AbstractObjectiveThe aim of this study is to optimize tomato as a suitable plant system for transient gene transformation for the production of pharmaceutical and industrial compounds.Materials and methodsIn order to investigate transient gene expression, three concentrations of Agrobacterium in OD600 (0.4, 0.6 and 0.8), three times after inoculation (4, 7 and 10 days) and two conditions with the presence of p19 gene and without p19 were selected as factors affecting the gene expression. The vectors used in this research pXK2FS7 and pCAMBIA1304 contained GFP and P19 genes, respectively. For transient GFP transformation, leaves of seedlings were inoculated using agroinfiltration method. In this way, different concentrations of Agrobacterium containing pXK2FS7 vector and P19 vector were mixed together and the expression of GFP gene was investigated. All the leaves of tomato plants at the appropriate stage were injected by insulin syringe without needle on the lower surface of the leaf. In order to confirm the transfection of the GFP and its expression, RNA extraction, cDNA synthesis and the analysis of expression by Real-Time PCR were performed and the expression of the GFP protein with a fluorescence microscope was finally detected.ResultsThe results showed that the concentration of Agrobacteria, the number of days after inoculation, and the expression of the p19 had significant effects on the GFP expression. The expression of p19 as a suppressor of gene silencing mechanism led to higher GFP expression in comparison with the absence of p19. Additionally, it was found that dilution of 0.6OD600 Agrobacterium was the most suitable concentration for high expression of GFP agroinfiltration. Furthermore, the highest level of GFP expression was obtained 7 days after leaf inoculation. The best combination for maximum GFP expression at transcript level obtained at 0.6 OD600, 7 days after inoculation and expression of p19. ConclusionsThe results of this research can be used in preliminary studies or optimization of recombinant protein production in tomato leaves.

Among the 26 human claudin proteins, the food-poisoning bacterium Clostridium perfringens produces an enterotoxin (~ 35.00 kDa) that specifically targets human claudin 4, causing diarrhea by fluid accumulation in the intestinal cavity. The Clostridium perfringens enterotoxin (CPE) C-terminal domain (cCPE ~ 15.00 kDa) tightly binds to claudin 4 and disrupts the tight junction barriers in the intestines. In this study, we aimed to determine the contribution and type of amino acid interactions involved in association between claudin 4 and the C-terminal CPE. First, the three-dimensional format of claudin 4 was downloaded from RCSB. Then, during 60.00 nanoseconds (nsec), molecular dynamics simulation was conducted using the GROMACS package on CPE of crystallographic structure. The results indicated that the simulations performed well during the simulation times and there were no noticeable problems or artifacts. We found that Coulombic (glycine 317, proline 311 and serine 313) and Lennard-Jones (tyrosine 310, leucine 315, serine 313 and glycine 317) interactions played a significant role in complex stability. This information localized the C-terminal of CPE as a linear sequence sufficient for recognition and binding to the eukaryotic CPE receptor. A detailed description of the dissociation process brings valuable insight into the interaction of the claudin 4-cCPE290-319 complexes, which could help in the future to design more potent drugs.

Objective Tissue plasminogen activator is one of the most important drugs in the treatment of heart disease. This drug is produced in the expression system as a recombinant protein that has high production costs. Transient expression system is very suitable for protein expression because of its high expression, high speed, low cost and no spatial effect. Post-transcriptional silencing has been shown to affect expression levels. Therefore, the aim of this study was to investigate the effect of simultaneous expression of P19 silencing suppressor gene on transient expression of recombinant tissue plasminogen activator (rtPA) at transcriptional and protein levels in Nicotiana benthamiana. Material and methods To serve this purpose, the expression proportion of injected Agrobacterium tumefaciens containing a binary vector pCAMBIA1304-rtPA with agrobacterium containing pCAMBIA1304-P19 have been studied comparison with the expression level from Agrobacterium containing only the binary vector pCAMBIA1304-rtPA. Leaf samples were prepared on 4, 7, and 10 day post-inoculation with Agrobacterium. Transcription and then protein levels were calculated using the Real Time PCR and ELISA tests. Results The results of Real Time PCR test showed that rtPA transcript increased in the presence of P19. Also, 4 days after plant inoculation, the highest transcript levels were obtained from p19 and rtPA genes. ELISA results showed that the expression of rtPA protein in the presence of P19 was 89 and 84 µg.g-1 leaf weight at the 7 and 10 day after inoculation, respectively. This expression was 12 and 15% higher than of when agrobacterium-containing pCAMBIA1304-rtPA vector alone was used, respectively. Conclusion The results showed that the use of transient expression method could be a suitable method for rtPA protein production.

Seeds of 20 accessions of six Brassica species including cultivated and five wild relatives were analyzed for oil and fatty acid composition. The results showed that oil content varied from 21 (B. nigra) to 46% (B. napus). Among wild species, B. rapa and B. oleracea had highest oil content (31 and 28%, respectively). The main fatty acids of oleic, linoleic, linolenic, erucic, palmitic, and stearic acids accounted for 89-94% of the total fatty acids in all species. Cultivated species of B. napus had highest oleic acid (61%) and lowest erucic acid (1%) content compared to other studied species. Brassica rapa and B. oleracea had the highest content of erucic acid (41 and 46%, respectively). The highest content of linolenic (20%) and linoleic (19%) acid was observed for B. juncea. The results showed that there was high genetic variation among the studied species for oil content and fatty acids composition. This indicates that seed oil of these species is possibly suitable for both human consumption and industrial purposes.