مجله پژوهش های سلولی مولکولی (زیست شناسی ایران)
سال بیست و هفتم شماره 1 (بهار 1393)

The rising popularity of natural aroma products such as vanillin and vanillic acid has triggered off significant research activities to use biocatalysts for the production of natural flavor compounds. We conducted this study to screen moderately halophilc bacteria which are able to degrade ferulic acid and to study the possibility of forming vanillic acid via conversion of ferulic acid under resting cells conditions. 22 different strains of bacteria were isolated from different samples collected from the salty environments of Iran. Primary screening was performed by HPLC analyse. The selected strains were identified based on physiochemical characteristics as well as molecular phylogenetic analysis. Biotransformation mixtures were quantified for vanillic acid content and the other produced methoxyphenols by HPLC analyse. Based on the HPLC results obtained، among the 22 isolated strains، resting cells of Halomonas salina HSL5 (GenBank accession number JQ327041) produce the greatest quantity of vanillic acid (397 mg/l، molar yield of 46. 8%) after a 24-h reaction time، without further optimization The current study brings the first report for bioconversion of ferulic acid to vanilic acid in the Halomonas salina.

Periwinkle (Catharanthus roseus (Linn.) G. Don) is known as an important medicinal plant because it contains vincristine and vinblastine alkaloids. Therefore development of micro-propagation in vitro culture has high importance. The present study describes two shoot induction and one root induction optimization experiments. The experimental designs were 3×5 factorial (BAP × NAA و KN × NAA) for shoot induction trails and 3×5 factorial (IBA × NAA) for root induction trail based on completely randomized design with four replications. The best proliferation of shoot induction percent and shoot per explants was shown on the medium supplemented with high KN and NAA، respectively. In overall، KN (0. 1 mg/l) + NAA (1 mg/l) combination proved to be optimal for the production of maximum number of shoots. Mean comparison showed that the highest multiplication rate was obtained with 0. 1 mg/l of 6-benzylaminopurine (87%) and with 0. 1 mg/l 6-benzylaminopurine + 2 mg/l naphthalene acetic acid (100%). The best condition for rooting (root number and length) was 0. 4 mg/l IBA + 4 mg/l naphthalene acetic acid and the maximum root length was observed on medium supplemented with higher NAA concentration.

One of the most important substrate for producing of the edible white button mushrooms is compost. Thermophilic actinomycetes are one of the most important microflora in composting. So these actinomycetes were isolated from different steps in composting process by dilution technique. The isolates were incubated at 40° C. The chalky colonies were selected and transformed to new solid media to prepare pure culture of each isolate. Bacterial isolates were identified based on biochemical tests and amplified 16SrRNA restriction analysis technique. Ability of the isolates to break down cellulose were assessed by using Filter paper in different temperature and pH. Maximum enzyme activity for each isolate were measured based on milligrams of glucose released from one milliliters extract. The isolates with high cellulase activity were screened for determination of the optimum pH and temperature. Isolate 23 had 1/10 mg/ml enzyme activity and it demonstrated a higher cellulase activity at 45°C.

Chamomile (Anthemis sp) is an important medicinal plant in the world that is widely used today. In order to investigate the proxidase diversity of three species of Anthemis (A. pseudocotula، A. haussknechtti and A. triumfetti) nine population were studied. From eleven abserved- alleles، there were three rare alleles in A triumfetti species. Four private alleles were seen in pseudocutula and triumfetti species (in PXA and PXB locus). The higher and lower genetic distance were obtained between pseudocutula and triumfett and between triumfetti and haussknechtti respectively. Using principal components analysis. Result showed that the psudocutula had the most distance from other species and cluster analysis was performed in a separate cluster. There was no correlation between ecological data and genetic character but correlation between latitude and longitude with genetic characters was significant at the 1% level. Analysis of molecular variance among populations was 18% and within of the population each species was 44%.

Incheh Borun hypersaline wetland is located near the border of Turkmenistan،in north of Iran. This wetland is remarkable because of salinity and variation in pH range. sampling was carried out from soil، water and salt in September 2010. 400 strains were purified and 55 strains were selected randomly for PCR. Genera: Bacillus (18%)، Marinobacter (16%)، Halomonas (16%)، Kocuria (9%)، Oceanobacillus (7%)، Dietzia (7%)، Virgibacillus (6%)، Chromohalobacter (5%)، Rhodococcus (2%)، Micrococcus (3%)، Paenibacillus (2%)، Halobacillus (3%)، Thalassobacillus (2%)، Arthrobacter (2%) and Desmospora (2%) were isolated. 13 strains showing 97- 98. 4 % similarity and 7 strains had less than 97% similarity in 16S rRNA sequencing which is showed significant differences in the level of species or even genus. Optimum growth for salt was evaluated and 22 strains were moderate halophile and 33 strains were halotolerant. Producing 4 hydrolytic enzymes was evaluated which the main producers of hydrolytic enzymes were Gram-positive bacilli and the most frequent enzyme was Gelatinase and Protease.

Desulfurization of thiophenic compounds is one of the important issues in refineries and environmental pollutions control. A bacterium was isolated from MTBE enriched soil was able to desulfurize of the thiophene. It was identified as Pseudomonas stutzeri based on phylogenetic analysis of 16S rRNA gene sequence. Desulfurization reactions from thiophene as the model of thiophenic compounds in aqueous phase by resting cells of this strain carried out and the concentration of thiophene was determined by UV-visible spectrophotometer in 230 nm wavelength. The results showed that the reduction of thiophene was 95% (from 12. 4 mM to 0. 3 mM) after 15 hours. Gas chromatography analysis showed the same results of thiophene desulfurization. Furthermore، The desulfurization activity of resting cells in a biphasic system containing crude oil by determination of released sulfate content turbidimetrically with BaCl2 was investigated.

One of the basic needs of different techniques in molecule biology is the production of genomic DNA with desired quality and quantity. In some researches the plant samples are collected from nature and these plant tissues are usually adult and due to presence of carbohydrate، polyphenols complex and proteins in their components، we cannot extract DNA with high quality and quantity. In other hand، these complexes are bond with nucleic acids and separation of them from acid nucleic is difficult. In this study، genomic DAN extraction from the leaf of adult Thymus plant was carried out by Doyle and Doyle، Dellaporta، Kang and Yang and Khanuja methods. The results showed that Khanuja method with some modifications is better than others. In this extraction method، brown-sticky sediment was separated from DNA. Analysis by GC-Mass، IR and UV revealed that this sediment contain Thymol، Carvacrol، Nerolidol and Borneol. Thymol substance has the greatest amount in GC (%65). So، the improved Khanuja method can separate the main secondary metabolites of Thymus and this method could be advised for DNA extraction from adult and mature plant.

Pseudomonas bacteria are the most important plant growth promoting rhizobacteria (PGPR) in different crop plants. In this study، thirty eight Pseudomonas bacteria isolated from corn rhizosphere grown in different parts of Khorasan Razavi province. Initially، all isolates were identified by using recommended physiological and biochemical tests. The results showed that most of Pseudomonas isolates were similar to Pseudomonas fluorescens and Pseudomonas aeruginosa. The potential sidrophore production of all Pseudomonas isolates was evaluated using CAS-agar solid medium. The results of this part of experiment showed that the ratio of halo to colony diameter varied from 0. 7175 to 4 after four days of incubation at 27 oC. The quantity of sidrophore production of Pseudomonas isolates was also measured by using spectrophotometry method. The results revealed that the amount of sidrophore production changed from 0. 96 to 132. 5 µML-1 among Pseudomonas isolates. The effect of sidrophore–iron complexes on growth of corn plant was investigated in a hydroponic system under green house condition. The results showed that chlorophyll content، shoot and root dried weights significantly increased when sidrophore–iron complexes was applied to corn plant. The effect of sidrophore-iron complex took out from Pseudomonas fluorescens on iron uptake in corn plant was similar to Fe–EDTA complex. The effect of sidrophore-iron complex extracted from P16 isolate on iron uptake was significantly higher compare to control treatment.

In this research، ultrastructure of rapeseed (Brassica napus L. cv. PF704) microspores following the gametophytic and embryogenic developmental pathways in vitro was compared with the gametophytic development in vivo via electron microscopy. In reprogramming of rapeseed microspores to the embryogenesis pathway، there are some defined changes influencing the cell activities and structure. Some of these changes can be considered as microspore embryogenesis pathway in rapeseed. In this study، rapeseed microspores were isolated at different days after in vitro culture and prepared for electron microscopy studies. During the different developmental stages of rapeseed microspores، Differences in specific cellular properties such as cell size and shape، nuclear architecture، starch accumulation، presence of vacuoles were studied to characterize different stages of microspore embryogenesis and other pathways occurring in vitro. Results، showed the presence of abundant starch grains in a defined cytoplasmic region appeared as a specific property of the gametophytic development in vitro، as well as of the non-induced microspores of in vitro cultures under embryogenic-inductive conditions.

The present study shows isolation and identification of thermophilic (60°C) and mesophilic (37°C) bacteria degrading the cellulose from the soil. after the growth environments enrichment with microcrystalline of cellulose as the sole source of carbon، Thermophilic and Mesophilic bacteria were isolated and purified by serial dilution method. Screening of purified bacteria was done to identify cellulase-producing bacteria by zymogram on plate method. Among the cellulolytic strains، 12 thermophilic and 12 mesophilic strains were selected and these bacteria were compared with each other based on cellulase activity، growth and extracellular protein amounts. Identifying the strains with highest enzyme activity based on gene sequences 16S rDNA suggested that these strains belong to genera Bacillus، Geobacillus و Chryseobacterium. the preferred thermophilic and mesophilic genera were assessed to identify the best temperature and pH inwhich the thermophilic and mesophilic genera showed the best function in temperature 50°C and pH 6. 5 and temperature 37°C and pH 8. 5 respectively. These bacteria have necessitated potential for biological conversion of urban and agricultural cellulolytic wastes to valuable materials.

Citrus canker bacterial disease is one of the most devastating citrus diseases and including a quarantine disease that severe damage to citrus، which made Iran. Is that why this is necessary to study about inhibition of this disaster. Xanthomonas citri subsp. citri (xcici) is agent of this disease. In recent years، many efforts have been focused on finding antibiotics that bacteria can not be resistant against it. Antimicrobial peptides produced by different organisms، are good candidates for this approach. We were collected soil، water and plant organs samples from variation regions of sistan and balluchestan (up to ten regions) then we moved them to the laboratory under suitable conditions. Lashar DGH2 a new bacteriocin، which is native and is a good candidate for controlling this disease. This bacteriocin is the first world Xanthomonas inhibitory bacteriocin were used against different bacterial strains Xanthomonas and able to inhibit strains of this bacterium in Iran and another countries. It is hoped that، with future research on this bacteriocin and optimization of its inhibitory effect be able an important step in controlling this disease.

In this study، for immunological analysis of the expression of the P5CS (Delta- 1- pyrrolin-5- carboxylate synthetase) gene in osmotic stress condition، embryos of the olive plant cv. Zard were cultured on the solid ½ MS media، after than 4 weeks، half of the plantlets were transferred to the solid ½ MS containing 200 mM NaCl and they were sub- cultured about 2 weeks، then total protein was extracted from fresh leaves of both plantlets under stress and non stress conditions and was measured the content of protein by brad ford test. Then they were compared by SDS-PAGE gel electrophoresis. To probe the effect of p5CS over expression on salinity stress tolerance، poly clonal antibody developed in rabbit against P5CS protein was used. The result of immunological methods for specific detection of P5CS gene، strongly indicated P5CS up – regulation in stressed plantlets versus non stressed plantlets.

Cigarette smoke contains a considerable number of dangerous chemicals which are toxic to all parts of human body، especially to oral cavity. Human saliva is the first line of defense that encounters these toxic agents. Therefore، saliva could be thought as a non-invasive research tool to monitor alternations in biochemistry of body fluids due to toxic chemicals. The aim of this research was to investigate the effect of cigarette smoke on activity of enzymatic antioxidants of saliva including peroxidase and superoxide dismutase. In practical section، a group of 20-25 years old smoker men entered the study and their salivary antioxidants were compared with a similar group of non-smokers. Un-stimulated saliva was collected in sterile tubes and kept frozen at -70°C after being centrifuged. The activity of peroxidase and superoxide dismutase was the measured using their specific substrates under assay conditions. The results showed that، within experimental errors، the activity of peroxidase was decreased significantly in the smoker group (P≤0. 001) as compared to non-smokers. Super oxide dismutase was also less active in the saliva of smokers. It is suggested that toxic chemicals in cigarette smoke could have acted as inhibitors of mentioned enzymes in saliva of smokers. A significant decrease in saliva flow rate and pH was also observed among smokers with respect to non-smokers. Calling the important contributing role of antioxidant enzymes in the defense system of saliva opens a novel new insight into smoking habit.

MYB gene family as one of the largest transcription factors in plants have various roles. One the most important of them is regulation of anthocyanin level in plant. In apple (Malus ×domestica)، three genes MdMYB1،MdMYBA and MdMYB10 are responsible for developing red color by controlling anthocyanin level. In this study a variant of MdMYB10، named MdMYB10b، was isolated from an Iranian domestic red flesh apple and its expression pattern in different tissues described. This gene was expressed in all red tissues of plant including leaves، seed، fruit skin and flesh while no expression was detected in control cultivar. Regarding these results and bioinformatics examination of the red color responsible genes as well as using the whole genome sequence of apple، it was suggested that these three homologues MYBs (10،1،A) are alleles of one locus. Furthermore، a tissue and genotype specific expressionpattern was presented based on that model alleles MdMYB1and MdMYBA are located in both red and white flesh genotypes and function only in the skin while MdMYB10 is located merely in red flesh genotype and function in all tissues.

Nitric oxide (NO) can both induce and suppress apoptosis according to the physiological conditions. Recent investigations have indicated that pectin derivatives، which are polysaccharides in the cell wall of plants، are able to induce apoptosis in cancerous cells. In the present study the amount of NO release from rat pituitary tumor cell line GH3/B6 after treatment with deferent concentration of modified citrus pectin (MCP)، pectic acid (PA)، SNP (as positive control) and L-NAME (as negative control) is under investigation. The cells were cultured in Ham’s F12 medium which was completed with 10% FBS and treated with different concentration of MCP and AP. The cell proliferation inhibition rate was determined with MTT assay. The amount of NO release was assessed by an indirect method of using Griess reagent. Our results demonstrated that incubation of GH3/B6 cells with 1mg/ml PA for 4 hours increased the amount of NO release up to 40% more than control otherwise none of MCP concentrations affect the secretion of NO. In addition، MTT assay determined significant proliferation inhibition rate for the cells with 2. 5، 5 mg/ml of PA and 3، 5 mg/ml of MCP after 24 and 48 hours incubation. So these results suggest that PA may inhibit cell proliferation via induction of NO release in GH3/B6 cells but MCP shows its inhibitory effect on cell proliferation by another pathway.