نشریه مهندسی ژنتیک و ایمنی زیستی
سال ششم شماره 2 (پیاپی 12، پاییز و زمستان 1396)

Native chicken breeds are important genetic resource and well adapted to the local environmental conditions. However, growth rate and feed efficiency of these breeds are not appropriate. Comparative studies of commercial and native chickens provide an opportunity to characterize the biological basis of differences between them. Here, RNA-Seq technology was used to investigate differences in the transcriptomes of breast muscle-related genes and associated pathways between Ross and Isfahani's native breed. In this study, 200 birds of the Ross 708 strain and 200 native chicks of Isfahani native were reared in a similar standard management and feeding manner. We extracted total RNA from two native and two commercial breast muscle samples of the 28 days old chickens. Compared with native breed, 606 significantly DEGs (differentially expressed genes) (357 up regulated and 249 down-regulated, P-adjusted ≤ 0.05) were obtained in commercial breed. PPI (Protein-protein interaction) network of DEGs (up regulated in commercial) was constructed and 267 genes were included in the resulted PPI network of which 75 genes were identified in the form of three modules with a maximum level of significance. Gene ontology studies showed that all three modules played an effective role in the growth of breast muscle and their hub genes with maximum degree were JUN, EGR1, THBS1, ITGA4 and ACTA2.

Nitrogen is one of the most important elements needed by plants, but plants are unable to reduce atmospheric nitrogen into ammonia and use it directly for its growth. Nitrogen fixing microorganisms, with the process of nitrogen fixation, provide the nitrogen required for plant utilization and play an important role in soil fertilization. The aim of this study was to evaluate nitrogen fixation in endophytic microorganisms isolated from root nodules of alder (Alnus subcordata) and Russian olive (Elaeagnus angustifolia) in Golestan province. A total of 50 nodule specimens were collected and cultured on nitrogen-free medium. Microorganisms were identified using biochemical and molecular tests. Nitrogen fixation of isolates was also evaluated using acetylene reduction and gas chromatography (GC). The two genera of Streptomyces badius strain BL225 and Actinomadura citrae strain DSM 43461 were identified as endophytic actinobacteria, Cladosporium cladosporioides strain F1 and Cryptococcus victoriae strain p41A001 were found as Ascomycota and Basidiomycota phyla of fungal endophytes, respectively. Cladosporium cladosporioides strain F1 and Cryptococcus victoria strain p41A001 had a high ability in nitrogen fixation. The root nodules of alder (A. subcordata) and Russian olive (E. angustifolia) carry the endophytic microorganisms with nitrogen fixation ability, therefore, the endophytes can be used as biocontrol agents in agriculture.

Probiotics are bacteria with beneficial effects to humans and animals. Gram-positive Lactococcus lactis can be genetically engineered to efficiently produce a large variety of proteins. Glucanase enzymes, are capable to hydrolyze β-glucans (a non-starch polysaccharide). β(1,3)-(1,4) glucan (Lechinan) is a characteristic hemicellulose in primary cell walls of grasses such as barley, rye, sorghum, rice and wheat. The purpose of this study is to express the lechinase enzyme in Lactococcus lactis to achieve a feed supplement for hydrolyzation of barley β-glucan to replace corn by barley in poultry diets. licBM2 gene encoding lechinas enzyme with a secretion signal peptide was cloned in pNZ8149 plasmid. Then recombinant plasmids were transferred to Lactococcus lactis using electroporation method and the transformed colonies were selected via their ability to grow on lactose containing medium. The expression of transgene was induced using of Nisin in the medium. Our results showed that by increasing of the induction time, the expression of lechinase gene and consequently the production of enzyme in the medium were increased.

Cucumber mosaic virus (CMV) is one of the most important viruses which causes yield reduction in tomato. In this experiment, the role of salicylic and jasmonic acids was investigated in the gene expression of antioxidant enzymes and induction of resistance to oxidative stress due to CMV inoculation in tomato (Falat cultivar). Results showed that in control plants expression of genes encoding catalase (CAT) and peroxidase (POX) enzymes gradually decreased to 15 days post inoculation (dpi), but expression of superoxide dismutase encoding gene (SOD) was increased at 15 dpi. In CMV inoculated plants, CAT gene was down-regulated, but POX gene was up-regulated to 8 dpi. SOD gene expression also increased in CMV inoculated plants to 15 dpi. The highest expression rate of POX observed at 15 dpi in SA treated plants which was 120% greater than before hormone application. Hormone application decreased the disease severity and symptoms to 80%. In Falat cultivar, peroxidase role in scavenging of H2O2 was greater than catalase and in order to inducing resistance, we can use SA and JA just before virus infection.

Because of several advantages such as low cost, simple culture, and high growth rate, bacterial systems, in particular Escherichia coli, are the main expression system for production of recombinant pharmaceutical proteins. In this research, we used this system to produce a valuable human growth factor, somatomedin C. cDNA of this gene was prepared and cloned into an expression vector using NdeI and BamHI restriction sites. The cloning process resulted in the generation of a recombinant vector in which the somatomedin cDNA was placed under control of the T7 inducible promoter. This recombinant vector was introduced into the E. coli and the protein expression was evaluated following induction with 1 M IPTG solution. The results showed that transgenic bacteria produce noticeable amount of human protein after 22 hrs induction at 25 degree centigrade.

Stevia rebaudiana is a medicinally important plant, free-calorie with sweet test and beneficial for diabetic patient. In order to optimization of the micropropagation of this plant, three experiments were conducted by factorial experiment based on completely randomized design (CRD) with three replications. Shoot tip explants were cultured on MS media supplemented with Kin (0, 1 & 2 mg/l) IAA (0, 0.5, 1 & 1.5 mg/l) (test 1), Kin (3 & 4 mg/l) with IAA (0.5, 1 & 1.5 mg/l) (test 2) and BAP (0, 1, 2, 3 & 4 mg/l) IBA (0, 0.5, 1 & 1.5 mg/l) (test 3). The shoot number increased with increasing concentrations of Kin in media and in second experiment, the highest shoot number was produced in MS medium supplemented with 4 mg/l Kin 0.5 mg/l IAA (with an average of 7 shoots per explant). However, the best result of micropropagation was obtained from MS medium containing 2 mg/l BAP 1.5 mg/l IBA in test 3 (ith an average of 9 shoots per explant). Also, increasing BAP concentrations from 2 mg/l resulted to a significant reduction in shoot number and shoot length and quality of plantlets as well as increase in callusing. In addition, shoot number was dramatically increased by incubation the cultured explants in culture room for two months. The maximum rooting of developed shootlets was achieved from MS medium supplemented with1 mg/l IBA. The rooted plantlets were hardened in culture room and with high successfully established in soil.

This experiment was conducted to evaluate the effects of different concentrations (0, 1, 2, 3 and 4 mg L-1) of lead (Pb) and cadmium (Cd) in vitro on the growth characteristics, enzymatic activity and Pb and Cd compositional content of Spinacia oleracea as CRD with three replications. The seeds were cultivated on MS medium and the traits were measured after 40 days. The results revealed that the highest malondialdehyde, H2O2 and the highest Cd content in leaves and roots were observed with 3 and 4 mg L-1 Cd treatments. The greatest data for catalase and SOD were recorded with four mg L-1 Cd and control plants, respectively. The highest data for the leaves number and width was recorded in control plants not receiving Cd and Pb treatments. The results also showed that with any increase in Pb treatment levels, the amounts of MDA, CAT, SOD and H2O2 were increased, correspondingly. The highest lead content was measured in the leaves and roots of plants treated with 4 mgL-1 of Pb. The results for bio-concentration factor showed that spinach could be considered as an excellent candidate for the phytoremediation of both Cd and Pb.

Trachyspermum ammi is one of the most important medicinal herbs in Iran. This plant contains important medicinal properties, including anti-digestive, antispasmodic and relieving rheumatic pains and has been used as a spice and food preservative. To overexpress some effective ingredients in this plant, it is necessary to optimize the gene transfer process. For this purpose, the explants of hypocotyl, two strains of Agrobacterium tumefaciens were studied along with factors such an inoculation times (5, 10, 15, 20 min) co-cultivation times (1, 2 and 3 days). In this study, pBI121 vector harboring GUS as the reporter gene, and nptII gene as the plant selected marker were used. To confirm the gene transfer, PCR, histochemical GUS staining and RT-PCR tests were used. According to the results, the time of inoculation of 5 minute and the time of one day for co- cultivation increased the efficiency of gene transfer. Both bacterial strains showed a relatively good performance in gene transfer, but the LBA4404 strain showed higher efficacy than GV3101. In this study, 25 mg/L of kanamycin was selected as the appropriate concentration for determining the probable transgenic plant by Agrobacterium. For the first time, the present study presents an effective and efficient method for gene transfer to Trachyspermu ammi that can be effectively used for gene transfer.

In this research, in order to study some physiological and molecular traits such as the expression level of Catalase (CAT1) and Ascorbate Peroxidase (APX1) genes in two, CH-Falat and Rio Grande-S, varieties of tomato plant (Solanum lycopersicum L.), an experiment was designed in randomized complete block design. Salinity with the concentration of 0, 25, 50, 75 and 100 mM NaCl was applied in hydroponic culture. Plants RNA was extracted and cDNA was synthesized, then Real–Time PCR was carried out. The results showed that the relative expression of CAT1 has been increased in Rio Grande-S at all levels of salinity and in Falat-CH variety only up to 50 mM NaCl. The relative expression of APX1 has been decreased in Rio Grande-S up to 50 mM of salinity level and has been increased in 75 and 100 mM salinity levels. The relative expression of APX1 in Falat-CH variety has been increased up to 50 mM salinity level. Some physiological traits in salinity treatments were also studied and the results showed that in both varieties, by salinity increase from 0 to 100 mM NaCl, the amount of Na, Prolin, the soluble sugers were increased and the amount of K, and K/Na were decreased. The membrane stability index was decreased in salinity treatments comparing to control. The results of the molecular and physiological analysis showed that Rio Grande S variety in comparison to CH-Falat is more resistant under high level of salinity.

Studying the effects of fungicides on rhizosphere and soil microbial communities is very important due to the critical role of microorganisms in soil and plant health. In this study, the effects of fungicide Rovral TS on dynamic and structure of cucumber rhizospheric bacteria was investigated by Next-Generation Sequencing based on 16S rRNA gene (V4 region) by means of Illumina MiSeq sequencing in three plant growth periods. Results showed that among 26 identified phyla and 352 identified bacterial genera for 18 samples, Proteobacteria and Pseudomonas were most dominate phylum and genus, respectively. Application of Rovral TS was resulted the changes in bacterial structure and dynamic. Decrease of relative abundance of Pseudomonas in middle stage of flowering in fungicide treatment and in the last stage of control treatment was observed. Moreover, the relative abundance of Sphingopyxis, Sphingobacterium and Chryseobacterim in fungicide-treated plants were more than control plant in all three stages. Analysis of diversity indices revealed that diversity in the rhizospheric soil of control plant was more than fungicide-treated plants (shanon=5.33), while species richness in control soil was greater than fungicide treated (Chao1=2324.17). In addition, the effect of plant developing stages on OUT number, diversity and richness indices (regardless type of treatment) was significant and rhizospheric soil belongs to before flowering stages benefit more diversity and richness.

Organic mercury is a sustainable form of mercury that enters the food chain of organisms and cause dangerous environmental hazards for natural ecosystem. Mercury stability and high cost conventional refining methods are disturbing factors. In this field, biological methods such as the use of bacteria or enzyme based for microbial remediation provided low-cost strategy and environmental lover of bioremediation. In this research, merB gene isolated from resistance bacteria by PCR technique by specific primers containing NcoI and NdeI restriction sites, so after amplification, it cut with restriction enzymes and after purification of gene, ligation was performed into PET28 a expression vector. PET28a+merB recombinant vector was transferred into E. coli strain TOP10 by heat shock. The recombinant bacteria were selected in kanamycin selective medium. The presence of gene in transformed bacteria was confirmed by PCR on plasmid and by enzymatic digestion. Results confirmed the accuracy of cloning of merB gene into this expression vector. In order to the expresse its protein, the recombinant vector was transferred into E. coli BL21 (DE3). Expression of the protein was confirmed by SDS-PAGE method. Finally, the growth of E.coli strain BL21 containing the recombinant vector with E coli BL21 bacteria including empty vector was measured by adding methyl mercury into the environment during 48 hours. Growth ability of E.coli containing the empty vector indicates lack of bacteria resistance to mercury, on the other hand; merB protein in transformed bacteria increased their resistance to methyl mercury in the environment. Therefore, bioremediation of organic mercury from pollutant environments could be possible by using this methodology.

In order to assessing the resistance to Fusarium oxysporum f.sp. Lentis in different genotypes of lentil and its relation to protein markers, a research was programmed using 38 genotypes of lentil in Greenhouse and laboratory conditions. Analysis of studied traits was performed as a factorial experiment with two levels for Factor A (conditions without disease and conditions for disease) and 38 levels (genotypes) for Factor B based on completely randomized design with three replications. Our data showed that the average mortality of plants in the eighth week was more than the fourth week dpi. The mean comparison of studied traits by using LSD test (p=0.01) indicated that they are in different demographic groups. According to the results of cluster analysis, genotypes were divided into three groups, which were differed in the average mortality rate. Analysis of water and salt-soluble proteins showed that the average genetic variation for all loci is 0.5 and the average of Shannon index is 0.34. In the SDS-PAGE analysis for seed storage protein, genotypes were divided into three groups in cluster analysis. Regression analysis of data from proteins and water-soluble salt with morphological traits showed that regression coefficient for the average mortality of plants in the fourth and eighth week with marker 12 was significant (p=0.01). Markers 4 and 12 because of having ties with more than one trait were identified as the most effective markers. It seems that in breeding programs for resistance to the disease, protein markers can be utilized in addition to morphological traits.

Cotton boll worm, Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) is an important polyphagous pest throughout the world. Due to the adverse effects of chemical pesticides, alpha-amylase enzyme inhibitors in insects have been noticed as an alternative method for the pest management programs. In this project, whole gut of the fourth instar larva was dissected and the enzyme extract was pulled out. After removing seed protein extracts of Prosopis farcta and Cicer arietinum by sodium chloride 0.1 M, the effect of four different concentrations of both seed extracts (1.48, 1.2, 0.87 and 0.42 μg) were studied on alpha-amylase activity of boll worm. According to the data, the protein extract of P. farcta had greater ability to inhibit cotton boll worm amylase. Enzyme assays in the presence of different concentrations of C. arietinum and P. farcta extracts showed enzyme inhibition in a dose-dependent manner. The maximum enzyme inhibition was in pH 9. Zymogram analysis confirmed quantitative enzyme assay and showed a sharp reduction in the band thickness during the amylase inhibition. Consequently, the seeds of both P. farcta and C. arietinum, had the potential to be considered as effective and important sources of alpha-amylase inhibitors and noticed as substitutes for chemical pesticides in the near future.

Innate immunity considered as the first defense line in insects and vertebrates against microbial invasion. Vast researches in recent years have shown that there are considerable similarities between the molecular organization of animal and plant systems for non-self-recognition and anti-microbial defense. Like animals, plants are able to recognize microbe associated molecular patterns (MAMPs) which are specific to microbes and are not found in plants. Such structures, also called general elicitors of plant defense, are often inevitable for microbial vita and their perception via specific receptors, aware the plant from microbial invasion. The immunity triggered in plant after perception of MAMPs is called MAMP Triggered Immunity (MTI). On the other hand, existence of such surveillance system in plants has resulted in coevolution of microorganisms with plants to develop effectors to disrupt and pass the MTI defense line. In the continuance of this competition, plants have developed other mechanisms to recognize these effectors and created another immunity called effector triggered immunity (ETI). The goal in this paper is to review the last findings about different mechanisms of immunity in plant.

Since the discovery of P53 in 1979 as a tumor antigen until today that has been established as an important tumor suppressor gene, over sixty four thousand paper and research reports were published in PubMed including this protein name. Although the results of these investigations have been helped to clarify the structure, function and regulation of P53, they also led to more ambiguities and questions about this protein. Identification of different isoforms of P53, the discovery of a P53 natural antisense (Wrap53) and recent findings about its different roles in processes such as longevity, aging and metabolism have made P53 remain attractive for study. Delivering the wild type P53 gene to p53 mutant cancer cells, introduction of small molecules that reactivate mutant p53 or suppress its important regulator, MDM2, are among the cancer therapies developed based on p53. The present article attempts to review the history and functions of p53 and discuss about the treatment strategies have been developed based on its antitumor properties.