Separation of anti-TMV active components and modes of action of Omphalia lapidescens.

BACKGROUND: Omphalia lapidescens is a saprophytic and parasitic fungus belonging to the Polypora genus of Tricholomataceae. It has repellent, insecticidal, anti-inflammatory and immunomodulatory effects. RESULT: This study found that the extract of O. lapidescens had significant anti-TMV activity, and the main active component was homopolysaccharide LW-1 by Bioassay-guided fractionation. LW-1 is a glucan with ß-(1,3) glucoside bond as the main chain and ß-(1,6) glucoside bond as the branch chain, with molecular weight in the range of 172,916-338,827 Da. The protective and inactive efficacies of LW-1(100 mg/L) against TMV were 78.10% and 48.20%, but had no direct effect on the morphology of TMV particles. The results of mechanism of action showed that LW-1 induced the increase of the activity of defense enzymes such as POD, SOD and PAL in Nicotiana glutinosa. The overexpression of resistance genes such as NPR1, PR1 and PR5, and the increase of SA content. Further transcriptome sequencing showed that LW-1 activated MAPK signaling pathway, plant-pathogen interaction pathway and glucosinolide metabolic pathway in Arabidopsis thaliana. Besides, LW-1 induced crops resistance against plant pathogenic fungi. CONCLUSION: Taken together, the anti-TMV mechanism of LW-1 was to activate MAPK signaling pathway, inducing overexpression of resistance genes, activating plant immune system, and improving the synthesis and accumulation of plant defencins such as glucosinolide. LW-1-induced plant disease resistance has the advantages of broad spectrum and long duration, which has the potential to be developed as a new antiviral agent or plant immune resistance inducer.

Natural α-Methylene-γ-Butyrolactone Analogues: Synthesis and Evaluation of Isoaurone Derivatives as Antifungal Agents.

In order to develop novel, efficient and green fungicides, a series of novel isoaurone derivatives were designed and synthesized, which were characterized by 1H and 13C NMR, high-resolution mass spectra and melting points. The target compounds showed different inhibitory activities against seven plant pathogenic fungi. Compounds 1, 12, 17, 20, 22, 24 and intermediate A showed more than 90% inhibition rates against S. s at 50 mg/L. Interestingly, compound 22 and intermediate A showed the great inhibitory effect against S. s with EC50 values of 4.65 and 4.24 mg/L, which were better than the lead compound isoaurone (EC50 = 15.62 mg/L). The EC50 values of compounds 17 and 24 against B. c were 13.94 and 22.13 mg/L. Moreover, compound 19 displayed significant antifungal activity against G. g with the EC50 value of 11.88 mg/L. Theoretical calculations by DFT revealed that the α, ß-unsaturated carbonyl bond and the benzyl ring are very importantly linked to the strength of the fungicidal activity. Therefore, this study identified a valuable antifungal lead compound for further development of green fungicides.

Bioactivity evaluation and active compounds identification of Symphoricarpos orbiculatus as potential botanical herbicide.

BACKGROUND: Evaluation of herbicidal activity and identification of active compounds are important bases for the development of new botanical herbicides. RESULTS: This study confirmed that Symphoricarpos orbiculatus has high herbicidal activities against mono-dicotyledonous weeds, including Echinochloa crusgalli, Digitaria sanguinalis, Amaranthus retroflexus and Portulaca oleracea. By bioassay-guided isolation, 12 compounds were isolated and identified from S. orbiculatus for the first time, including iridoids: naucledal (K1), loganin (K2), loganigenin (K3), loganin acid (K4), glucologanin (K5) and vogeloside (K6), as well as flavonoids: quercetine (K7), luteolin (K8), nobiletin (K9), astragalin (K10), isorhamnetin 3-d-glucoside (K11) and rutin (K12). Biological assays showed that iridoids are the main active ingredients of S. orbiculatus. The compounds of K5 and K6 could inhibit both the root (IC50 = 37.54 and 38.91 µg mL-1, respectively) and shoot (IC50 = 42.78 and 45.72 µg mL-1, respectively) of Portulaca oleracea, which have a weeding toxicity similar to that of the commercialized plant-based herbicide pelargonic acid. In addition, the results of pot culture assay showed that S. orbiculatus ethanol extracts had high fresh weight control effect against Digitaria sanguinalis and P. oleracea at the concentration of 40 g L-1. After 7 days, both the soil treatment and the stem and leaf spray method resulted in severe leaf necrosis and significant leaf etiolation. CONCLUSION: Symphoricarpos orbiculatus and its herbicidal active compounds have the potential to develop into botanical herbicides, and are first reported in the present study. © 2024 Society of Chemical Industry.

Chitosan-thymol nanoparticle with pH responsiveness as a potential intelligent botanical fungicide against Botrytis cinerea.

The practical application of essential oils (EOs) as an alternative for synthetic pesticides in agricultural production is severely limited because of their instability, high volatility, and water insolubility. Nanoencapsulation of EOs is an important strategy to overcome these limitations. In view of this, this study aimed to develop chitosan-thymol nanoparticle (NCS-Thy) with pH-responsive which can be used as an intelligent botanical fungicide to control Botrytis cinerea. The NCS-Thy nanoparticle was prepared by ionic crosslinking method with the loading capacity and encapsulation efficiency of 29.87% and 41.92%, respectively. The synthesized NCS-Thy nanoparticle was further characterized by Fourier transform infrared spectroscopy analysis, transmission electron microscopy observation, and dynamic lights scattering. The results of release kinetics and antifungal activity of NCS-Thy under different pH conditions were determined. The results showed that the NCS-Thy nanoparticle had excellent pH-responsiveness and can release more thymol under acidic conditions formed by B. cinerea, thereby achieving higher antifungal effects. Therefore, compared with unencapsulated thymol, the NCS-Thy nanoparticle had higher antifungal activity against B. cinerea in vitro. In addition, both the protective and curative efficacies of detached leaf test and pot experiment were significantly higher than those of unencapsulated thymol. Among them, the protective efficacy of NCS-Thy in the pot experiment was 78.73%, which was significantly higher than that of unencapsulated thymol with 61.13%. Therefore, the pH-responsive chitosan-thymol nano-preparation had a promising prospect of application in practical management of gray mold as an intelligent botanical fungicide.

Cloning and expression of the mitochondrial cytochrome c oxidase subunit II gene in Sitophilus zeamais and interaction mechanism with allyl isothiocyanate.

In the United States, allyl isothiocyanate (AITC) has been registered as an insecticide, bactericide, and nematicide. And it has been confirmed that AITC has significant insecticidal activities against four stored product pests including Sitophilus zeamais Mostchulky (Coleoptera: Curculionidae). This study aimed to verify the mechanism of action of AITC on cytochrome c oxidase core subunits II in S. zeamais. Enzyme - catalyzed reactions and Fourier transform infrared spectrometer (FTIR) analysis revealed that the expressed COX II proteins could competitively bind and inhibit the activity of COX II. Furthermore, molecular docking results showed that a sulfur atom of AITC could form a 2.9 Å hydrogen bond with Ile-30, having a binding energy of -2.46 kcal/mol.

Limonene anti-TMV activity and its mode of action.

The main component of orange peel essential oil is limonene. Limonene is a natural active monoterpene with multiple functions, such as antibacterial, antiseptic and antitumor activity, and has important development value in agriculture. This study found that limonene exhibited excellent anti-tobacco mosaic virus (TMV) bioactivity, with results showing that its protection activity, inactivation activity, and curative activity at 800 µg/mL were 84.93%, 59.28%, and 58.89%, respectively-significantly higher than those of chito-oligosaccharides. A direct effect of limonene on TMV particles was not observed, but limonene triggered the hypersensitive response (HR) in tobacco. Further determination of the induction activity of limonene against TMV demonstrated that it displayed good induction activity at 800 µg/mL, with a value of 60.59%. The results of physiological and biochemical experiments showed that at different treatment days, 800 µg/mL limonene induced the enhancement of defense enzymes activity in tobacco, including of SOD, CAT, POD, and PAL, which respectively increased by 3.2, 4.67, 4.12, and 2.33 times compared with the control (POD and SOD activities reached highest on the seventh day, and PAL and CAT activities reached highest on the fifth day). Limonene also enhanced the relative expression levels of pathogenesis related (PR) genes, including NPR1, PR1, and PR5, which were upregulated 3.84-fold, 1.86-fold and 1.71-fold, respectively. Limonene induced the accumulation of salicylic acid (SA), and increased the relative expression levels of genes related to SA biosynthesis (PAL) and reactive oxygen species (ROS) burst (RBOHB), which respectively increased by 2.76 times and 4.23 times higher than the control. Systemic acquired resistance (SAR) is an important plant immune defense against pathogen infection. The observed accumulation of SA, the enhancement of defense enzymes activity and the high-level expression of defense-related genes suggested that limonene may induce resistance to TMV in tobacco by activating SAR mediated by the SA signaling pathway. Furthermore, the experimental results demonstrated that the expression level of the chlorophyll biosynthesis gene POR1 was increased 1.72-fold compared to the control in tobacco treated with 800 µg/mL limonene, indicating that limonene treatment may increase chlorophyll content in tobacco. The results of pot experiment showed that 800 µg/mL limonene induced plant resistance against Sclerotinia sclerotiorum (33.33%), Phytophthora capsici (54.55%), Botrytis cinerea (50.00%). The bioassay results indicated that limonene provided broad-spectrum and long-lasting resistance to pathogen infection. Therefore, limonene has good development and utilization value, and is expected to be developed into a new botanical-derived anti-virus agent and plant immunity activator in addition to insecticides and fungicides.

Linalool Induces Resistance Against Tobacco Mosaic Virus in Tobacco Plants.

The essential oil of Cinnamomum camphora is the most widely consumed and used spice in the world today. It has therapeutic effects in medicine and has been shown to have good antibacterial and bacteriostatic effects in agriculture. This study found that C. camphora oil significantly induced plant disease resistance activity. Linalool, its main active component, significantly induced plant disease resistance activity (67.49% at a concentration of 800 µg/ml) over the same concentration of the chitosan oligosaccharide positive control but had no direct effect on tobacco mosaic virus (TMV). In this study of its antiviral mechanism, linalool induced hypersensitive reaction (HR); the overexpression of related defense enzymes SOD, CAT, POD, and PAL; and the accumulation of H2O2 and SA content in N. glutinosa. Besides, linalool induced crops resistance against Colletotrichum lagenarium, Botrytis cinerea, Sclerotinia sclerotiorum, and Phytophthora capsica. Taken together, the anti-TMV mechanism of linalool involved the induction of plant disease resistance through activation of a plant immune response mediated by salicylic acid. Linalool-induced plant disease resistance activity has a long duration, broad spectrum, and rich resources; linalool thus has the potential to be developed as a new plant-derived antiviral agent and plant immune activator.

Effects of Three Essential Oil Fumigation Treatments on the Postharvest Control of Botrytis cinerea and Their Efficacy as Preservatives of Cherry Tomatoes.

Cherry tomatoes (Solanum lycopersicum) are becoming increasingly popular due to their nutrition and delicious flavor. However, cherry tomatoes are highly perishable and susceptible to various pathogenic microorganisms after harvest, such as Botrytis cinerea. In the pretest experiment, we screened out three kinds of plant essential oils (EOs) (Torreya grandis oil, Eriobotrya japonica oil, and Citrus medica oil) that have strong fungicidal activity on B. cinerea from cherry tomatoes. To further evaluate the postharvest preservation application prospect of these three oils for cherry tomatoes, the oils were extracted from different parts of three plants by hydrodistillation, and their chemical constituents were analyzed by gas chromatography-mass spectrometry. The main representative components of T. grandis oil, E. japonica oil, and C. medica oil were δ-cadinene (11.76%), transnerolidol (9.70%), and 5,7-dimethoxycoumarin (23.22%), respectively. These three EOs effectively inhibited the mycelial growth of B. cinerea in vitro, with EC50 values of 81.672, 144.046, and 221.500 µl/liter, respectively. Compared with the blank control and other oil treatments, the T. grandis oil (at a concentration of 200 µl/liter) fumigation treatment was more effective at inhibiting the growth rate of the pathogen. In addition, the phenolic content and phenylalanine ammonia lyase, ß-1,3-glucanase, chitinase, and peroxidase activities of tomatoes significantly increased on the seventh day due to the T. grandis oil treatment. The present study shows that these three oils with high extraction rates have preservation potential for cherry tomatoes. Among these three EOs, T. grandis oil can be used to further develop preservative products as a fumigant.

An Asymmetric Hydrogenation/N-Alkylation Sequence for a Step-Economical Route to Indolizidines and Quinolizidines.

The direct catalytic asymmetric hydrogenation of pyridines for the synthesis of piperidines remains a challenge. Herein, we report a one-pot asymmetric hydrogenation of pyridines with subsequent N-alkylation using a traceless Brønsted acid activation strategy. Catalyzed by an iridium-BINAP complex, the substrates undergo ketone reduction, cyclization and pyridine hydrogenation in sequence to form indolizidines and quinolizidines. The absolute configuration of the stereocenter of the alcohol is retained and influences the formation of the second stereocenter. Experimental and theoretical mechanistic studies reveal that the chloride anion and certain noncovalent interactions govern the stereoselectivity of the cascade reaction throughout the catalytic process.

Insecticidal action of the botanical insecticide wilforine on Mythimna separata (Walker) related with the changes of ryanodine receptor expression.

The detailed molecular mechanism of wilforine, a novel botanical insecticidal component, remains unclear, except for the knowledge that it affects the calcium signaling pathway. The aim of the current study was to examine the underlying molecular mechanism of wilforine in Mythimna separata (Walker) by transcriptome and RNA interference (RNAi), with chlorantraniliprole as control. RNA sequencing showed that the relative expression of genes related to the calcium signaling pathway and muscle contraction in M. separata treated with wilforine significantly changed and was further validated by qRT-PCR. Interestingly, the expression level of the ryanodine receptor (MsRyR) gene was downregulated by wilforine at relatively high concentrations and long treatment time, contrary to that observed using chlorantraniliprole. Furthermore, a putative MsRyR was cloned using a 16,258-bp contiguous sequence containing a 308-bp 5'-untranslated region and 578-bp 3'-untranslated region by RT-PCR and RACE. The results of the RNAi experiment showed that injection of dsMsRyR significantly reduced MsRyR mRNA levels, and growth and development were inhibited. Importantly, silencing of the MsRyR gene resulted in decreased susceptibility to both wilforine and chlorantraniliprole. Together with the results of our previous studies on toxic symptoms and muscle tissue lesions between wilforine and chlorantraniliprole, we propose that RyR Ca2+ release channel dysfunction is closely related with significant lethal mechanisms of wilforine.

Effects of RNAi-mediated plasma membrane calcium transporting ATPase and inositol 1,4,5-trisphosphate receptor gene silencing on the susceptibility of Mythimna separata to wilforine.

Wilforine, a compound of sesquiterpene alkaloids isolated from Tripterygium wilfordii, exhibits excellent insecticidal activity against Mythimna separata. In order to clarify the action mechanism of wilforine, the plasma membrane calcium transporting ATPase (PMCA) and inositol 1,4,5-trisphosphate receptor (IP3R) from M. separata were studied. Results showed that the open reading frame of MsIP3R and MsPMCA were 8118 bp and 3438 bp in length, as well as encoded 2706 and 1146 amino acids, respectively. Multiple sequence alignment and phylogenetic analysis revealed that the MsIP3R and MsPMCA had high homology with the IP3R and PMCA of other insects, but had low similarity with those of mammals, which means the IP3R and PMCA have potential to be the novel targets of insecticides with high selectivity between mammals and insects. Both MsIP3R and MsPMCA genes existed throughout the life cycle of M. separata, and were all predominantly expressed in somatic muscle of fifth-instar larvae and the adults. The susceptibilities of PMCA-silenced M. separata to wilforine were significantly lower than that of the normal M. separata, which illustrates that PMCA could be one of the targets of wilforine. However, the susceptibilities of IP3R-silenced M. separata to wilforine did not change significantly compared with the susceptibilities of normal M. separata, which shows that wilforine may not interact with the IP3R protein. These findings provide clues for elucidating the insecticidal mechanism of wilforine.

Function of four mitochondrial genes in fumigation lethal mechanisms of Allyl Isothiocyanate against Sitophilus zeamais adults.

Allyl isothiocyanate (AITC) is a promising alternative to chemical fumigants, and mitochondrial dysfunction has been proposed to play a crucial role in its lethal mechanisms; however, the specific lethal mechanisms of AITC remain unknown. Four mitochondrial electron transport chain genes, nd5, nd6, cox1, and cox5, were selected from adult Sitophilus zeamais and processed with RNA interference experiments. Then, the biochemical and biophysical effects were compared between double-stranded RNA (dsRNA)-mediated insects and wild-type insects after AITC fumigation at the concentration of LC50 values. The bioactivity of AITC against dsnd6-mediated insects increased, while the bioactivity against dcox1-mediated insects decreased. Compared with the wild-type insects, the increase of reactive oxygen species (ROS) levels by AITC in mitochondria from dsnd6-mediated insects increased by 18.95%, while that of dscox1-mediated insects decreased by 27.45%. The effects of AITC on mRNA expression levels of detoxifying enzymes including CAT (down-regulation effect) and CuZnSOD (overexpression effect) partly recovered in the dsnd5-mediated insects, while a greater effect was observed for dscox1-mediated insects. Molecular docking results indicated that ASN511 at the cox1 subunit was the binding site of AITC by one hydrogen bond, with a bond distance of 2.1 Å. These findings provide insight for further applications of AITC and could provide a novel strategy to investigate lethal mechanisms of insecticides.

Potential value of small-molecule organic acids for the control of postharvest gray mold caused by Botrytis cinerea.

In the present study, a total of 21 natural or synthetic small-molecule organic acids were selected and determined for their activity against postharvest gray mold caused by B. cinerea. Overall, cuminic acid, which was extracted from the seed of Cuminum cyminum L, showed the most promising antifungal activity against B. cinerea both in vitro and in vivo. The study on action mechanism showed that cuminic acid could inhibit the development of sclerotia and the secretion of oxalic acid, destroy the cell membrane integrity, and down regulate the expression of several key genes involved in sclerotia development and pathogenicity of B. cinerea. Furthermore, cuminic acid could potentially reduce the degradation of TSS and TA content, while it had no significant effect on the weight loss, firmness, and VC content of apple and tomato. Importantly, cuminic acid could enhance the antioxidant enzyme activities of the fruits. All these results demonstrate the antifungal activity and highlight the great potential of cuminic acid as an alternative environmental-friendly agent for the control of postharvest gray mold both on fruits and vegetables.

Two single mutations in carboxylesterase 001C improve fenvalerate hydrolase activity in Helicoverpa armigera.

Carboxylesterases (CarEs) usually play critical roles in the detoxification of toxic chemicals and therefore may be involved in insecticide resistance in agricultural pests. Previous work has shown that CarE 001C from Helicoverpa armigera was able to metabolize the isomers of cypermethrin and fenvalerate. In this study, seven mutants of CarE 001C with single amino acid substitution were produced and expressed in the Escherichia coli. Enzyme kinetic analysis indicated that all seven mutations dramatically reduced enzymatic activities toward the generic substrate α-naphthyl acetate, but in vitro metabolism assay showed that two of the mutations, H423I and R322L, significantly improved hydrolase activities toward fenvalerate, with their recorded specific activities being 3.5 and 5.1 nM·s-1·mg -1 proteins, respectively. Further, thermostability assay showed that the stability of one mutant enzyme was enhanced. This study will help us better understand the potential of CarEs in insecticide detoxification and resistance in H. armigera.

Acting target of toosendanin locates in the midgut epithelium cells of Mythimna separate Walker larvae (lepidoptera: Noctuidae).

Toosendanin (TSN), which is extracted from the root bark of Melia toosendan Siebold and Zuccarini, has multiple modes of action against insects. Especially, this compound has a potent stomach poisoning activity against several lepidoptera pests. In this paper, the signs of toxicity, digestive enzymes activity, the histopathological changes and immuno-electron microscopic localization of TSN in the midgut epithelium of Mythimna separate Walker larvae were investigated for better understanding its action mechanism against insects. The bioassay results indicated that TSN has strong stomach poisoning against the fifth-instar larvae of M. separata (LC50 = 252.23 µg/mL). The typical poisoned symptom were regurgitation and paralysis. Activities of digestive enzymes had no obvious changes after treatment with LC80 dose of TSN. The midgut epithelial cells of insect were damaged by TSN, showing the degeneration of microvilli, hyperplasia of smooth endoplasmic reticulum and condensation of chromatin. Immunohistochemical analysis revealed that the gold particles existed on the microvilli of columnar cells and goblet cells, and gradually accumulated with the exacerbation of poisoning symptoms, showing that TSN targets on the microvilli of the midgutcells. Therefore, TSN acts on digestive system and locates in the microvilli of midgutcells of M. separata.

Validation of suitable reference genes for quantitative gene expression analysis in Tripterygium wilfordii.

Validation of suitable reference genes is critical in quantitative real-time polymerase chain reaction (qRT-PCR) analysis. Suitable and reliable reference genes for the normalization of gene expression data are characterized by high gene expression stability across tissues and different experimental conditions. This study evaluated the gene expression stability of ten reference genes commonly used in Arabidopsis thaliana for their suitability in qRT-PCR analysis in Tripterygium wilfordii Hook.f. The orthologous sequences of these ten candidate genes were identified from T. wilfordii transcriptomic data (Project No. SRX472292). Five algorithms including GeNorm, NormFinder, BestKeeper, ΔCt, and RefFinder were used to assess the gene expression stability of these putative reference genes in different plant tissues and different stress conditions. The results identified ACTINT7 and TBP as the most suitable reference genes across all samples. The gene expressions of TwHMGR (3-hydroxy-3-methylglutaryl coenzyme A reductase, KU246037.1) and of TwDXR (1-deoxy-D-xylulose-5-phosphate reductoisomerase, KJ174341.1) were investigated to validate the suitability of the reference genes. The validation analysis confirmed the suitability of ACTINT7 and TBP as the best reference genes for elucidating secondary metabolite biosynthesis pathway in T. wilfordii. In summary, this study identified the most suitable and reliable reference genes for future qRT-PCR- based studies in T. wilfordii.

Identification and biochemical characterization of carboxylesterase 001G associated with insecticide detoxification in Helicoverpa armigera.

Carboxylesterases (CarEs) are a major class of detoxification enzymes involved in insecticide resistance in various insect species. In this study, a novel CarE 001G was isolated from the cotton bollworm Helicoverpa armigera, one of the most destructive agricultural insect pests. The open reading frame of 001G has 2244 nucleotides and putatively encodes 747 amino acid residues. The deduced CarE possessed the highly conserved catalytic triads(Ser-Glu-His) and pentapeptide motifs (Gly-X-Ser-X-Gly), suggesting 001G is biologically active. The truncated 001G was successfully expressed in Escherichia coli, and the recombinant proteins were purified and tested. The enzyme kinetic assay showed the purified proteins could catalyze two model substrates, α-naphthyl acetate and ß-naphthyl acetate, with a kcat of 8.8 and 2.3 s-1, a Km of 9.6 and 16.2 µM, respectively. The inhibition study with pyrethroid, organophosphate and neonicotinoid insecticides showed different inhibition profile against the purified CarE. The HPLC assay demonstrated that the purified proteins were able to metabolize ß-cypermethrin, λ-cyhalothrin and fenvalerate insecticides, exhibiting respective specific activities of 1.7, 1.4 and 0.5 nM/min/mg protein. However, the purified proteins were not able to metabolize the chlorpyrifos, parathion-methyl, paraoxon-ethyl and imidacloprid. The modeling and docking analyses consistently demonstrated that the pyrethroid molecule fits snugly into the catalytic pocket of the CarE 001G. Collectively, our results suggest that 001G may play a role in pyrethroids detoxification in H. armigera.

Comparative studies on muscle microstructure and ultrastructure of Mythimna separata Walker treated with wilforgine and chlorantraniliprole.

We attempted to elucidate the comparative effects between wilforgine and chlorantraniliprole on the microstructure/ultrastructure of muscle tissue in Mythimna separate larvae. The typical toxicity symptoms of M. separata larvae upon wilforgine treatment was feeding cessation and flaccid paralysis, whereas feeding cessation and contraction paralysis were the main poisoning symptoms wrought by chlorantraniliprole. Light-microscopy observations showed that the microstructure of muscle tissue could be damaged by wilforgine and chlorantraniliprole, and the death of insects was associated with muscle lesions. Muscle tissue was loose after wilforgine treatment but constricted muscle tissue was observed upon chlorantraniliprole treatment. Transmission electron microscopy showed that wilforgine and chlorantraniliprole could disrupt endomembranes and plasma membranes. These results suggest that wilforgine can induce microstructural and ultrastructural changes in the muscles of M. separata larvae; the sites of action are proposed to be calcium receptors or channels in the muscular system.

Cloning, Expression Analysis and Functional Characterization of Squalene Synthase (SQS) from Tripterygium wilfordii.

Celastrol is an active triterpenoid compound derived from Tripterygium wilfordii which is well-known as a traditional Chinese medicinal plant. Squalene synthase has a vital role in condensing two molecules of farnesyl diphosphate to form squalene, a key precursor of triterpenoid biosynthesis. In the present study, T. wilfordii squalene synthase (TwSQS) was cloned followed by prokaryotic expression and functional verification. The open reading frame cDNA of TwSQS was 1242 bp encoding 413 amino acids. Bioinformatic and phylogenetic analysis showed that TwSQS had high homology with other plant SQSs. To obtain soluble protein, the truncated TwSQS without the last 28 amino acids of the carboxy terminus was inductively expressed in Escherichia coliTransetta (DE3). The purified protein was detected by SDS-PAGE and Western blot analysis. Squalene was detected in the product of in vitro reactions by gas chromatograph-mass spectrometry, which meant that TwSQS did have catalytic activity. Organ-specific and inducible expression levels of TwSQS were detected by quantitative real-time PCR. The results indicated that TwSQS was highly expressed in roots, followed by the stems and leaves, and was significantly up-regulated upon MeJA treatment. The identification of TwSQS is important for further studies of celastrol biosynthesis in T. wilfordii.

New Cytotoxic Secondary Metabolites from Marine Bryozoan Cryptosula pallasiana.

A new sterol, (23R)-methoxycholest-5,24-dien-3ß-ol (1), two new ceramides, (2S,3R,4E,8E)-2-(tetradecanoylamino)-4,8-octadecadien-l,3-diol (6) and (2S,3R,2'R,4E,8E)-2-(tetradecanoylamino)-4,8-octadecadien-l,3,2'-triol (7), together with three known sterols (2-4), a lactone (5) and two ceramides (8,9), were isolated from the marine bryozoan Cryptosula pallasiana, collected at Huang Island of China. The structures of the new compounds were elucidated by extensive spectroscopic analyses, chemical methods and quantum electronic circular dichroism (ECD) calculations. Among the isolated compounds, sterol 1 possessed a rare side chain with a methoxy group at C-23, and a double bond between C-24 and C-25. Ceramides 6 and 7 possessed 14 carbons in their long-chain fatty acid base (FAB), which were different from the normal ceramides with 16 carbons in the FAB. Moreover, compounds 5 and 8 were isolated for the first time from marine bryozoans. Compounds 1-9 were evaluated for their cytotoxicity against human tumor cell lines HL-60, Hep-G2 and SGC-7901. The results showed that lactone 5 appears to have strong cytotoxicity against the test tumor cell lines, with IC50 values from 4.12 µM to 7.32 µM, and sterol 1 displayed moderate cytotoxicity with IC50 values between 12.34 µM and 18.37 µM, while ceramides 6-9 showed weak cytotoxicity with IC50 ranging from 21.13 µM to 58.15 µM.