نشریه مهندسی ژنتیک و ایمنی زیستی
سال هشتم شماره 2 (پیاپی 16، پاییز و زمستان 1398)

Laccase (EC 1.10.3.2) are multi-copper oxidase which catalyze the oxidation aromatic and non- aromatic compounds with electron reduction of molecular oxygen to water. Nucleotide sequence of laccase (accession number : ) was optimized according codon preference of Pichia pastoris. Gene was synthesized and cloned into pPICZalpha A. laccase under control of AOX1 promoter was transformed to P.pastoris by electroporation and integrated to P.pastoris genome. Methanol concentration, copper salts concentration and temperature were varied in order to enhance laccase expression in the heterologous system. Optimal fermentation condition for laccase production in shake flask cultivation using BMGY medium were obtained: presence of 0.4 mM Cu+2, culture temperature 25°C, 0.8% methanol added into culture every 24 h. the volumetric activity was achieved 9600 U/L after 12 day culture in fernbach flask. The biochemical properties of recombinant laccase and kinetic parameters were studied. The laccase activity was optimal at pH 4 and 40°C. Its Km value was 0.089 mM For ABTS [2, 2´azino-bis (3-ethylbenzthiazoline-6-sulphoric acid)]. The result of this study was showed Pichia pastoris can be useful tool for heterologous laccase expression in order to use in industrial application such as: waste water treatment, biosensor & etc.

Agrobacterium is one of the gram negative genuses of soil bacteria that causes a number of diseases in plants through the use of horizontal gene transfer. The rol genes of Agrobacterium rhizogenese, rolA, rolB and rolC, are the major causes of ‘hairy root’ symptomes in infected plants. It has been demonstrated that rol genes could have a possitive effect on secondary metalbolism in plants. The homologue of rol genes has also been found in some plants such as the genus Nicotiana and in a member of the Scrophulariacceae, Linaria vulgaris. Here we examined the effect of A. rhizogeneses rolC gene and its homologue in tobacco, trolC gene, on pigmentats content, production of some secondary metabolites and antioxidant activity in ‘in vitro’ cultered 4 weeks old seedlings. In these plants, the transgenes were located under the contol of dexamethasone inducible prompter. Methanol extracts of aerial parts of seedlings were prepared and DPPH radical scavenging method was used to evaluate the antioxidant activity. Experiments were done in a completely randomized design with 3 replications. Results showed that in induced transgenic lines, pigments content (chlorophyll a, chlorophyll b, caretenoid and anthocyanin) were decreased and total phenols contents were significantly increased compared to non induced plants (control) and DPPH radical scavenging activity exhibited a remarkable increase in induced plants. However the level of flavonoids didn’t show a significant change. These results indicate that plant homologue of bacterial rolC gene, trolC, functions as rolC and can be used as an alternative to enhance the level of secondary metabolite in plants.

Different concentrations of Zn and Cd (0, 2 and 4 mg L-1) were tested on the growth and some physiological traits of two spinach cultivars (common seed and Thorny seeds) as factorial based on Completely Randomized Design. The results revealed that the highest plant fresh weight was attained by 2 mg L-1 of Zn treatment. For dry weight, the highest data was belonged to Thorny seeds and the tallest plants were belonged to common seed spinaches. The top catalase content was belonged to 4 mg L-1 Zn with thorny seed. H2O2 content was affected by 2 mg L-1 of Cd in common seed cultivars. The highest Cd content in plant was measured with 4 mg L-1 of Cd at both cultivars. The greatest Zn content was recorded by Thorny seed spinach × leaf sample of 4 mg L-1 Zn and with common seed × leaf sample of 2 mg L-1 Zn.  For K+, the highest data was gained by Thorny seeds spinach × leaf sample of 4 mg L-1 of Zn, Thorny seeds spinach × root sample of 2 mg L-1 Zn and common seed × root sample of 2 and 4 mgL-1 of Zn. For Ca 2+ concentrations, Thorny seeds spinach × leaf sample of controls had the highest content. The highest Mn2+content was recorded by common seeds × leaf sample of 2 and 4 mg L-1 of Zn treatment. The evaluation of the tolerant plants having reasonable growth potential under the excess metal contaminated soils is quite emergent. Since, spinach is a fast growing and multi harvest plants during a year and, there is some reports on the hyper-accumulative behavior of the plant. So, it seems that the plant would be a nice candidate for the phytoremediation ideas and applications.

The most destructive viral disease in vine plants, grapevine fan leaf virus each year is causing severe losses in vineyards. The disease is caused by grapevine fanleaf virus. Since the cultivation of grapes to other great products impressive, and on the other hand the transmission and spread it through the material vegetative to do. Accurate identification of the disease in vineyards using a fast method with high sensitivity, the lowest level of contamination in the plant show is essential. In this research is to design a nanobiosensor graphene oxide -based from cDNA sequence of the gene cp has been used fanleaf virus. Based on this sensor, fluorescence emission from DNA Probe is labeled that if hybrid of DNA probe with the DNA of the cp gene, fluorescence emission observed and measured using fluorimetry. In order to optimize the nanosensor all factors connection time DNA probe to graphene oxide, graphene oxide concentration, the DNA hybridization time DNA pattern to labeled DNA Probe and the concentration of DNA template to hybridization attached probe to graphene oxide evaluated. The results showed that the nanosensor designed to detect the cp gene and can grapevine fanleaf virus with high efficiency to identify 10-13 mol/l

The method of barcoding DNA has a great potential for the revival of archeology and traditional biodiversity studies; therefore, this method has so far been widely used in studies on plants and for purposes of classification and archeology. In this research, 96 plants were planted in greenhouse according to a completely randomized design, and in 4 leaf stage, young leaves were harvested as samples. In the next step, DNA extraction, quantitative and qualitative evaluation, PCR and PCR sequencing were performed and data analysis was performed using Clastal w2 and BioEdit software. The results showed that the nucleotide sequences of the ITS gene and rbcl in the pumpkin family, Guanine were highest (26.9 and 22.6, respectively), adenine was the least frequent (20.3 and 27.4). Estimation of the nucleotide replacement of RBCL and ITS gene in the pumpkin family had high levels of pyrimidine replacement, resulting in the conversion of thymine to cytosine 31.99% and 20.43%, respectively, and for the conversion of cytosine to thymine was 47.05 and 16.04%, respectively The primer of its, 5 rbcl samples identified all 8 specimens, so the rbcl primer is more suitable for suckling.

Sunflower is one of the most important sources of plant oil in the world and in Iran. This plant is classified as semi-resistant to salt stress; however, its performance is negatively affected by salinity. Transcription factors are molecules that play an important role in the understanding and transmission of stress messages as well as many physiological processes. In this study, using Real Time PCR technology, transcript variations of two transcription factors WRKY and AP2Domain were examined in two oily sunflower lines; AS5305 (salt tolerant line) and 9CSA3 (salt susceptible line) at different salinity levels (2, 5, 8, 11, 14 ds/m). Sampling from leaves of plants was performed at 8-leaf stage in 6, 12, 24, 48 hours after applying stress. The results of statistical analysis showed that the gene expression pattern of studied transcription factors was different in sunflower lines. So that, the rate of expression of both studied transcription factors (WRKY and AP2Domain) was significantly more in tolerant line (AS5305) than susceptible one in sever salt stress level (14 ds/m) 24 hours after applying stress.

In order to examine the role of this regulatory factor and its gene expression pattern during stress in wheat plant as well as the physiological characteristics and expression of wdhn5 gene under drought stress as a  factorial experiment was carried out in completely randomized design in pot with three replications on  The wheat gemoyypes including two hexaploide cultivars (Bolani, Sistan) , two tetraploide cultivars(, Behrang, Shabrang) and wild (Urartu, Tauschii, Speltoides) were as the first factor and irrigation treatment with three levels consisted of normal (90% of field  capacity), mild stress (60% of field  capacity) and severe stress (40% of field  capacity) was as the second factor. Analysis of variance for physiological characteristics showed that the effect of water stress on all traits were significant, and the sensitive cultivar Speltoides had significantly lower dry mater production, water use efficiency and relative water content than the other cultivars. The result of the quantitave expression of wdhn5 gene expression by  real time PCR showed that expression of this gene in Tauschii and Bolani tolerant cultivars in 40% of field  capacity increased significantly which could indicte the importance of this gene in drought stress conditions. Based on these results, Bolani cultivar and Tauschii wild cultivar are relatively tolerant to drought and Splotheids is sensative to drought.

Water stress is one of the most important environmental factors that regulate plant growth and development, and limit production. Plants can respond and adapt to water stress by altering their cellular metabolism and invoking various defense mechanism. Manipulating the levels of antioxidative defence system may affect yield. Therefore, the aim of this study was to find out the relationship between gene expression of antioxidant enzymes and proline as an osmotic regulator in wheat cultivars under drought stress. For this purpose, a factorial experiment based on randomized complete block design was carried out with three replications. Treatments were two Iranian wheat cultivars (Pishtaz and Niknejad) and three levels of drought stress. Drought stress resulted in a reduction of Chl a, Chl b, total Chl and yield in both cultivars. This reduction was lower in tolerant cultivar Niknejad. Drought stress led to an enhancement in proline content, the activity of CAT (Catalase), APX (Ascorbate Peroxidase) and SOD (Superoxide Dismutase) enzymes. The tolerance of Niknejad cultivar under drought stress is associated with high chlorophyll (Chlorophyll), proline contents which is closely related to its enzymatic antioxidants activity (including CAT, APX and SOD). Our results showed that drought stress resulted in an increase in the gene expression of CAT, APX and SOD enzymes as well as enhanced the expression of gene for enzymes involved in the biosynthesis of proline (P5C5). Therefore, antioxidant enzymes and compatible osmolytes regulate plant yield mainly by modulating ROS with different mechanism.

Fluctuations in environmental water potential caused by drought and salinity are the most important abiotic stress factors in many natural habitats of organisms. Compatible solutes stabilize biomolecules and whole cells and play as stress-protective agents. Ectoine and its derivative 5-hydroxyectoine as compatible solutes are synthesized widely by the members of the bacteria, a few archaea, and eukarya in response to high salinity and osmolarity. Previously reported that the genetically engineered ectoine synthesis using ectA, ectB or ectC has resulted in increased hyperosmotic tolerance of model plants. In this review, the importance of current and potential applications of ectoines as protecting agents for macromolecules, cells, and tissues, are discussed. Besides, the leading theory for the protection mechanism of ectoines including the exclusion of ectoines molecules from the protein surface, slows the diffusion of water molecules and preserve the native state of the protein were explained. The experimental data of the past decades concerning alleviating drought and salinity stresses by ectoines and the prospects for their use in different fields of modern biotechnology, including genetic engineering techniques, have been summarized in this review article.

In a situation, where conflicts and contradictions on transgenic products are increasing day by day. Under such conditions, it appears that supplying documentation on the lack of side effects of these products and improving scientific knowledge will lead to a debate about the transgenic products in a rational way, and the only way to eliminate these contradictions is to investigate further on transgenic crops Is. In this regard, the present study was conducted on the basis of qualitative paradigm and using qualitative content analysis to analyze the researchers' views on the application of technology of genetically modified crops in Iranian agricultural research centers. In order to collect the data, a semi-structured interview method with 23 researchers was used with purposeful sampling and snowball sampling. According to the findings of this research, researchers' viewpoints on the use of transgenic technology around three themes, "The individual capability of activity in two institutions of the university and research center", "The impact of technology of transgenic products on occupational development and economic development of the country" And "the impact of political and social flows on the individual".