فهرست مطالب hooshang alizade

Peroxidases are used in a wide range of biotechnological processes, most of which are carried out at high temperatures and high pH levels. Since most of the commonly used peroxidases are unstable and inactive in alkaline conditions and high temprature, it is necessary to find thermoalkalophilic peroxidases for practical purposes.

In this study, extracellular production of peroxidase in the native strain Bacillus tequilensis was studied. for this purpose, Enzyme activity was evaluated using two substrates 2,4-DCP and pyrogallol in bacterial liquid culture and the effect of culture time on enzyme production, as well as the effect of parameters such as pH and temperature on enzyme activity investigated. The relative purification of the enzyme was performed using ion exchange chromatography with sephadex DEAE A50 and the kinetic parameters of enzyme activity were evaluated. In this study, kinetic parameters such as Km and Vmax were calculated.

Measurement of enzyme activity at different times of culture indicated that the highest amount of peroxidase production was obtained 72 h after bacterial culture.

The most common cause of diarrhea is Shigella and no vaccine has been found so far. IpaD and B subunit of Shiga toxin proteins (STxB) play an important role in invasion, infection and pathogenesis caused by Shigella. On the other hand, Co1 ligand has introduced as one of the antigen delivery systems to M-cells in mucosal layer. In this study, nasal administration of antigen in mice, used to evaluation and comparison of immunogenicity of IpaD, IpaD-STxB and IpaD-Co1 proteins. The coding sequences of proteins cloned in pET28a vector and transformed to bacterial strain of E.coli BL21(DE3). Recombinant protein expression confirmed by using of Western bloting technique. The each one of the recombinant protein purified by affinity chromatography column and used for nasal administration in four times consecutively on mice. After the second nasal administration (first booster), a week after each administration, blood samples were collected and ELISA performed on serum. The ELISA results of serum titration showed that the highest amount of IgG has been produced against IpaD-STxB protein; and IpaD-Co1 was in next step. The immunogenicity was evaluated using active toxin E. coli O157:H7. The challenge results showed that immunized mice could endure 5 times the LD50 Shiga toxin E. coli O157: H7; but after injection of 10 times the LD50, the immunized mice by IpaD, IpaD-Co1 and IpaD-STxB were death after 24, 24 and 72 hours, respectively. According to these results recombinant protein IpaD-STxB founded to be more immunogenic than IpaD-Co1 against shiga toxin in mice.

Insulin is a hormone exclusively produced by pancreatic beta cells. The production of recombinant proteins in microorganisms has some disadvantages such as high cost, the possibility of contamination with toxic proteins, and costly purification steps; so, the production of recombinant proteins in plants can be investigated. Development of transient expression system based on a deleted version of Cowpea mosaic virus RNA-2, Cowpea Mosaic Virus-Hyper Translatable (CPMV-HT), has provided the extremely high-level and rapid production of proteins without viral replication. In this study, two constructions were prepared; pBI121-Proinsulin-Zera (pBI-ProZ) containing human proinsulin gene and Zera (N-terminal proline-rich domain of γ-zein) and pCAMBIA1304-Proinsulin-Extensin (pCAMBIA-ProE) containing CPMV-HT expression system for improvement of translation of human proinsulin gene and carrot extensin signal peptide. Both structures were transiently transferred in to lettuce and alfalfa leaves using Agrobacterium tumefasiens pv. C58. Statistical analysis of this study was conducted on the concentration of produced proinsulin per each gram of transgenic leaf using factorial arrangement of treatment in a complete randomized design. Gene expression was confirmed in transcription level using reverse transcription polymerase chain reaction (RT-PCR) method and in translation level using enzyme-linked immunosorbent assay (ELISA) and Dot blot assay. Protein accumulation for pCAMBIA-ProE and pBI-ProZ constructs were 6.82 and 4.32 ng/g in recombinant alfalfa leaves and 6.6 and 3.8 ng/g in recombinant lettuce leaves, respectively. Results showed that the expression of proinsulin in pCAMBIA-ProE contains CPMV-HT expression system was more than pBI-ProZ.

Abiotic stress are the main factors causing low growth and yield in crops. Osmotic stress is an important stress and in case of salinity stress is presuming at the first step. There are different experimental and computational methods to study plant response to stress and lead to better understanding of mechanism involved in these conditions and improvement of plant tolerance to stress. Upstream analysis of genes including regulatory sequences can improve our relative knowledge about gene response to stress. To study the scope of adaptation between experimental and computational analysis and also co-regulation and co-expression ability of interested salt-response genes, we analyzed upstream regions of eight salt-response genes TSA,GCS,SOD,Tpis,AtpE,FBA,ps16 and 14-3-3p in rice and examined their expression profiling via semi-quantitative RT-PCR in bread wheat under salt stress. Our results showed two distinct groups of genes according to their expression pattern similarities. In spite of deficiency in regulatory element databases and different analyzer programs, our results showed expective adaptation between the methods.

Drought and salinity stress are adverse environmental factors that affect crop growth and yield. Abiotic stress cause some reaction in plants such as change in gene expression, cell metabolism, growth and yield. Moreover, stress condition resulted in increase of reactive oxygen species and disturbe macromolecules such as proteins, fatty acids, nucleic acid and pigments. In order to evaluate salinity effect on leaf proteome pattern and changes in antioxidant activity of two barley (Afazal, L.527) genotypes, an experiment was carried out using factorial experiment in a completely randomized design with three replications under controlled conditions and salinity (200 and 300 mM) stress. Salinity stress was imposed in seedling with 4-5 leaves stage for 24 hours. Salt stress decreased the catalase activity while increased the other antioxidant activity such as peroxidase, ascorbate peroxidase and glutathione reductase. Although the response of genotypes was different. Analysis of gels revealed that in tolerant genotype 97 protein spots (%78 up-regulated, %22 down-regulated) and in susceptible genotype 94 protein spots (%37 up-regulated, %63 down-regulated) showed significant changes in compare with control. Result of MS/MS led to the identification of some proteins involved in antioxidant activity, energy generating and other mechanisms.

As regards the kind of use and production of biomass, lettuce benefits from a potential of acting as a bioreactor in producing recombinant proteins as well as edible vaccines. For the propose, one needs to optimize tissue culture and gene transformation in Lactuca sativa. Seeds of two cultivars TN-96-39 and TN-96-41 were disinfected in a solution of 2.5% sodium hypochlorite containing 0.1% Tween 20 for 25 minutes. This was followed by three rinses of sterile distilled water. The seeds were then germinated in Petri dishes on filter paper soaked with sterile distilled water. The cultures were continuously exposed to white fluorescent light at a constant temperature of 25oC, for a minimum of 16 hours. When aseptically germinated seedlings were 48 to 72 hours old, cotyledons were excised near the cotiledonary node, and cut into either 6 or 8 pieces to expand the already existing wound on the explants for callus growth. The most suitable growth regulators for callus production and embryogenesis were 0.05 mg/l NAA, and 0.2 mg/l BA. The most appropriate growth regulator for direct regeneration and proliferation was found out as 0.05 mg/l NAA, 0.4 mg/l BA. To produce roots, shoots were transferred to MS medium culturing 0.2 mg/l of NAA. Strong root bearing plantlets were planted in pots to produce seeds. Transgenic plants of lettuce cultivars (TN-96-39, TN-96-41) were produced by use of Agrobacterium tumefaciens vectors containing the β- glucuronidase (GUS) reporter gene and the nptII gene for kanamycin resistance as a selectable marker. High frequency of transformation based on kanamycin resistance and GUS expression, was obtained with 72-h-old cotyledon explants cocultivated for 48 h with Agrobacterium tumefaciens strain LBA4404 & 2 minute dive for infection at cultivar TN-96-39. Progenies of T0 and T1 plants along with PCR & assay demonstrated linked monogenic segregation for kanamycin resistance and for GUS activity.