ABSTRACT
Isopentenyl diphosphate isomerase (IDI) is a key enzyme in the regulation of triterpenes biosynthesis and plays an important role in ginsenoside biosynthesis. In this study, two IDI genes, PvfIDI1 (GenBank No. MZ736417) and PvfIDI2 (GenBank No. MZ736418) were cloned from Panax vietnamensis var. fuscidiscus. The open reading frame of both PvfIDI1 and PvfIDI2 was 924 bp encoding 307 amino acids. The molecular weights of PvfIDI1 and PvfIDI2 were 34.84 kDa and 34.66 kDa, respectively, with theoretical pIs of 6.01 and 5.66. Bioinformatic analysis indicated that PvfIDI1 and PvfIDI2 contained two conserved sequences: TNTCCSHPL and WGEHELDY. Phylogenetic analysis showed that PvfIDI1 and PvfIDI2 were closely related to Panax notoginseng IDI. Expression analysis showed that both PvfIDI1 and PvfIDI2 genes are expressed in root, rhizome, stem and leaf of P. vietnamensis var. fuscidiscus. However, PvfIDI1 is highly expressed in the rhizome and PvfIDI2 is highly expressed in the stem. PvfIDI1 and PvfIDI2 recombinant proteins were expressed in E. coli; a functional coloration experiment showed that PvfIDI1 and PvfIDI2 could promote the accumulation of lycopene, indicating that both PvfIDI1 and PvfIDI2 encode functional IDI enzymes. The cloning and functional studies on PvfIDI1 and PvfIDI2 provide a foundation for the further study of IDI and the regulation of ginsenoside biosynthesis in P. vietnamensis var. fuscidiscus.
ABSTRACT
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.
ABSTRACT
OBJECTIVE@#To evaluate the impact of plant growth regulators including kinetin (KN), benzyl adenine and naphthalene acetic acid, yeast extract and casein hydrolyzate on biomass accumulation of Vietnamese ginseng Panax vietnamensis (P. vietnamensis) in cell suspension culture.@*METHODS@#Cell suspension cultures were established from friable calluses derived from leaves and petioles of 3-year-old in-vitro P. vietnamensis plants. The cell suspension cultures were grown in Murashige and Skoog basal media supplemented with various concentrations of KN, benzyl adenine, naphthalene acetic acid, and yeast extract and casein hydrolyzate.@*RESULTS@#All tested factors generated an increase in the cell biomass of P. vietnamensis in suspension culture, but the impact of each varies depended on the factor type, concentration, and incubation period. Addition of 2.0 mg/L KN resulted in the largest biomass increase after 24 d, (57.0 ± 0.9) and (3.1 ± 0.1) mg/mL fresh and dry weight, respectively, whereas addition of benzyl adenine or naphthalene acetic acid produced optimum levels of Panax cell biomass at 1.0 and 1.5 mg/L, respectively. Addition of the elicitor yeast extract led to a 1.4-2.4 fold increase in biomass of P. vietnamensis, while addition of casein hydrolyzate enhanced biomass accumulation 1.8-2.6 fold.@*CONCLUSIONS@#The addition of each factor causes significant changes in biomass accumulation of P. vietnamensis. The largest biomass accumulation is from cultures grown in MS media containing 2.0 mg/L KN for 24 d. The outcome of the present study provides new insights into the optimal suspension culture conditions for studies on the in vitro cell biomass production of P. vietnamensis.
ABSTRACT
Objective To evaluate the impact of plant growth regulators including kinetin (KN), benzyl adenine and naphthalene acetic acid, yeast extract and casein hydrolyzate on biomass accumulation of Vietnamese ginseng Panax vietnamensis (P. vietnamensis) in cell suspension culture. Methods Cell suspension cultures were established from friable calluses derived from leaves and petioles of 3-year-old in-vitro P. vietnamensis plants. The cell suspension cultures were grown in Murashige and Skoog basal media supplemented with various concentrations of KN, benzyl adenine, naphthalene acetic acid, and yeast extract and casein hydrolyzate. Results All tested factors generated an increase in the cell biomass of P. vietnamensis in suspension culture, but the impact of each varies depended on the factor type, concentration, and incubation period. Addition of 2.0 mg/L KN resulted in the largest biomass increase after 24 d, (57.0 ± 0.9) and (3.1 ± 0.1) mg/mL fresh and dry weight, respectively, whereas addition of benzyl adenine or naphthalene acetic acid produced optimum levels of Panax cell biomass at 1.0 and 1.5 mg/L, respectively. Addition of the elicitor yeast extract led to a 1.4–2.4 fold increase in biomass of P. vietnamensis, while addition of casein hydrolyzate enhanced biomass accumulation 1.8–2.6 fold. Conclusions The addition of each factor causes significant changes in biomass accumulation of P. vietnamensis. The largest biomass accumulation is from cultures grown in MS media containing 2.0 mg/L KN for 24 d. The outcome of the present study provides new insights into the optimal suspension culture conditions for studies on the in vitro cell biomass production of P. vietnamensis.
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Objective: The SSR loci information in the transcriptome of Panax vietnamensis var. fuscidiscus (PVF) was analyzed in this study to provide more powerful tools for molecular marker-assisted breeding in this plant. Methods: Simple sequence repeats (SSR) loci were searched in all 126 758 unigenes by using MISA. SSR loci information was analized and SSR primers were designed by Primer3. Furthermore, 30 pairs of primers were randomly selected for the polymorphic analysis on 13 PVF plants collected from different habitats. Results: A total of 21 320 SSRs were found in the transcriptome of PVF, distributed in 17 780 unigenes with the distribution frequency of 16.82%. Di-nucleotide repeat was the main type, accounted for as much as 52.52% of all SSRs, followed by tri-nucleotide repeat motif (28.08%). The dinucleotide repeat motifs of AG/CT and AT/AT were the predominant repeat types (46.25%). Using Primer3, a total of 39 336 pairs of SSR primers were designed. For validating the availability of those SSR primers, we randomly selected 30 pairs of primers for PCR amplification. Among them, 29 pairs of primers (96.67%) produced clear and reproductive bands, 15 pairs of primers (50.00%) showed polymorphism, and 13 PVF plants were divided into two groups by UPGMA. Conclusion: There are numerous SSRs in PVF transcriptome with high frequency and various types, this will provide the abundant candidate molecular markers for genetic diversity study and genetic map for this plant.
ABSTRACT
The present work describes a procedure that allows for the easy and rapid induction of somatic embryos, calli, shoots and adventitious roots of Vietnamese ginseng (Panax vietnamensis Ha et Grushv.) from longitudinal thin cell layers (lTCLs). In order to investigate the morphogenesis of this medicinal plant, the effect of separately–supplemented plant growth regulators and combinatorial effect of co–supplemented auxins and cytokinins in dark or under 16-hour photoperiod was examined. After eight weeks of culture, the lTCL explants excised from petiole of three-month-old in vitro plants and cultured on a semi solid basal Murashige and Skoog (MS) media supplemented with 1.0 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.1 mg/l thidiazuron (TDZ) in dark, 2.0 mg/l α-napththaleneacetic acid (NAA) in dark and 1.0 mg/l 2,4-D under light gave the highest rate of callogenesis (100%), embryogenesis (53.3%) and adventitious root formation (100% with a mean of 16.7 roots), and shoot formation (26.7%), respectively. The metabolite of petiole lTCL-derived calli qualitative and quantitative analyses were performed by using high-performance liquid chromatography and thin layer chromatography. The simple procedure, together with similar saponin profiles between the resulted in vitro tissues and plants grown in nature, suggest its potential use in generating Vietnamese ginseng in large amount for medicinal purpose.