Gene excavation and expression analysis of CYP and UGT related to the post modifying stage of gypenoside biosynthesis in Gynostemma pentaphyllum (Thunb.) Makino by comprehensive analysis of RNA and proteome sequencing.

Previous studies have revealed that gypenosides produced from Gynostemma pentaphyllum (Thunb.) Makino are mainly dammarane-type triterpenoid saponins with diverse structures and important biological activities, but the mechanism of diversity for gypenoside biosynthesis is still unclear. In this study, a combination of isobaric tags for relative and absolute quantification (iTRAQ) proteome analysis and RNA sequencing transcriptome analysis was performed to identify the proteins and genes related to gypenoside biosynthesis. A total of 3925 proteins were identified by proteomic sequencing, of which 2537 were quantified. Seventeen cytochrome P450 (CYP) and 11 uridine 5'-diphospho-glucuronosyltransferase (UDP-glucuronosyltransferase, UGT) candidate genes involved in the side chain synthesis and modification of gypenosides were found. Seven putative CYPs (CYP71B19, CYP77A3, CYP86A7, CYP86A8, CYP89A2, CYP90A1, CYP94A1) and five putative UGTs (UGT73B4, UGT76B1, UGT74F2, UGT91C1 and UGT91A1) were selected as candidate structural modifiers of triterpenoid saponins, which were cloned for gene expression analysis. Comprehensive analysis of RNA sequencing and proteome sequencing showed that some CYPs and UGTs were found at both the transcription and translation levels. In this study, an expression analysis of 7 CYPs and 5 UGTs that contributed to gypenoside biosynthesis and distribution in G. pentaphyllum was performed, providing consistent results that will inspire more future research on vital genes/proteins involved in gypenoside biosynthesis.

Effects of elevated CO2 and temperature on Gynostemma pentaphyllum physiology and bioactive compounds.

Recently, an important topic of research has been how climate change is seriously threatening the sustainability of agricultural production. However, there is surprisingly little experimental data regarding how elevated temperature and CO2 will affect the growth of medicinal plants and production of bioactive compounds. Here, we comprehensively analyzed the effects of elevated CO2 and temperature on the photosynthetic process, biomass, total sugars, antioxidant compounds, antioxidant capacity, and bioactive compounds of Gynostemma pentaphyllum. Two different CO2 concentrations [360 and 720µmolmol(-1)] were imposed on plants grown at two different temperature regimes of 23/18 and 28/23°C (day/night) for 60days. Results show that elevated CO2 and temperature significantly increase the biomass, particularly in proportion to inflorescence total dry weight. The chlorophyll content in leaves increased under the elevated temperature and CO2. Further, electron transport rate (ETR), photochemical quenching (qP), actual photochemical quantum yield (Yield), instantaneous photosynthetic rate (Photo), transpiration rate (Trmmol) and stomatal conductance (Cond) also increased to different degrees under elevated CO2 and temperature. Moreover, elevated CO2 increased the level of total sugars and gypenoside A, but decreased the total antioxidant capacity and main antioxidant compounds in different organs of G. pentaphyllum. Accumulation of total phenolics and flavonoids also decreased in leaves, stems, and inflorescences under elevated CO2 and temperature. Overall, our data indicate that the predicted increase in atmospheric temperature and CO2 could improve the biomass of G. pentaphyllum, but they would reduce its health-promoting properties.

Toxicity evaluation of standardized extract of Gynostemma pentaphyllum Makino.

ETHNOPHARMACOLOGICAL RELEVANCE: To evaluate the safety of standardized extract of Gynostemma pentaphyllum in rats. MATERIALS AND METHODS: The water extract of Gynostemma pentaphyllum was prepared and standardized, the dry powder yielded 6% gypenosides. In the acute oral toxicity test, the single oral dose of 5000 mg/kg of Gynostemma pentaphyllum extract was given to female Sprague-Dawley rats. In subchronic toxicity test, the oral dose of 1000 mg/kg/day of the extract was given to rats in treatment and satellite groups for 90 days. Satellite groups of both sexes were kept for additional 28 days after 90-day treatment. Control rats received distilled water. RESULTS: Standardized extract of Gynostemma pentaphyllum did not cause death or any toxic signs in rats. The daily administration of the extract for 90 days did not produce lethal or harmful effects. Although certain hematological and blood chemistry values (i.e., neutrophil, monocyte, glucose, and serum alkaline phosphatase levels) were found to be statistically different from the control group, however; these values were within the ranges of normal rats. CONCLUSION: Standardized extract of Gynostemma pentaphyllum did not produce mortality or any abnormality in rats.

[Molecular diversity of arbuscular mycorrhizal fungi in wild and cultured Gynostemma pentaphyllum roots in Xishuangbanna, Southwest China].

By using nested-PCR, DNA cloning, and sequencing techniques, this paper studied the diversity of the community structure of arbuscular mycorrhizal fungi (AMF) in wild and cultured Gynostemma pentaphyllum roots. A total of 551 clones containing 18S rDNA genes of AMF were obtained from the roots. After the analysis of the restriction fragment length polymorphism, 100 different RFLP types were obtained, which were further divided into 20 AMF phylotypes belonging to seven families. The comparison of the sequences of 20 AMF phylotypes with the GenBank database showed that there were 5 AMF phylotypes having high similarity to the sequences of reported AMF species Glomus viscosum, Claroideoglomus etunicatum, Racocetra tropicana, Acaulospora spinosa, and Acaulospora mellea, respectively. These sequences were then assessed for the similarities against the MaarjAM database, and 12 phylotypes showed high similarity to the corresponding molecular virtual taxa, of which, 7 phylotypes were not obtained by the morphological identification of soil asexual spores. Statistical analysis indicated that there were significant differences in the AMF community between wild and cultured G. pentaphyllum roots. The analysis of relative abundance data indicated that Glo-2, Amb-1, and Para-1 were the dominant phylotypes in wild G. pentaphyllum roots, while Glo-3, Glo-8, Glo-10, and Div-1 were the prevalent phylotypes in cultured ones. Claroideoglomeraceae and Ambisporaceae were only detected in wild G. pentaphyllum roots, and Diversisporaceae was only identified in cultured ones.

[Effects of different factors on transformed hairy root in Gynostemm apentaphyllum].

OBJECTIVE: To induce hairy roots of Gynostemm apentaphyllum by Agrobacterium rhizogenes strains. METHODS: Hairy roots were induced by the co-culture method of explants and Agrobacterium rhizogenes strains. Effects of different Agrobacterium rhizogenes strains, explants, pre(co)-culture time, Bacterial concentration, infecting time, As concentration and antibiotic medium on the transformation frequency were studied. RESULTS: The highest induction frequency was obtained form leaf 2 days co-cultivation, which were induced by Agrobacterium rhizogenes OD 600 0. 8 for 10 min, 100 micromol/L As and MS + 300 mg/L Cab. CONCLUSION: Hairy roots were induced by co-cultivation and the optimum induced condition were determined.

[Studies on sex identification and variation of endogenous hormones in female and male plants of Gynostemma pentaphyllum].

OBJECTIVE: To distinguish female and male plants of Gynostemma pentaphyllum quickly and accurately in the stage of seedling and to understand the relation between sex differentiation and endogenous plant hormone. METHOD: Stained methods of BTB and enzyme linked immuno-sorbent Assay were used. RESULT: 10 Hours after being dyed, the liquid extract of female plants turned into yellow, while extract of male ones turned green. There were obvious differences on iPAs and GA(1 + 3) contents but no differences on IAA and ABA contents between male and female plants during the stages of seedling and vegetative growth. CONCLUSION: BTB can be used for distinguishing female and male plants and there were a certain relation between sex and endogenous plant hormones.

[Photosynthetic characteristics of Gynostemma pentaphyllum under shade].

The study showed that under summer shade condition, the diurnal variation of net photosynthetic rate of Gynostemma pentaphyllum presented nontypical double apex, the first apex being 13.8 micromol CO2 x m(-2) x s(-1) at 11:00, and the diurnal net photosynthetic rate was about 176.97 micromol CO2 x m(-2), 3.1 times of that under full sunlight. There was a positive correlation between net photosynthetic rate and photon flux density (PFD), and relative humidity had a small effect on net photosynthetic rate. Under full sunlight, the typical "midday depression" of photosynthesis was observed, and the diurnal variation of net photosynthetic rate presented double apex, with the first apex being 3.0 micromol CO2 x m(-2) x s(-1) at 10:00 and the second being 1.25 micromol CO2 x m(-2) x s(-1) at 14:00. There was a positive correlation between net photosynthetic rate and relative humidity, and the latter had a strong effect on net photosynthetic rate. When PFD was higher than 700 micromol CO2 x m(-2) x s(-1), it had a negative correlation with net photosynthetic rate. Stoma conductance was the main factor affecting the transpiration rate of Gynostemma pentaphyllum. Therefore, Gynostemma pentaphyllum was a typical sciophytic plant, and light factor should be considered firstly in its cultivation.

[Light environment characteristics of forest gap in deciduous broad-leaved forest and its effects on growth features of Gynostemma pentaphyllum in Jianghuai watershed].

The daily total light intensity, various wave band light intensities, spectrum composition, and the growth characteristics and total saponia content of Gynostemma pentaphyllum in forest gap and under forest were studied in deciduous forest (Liquidambar formosana) in Jianghuai watershed. The results showed that the intensities of full wavelength daily total light, blue light(400-510 nm), yellow-green light(510-610 nm), red-orange light (610-720 nm) and near-infrared light(720-1100 nm) in forest gap were higher than those under forest. The intensities and spectra compositions of blue light and red-orange light in forest gaps were significantly higher than those under forest. Additionally, the differences between those in forest gap and under forest varied seasonally. The relative intensity of blue light in April, July and October in forest gap was 8.32%-28.83%, 30.00%-579.17% and 6.70%-88.74% more than those under forest, respectively, and that of red-orange light in April, July and October in forest gap was 8.72%-28.83%, 30.19%-502.69%, 6.76%-91.57% more than those under forest, respectively. The spectrum composition of blue light in forest gap in these 3 months was 5.76%, 33.14% and 17.29% higher than those under forest, respectively, and that of red-orange light in forest gap in these 3 months was 5.04%, 53.34% and 10.23% higher than those under forest, respectively. The net photosynthetic rate, total dry-matter weight, and total saponia content of Gynostemma pentaphyllum growing in forest gap were higher than those under forest. Gynostemma pentaphyllum growing in forest gap had more dry-matter distributed in stem. The results could be available to utilize the light environment of deciduous broad-leaved forest and further building comprehensive management model of Gynostemma pentaphyllum in Jianghuai watershed zone.