Optimizing the extract yield of bioactive compounds in Valeriana officinalis root: a D-optimal design.

It is estimated that 80% of all synthetic drugs are derived from medicinal plants, and nowadays, many synthetic drugs are derived from medicinal plants. Valeriana officinalis can treat many diseases of the nervous system. A crucial aspect of valerian extract is that it inhibits the proliferation of breast cancer cells. To optimize the yield of bioactive compounds in the V. officinalis root extraction, a response surface methodology-based D-optimal design was used. To fulfill this aim, the effects of various factors such as solvent type and concentration, mixing temperature, ultrasound time, and drying method were examined. The optimal conditions for solvent percentages, mixing temperature, ultrasound time, solvent type, and drying methods were determined to be 94.88%, 25 °C, 48.95 min, methanol, and microwave, respectively, with a desirability of 0.921. The predicted valerenic acid, total phenols, total flavonoids, and antioxidant activity in V. officinalis extract were 1.19 (mg/g DW), 8.22 (mg/g DW), 5.27 (mg/g DW), and 92.64%, respectively. In optimal conditions, the extracted amounts of valerenic acid, total phenols, total flavonoids, and antioxidant activity were 2.07 mg/g DW, 7.96 mg/g DW, 5.52 mg/g DW, and 78.68%, respectively, which were consistent with the model predicted amounts (based on 95% prediction interval). This study could be useful as a model for demonstrating the efficacy of microwave drying to maximize the biochemical content of V. officinalis, as well as the antioxidant activity of the root extracts of V. officinalis on industrial scale.

Graphene Quantum Dots (GQD)-Mediated dsRNA Delivery for the Control of Fusarium Head Blight Disease in Wheat.

Graphene quantum dots (GQDs), a class of fluorescent carbon materials, have displayed significant potential in various fields such as energy devices, catalysis, sensing, bioimaging, and drug delivery. Because of their extremely small size, generally less than 100 nm, they also have tremendous potential in plant science research, especially for the delivery of nucleic acids, breaking the barrier of cell walls. In this study, we synthesized GQDs with a size range of 2-5 nm, characterized them, and surface-functionalized them with branched polyethylenimine (bPEI). We then used the surface-functionalized GQDs as carriers to deliver double-stranded RNA (dsRNA) that target two growth-and-development-related genes in Fusarium graminearum─the causative organism of the Fusarium head blight disease of wheat. The successful binding of dsRNA to GQDs-bPEIs was demonstrated through gel-shifting assays, showcasing the potential for efficient dsRNA delivery. We designed dsRNAs targeting the MGV1 and RAS1 genes of F. graminearum by using the pssRNAit pipeline, ensuring high specificity and no off-target effects. The coding sequences of the designed dsRAS1 and dsMGV1 were cloned into the L4440 vector and transformed into the Escherichia coli HT115 strain for dsRNA production. Fungal culture analysis revealed that the inclusion of dsRNAs in potato dextrose agar (PDA) media effectively slowed down the growth. Exogenous spraying experiments both in plate cultures and in intact wheat spikes demonstrated that the dsRNA:GQDs-bPEIs treatment was more effective in restricting fungal mycelium growth or the number of infected spikelets compared to naked dsRNA treatment. Our study demonstrates the promising potential of graphene quantum dots as carriers for dsRNA-based fungal disease management in wheat and other crops.

Application of machine learning algorithms and feature selection in rapeseed (Brassica napus L.) breeding for seed yield.

BACKGROUND: Studying the relationships between rapeseed seed yield (SY) and its yield-related traits can assist rapeseed breeders in the efficient indirect selection of high-yielding varieties. However, since the conventional and linear methods cannot interpret the complicated relations between SY and other traits, employing advanced machine learning algorithms is inevitable. Our main goal was to find the best combination of machine learning algorithms and feature selection methods to maximize the efficiency of indirect selection for rapeseed SY. RESULTS: To achieve that, twenty-five regression-based machine learning algorithms and six feature selection methods were employed. SY and yield-related data from twenty rapeseed genotypes were collected from field experiments over a period of 2 years (2019-2021). Root mean square error (RMSE), mean absolute error (MAE), and determination coefficient (R2) were used to evaluate the performance of the algorithms. The best performance with all fifteen measured traits as inputs was achieved by the Nu-support vector regression algorithm with quadratic polynomial kernel function (R2 = 0.860, RMSE = 0.266, MAE = 0.210). The multilayer perceptron neural network algorithm with identity activation function (MLPNN-Identity) using three traits obtained from stepwise and backward selection methods appeared to be the most efficient combination of algorithms and feature selection methods (R2 = 0.843, RMSE = 0.283, MAE = 0.224). Feature selection suggested that the set of pods per plant and days to physiological maturity along with plant height or first pod height from the ground are the most influential traits in predicting rapeseed SY. CONCLUSION: The results of this study showed that MLPNN-Identity along with stepwise and backward selection methods can provide a robust combination to accurately predict the SY using fewer traits and therefore help optimize and accelerate SY breeding programs of rapeseed.

Economic purification of recombinant uricase by artificial oil bodies.

Rasburicase is an expensive treatment used to control hyperuricemia caused by tumour lysis syndrome (TLS). In this study, a non-chromatographic method was designed based on nano-oil bodies for convenient and economical purification of the recombinant uricase. For this purpose, two chimaeras were synthesized with a different arrangement of the uricase, caleosin and intein fragments. After confirming the protein expression by measuring the uricase activity at 293 nm, purification was conducted through oil-body construction. The results were resolved on the 12% SDS-PAGE gel. Finally, the stability of the oil bodies was examined against different salts, surfactants, temperatures, and pH values. According to our results, the overexpression of uricase-caleosin chimaera under the T7 promoter in Escherichia coli led to the production of soluble protein, which was successfully purified by artificial oil bodies. The active uricase was subsequently released through the self-splicing of intein. Further investigations highlighted the importance of the free C-terminus of caleosin in constructing artificial oil bodies. Moreover, surfactants and low temperature, in contrast to salts, improved the stability of oil bodies. In conclusion, caleosins are an efficient purification tag reducing the cost of purification compared to conventional chromatography methods.

pMOX: a new powerful promoter for recombinant protein production in yeast Pichia pastoris.

AOX1 promoter (pAOX1) is a robust inducible promoter highly preferred for the production of recombinant proteins in Pichia pastoris (P. pastoris). However, repression by other carbon sources and induction by methanol, which is a fire hazard chemical and undesirable for industrial production, are remarkable drawbacks in large-scale use of this promoter. Hence, novel strong regulatory promoters are highly desired. In the present study, the promoter region of methanol oxidase gene (pMOX), from Hansenula polymorpha, was explored for the heterologous expression of foreign proteins in protease deficient and wild type P. pastoris strains. The promoter region of MOX was isolated and replaced with the pAOX1 in the pPINK-HC plasmid. The activity of pMOX and pAOX1 was compared using endoglucanase 3 (CMC3) and endoglucanase II (EgII) enzymes as the reporter proteins. Evaluation of carbon sources on pMOX activity showed complete inactivation in the presence of xylose and sorbitol and high activity by glycerol, glucose and methanol feeding. Furthermore, the results indicated that increasing the gene dosage and using protease deficient-trait significantly increased CMC3 and EgII expression under the control of pMOX. In conclusion, in this study, a new small powerful and methanol-free promoter is introduced for recombinant protein production in yeast P. pastoris.

Predictive and fluorescent nanosensing experimental methods for evaluating anthrax protective antigen and lethal factor interactions for therapeutic applications.

Recently, specific interaction of anthrax protective antigen domain 4 (PAD4) and lethal factor domain 1 (LFD1) have been considered for the design of novel diagnostic and therapeutic systems in medicine. In this study, theoretical and experimental approaches were used to monitor the interactions of PAD4 and LFD1. CLusPro server and Dimplot software were used to predict the interaction of these domains. Results, revealed interactive sites between PAD4 and LFD1 on loop regions of both C and N terminal of PAD4. In experimental methods, PAD4 and LFD1 were expressed in Escherichia coli and purified for usage in Magnetic Bead (MB) and Multi-Walled Carbon Nanotubes (MWCNTs) based bio-sensing platforms. In the magnetic-based system, the magnetic sedimentation of QD-PAD4 by MBs-LFD1 and the observation of the fluorescence spectrum related to QD-PAD4 in the precipitated materials confirmed the interaction of PAD4 with LFD1 protein. In the MWCNTs-based method, the QD-PAD4 fluorescence was quenched by absorption on MWCNTs. Upon the addition of LFD1, fluorescence emission was recovered, indicating interaction of LFD1 with QD-PAD4, which results the separation of QD-PAD4 from MWCNTs surfaces and fluorescence restoration. Finally, new approaches showed the interaction of PAD4 and LFD1, which can be used as an attractive model in medicine.

Differential expression of heat shock proteins and antioxidant enzymes in response to temperature, starvation, and parasitism in the Carob moth larvae, Ectomyelois ceratoniae (Lepidoptera: Pyralidae).

Insects face diverse biotic and abiotic stresses that can affect their survival. Many of these stressors impact cellular metabolism, often resulting in increased accumulation of reactive oxygen species (ROS). Consequently, insects will respond to these stressors by increasing antioxidant activity and increased production of heat shock proteins (HSPs). In this study, the effect of heat, cold, starvation, and parasitism by Habroacon hebetor wasps was examined in the carob moth, Ectomyelois ceratoniae, to determine which responses were common to different stresses. For all stressors, malondialdehyde levels increased, indicative of oxidative stress in the insects. The activity of two antioxidant enzymes, superoxide dismutase (SOD) and catalase (CAT), increased with each stress, suggesting that these enzymes were serving a protective role for the insects. Heat (46°C for 100 min) and cold (-15°C for 30 min) treatments caused significant mortalities to all developmental stages, but pretreatments of moderate heat (37°C for 10 min) or cold (10°C for 10 min) induced thermotolerance and reduced the mortality rates when insects were subsequently exposed to lethal temperatures. Quantitative RT-PCR confirmed that heat and cold tolerance were associated with up-regulation of two HSPs, HSP70 and HSP90. Interestingly, HSP70 transcripts increased to a greater extent with cold treatment, while HSP90 transcripts increased more in response to high temperatures. RNA interference (RNAi)-mediated knockdown of either HSP70 or HSP90 transcripts was achieved by injecting larvae with dsRNA targeting each gene's transcripts, and resulted in a loss of acquired thermotolerance in insects subjected to the heat or cold pretreatments. These observations provide convincing evidence that both HSP70 and HSP90 are important mediators of the acquired thermotolerance. Starvation and parasitism by wasps caused differential expression of the HSP genes. In response to starvation, HSP90 transcripts increased to a greater extent than HSP70, while in contrast, HSP70 transcripts increased to a greater extent than those of HSP90 during the first 48 h of wasp parasitism. These results showed the differential induction of the two HSPs' transcripts with variable stresses. As well as, heat, cold, starvation, and parasitism induce oxidative stress, and antioxidant enzymes likely play an important role in reducing oxidative damage in E. ceratoniae.

Host-Induced Silencing of Some Important Genes Involved in Osmoregulation of Parasitic Plant Phelipanche aegyptiaca.

Broomrape is an obligate root-parasitic weed that acts as a competitive sink for host photoassimilates. Disruption of essential processes for growth of broomrape using host plant-mediated systemic signals can help to implement more specific and effective management plans of this parasite. Accordingly, we tested the possibility of transient silencing three involved genes (PaM6PR, PaCWI, and PaSUS1) in osmoregulation process of broomrape using syringe agroinfiltration of dsRNA constructs in tomato. The highest decrease in mRNA levels, enzyme activity, and amount of total reducing sugars was observed in Phelipanche aegyptiaca when grown on agroinfiltrated tomato plants by PaM6PR dsRNA construct than control. In addition, PaSUS1 dsRNA construct showed high reduction in mRNA abundance (32-fold fewer than control). The lowest decrease in mRNA levels was observed after infiltration of PaCWI dsRNA construct (eightfold fewer than control). While the highest reduction in PaM6PR and PaSUS1 expression levels was detected in the parasite at 3 days post-infiltration (dpi), the maximum reduction in both of the total reducing sugars amount and M6PR and SUS1 activities was observed at 8 dpi. On the contrary, CWI activity, PaCWI expression level, and amount of total reducing sugars in broomrape shoots simultaneously decreased at the day 3 after the dsRNA construct infiltration against PaCWI. On the whole, our results indicated that the three studied genes especially PaM6PR may constitute appropriate targets for the development of transgenic resistance in host plants using silencing strategy.

Developmental patterns of enzyme activity, gene expression, and sugar content in sucrose metabolism of two broomrape species.

A better understanding of broomrape physiological features opens up new perspectives for developing specific management strategies. For this purpose, activities of key enzymes involved in osmoregulation (SAI1, CWI, M6PR, and SUS1) were considered at developmental stages of two important broomrape species (Egyptian and branched broomrape) on tomato. While Egyptian broomrape tubercles had high activities of invertases, branched broomrape shoots revealed high activities of M6PR and SUS1 during both pre- and post-emergence stages except for M6PR at post-emergence stages of P. aegyptiaca. Interestingly, the main accumulation of total reducing sugars was detected in tubercle during pre- and in shoot during post-emergence. Unlike low levels of genes expression (except for CWI) before parasite emergence, significantly higher expression levels of SAI1, SUS1 and M6PR were detected after parasite emergence. Matching the expression levels of SAI1 and SUS1 genes with their corresponding enzymes activities makes them as the suitable candidates for gene silencing strategies.

Molecular cloning, functional characterization and expression of a drought inducible phenylalanine ammonia-lyase gene (ObPAL) from Ocimum basilicum L.

Phenylalanine ammonia-lyase (PAL) is a control point for branched phenylpropanoid and terpenoid pathways. It represents the first regulatory step to provide a metabolic flux to produce of the precursors needed for biosynthesizing main volatile phenylpropanoid compounds (methyleugenol and methylchavicol) in basil. It is crucial during the stage of the environmental and development stimulants. To obtain better knowledge of the biosynthesis of these phenylpropene compounds, characterization and cloning of Ocimum basilicum PAL (ObPAL) cDNA and its heterologous expression and enzyme activity were assessed. The almost full-length ObPAL was 2064 bp in size encoding a 687-amino-acid polypeptide with molecular weight of 74.642 kDa and theoretical pI of 8.62. Phylogenetic analysis revealed a significant evolutionary relatedness of ObPAL with the PAL sequence reported in different species of Lamiaceae. To further confirm its function, ObPAL was cloned into pET28a (+) vector and expressed in E. coli. The recombinant protein exhibited high PAL activity and could catalyze the L-Phe conversion to trans-cinnamic acid. Expression analysis of PAL gene showed that ObPAL manifested various transcription ratios exposed to drought stress. Overall, our results demonstrated the ObPAL regulation gene is possibly a mechanism dependent on cultivar and drought stress.

Changes in Cytomorphology, Expression of Retinoblastoma Related Gene, and Superoxide Dismutase Enzyme Activity in Maize Cell Culture Exposed to Silver Nanoparticles.

The ever-increasing use of silver nanoparticles (nAg) in various products necessitates investigation of the behavior of biological systems encountering these particles. In this paper, considering maize as a biological model, the effects of colloidal nAg (<80nm) on its cell culture were investigated. For comparison purposes, silver nitrate was used as a representative of silver ion (Ag+). After stabilization of cell suspensions, they were treated with nAg and Ag+ (1 mg/l), then cell suspension growth was measured and the microscopic analysis and a cell viability test were performed. In addition, the activity of superoxide dismutase (SOD) enzyme was explored. Owing to the key role of retinoblastoma-related protein (RBR) in cell cycle as well as in development and differentiation processes, the relative expression of ZmRBR1 was studied in nAg and Ag+ exposure. Microscopic analyses revealed that cells in suspensions treated by nAg and Ag+ were morphologically classified into five types: embryogenic; larvae-like; long; swollen; and polarized. The results showed an increase in percentages of large and live cells in the treated suspensions. Remarkably, we observed some cells which were differentiated into trichomes along with some stages of trichome development in the treated cell suspensions. Moreover, exposure to nAg and Ag+ did not elevate the activity of SOD enzyme in the treated cells. Also, the relative expression of ZmRBR1 was slightly reduced in the treated cells. The findings of these experimentations indicated that nAg affected maize suspension-cultured cells in the same manner as Ag+.

Covalent-display of an active chimeric-recombinant tissue plasminogen activator on polyhydroxybutyrate granules surface.

OBJECTIVE: To develop a deliberately engineered expression and purification system for an active chimeric-recombinant tissue plasminogen activator (crtPA) using co-expression with polyhydroxybutyrate (PHB) operon genes. RESULTS: Fusion of crtPA with PhaC-synthase simplified the purification steps through crtPA sedimentation with PHB particles. Moreover, the covalently immobilized crtPA was biologically active as shown in a chromogenic assay. Upon WELQut-protease activity, the released single-chain crtPA converted to the two-chain form which produced a pattern of bands with approx. MW of 32 and 11 kDa in addition to the full length crtPA. CONCLUSION: Fusion of crtPA with PhaC-synthase not only simplifies purification from the bacterial host lysate, but also co-expression of PHB operon genes creates an oxidative environment, thereby reducing the inclusion body formation possibility. The isolated crtPA-PHB granules exhibited crtPA serine protease activity. Thus, fusion with the PhaC protein could be used as a scaffold for covalent displaying of functional disulfide-rich proteins.

Water deficit stress fluctuates expression profiles of 4Cl, C3H, COMT, CVOMT and EOMT genes involved in the biosynthetic pathway of volatile phenylpropanoids alongside accumulation of methylchavicol and methyleugenol in different Iranian cultivars of basil.

Here, for the first time, the accumulation ratio of methylchavicol and methyleugenoland compounds together with the expression profiles of five critical genes (i.e., 4Cl, C3H, COMT, CVOMT and EOMT) in three Iranian cultivars of basil were assessed under water deficit stress at flowering stage. The highest value of methylchavicol was detected for Cul. 3 under severe stress (S3; 7.695µg/mg) alongside Cul. 2 under similar circumstances (S3; 4.133µg/mg), while regarding Cul. 1, no detectable amounts were acquired. Considering methyleugenol, Cul. 3 (0.396µg/mg; S0) followed by Cul. 1 (S3; 0.160µg/mg) were the capable plant samples in producing some detectable amounts of methyleugenol. Apart from some expectations, all the genes under study exhibited also different transcription ratios under deficit stress. Our results, overall, demonstrated that the regulation of the above-mentioned genes and production of methychavicol and methyleugenol seems to be a cultivar- and drought stress-dependent mechanism.

Production of Recombinant Antimicrobial Polymeric Protein Beta Casein-E 50-52 and Its Antimicrobial Synergistic Effects Assessment with Thymol.

Accelerating emergence of antimicrobial resistance among food pathogens and consumers' increasing demands for preservative-free foods are two contemporary challenging aspects within the food industry. Antimicrobial packaging and the use of natural preservatives are promising solutions. In the present study, we used beta-casein-one of the primary self-assembly proteins in milk with a high polymeric film production capability-as a fusion partner for the recombinant expression of E 50-52 antimicrobial peptide in Escherichia coli. The pET21a-BCN-E 50-52 construct was transformed to E. coli BL21 (DE3), and protein expression was induced under optimized conditions. Purified protein obtained from nickel affinity chromatography was refolded under optimized dialysis circumstances and concentrated to 1600 µg/mL fusion protein by ultrafiltration. Antimicrobial activities of recombinant BCN-E 50-52 performed against Escherichia coli, Salmonella typhimurium, Listeria monocytogenes, Staphylococcus aureus, Aspergillus flavus, and Candida albicans. Subsequently, the synergistic effects of BCN-E 50-52 and thymol were assayed. Results of checkerboard tests showed strong synergistic activity between two compounds. Time-kill and growth kinetic studies indicated a sharp reduction of cell viability during the first period of exposure, and SEM (scanning electron microscope) results validated the severe destructive effects of BCN E 50-52 and thymol in combination on bacterial cells.

Seasonal-based temporal changes fluctuate expression patterns of TXS, DBAT, BAPT and DBTNBT genes alongside production of associated taxanes in Taxus baccata.

KEY MESSAGE: Environmental cues have synergistic or antagonistic regulatory roles on transcription activity and taxanes accumulation in yew, though DBAT activity is less influenced, could be accordingly a rate-limiting enzyme. The current work was undertaken to elucidate the consequences of some environmental cues (i.e., day length, temperature, sunlight and relative humidity) on the expression patterns of TXS, DBAT, BAPT and DBTNBT genes contributed to the taxol biosynthetic pathway along with the accumulation of some taxanes in needles and stems of Taxus baccata over year 2013-2014. In both tissues, light intensity and temperature correlated with the production of 10-DAB III and total taxanes, and TXS activity, while a lack of significant association was deduced for day length and relative humidity. Furthermore, in both tissues, a weak correlation was observed between BAC III and light intensity, temperature, day length and relative humidity, and the corresponding gene, DBAT. Surprisingly, DBAT activity was not co-induced with TXS in both tissues, and remained expressed at basal levels over year, supporting that the conversion of 10-DAB III into BAC III could presumably be a rate limiting step in the taxol biosynthetic pathway. Similar to BAC III, no strong correlation was detected between production of taxol in both tissues and all the meteorological data, while the corresponding genes BAPT and DBTNBT, in some cases, exhibited significant correlated results. Notably, despite higher activities of BAPT and DBTNBT in both tissues over year, taxol production was still in small quantities, probably owing to the low amounts of its precursors rather than low volumes of BAPT and DBTNBT transcripts. The results, altogether, could provide us new insights towards the potential regulatory roles of environmental cues on the production of taxanes in yew trees.

MOLECULAR IDENTIFICATION OF CYSTEINE AND TRYPSIN PROTEASE, EFFECT OF DIFFERENT HOSTS ON PROTEASE EXPRESSION, AND RNAI MEDIATED SILENCING OF CYSTEINE PROTEASE GENE IN THE SUNN PEST.

Sunn pest, Eurygaster integriceps, is a serious pest of cereals in the wide area of the globe from Near and Middle East to East and South Europe and North Africa. This study described for the first time, identification of E. integriceps trypsin serine protease and cathepsin-L cysteine, transcripts involved in digestion, which might serve as targets for pest control management. A total of 478 and 500 base pair long putative trypsin and cysteine gene sequences were characterized and named Tryp and Cys, respectively. In addition, the tissue-specific relative gene expression levels of these genes as well as gluten hydrolase (Gl) were determined under different host kernels feeding conditions. Result showed that mRNA expression of Cys, Tryp, and Gl was significantly affected after feeding on various host plant species. Transcript levels of these genes were most abundant in the wheat-fed E. integriceps larvae compared to other hosts. The Cys transcript was detected exclusively in the gut, whereas the Gl and Tryp transcripts were detectable in both salivary glands and gut. Also possibility of Sunn pest gene silencing was studied by topical application of cysteine double-stranded RNA (dsRNA). The results indicated that topically applied dsRNA on fifth nymphal stage can penetrate the cuticle of the insect and induce RNA interference. The Cys gene mRNA transcript in the gut was reduced to 83.8% 2 days posttreatment. Also, it was found that dsRNA of Cys gene affected fifth nymphal stage development suggesting the involvement of this protease in the insect growth, development, and molting.

A proteomic analysis to identify cold acclimation associated proteins in wild wheat (Triticum urartu L.).

To gain a better understanding of cold acclimation process in wheat, we applied a 2-DE based proteomic approach to discover changes in proteome profile of a diploid wild wheat (Triticum urartu L.) during prolonged cold stress treatment. To this end, plants were grown in pots and the growing seedlings (4-leaf stage) were exposed to cold stress. After 4 weeks of cold acclimation (4-6 °C) and subsequent treatment for 12 h at -2 °C, samples were collected from control and stressed plants and were subjected to proteome pattern analysis. Among approximately 450 reproducible protein spots displayed in each given 2-DE gels, 34 proteins changed significantly in abundance in response to cold stress. Among them, 25 and 9 proteins were up and down-regulated under stress condition, respectively. Analysis by matrix-assisted laser desorption ionization time of flight/time of flight mass spectrometry coupled with non-redundant protein database search allowed the identification of 20 cold-induced proteins. Integrated proteomic and database survey resulted in identification of several cold stress related proteins such as pathogenesis related protein, cold regulated protein, cold-responsive LEA/RAB-related COR protein, oxygen-evolving enhancer protein and oxalate oxidase. The presumed functions of the identified proteins were mostly related to cold acclimation, oxidative stress and photosynthesis. The possible implications of differentially accumulated proteins in acquiring systemic tolerance to freezing stress following exposure to prolonged low temperature will be discussed.

Overexpression of Arabidopsis Dehydration-Responsive Element-Binding protein 2A confers tolerance to salinity stress to transgenic canola.

Stress responsive transcriptional regulation is an adaptive strategy of plants that alleviates the adverse effects of environmental stresses. The ectopic overexpression of Dehydration-Responsive Element Binding transcription factors (DREBs) either in homologous or in heterologous plants are the classical transcriptional regulators involved in plant responses to drought, salt and cold stresses. To elucidate the transcriptional mechanism associated with the DREB2A gene after removing PEST sequence, which acts as a signal peptide for protein degradation, 34 transgenic T0 canola plants overexpressing DREB2A were developed. The quantitative Real time PCR of transgenic plants showed higher expression of downstream stress-responsive genes including COR14, HSF3, HSP70, PEROX and RD20. The transgenic plants exhibited enhanced tolerance to salt stress. At the high concentration of NaCl the growth of non-transformed plants had been clearly diminished, whereas transgenic line was survived. These results indicated that transformed DREB2A gene might improve the plant response to salinity in transgenic canola plants.

Expression of the truncated tissue plasminogen activator (K2S) gene in tobacco chloroplast.

As because the plant plastid genome is highly polyploid, the transformation of chloroplasts permits the introduction of thousands of copies of foreign genes per plant cell and generates extraordinarily high levels of recombinant protein. Human tissue-type plasminogen activator is one of the most important pharmaceutical proteins involved in the breakdown of blood clots in brain and heart blood vessels. We report the introduction and expression of the truncated human tissue plasminogen activator (K2S) gene in tobacco chloroplasts. The K2S-containing vector pKCZK2S was successfully transferred to tobacco plastomes using the biolistic delivery procedure. Transplastomic plants were selected on RMOP medium containing spectinomycin (500 mg/l). In order to achieve homoplasmy, several rounds of selection and regeneration were performed. The presence, site-specific integration, homoplasmy, expression and activity assay of the transgene were confirmed in the transplastomic plants by PCR, Southern-blot, RT-PCR, SDS-PAGE, ELISA, Dot-blot, Western-blot and zymography analysis. Our results show that the tissue plasminogen activator (K2S form) protein to be expressed in tobacco chloroplasts in active form.

Expression and large-scale production of human tissue plasminogen activator (t-PA) in transgenic tobacco plants using different signal peptides.

An attempt was made to assess the expression level and targeting of a human protein entitled recombinant tissue plasminogen activator (rt-PA) through accumulation in three cellular compartments including the endoplasmic reticulum and cytosolic and apoplastic spaces in transgenic tobacco plants. In this context, three chimeric constructs pBI-SP-tPA, pBI-tPA-KDEL, and pBI-Ext-tPA were employed and transferred into the tobacco plants through a popular transformation-based system called Agrobacterium tumefaciens. As an initial screening system, the incorporation of the rt-PA gene in the genomic DNA of tobacco transgenic plants and the possible existence of the rt-PA-specific transcript in the total RNAs of transgenic plant leaves were confirmed via PCR and reverse transcription (RT)-PCR, respectively. Southern blot analysis, in addition, was used to determine the copy number of the corresponding gene (i.e., t-PA) transformed into the each transgenic plant; one or more copies were detected regarding transformants derived from all three abovementioned constructs. According to the enzyme-linked immunosorbent assay, the mean values of t-PA expression were calculated as 0.50, 0.68, and 0.69 µg/mg of the total soluble protein when a collection containing 30 transgenic plants transformed with pBI-SP-tPA, pBI-tPA-KDEL, and pBI-Ext-tPA was taken into account, respectively. The zymography assay was lastly performed and concluded the expression of the properly folded rt-PA in this expression system. Our results, altogether, revealed that tobacco plants could be utilized as a bioreactor system for the large-scale production of enzymatically active t-PA and presumably other therapeutic recombinant proteins in large quantities.