Cloning and characterization of squalene synthase and cycloartenol synthase from Siraitia grosvenorii

AbstractMogrosides and steroid saponins are tetracyclic triterpenoids found in Siraitia grosvenorii. Squalene synthase (SQS) and cycloartenol synthase (CAS) are key enzymes in triterpenoid and steroid biosynthesis. In this study, full-length cDNAs of SgSQS and SgCAS were cloned by a rapid amplification of cDNA-ends with polymerase chain reaction (RACE-PCR) approach. The SgSQS cDNA has a 1254bp open reading frame (ORF) encoding 417 amino acids, and the SgCAS cDNA contains a 2298bp ORF encoding 765 amino acids. Bioinformatic analysis showed that the deduced SgSQS protein has two transmembrane regions in the C-terminal. Both SgSQS and SgCAS have significantly higher levels in fruits than in other tissues, suggesting that steroids and mogrosides are competitors for the same precursors in fruits. Combined in silico prediction 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 of SgSQS and SgCAS gene functions in S. grosvenorii, and may facilitate improvements in mogroside content in fruit by regulating gene expression

Methyl jasmonate-induced accumulation of metabolites and transcriptional responses involved in triterpene biosynthesis in Siraitia grosvenorii fruit at different growing stages

The cucurbitane-type triterpenoid glycosides, mogrosides, are the main active components of Siraitia grosvenorii fruit. Squalene and cucurbitadienol are among the intermediates of the biosynthetic pathway for the formation of cucurbitane-type triterpenoid backbones of mogrosides. It is recognized that the exogenous application of methyl jasmonate (MeJA) increases the accumulation of secondary metabolites in various plant species. Here, the effect of MeJA (50, 200, and 500 μM) on the accumulation of squalene and cucurbitadienol in the fruits of S. grosvenorii at 10, 20, and 30 days after flowering (DAF) was tested for the first time. Since mogroside II E is the main cucurbitane-type triterpenoid present at this time, its concentration was also determined. The results show that MeJA can indeed promote squalene and cucurbitadienol accumulation, the application of 500 μM MeJA at 30 DAF being optimal. The concentration of squalene and cucurbitadienol increased up to 0.43 and 4.71 μg/g dry weight (DW), respectively, both of which were 1.2-fold greater than that of the control. The content of mogroside II E increased by 15% over the untreated group. We subsequently analyzed the expression of key genes involved in the mogroside biosynthetic pathway, including the 3-hydroxy-3-methylglutaryl-coenzyme A reductase gene (SgHMGR), squalene synthetase gene (SgSQS), cucurbitadienol synthase gene (SgCS), and cytochrome P450 (SgCYP450) with quantitative real-time PCR. The results showed that transcriptional levels of these genes were upregulated following the treatment described above. Additionally, their responses in the presence of MeJA was related to the concentration and timing of MeJA treatment

Methyl jasmonate-induced accumulation of metabolites and transcriptional responses involved in triterpene biosynthesis in Siraitia grosvenorii fruit at different growing stages

The cucurbitane-type triterpenoid glycosides, mogrosides, are the main active components of Siraitia grosvenorii fruit. Squalene and cucurbitadienol are among the intermediates of the biosynthetic pathway for the formation of cucurbitane-type triterpenoid backbones of mogrosides. It is recognized that the exogenous application of methyl jasmonate (MeJA) increases the accumulation of secondary metabolites in various plant species. Here, the effect of MeJA (50, 200, and 500 μM) on the accumulation of squalene and cucurbitadienol in the fruits of S. grosvenorii at 10, 20, and 30 days after flowering (DAF) was tested for the first time. Since mogroside II E is the main cucurbitane-type triterpenoid present at this time, its concentration was also determined. The results show that MeJA can indeed promote squalene and cucurbitadienol accumulation, the application of 500 μM MeJA at 30 DAF being optimal. The concentration of squalene and cucurbitadienol increased up to 0.43 and 4.71 μg/g dry weight (DW), respectively, both of which were 1.2-fold greater than that of the control. The content of mogroside II E increased by 15% over the untreated group. We subsequently analyzed the expression of key genes involved in the mogroside biosynthetic pathway, including the 3-hydroxy-3-methylglutaryl-coenzyme A reductase gene (SgHMGR), squalene synthetase gene (SgSQS), cucurbitadienol synthase gene (SgCS), and cytochrome P450 (SgCYP450) with quantitative real-time PCR. The results showed that transcriptional levels of these genes were upregulated following the treatment described above. Additionally, their responses in the presence of MeJA was related to the concentration and timing of MeJA treatment

Factors influencing householder self-evacuation in two Australian bushfires

The thesis investigated householder self-evacuation decision-making during bushfires in the Perth and Adelaide Hills in 2014 and 2015. It explored the factors that influenced householders’ decisions to evacuate, identified factors that predict self-evacuation and established the characteristics of self-evacuators. The Protective Action Decision Model (PADM) provided a conceptual framework for the research. Its theoretical and analytical usefulness in an Australian context, was assessed. A mixed methods research strategy was used involving quantitative telephone surveys of 457 bushfire-affected participants and face-to-face interviews of 109 participants in 59 households. The study concluded that environmental and social cues and warnings and householders’ perceptions of the threat, of hazard adjustments and of other stakeholders, influenced self-evacuation decision-making. Protective action perceptions, particularly the effectiveness of evacuating or not evacuating in protecting personal safety or property, were most important in predicting self-evacuation. Receipt of official warnings and the perception of likely impact of the bushfire on property were also important predictors. Undertaking long-run hazard adjustments, although not predictive of self-evacuation, was pivotal in shaping perceptions of the effectiveness of evacuating and remaining in protecting personal safety and property and indirectly influenced evacuation decisions. Seven archetypes that characterised householders’ self-evacuation attitudes and behaviour were identified. These included Threat, and Responsibility Deniers, Dependent, and Considered Evacuators, Community Guided and Experienced Independents all who took different decisional ‘rules of thumb’ and routes toward evacuating or remaining . The PADM needs to be split into two separate models to incorporate the influence of long-run hazard adjustments on protective action decision-making in an Australian bushfire. The findings suggest that future research on those who wait and see during a bushfire should take account of their decisional rules of thumb and that design and targeting of Australian bushfire safety policy should better account for self-evacuator characteristics

An extra-large Stokes shift near-infrared fluorescent probe for specific detection and imaging of cysteine

Cysteine (Cys) plays a crucial role in numerous physiological and pathological processes. Therefore, it is imperative to design a highly selective and sensitive near-infrared (NIR) fluorescent probe to monitor Cys. In this study, we have developed a novel NIR fluorescent probe XA based on Xanthene hybrid tetrahydro-acridine salt dye for specifically tracking of Cys, where a chlorine-substituted tetrahydro-acridine acts as a high Cys-reactive site and water-soluble group. Probe XA exhibits a remarkable turn-on NIR emission (830 nm) with an extra-large Stokes shift (305 nm) for monitoring Cys. It also has a high selectivity, rapid response time (6 min) and high sensitivity (LOD as 0.5 μM). We fully characterized and discussed the sensing mechanism of XA toward Cys using HPLC and MS spectrums, as well as quantum theory calculations. Furthermore, the excellent properties of NIR fluorescent detection allow this novel probe to successfully monitor fluctuations of exogenous and endogenous Cys concentration levels in living cells and in vivo

Magnetron sputtered zinc oxide nanorods as thickness-insensitive cathode interlayer for perovskite planar-heterojunction solar cells

Suitable electrode interfacial layers are essential to the high performance of perovskite planar heterojunction solar cells. In this letter, we report magnetron sputtered zinc oxide (ZnO) film as the cathode interlayer for methylammonium lead iodide (CH3NH3PbI3) perovskite solar cell. Scanning electron microscopy and X-ray diffraction analysis demonstrate that the sputtered ZnO films consist of c-Axis aligned nanorods. The solar cells based on this ZnO cathode interlayer showed high short circuit current and power conversion efficiency. Besides, the performance of the device is insensitive to the thickness of ZnO cathode interlayer. Considering the high reliability and maturity of sputtering technique both in lab and industry, we believe that the sputtered ZnO films are promising cathode interlayers for perovskite solar cells, especially in large-scale production

Magnetron Sputtered Zinc Oxide Nanorods as Thickness-Insensitive Cathode Interlayer for Perovskite Planar-Heterojunction Solar Cells

Suitable electrode interfacial layers are essential to the high performance of perovskite planar heterojunction solar cells. In this letter, we report magnetron sputtered zinc oxide (ZnO) film as the cathode interlayer for methylammonium lead iodide (CH₃NH₃PbI₃) perovskite solar cell. Scanning electron microscopy and X-ray diffraction analysis demonstrate that the sputtered ZnO films consist of <i>c</i>-axis aligned nanorods. The solar cells based on this ZnO cathode interlayer showed high short circuit current and power conversion efficiency. Besides, the performance of the device is insensitive to the thickness of ZnO cathode interlayer. Considering the high reliability and maturity of sputtering technique both in lab and industry, we believe that the sputtered ZnO films are promising cathode interlayers for perovskite solar cells, especially in large-scale production