نشریه مهندسی ژنتیک و ایمنی زیستی
سال دهم شماره 2 (پیاپی 20، پاییز و زمستان 1400)

At present, the use of plant growth-promoting bacteria has been considered as a suitable alternative to the use of chemical fertilizers to improve wheat growth. The objectives of this study was to identify the best and most effective rhizobacteria on wheat growth. First, Bacillus strains were isolated from wheat rhizosphere and their ability to produce IAA was assayed. Then, Triticum aestivum (Roshan) plants were treated in a completely randomized design by eight heighest IAA-producing bacterial strains at three different bacterial concentrations. By examining morphological traits, the best treatment was selected and the expression of genes involved in the auxin pathway was studied by RT-qPCR technique. Here, twenty Bacillus strains were isolated. The amount of IAA production varied among the strains (the minimum and maximum IAA production were 0 and 95.89 μg/ml, respectively). Twenty strains were isolated, eight strains with the highest levels of IAA production were selected for applying to the plants. Results of morphological traits demonstrated that strain C6 at concentration 1 (OD=1) had a significantly greater positive effect on Roshan plants. This strain was chosen for genes expression analysis. Analysis of expression of TaTAR2.1 and TaTAR2.4 genes showed that these genes have been influenced by strain C6 and they were significantly down-regulated as compared to the control.

Parthenolide is a secondary metabolite, which naturally occurs in the feverfew plant (Tanacetum parthenium) and is responsible for its healing power. The potential of parthenolide in inhibition of cancer cell growth, alone or in combination with other anti-cancer therapeutics, has been studied in several laboratories. In this study, the effect of extracted parthenolide on the expression of seven pro-apoptotic genes (BID, P21, PUMA, BAX2, P53, CASP8 and BIM), all of them were influenced by NF-κB signaling pathway, in MDA-MB- 231, a breast cancer cell line was investigated. The results indicated that in response to the parthenolide treatment, all of the selected genes were induced and up-regulated significantly with different degrees. We proved that parthenolide alters the expression of a variety of genes involved in apoptosis pathway in MDA-MB- 231 cell line. In conclusion, we provided evidence that parthenolide alters the expression of a variety of genes involved in apoptosis pathway in MDA-MB- 231 breast cancer cells. The selected genes are directly or indirectly were regulated by NF-κB and it is confirmed that NF-κB is an important target of parthenolide.

In order to investigate different lines of durum wheat for water stress tolerance, an experiment was carried out in the greenhouse in the form of a split plot design in a Randomized complete block design with three replications using 20 lines of durum wheat at two levels of water stress (40% and 70% of the amount of available water was completely drained in 0.4 and 0.7 levels of stress, respectively and re-irrigation was performed) and a control level (complete irrigation). Also, water-soluble and salt-soluble proteins were extracted and SDS-PAGE was done. Measured traits were photosynthetic pigments (chlorophyll a, chlorophyll b, chlorophyll a/b, total chlorophyll and carotenoids), soluble sugar and prolin content. The results of the analysis of variance showed that there was a significant difference between the studied lines at different levels of stress. The results of comparing the mean of the studied traits showed that in total, lines 16 and 5 have the highest value in most of the traits and lines 10 and 18 have the lowest value in most of the traits. Also, the results obtained from the cluster analysis indicated that due to the higher mean values compared to the other lines both in stress-free and stress conditions, lines 16 and 5 (Mra-1/4/Aus1/3/Scar/GdoVZ579//BitI, TRN//21563/AA/3/BD2080/4/BD2339/5/RASCON_37/TARRO_2//) have a higher performance stability in the different environmental conditions. Furthermore, the results of molecular analysis of the studied proteins showed that in water and salt soluble proteins based on matrix similarity and genetic distance of straw, the lowest genetic distance was between genotypes 18 and 19 (0.32) and the highest genetic distance was between genotypes 16 and 8 (0.98). Finally, it can be concluded that there is a significant genetic diversity between the studied cultivars and this diversity can be used in plant breeding.

Acquired systemic resistance (SAR) is one of the defenses that can make plants resistant to systemic viral infections. Acquired systemic resistance is associated with a hypersensitivity response in the host, and salicylic acid is known as its signal. In this study, the effect of plant inducers such as salicylic acid and jasmonic acid on cucumber viral disease with cucumber mosaic virus, CMV and the expression of some defense genes were investigated. Treatments included plants treated with CMV, salicylic acid (with a concentration of 1.5 mmol), jasmonic acid (with a concentration of 1.5 mmol), as well as salicylic acid and jasmonic with CMV. The results of this study showed that the maximum severity of CMV-induced disease was observed in plants treated only with CMV (68.85%). In the treatment of salicylic acid and jasmonic acid with CMV, the severity of symptoms was significantly reduced (10.23%). Then, the effect of these treatments on the expression of four genes Peroxidase (POX), Catalase (CAT), Superoxide Dismutase (SOD) and Phenylalanine Ammonialyase (PAL) was investigated. The results showed that the expression of these genes in treated plants had an upward trend until the ninth day after treatment. But this upward trend was much less observed in control plants (treated with water and virus). The highest level of gene expression in the studied enzymes compared to the control plant was related to the catalase enzyme (401.7 times), followed by peroxidase enzymes (266.8), superoxide dismutase (152.7) and phenylalanine ammonialyase (101.8). According to these results, salicylic acid and jasmonic acid, increased plant resistance to CMV and reduced the symptoms of the virus in cucumber plant by inducing plant defense mechanisms.

Bovine chymosin enzyme is one of the most commonly used enzymes in the dairy industry. The production of this enzyme from its natural source does not meet the needs of this huge industry. The production of recombinant bovine chymosin in plants can be a good alternative to native enzyme. Insertion and expression of foreign genes in plants can occur in the nucleus and chloroplast organelles. The expression of foreign genes in eukaryotic heterologous hosts can be increased by optimizing its codons as well as translocation of the heterologous protein after expression to encapsulated organs such as chloroplasts. In this study, codons of the bovine prochymosin gene were optimized to be suitable for expression in the nucleus and chloroplasts of tobacco plants. After optimization, the CAI index of this gene for nucleus and chloroplasts was 0.929 and 0.947, respectively. The optimized sequence was cloned in the pUC57 basic vector after artificial synthesis, and was confirmed by sequencing. The function of the synthesized gene was confirmed by expression in E. coli and milk coagulation test. To provide a suitable expression vector for gene transfer to the plant genome, the GFP gene was replaced by the prochymosin gene in the p35S-TP-GFP expression vector. This vector is suitable for gene transfer by gene gun method, and with the presence of chloroplast signal peptide, it causes protein accumulation in chloroplasts. The gene was also replaced with the GUS gene in the pBI121 vector, which is suitable for gene transfer by the Agrobacterium method. The recombinant vectors constructed in this study can be used to effectively transfer and express this industrial enzyme in plants.

Optimization of the effective parameters in the biolistic method for high-performance DNA transferring into the chloroplasts without tissue damage is a necessary requirement for developing transplastomic plants. Due to the relative similarity of the physical barriers at the entry of microcarriers into the nucleus and chloroplasts, the nuclear gus reporter gene is usually used, but the physical and biological parameters involved in plastid engineering have not been directly reported. In this study, to optimize gene transfer to tobacco chloroplasts, some parameters such as microcarrier type, DNA concentration, helium accelerating pressure, leaf arrangement and shooting distance, and pretreatment duration of explants before bombardment were investigated. After transferring the chloroplast's vector to explants, regeneration of transplastomic plants was performed in a selection medium containing 500 mg/l spectinomycin. The highest number of transplastomic plants (2.5 plants per bombardment) was obtained with Tungsten particles with a diameter of 0.7 μm, with 1 μg DNA/shooting, accelerating helium pressure 1100 psi, shooting distance of 6 cm and 24h pre-treatment period. Homoplasmic plants were obtained after three re-regeneration periods at the presence of both strepto/spectinomycin antibiotics. Molecular confirmation of the correct insertion of expression cassette at the targeted place of the genome and the confirmation of homoplasmy were performed using PCR analysis and southern blotting. Maternal inheritance of the transferred genes into the chloroplasts was confirmed by green and albino seedlings resulted from seed germination, product of reciprocal crosses between control and transplastomic tobacco plants.

Among the various diseases that threaten the green space plants, the Fusarium genus is of great importance. Due to time consuming, requiring high expertise and low reliability in the morphological methods, it would be recommended to use species-specific primers for identification of Fusarium species as a fast and cost-effective method without need to sequencing that could be easily used to identify the genus and species of Fusarium. In this study, in order to molecularly confirming of Fusarium isolates associated with Ash, Black locust and Populus decline, and turfgrass yellowing, in addition to verifying species-specific primers with standard isolates, Fusarium isolates obtained from Ardabil’s green space plants were identified using these specific primers by PCR. Totally, the 24 isolates belonging to F. solani using fFuso1 and rFuso2, 11 isolates belonging to F. oxysporum species using CLOX1-F and CLOX2-R primers, 10 isolates belonging to F. acuminatum species with using FAC-F and FAC-R primers, 8 isolates belonging to F. equiseti were identified using FEF1 and FER1 primers and 8 isolates belonging to F. graminearum were identified using FgCTPSf024 and FgCTPSrR306 primers. These primers did not react with isolates belonging to other Fusarium species. Also, the sequencing and phylogenetic analysis results lead to identify 2 and 1 isolates as F. solani and F. redolens, respectively.

Germanders with the scientific name of Teucrium polium has antimicrobial properties and has long been used in traditional medicine to treat many infections. The aim of the present study is to investigate the antifungal properties of methanolic extract of Teucrium polium against different Candida species. Briefly, after collecting from Borujen hills in Isfahan province in 2011 and preparing isolates of Candida albicans (NCPF 3153, ATCC 1677), Candida cruzi (CBS 537) and Candida globrata (CBS 2175), the extraction process was performed by soxhlet apparatus. Then the antifungal activity of methanolic extract of Teucrium polium was evaluated by well and disk agar diffusion method. Minimum inhibitory concentration (MIC) and minimum fungicide concentration (MFC) were determined. Data analysis was performed using SPSS 18 software using two-way analysis of variance (P <0.001). In the well and disk diffusion method, all concentrations of methanolic extract of Teucrium polium had an inhibitory effect on Candida albicans (NCPF 3153) and Candida cruzi species. The lowest inhibitory concentration (MIC) of methanolic extract of Teucrium polium on Candida albicans NCPF 3153 and ATCC 1677, Candida glabrata, and Candida cruzi isolates were 6.25, 6.25, 12.5, and 0.78 mg/ml, respectively. The lowest fungicide concentration (MFC) against both Candida albicans tested was 12.5, on Candida glabrata and Candida cruzi were 25 and 1.56 mg/ml, respectively. According to the results, methanolic extract of Teucrium polium plant had significant inhibitory activity against different species of Candida.

Despite the significant benefits of transgenic crops, there is a general concern that if these crops, like their non-transgenic counterparts, are safe for human and animal nutrition. One way to investigate the food safety of these crops is to compare the molecular properties of transgenic crops with their natural parents as well as crops resulted from conventional breeding which have a history of safe use. This research pursued to determine whether the set of changes that occurred in the two transgenic events of rhizomania-resistant sugar beet is comparable to the parental non-transgenic plants and was within the range of natural changes? Whether genetic engineering led to the generation of compounds that are not safe for humans and animals? Investigations have shown that proteomic alterations in both transgenic lines were minimal and within the range of natural variations. Metabolomic changes in a selected S6 event were very low while there were remarkable alterations in the metabolome of S3 event, although these were within the range of natural deviations too.

In order to provide the nutritional needs of the people and increase productivity in agriculture and gain high crop yields from existing cultivable lands while controlling pests and diseases, has led agricultural experts to use plant breeding methods and as the traditional methods are time consuming, therefore, with the help of genetic engineering, they have sought to transfer genes to plants to increase crop yield by creating resistance to pests and diseases and a variety of abiotic stresses. The engineered plants are called Genetically Modified Organisms (GMOs) or recombinant plants. On the other hand, some believe that man-made transgenic plants may be harmful and problematic for humans, and there are concerns in this regard in society. In many cases, products on the market are considered transgenic without sufficient reason. Obviously, it is possible to use genetic engineering to create transgenic plants with genes that are harmful and dangerous to humans for the purpose of bioterrorism. Therefore, field study and transgenicity of various products available in the market is necessary with the help of biotechnological and precise molecular methods. For this purpose, sampling (more than 100 samples) of different agricultural products from markets, fruit gardens and vegetable fields of different cities of East and West Azerbaijan in Iran was performed. After DNA extraction with CTAB buffer, their quantity and quality were evaluated and their functionality was confirmed by testing on the conserved house-keeping Actin gene. Four genes, a promoter and a terminator, common in plant transformation, were selected and their sequences were obtained from the NCBI and specific primers were designed for them. DNAs were used for PCR on these target genes. Their PCR results showed that the Cry, Hyg and Bar genes and the 35S promoter sequence and the Nos terminator sequence were not present in any of the samples. Among 112 tested samples, NptII gene was detected in 13 samples. This diagnosis alone cannot be a reason for the transgenicity of these samples, because this gene is also present in E. coli, and if a sample is infected or contaminated with it, when DNA is extracted, its genetic material is also extracted and shows positive in PCR test on bacterial DNA. The results of this study showed that, contrary to popular belief, the products on the market are not transgenic and people can consume them safely.

Risk assessment for a transgene is one of the key steps in the genetic transformation. In order to use cryIAc gene in production of transgenic plants, a library and in-silico research was performed to confirm the safety of the gene product for human consumption, animal feed and the environment. In the first step, the molecular mechanism of action of the CryIAc protein and its specific receptors in the midgut cells of the target organism was explored. Digestibility of the protein in the stomach and intestine liquids have been investigated based on the peer reviewed documents. It was demonstrated that the CryIAc protein could be completely digested in the stomach liquid of vertebrates, especially mammalians including the human. It was shown that addition of the pure protein into the animal diets did not result to any abnormalities in the growth pattern and also blood histochemistry of the experimental animals. Also, whole plant assays using the different diets contained the transgenic and non-transgenic plant materials in different experimental animals was discussed. These experiments did not result to any differences between the groups. Possibility of the toxic effect of the CryIAc protein was investigated via in-silico tools. The results did not show any similarity between the known toxic protein and the CryIAc protein. Allergenicity of the CryIAc protein was assessed in-silico in different ways. It was proven that there were any similarities between the protein and the other known allergens. At the end, the persistency of the CryIAc protein in the soil was explored. Since the different experiments on exposure of the pure protein or the transgenic plant tissues into the environment proved that the CryIAc protein is degraded easily in the soil, it could be concluded that the CryIAc protein is an environmental friendly protein. Eventually, the final conclusion is that the CryIAc protein is a safe protein for human consumption, animal use and the environment, and could be used safely in genetic engineering programs.

Viruses are the amazing particles in nature that is designed to reproduce with a minimal genetic set. This vital process is very complex and requires the time and coordinated activities of a number of virus and host proteins. In order to the genome packaging, the virus protein subunits interact with each other and with viral nucleic acids. The general principle in packaging is the interaction between the structure of coat protein and a specific signal in RNA. Replication of +ssRNA viruses and their nucleocapsid assembly occurs in the cytoplasm and requires Pro-Pro and Pro-RNA interactions. The packaging process in them is spontaneous and does not require ATP. The interaction between the coat protein subunits and the genomic RNA is controlled by a unique structure in the sequence that is highly desired to interaction with the coat protein. The presence of these unique structures in the viral nucleic acid, called the packaging signal or OAS (origin of assembly sequence), causes viral nucleic acid to be distinguished from other cellular RNA molecules in the region which assembly takes place. General knowledge of the precise mechanisms of viruses, the configuration and aggregation of viruses, and the packing of their genomes that lead to the formation of stable viral structures is an important prerequisite for understanding the overall biology of viruses.