مجله پژوهش های سلولی مولکولی (زیست شناسی ایران)
سال سی‌ام شماره 3 (پاییز 1396)

Infections, which are caused by multiple drug resistance bacteria is an increasing problem. This is due to emergence and distribution of microbial drug resistance, and none development of new antibiotics. The use of phytochemical products and plant extract as a resistance reducing agent is an increasingly active subject of research. This study was aimed to study the effect of total alkaloides extract of Sophora alopecuroides on MIC value and intracellular accumulation of ciprofloxacin in an Escherichia coli ciprofloxacin resistant mutant. For this purpose serial dilution method for determining MIC value and spectrofluorometric assay assays for measuring intracellular accumulation of ciprofloxacin antibiotic were used. Results showed that plant extract decreased ciprofloxacin MIC. This extract also increased intracellular accumulation of antibiotic in the Escherichia coli ciprofloxacin resistant mutant. In conclusion, extract containing alkaloids of Sophora alopecuroides can increase sensitivity to ciprofloxacin in resistant mutants through its effect on membrane efflux pump, AcrAB-TolC.

Deep eutectic solvents (DES) are significantly important in enzymatic reactions. These solvents increase the efficient contact between substrate and enzyme and therefore, may increase enzyme stability and activity. Bioluminescence is the production and emission of light by a living organism. The principal chemical reaction in bioluminescence involves the light-emitting pigment luciferin and the enzyme luciferase. The enzyme catalyzes the oxidation of luciferin, which requires the energy-carrying molecule adenosine triphosphate (ATP). Because of its easily detectable product, luciferase has found a wide range of application in biotechnology and molecular biology. However, its low thermostability, low turnover number and high Km for ATP restrict further use of luciferase based systems in commercial application. Newly developed method which can be used to increase the stability of proteins and biocatalysts is to take advantage of Deep eutectic solvents (DESs). One group of Deep eutectic solvents are generally composed of organic salts with hydrogen bond donors, as a result of which hydrogen bonds are formed. In this study, we investigated the effects of these solvents on kinetic properties of wild type and E354R/Arg356 mutant Lampyris turkestanicus luciferases in the presence of choline chloride: glycerol with molar ratio of 2:1 as DES. For this, both wild type and mutant, expressed in Escherichia coli BL21, the protein of interest purified through affinity chromatography and used for kinetic studies. Based on the obtained results, DES has positive effect on the thermostability of wild type and mutant luciferases.

At the present research, 30 isolates of Actinomycetes have been isolated from agricultural soils of Kerman Province of Iran and assayed for antagonistic activity against Sclerotinia sclerotiorum. 10 isolates showed antagonistic effect of In Disc-Agar method, Among selected isolates, Streptomyces isolate 363 UK showed high antagonistic activity. Also, in order to determine genetic diversity of 30 isolates of Streptomyces, the DNA was extracted using CTAB method in laboratory. To do molecular investigation 10 RAPD primers were used for PCR. After doing electrophoresis, 128 sharp bands between 250 and 2800 base pair were recognized. The experiment results were analyzed using NTSYS software and UPGMA method with Dice coefficient. The analyzed cluster found from Dice coefficient divided the 30 Streptomyces isolates in two major groups. In the first group, 10 isolates were antagonistic properties,and in the second group of 20 isolates were no antagonistic effect. Grouping isolates using principal components analysis was performed and two-dimensional and three-dimensional plots respective was drawn. The isolates were divided into eight groups.

The hairy root formation is performed by transferring of special genes of Agrobacterium rhizogenes plasmid's to the explants. These roots are used to increase of valuable pharmaceutical secondary metabolites. One of the most important factors that inhibits the hairy root formation after induction of explants by A. rhizogenes is browning. The cause of browning in leaf explants is probably release of phenolic compounds from the wound site or defense mechanisms performed in plant cells against infection by A. rhizogenes. In this study, the root explants of four species of solanaceae family including Atropa belladonna, Hyoscyamus niger, Datura stramonium and Datura metel were induced by different strains of A. rhizogenes (AR318, AR15834, AR9543, A7, AR9402, and A4) and the percent of browning evaluated in these explants. For the results, the minimum percent (2% and 6%) of browning observed in leaf explants that induced by strains of A4 and AR15834 respectively. Hence, the use of these strains is appropriate for induction of hairy roots in the solanaceae family.

Medical Plants like Salix alba are potential sources of natural antioxidants and produce various antioxidative compounds to counteract reactive oxygen species.
The aim of the present study was to determine total phenol and flavonoid and antioxidant activity of four various extracts of stem and branch barks of Salix alba from Hamedan.
In this study Phenol and flavnoid tests were done To determine antioxidant compounds. 2, 2-diphenyl-1-picrylhydrazyl (DPPH) assay was used to determine the free radical quenching capacity.The collected data was analyzed by SPSS software.
The highest value of total phenol were relevant to ethanolic extracts of branch and stem barks that 60.87± 1.19 and 69.98 ± 1.41 mg Gallic acid/g dw was measured, respectively and the lowest value of total phenol were determined to chloroformic extracts. The highest level of total flavnoid in branch and stem barks were relevant to ethanolic extract amount 71.20± 4.08 and 60.87± 1.19 respectively and also the lowest level were determined to chloroformic extracts of stem and branch barks amount 12.56± 0.29 and 13.80± 0.57 respectively. Furthermore the most activity of the free radical quenching capacity DPPH after Ascorbic Acid were determined to ethanolic extracts of branch and stem bark 0.129 and 0.130 mg /ml. There were significant differences between means of total flavnoid and total phenol of stem and branch barks.In conclusion,The best antioxidant activity were relevant to ethanolic extracts of branch and stem barks.

Chavicol -O-methyl transferase (CVOMT) is a key enzyme in phenylpropanoid pathway in basil (Ocimum basilicum L.), which is a medicinal and aromatic plant of the Lamiaceae family. In this study, the effect of chitosan elicitor on essential oils yield, phenylpropanoid components and chavicol -O- methyl transfrase gene expression under water deficit was evaluated. The plants under different irrigation regimes were treated with 0.2 and 0.4 g/L chitosan in three stages of growth. Phenylpropanoid compounds were identified by Gas Chromatography and Gas Chromatography/Mass Spectrometry (GC/MS) and the level of gene expression was monitored by Semi quantitative RT-PCR. An analysis of variance indicated that oil yield and CVOMT gene expression and methylchavicol increased under water deficit and chitosan elicitor compared to untreated plants. The changes observed in methylchavicol components were in accordance with the changes of gene expression of chavicol -O-methyl transferase. Thus, chitosan as a biotic elicitor could increase phenylpropanoid components in purple basil by increased gene expression and activity of chavicol -O-methyl transferase.

Bitter melon is an anticancer medicinal plant that its saponins and flovonoids induce immunity system of body, thus it is one of the most important medicinal plants. Direct plantlet regeneration from Iranian accessions of this plant for its in vitro propagation will be very functional. For this, an experiment was conducted in factorial based on Complete Randomized Design. It`s factors included explants (stem and leaf), hormonal combinations (different levels of BAP ad NAA), culture medium (complete MS, ½MS and ¼MS) and population (Gachsaran and Dehdasht). In Dehdasht accession, stem explants were better than leaf explants, but in Gachsaran accession was equal for both explants. In respect to shoot regeneration percent, shoot number and shoot length, hormonal combination of 1.5 mg.L-1 BAP together with 1 mg.L-1 NAA in ½MS showed the best direct regeneration in both populations (44.4 and 33.3 percent, respectively). The best medium for rooting in plantlet obtained from regeneration was MS medium containing 0.5 mg.L-1BAP and 0.5 mg.L-1NAA (76.8 and 73.33 percent, respectively). The results of this research showed that these two assessed Iranian populations have good response to tissue culture and direct plantlet regeneration .

The endophytic fungus Piriformospora indica increases nutrient uptake in plants and allows plants to survive under biotic and abiotic stresses. This fungus stimulates growth and seed production of plants and increases the secondary metabolite contents of medicinal plants. There is little information on cultivation of symbiotic fungi therefore the development of methods to the mass culture of symbiotic fungi is very importance for practical applications. The aim of this study was to reveal a simple culture medium to higher biomass production for this fungus.
A comparison of YPG simple medium and Kafer Complex medium was carried out on the dry weight of fungus. Then Taguchi method was used to design experiments for medium optimization to determine the effect of four factors including glucose, yeast extract and peptone concentrations, and pH on the fungal biomass production in a non-continuous culture.
Comparison of the growth curve of the fungus in both the Kafer and YPG media showed that P. indica growth was better and higher in YPG medium. After optimization of YPG medium, biomass production was reached to 19/3 g.L-1 in compared with the initial amount i.e. 16/2 g.L-1.
Our results suggested that YPG medium can be used as a suitable medium to P. indica industrial production for agricultural application, due to the higher biomass production of P. indica in YPG medium by 2.5-fold and its low cost in compare to Kafer medium.

To optimize gene transformation condition in ajowan medicinal plant effect of individual parameters such as optical density of Agrobacterium cell culture, Agrobacterum strain, type of antibiotic to elimination of Agrobacterium, acetosyringone concentration and inoculation duration was investigated using p6U-Ubi-FVT1 plasmid contain HPT gene as selectable marker and fld gene. 20 mg/L of hygromaycin was identified as minimum concentration of this selectable agent to prevent of callus induction in non-transgenic cells of ajowan. Interaction effect of optical density of Agrobacterium, Agrobacterium strain and type of Agrobacterium killing antibiotic on number of regenerated plant was significant at 1% probability level and less inhibitory effect on regenerated plant was related to OD600=0.6-0.8×GV3101 Agrobacterium starin×160 mg/L of timentin antibiotic. More regenerated plant resistance to hygromaycin was related to 254.8 µm/L of acetosyringone. More regenerated plant was achieved from 30 minutes inoculation duration than 1and 20 minutes inoculation duration, nonetheless there was no statistically significant difference between means of regenerated plant from 20 and 30 minutes inoculation duration. The presence of the target gene was evaluated using PCR analysis on regenerated plants. Results of PCR analysis confirm entity of at least one copy of the target gene on plants genome. The superiority of transgenic plants against non-transformed plants for tolerance to -0.3 Mpa osmotic stress was proved by osmotic stress tolerance bioassay.