
17.
China Journal of Chinese Materia Medica ; (24): 2438-2440, 2009.
Article in Chinese | WPRIM | ID: wpr-315428

ABSTRACT

<p><b>OBJECTIVE</b>To clone and sequence the open reading frame of beta-amyrin synthase (bAS) from Glycyrrhiza uralensis.</p><p><b>METHOD</b>The primers were designed according to the cDNA sequence of beta-amyrin synthase from G. glabra reported by Hiroaki HAYASHI, and the open reading frame of beta-amyrin synthase was cloned by RT-PCR strategy with the template of total RNA extracted from roots of G. uralensis.</p><p><b>RESULT</b>The GubAS (GenBank Accession number: FJ627179) was 2 289 bp in length encoding one pelypeptide of 762 amino acid. Deduced amino acid sequence had 99%, 92%, 90%, 90% and 89% homology to the amino acid sequence of G. glabra, Lotus japonicus, Pisum sativum, Medicago truncatula, Glycine max, respectively.</p><p><b>CONCLUSION</b>The open reading frame of bAS from G. uralensis is cloned and reported for the first time. The conclusion will provide a foundation for exploring the mechanism of triterpenes biosynthesis.</p>


Subject(s)
Cloning, Molecular , Glycyrrhiza uralensis , Classification , Genetics , Intramolecular Transferases , Genetics , Metabolism , Molecular Sequence Data , Open Reading Frames , Phylogeny , Plant Proteins , Genetics , Metabolism , Plants , Classification , Genetics
18.
Braz. j. microbiol ; 36(4): 347-351, Oct.-Dec. 2005. tab, graf
Article in English | LILACS | ID: lil-433472

ABSTRACT

Saccharomyces cerevisiae deficientes nos genes da superóxido dismutase (mutantes sod1D, sod2De sod1Dsod2D) cultivados em fase estacionária sob condições de alta aeração foram submetidos ao estresse com peróxido de hidrogênio (H2O2). Todos os mutantes mostraram-se sensíveis após o tratamento com o H2O2. A enzima glutationa peroxidase (GPx) apresentou níveis significativamente mais baixos nos simples mutantes sod1D e sod2D que na cepa selvagem sem tratamento. Após, a exposição a diferentes concentrações de H2O2, os níveis da glutationa peroxidase aumentaram no duplo mutante sod1Dsod2D e no simples mutante sod2D, enquanto o mutante sod1D manteve baixa atividade da glutationa peroxidase. O mutante sod2D demonstrou atividade da catalase similar a da cepa selvagem sem tratamento, enquanto foi observado que a atividade da catalase decresceu em condições de baixa aeração. O duplo mutante sod1Dsod2D apresentou baixa atividade da catalase mesmo sem tratamento. Os níveis da catalase foram maiores em condições de alta aeração do que em condições microaerófilas, inclusive o duplo mutante sod1Dsod2D contém menos H2O2, visto que, a SOD catalisa a clivagem do superóxido produzindo H2O2 e oxigênio. Nós sugerimos neste trabalho que a catalase não é essencial para os mutantes sod sob condições normais, mas ela participa de uma importante via na aquisição da tolerância ao estresse oxidativo induzido por condições de alta aeração.


Subject(s)
Catalase , In Vitro Techniques , Intramolecular Transferases , Saccharomyces cerevisiae , Superoxide Dismutase , Aeration , Clinical Enzyme Tests , Methods
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