ABSTRACT
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.
ABSTRACT
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.
ABSTRACT
A rapid quantitative evaluation method for Siraitia grosvenorii cells was successfully developed based on plant cells' capacitance value detected by a viable cell mass monitor and the cryopreservation of S. grosvenorii suspension cells was optimized. The survival rate of S. grosvenorii cells was quantitatively measured by viable cell mass monitor and 2, 3, 5-triphenyltetrazolium chloride (TTC). An optimum cryoprotectant recipe is that the growth medium contained 10% sucrose and 10% DMSO. The experimental results also showed higher cell survival rates and cell viabilities were achieved when suspension cells were treated with pretreatment of 0.2 mol/L sucrose. With the increase of concentration of sucrose, however, the cell survival rate was decreased. And the cell survival rate represented a bell shape with the increase of pretreatment time. The highest cell survival rate and cell viability were obtained with the 9 h' s pretreatment. In addition, there was a good correlation between the cell survival rate measured by cell recovery test and that measured by viable cell mass monitor, while there were no significant differences in the cell morphology and the ability of mogrosides V production by S. grosvenorii cells cultured in suspension after cryopreservation. Therefore, the evaluation method developed based on the viable cell mass monitor has good feasibility and reliability.
ABSTRACT
CYP450 plays an essential role in the development and growth of the fruits of. However, little is known about thegene in. Here, based on transcriptome data, a full-length cDNA sequence ofwas cloned by reverse transcriptase-polymerase chain reaction (RT-PCR) and rapid-amplification of cDNA ends (RACE) strategies.is 1677 bp in length (GenBank accession No. AEM42985.1) and contains a complete open reading frame (ORF) of 1422 bp. The deduced protein was composed of 473 amino acids, the molecular weight is 54.01 kDa, the theoretical isoelectric point (PI) is 8.8, and the protein was predicted to possess cytochrome P450 domains.gene was highly expressed in root, diploid fruit and fruit treated with hormone and pollination. At 10 days after treatment with pollination and hormones, the expression of Sghad the highest level and then decreased over time, which was consistent with the development of fruits of. Hormonal treatment could significantly induce the expression of. These results provide a reference for regulation of fruit development and the use of parthenocarpy to generate seedless fruit, and provide a scientific basis for the production of growth regulator application agents.
ABSTRACT
Aim To study the apoptosis effect of Sir-aitia grosvenorii extract on human lung cancer cells A549 and its mechanisms.Methods MTT assay was applied to determine A549 cell proliferation.Hoechst 33258 staining was applied to investigate morphological changes in A549 cells.To find out the cause of cell growth inhibition,several experiments on cell cycle distribution and apoptosis were performed by flow cy-tometry analysis.The expression of p21 and Bcl-2 was determined by Western blot.Results Flow cytometry analysis showed that treatment with mogrol arrested A549 cells in the G0 /G1 phase and induced apoptosis. After treatment with Siraitia grosvenorii extract,West-ern blot experiment showed cell cycle regulator p21 was up-regulaed,while the apoptosis inhibitor Bcl-2 was down-regulated.Conclusion Treatment with Siraitia grosvenorii extract arrests the A549 cells at G0 /G1 phase and induces apoptosis that may contribute to the anti-proliferation activity of mogrol through the regula-tion of p21 and Bcl-2 expression.
ABSTRACT
To evaluate the genetic background of triploid female and diploid male strains of Siraitia grosvenorii and provide the biological reference for good varieties breeding of seedless S. grosvenorii. Inter simple sequence repeats (ISSR) marker was developed to analyze the genetic background among 28 samples of S. grosvenorii, and cluster analysis and double principal coordinate analysis were revealed by the NTSYS-pc software and GenAIEx software, respectively. Out of 100 ISSR primers selected, 13 primers were used for amplification and a total of 131 unambiguous bands were obtained, among which 99 (PPB = 75.57%) were polymorphic. The results of cluster analysis and double principal coordinate analysis showed that there was a certain rich of genetic background in triploid female and diploid male strains of S. grosvenorii. But the genetic similarity coefficients of majority were bigger and the genetic distance was closer. The complexity of the genetic background in triploid female and diploid male strains of S. grosvenorii is lower and germplasm innovation strategies should be carried out to enrich the genetic background of the parents of seedless S. grosvenorii.
ABSTRACT
Objective: To observe the effects of various factors on the antitussive effect of Siraitia grosvenorii. Methods: According to the sequential method (i.e., up and down method) for the principle of median effective dose, the antitussive effect of S. grosvenorii was observed and the median effective time (EDT50) of cough induced by NH3 aq. in mice was calculated. Results: S. grosvenorii from Hunan-Hengyang, Nanning-Tanluo, Yongfu-Longjiang, and Baishou have the antitussive effect, especially in Yongfu-Longjiang and Baishou (R>150%). The fruits obtained from cuttage seedlings and tissue culture seedlings have the antitussive effect, while the immature fruits have not (R130%) have the significant antitussive effect, and so first class fruit and ringing fruit do (R>130%). However secondary class fruit does not have the antitussive effect (R<130%). Conclusion: The factors, except the habitat, breeding technology, and whether drying or not, such as growth period and commercial specification may influence the antitussive effect of S. grosvenori.
ABSTRACT
Objective: To screen the reference genes of Siraitia grosvenorii for gene expression analysis, and to study the spatio-temporal expression characteristics of 3-hydroxy-3-methylglutaryl coenzyme A (HMGR) which was the key enzyme of mogroside V biosynthesis. Methods: In this study, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), microtubule associated protein (α-tubulin), actin (β-actin), and ubiquitin (UBQ5) fragments of S. grosvenorii were cloned, and the stabilities of the four housekeeping genes were evaluated in different positions (leaves, stems, and fruits) and different periods of fruit development. In addition, the spatio-temporal expression of HMGR gene was analyzed. Results: UBQ5 was the most suitable reference gene for spatio-temporal expression analysis in S. grosvenorii; The relative expression of HMGR was low in leaves, and that in stems and fruits was higher; The relative expression quantity of HMGR in fruits showed the fluctuation changes that increased firstly and then decreased, then increased and decreased again; The highest expression of HMGR was at 70 d of fruit development period, followed by 5, 30, 10, and 50 d. Conclusion: UBQ5 is the most suitable reference gene in S. grosvenorii. The relative expression of HMGR changes significantly in leaves, stems, and fruits. The relative expression quantity of HMGR in fruits shows the fluctuation changes, which is similar to synthetic accumulation pattern of mogroside V.
ABSTRACT
Objective To get a new approach for conserving the germplasm of Siraitia grosvenorii Methods Shoots, 0.8—1 cm in length, excised from test-tube plantlets which were subcultured 15 d, were precultured. Then its shoot-tips about 2—3 mm in length were dissected and loaded with 60% PVS2 at 25 ℃, and dehydrated with 100% PVS2 at 0 ℃, changed for fresh 100% PVS2 prior to directly plunging into liquid nitrogen. After cryopreservation for 24 h, the shoot tips were thawed, rinsed in MS+1.2 mol/L sucrose medium, blotted with filter papers, then plated on the MS + 1.0 mg/L 6-BA+0.05 mg/L NAA + 0.1 mg/LGA3 + 0.8% agar + 45 g/L sucrose medium at 25 ℃ for 7 d in dark prior to exposure to the light. The root medium for regeneration plantlets was 1/2 MS + 0.2 mg/L NAA + 30 g/L sucrose. Results Shoots were precultured for 3 d on MS + 0.7 mol/L sucrose medium, then its shoot-tips loaded for 40 min, dehydrated for 50 min. After cryopreservation, the shoot tips were rapidly thawed in water at 40 ℃, then rinsed for 40 min. The survival rate of shoot-tips plated on the recovering medium in one week was 100%, and regeneration rate after 30 d was 78.33%, which was the highest. The regeneration plantlets inoculated on root medium were reconstructed integrating plantlets. Conclusion The method of vitrification to cryopreserve the germplasm of S. grosvenorii is a simple way with high survival rate and normal regeneration plants.
ABSTRACT
Glycosides are the bioactive components of many famous Chinese medicines. Here reported are some bioactive glycosides we discovered from Chinese medicines in recent years. (1) Pheolic glycosides from Chinese medicines: Gastrodia elata, acontium austroynanense and Helicia erratica, three bioactive phenolic glycosides were discovered and two of them have been developed into new drugs. (2) Terpenoidal glycosides: a) Monoterpenoid: the sweroside from Swertia mollensis has been developed intro an anti-hepatitis drug; b) Diterpenoid: Phlomis betonicoides contains sweet glycoides; c) Triterpenoid: many biologically active triterpenoid glycosides were isolated from Panax plants and Siraitia grosvenorii. (3) Steroidal glycosides: a) C21-steroid: Cynanchum otophyllum and C. atratrum contain anti-epilepsy and-tumor glycosides; b) C27-steroid Hemostatic saponins were found in Paris polyphylla.