مجله پژوهش های سلولی مولکولی (زیست شناسی ایران)
سال بیست و نهم شماره 1 (بهار 1395)

Papaver somniferum L. is the most important commercial source of production of morphinan alkaloid component, such as codeine, thebaine, narcotine and papaverine that are exploited by the pharmaceutical industry as analgesics, antitussives and anti-spasmodics. Recently, metabolic engineering technology for genetic manipulation of alkaloid biosynthesis and metabolic products has been used via production of transgenic plants. Codeine O-demethylase (CODM) catalyzes the late step of morphine biosynthesis in opium poppy for conversion of thebaine to oripavine and codeinone and codeine to morphine via demethylation mechanism. This study was conducted in silencing of CODM gene using VIGS technique. cDNA was amplified using specific primers, and then a part of this cDNA at the 3́ UTR terminal was subcloned in TRV2 vector in Agrobacterium as silencing fragment. Plant leaves was inoculated via Agrobacterium containing silencing fragment. Initial screening of transformed plants was conducted via PCR on tobacco rattle viral coat protein gene (TRV-CP) and then, some positive TRV plants were analyzed using semi-quantitative RT PCR. The final analysis of VIGS was performed using real-time PCR. Based on the results 95 percent reduction in gene expression of CODM in transgenic plants compared to the control plants was achieved.

Proteases are one of the most important groups of industrial enzymes, constituting 65% of worldwide industrial enzyme marketing and around 25% of the total global enzyme production. Their use encompasses a great number of applications in different industrial sectors, such as detergent additives, in waste treatment processes, silver recovery and the food, leather, photographic and pharmaceutical industries. Protease producing bacteria were isolated from Kerman,s dairy industry sewage with clear zone surrounding colony in Skimmed milk agar plate. Its biochemically tests and 16S rRNA gene sequencing showed that it is Chryseobacterium indologenes strain BYK27. The extracellular protease secreted by this strain partially purified by a combination of ammonium sulfate precipitation, dialysis in Tris-HCl buffer 35 mM and ion-exchange chromatography. The temperature and pH optima for activity of produced protease were 40˚C, pH 8 and for stability were 40˚C, pH 8-9. This enzyme was activated by K, Triton X-100 and commercial detergent Tage. It was also active in 1% concentration of salinity. These results showed that protease BYK27 can be used in detergent industry, meat tenderization and peptide synthesis.

E-cadherin protein is a member of cadherin superfamily which expressed mainly at the surface of epithelial cells. Reduced expression of this protein results in cell adhesion weakening, EMT (Epithelial-mesenchymal transition), migration and metastasis of epithelial cancer cells. In traditional medicine, pomegranate (Punica granatum) is used to treat several diseases including diarrhea, heart diseases and blood pressure disorders. In this study, we examined the effect of pomegranate acetone extract on the expression of β-catenin and E-cadherin proteins in PC-3 cancer cells. The results of MTT experiments showed that acetone-derived extract of pomegranate inhibits viability of PC-3 cells, especially in concentrations higher than100 μg/ml. The IC50 value of pomegranate extract was estimated to be 86μg/ml. The results of western blotting experiments showed that concentrations of 20 and 50 μg/ml of pomegranate extract led to 1.5- and 2.2-folds increase in β-catenin and E-cadherin protein levels, respectively. The results of this study suggest that pomegranate fruit may have great potentials in inhibition of cancer cell metastasis.

In recent years, using the assisted hatching techniques such as puncture of blastocyst, are used in in-vitro fertilization (IVF) for increase the rate of implantation. This study was designed in order to evaluate of AC effect in survival and hatching rate of mouse blastocysts by expression of Wnt3a gene. In vivo and In vitro blastocysts are obtained. Some of them in in-vivo and In vitro groups were collapsed with a micro-needle and non-collapsed blastocysts in each group were used as a control. Evaluated survival and hatching rate and quantitative expression of Wnt3a gene in both groups was investigated by using Real time PCR. Our results revealed that AC of blastocyst in in-vivo and In vitro blastocysts did not improve survival rate although hatching rate had a significant difference in the collapsed blastocysts compared to non-collapsed blastocysts in both groups. The results real time PCR quantitative analysis showed that expression level of Wnt3a gene has increased in in-vivo blastocysts and it has decreased in in-vitro blastocysts comparing to control group in both treatment. AC could be effective way to improve the assisted hatching techniques.

In this study, the biological and molecular features of Pseudomonas fluorescens UTPF5 is evaluated as an important bacterial in IRAN. Effect of silver nanoparticles, effect of bacteria on laccase and also molecular aspect of UTPF5 (detection of phlD, phlA and hcnAB genes) examined. In this research, biocontrol ability of Pseudomonas fluorescens UTPF5 was studied on the root knot nematode Meloidogyne javanica in tomato Also inhibition of nematode pathogenicity is determined. Bacterial extract, suspension and volatile causes 35%, 25% and 85% reduction in nematode egg hatching respectively. J2 mortality induces 34-53% and 85% in effect of bacterial extract and volatile respectively, whereas suspension has no effect in j2 mortality. Biofilm formation is promoted by 0/25-2 µL silver nanoparticle. The strain UTPF5 has phlD, phlA and hcnAB genes. Culture filtrate of bacteria effected laccase enzyme significantly. Considering the disease measurement following seed treatment with the bacterium, gall index, the number of galls and all eggs in root system were considerably reduced. Biocontrol potential and plant growth promotion is described by application of the bacterium in greenhouse trials. Generally UTPF5 has good biocontrol effect and also molecular features study with detection important genes.

Initiation of flowering in plants is controlled by various factors such as photoperiod, cold, hormones and epigenetic effects. A major pathway in this process is the epigenetic control of FLC gene, whose expression inhibits the flowering initiation. Removing acetyl groups from FLC gene histons, by MSI4 protein represses FLC expression, and thus, flowering is initiated. However, the molecular mechanism of this protein is largely unknown. In this research, we used the Yeast Two Hybrid System (Y2HS) to study protein-protein interactions, to find proteins interacting with MSI4. Therefore, first, we cloned the MSI4 gene in proper Y2HS vector. Then, cDNA bank of Arabidopsis thaliana was screened by MSI4 as bait to prey the interactors of MSI4. The protein trapped by this method was PKT3 (Peroxisomal 3-Ketoacyl-CoA Thiolase 3), which is an acetylcoacyl transferase. The function of this protein is to release acetyl groups and deliver them to other molecules, and therefore, it has significant role in many crucial cell processes. This interaction was confirmed by cloning the complete cDNA of PKT3 as well as its homologue, PKT4. The interaction of MSI4-PKT3 is reported here for the first time and opens new horizons for more studies on MSI4 functional mechanism. This finding can be useful in regulation of flowering time in fruits and crops through genetic engineering of this pathway for example by alteration of PKT4 regulation.

The development of drug delivery nano-systems has improved the drug bioavailability while reducing their adverse side effects. Among them, iron oxide nanoparticles have gained lots of attention due to their applications as contrast agent in MRI technique as well as being targeted through an external magnetic field. The biological properties of these nanoparticles have been improved through surface modifications by different kinds of polymers. Hyperbranched polymers are of great interest due to existence of plenty of surface functional groups which could be utilized in conjugation of targeting and/or tracing agents. In this study, we have synthesized iron oxide nanoparticles using thermal decomposition method, modified its surface with hyperbranched polyglycerol through ring opening polymerization and compared its biocompatibility with naked iron oxide nanoparticles. The results confirmed the synthesis of iron oxide nanoparticles with average diameter of 11 nm coated with hyperbranched polyglycerol. The comparative biocompatibility tests revealed that naked nanoparticles show cytotoxicity when the concentration reaches above 200 µg/ml, whereas they are safe when coated with hyperbranched polyglycerol.

Plant defensins are a family of cationic antimicrobial peptides, small and cysteine rich that have a broad range of antibacterial effects. The aim of this study was designed, synthesis, cloning, expression of wheat defensin (Triticum aestivum) gene and evaluation of the antibacterial effect of recombinant protein. The defensin gene was fused to sumo gene for more expression and solubility and then was optimized by some software and was synthesized and cloned in pGH vector. Then sumo_defensin sequence was sub cloned into pET-32a() vector and transformed into bacterial cells (E.coli origami). Gene expression was induced by 1mM IPTG in 30°C and proteins were analyzed by SDS-PAGE and Western blotting.The fusion protein with a molecular weight of approximately 38 kDa was analyzed on SDS-PAGE gel and confirmed by western blotting. After digestion of protein by Sumo protease, the antibacterial effect of the recombinant wheat defensin was evaluated on the plant pathogenic Gram-negative bacteria Xanthomonase axonopodis, Pseudomonas syringae and some human pathogenic Gram-negative and Gram-positive bacteria such as E.coli and Klebsiella pneumoniae , Staphylococcus aureus and Bacillus cereus. In the end, recombinant wheat defensin had an antibacterial effect against Xanthomonase axonopodis, Staphylococcus aureus and Bacillus cereus. Based on the antimicrobial characteristics of wheat defensin, this study can be effective to introduce a new antimicrobial agent.