نشریه مهندسی ژنتیک و ایمنی زیستی
سال نهم شماره 2 (پیاپی 18، پاییز و زمستان 1399)

Potato virus Y (PVY) is one of the most damaging viruses of potato plants which infects most cultivars and causes a significant decrease in yield products quality and also economical losses annually. Recently, the application of pathogen derived resistance mechanisms has opened new horizons for the development of virus-resistant transgenic plants. This research was carried out to study RNA silencing to engineered potato plants that are resistant to potato virus Y (PVY). RNAi construct was designed from coat protein (CP) segment of PVY. Potato explants were transformed by Agrobacterium tumefaciens AGL1 with pCAMRNAiCP containing bar selectable gene. The rate of regenerated plants was evaluated in the selected medium containing the herbicide phosphinotricin after growth and propagation. Transgenic plants and their gene expression were investigated using polymerase chain reaction (PCR) and RT-PCR. Results showed that 42 percent of regenerated plants were transformed. The expression of CP hairpin construct was determined by RT-PCR analysis in most of the lines. PVY resistance bioassay showed that 82 percent of transgenic potato plants were resistance to PVY.

Soybean, one of the most important sources of edible oil and protein in the world, is exposed to various environmental biotic and abiotic stresses. These stresses can negatively impact the quality and quantity of soybean production. This study aimed to identify genes that express circular RNAs in response to low phosphorus and nitrogen stresses in soybean roots. Soybean seeds were grown under different low phosphorus and nitrogen treatments, and sampling was done from the root tissues for total RNA isolation. After measuring the quality and quantity of the isolated RNA, samples were sequenced using next-generation sequencing. After obtaining the sequencing data, the quality of RNA-seq data measured, and then circular RNAs were identified and characterized using five different programs. Finally, to identify the expression pattern and putative function of circular RNAs, differential expression analysis and functional analysis of circular RNAs’ parent genes was done using bioinformatics approaches. RNA-seq experiment produced more than 360 million reads, and in total, 2033 circular RNAs were identified in all treatments. Although some circular RNAs were co-expressed in two or more treatments, most of the circular RNAs had a stress-responsive expression. Our results were revealed that circular RNAs may play their roles by binding to their target molecules. Identified genes can be used in plant breeding programs for producing tolerant soybean cultivars to low phosphorus and nitrogen stresses.

Vaccine design from the past to the present is based on classical methods, which are time consuming, costly and have some risks, but with the discovery of the genome and gene sequencing, the modern methods for vaccine design have been developed, including Referred to the reverse method of vaccine design (vaccine polytop). In this study we used NCBI (ORF Finder) to find the coding sequences, PSORTb server to locate the protein, Algpred server to check the allergenicity of the protein and LBtope - ABCpred - IEDB CBTOPE servers - Disco Tope 2.0 - IEDB (Ellipro) to predict the linear and spatial epitopes. The final epitope was selected and then we analyzed the polypepe structure by ExPasy server (ProtParam) and finally obtained the optimized nucleotide sequence with JCat server. Using this method, 6074 reading frameworks as well as 43 surface and secretory proteins were identified, from which, finally, 10 suitable epitopes that are able to provide immunity against a wide range of strains were selected as a vaccine candidate. It is hoped that this will facilitate the development of this vaccine.

AKT1 gene, plays an important role in controlling cell survival and apoptosis and is therefore considered an effective factor in thyroid tumors, which is one of the most common tumors in Iran. Single nucleotide polymorphisms in rs1130233 (726 G / A) of Akt1 gene may be involved in the development and progression of cancer or response to treatment. In this study, the association of rs1130233 (726 G / A) polymorphism with a predisposition to thyroid tumor in northwest of Iran was investigated. In this study, 80 patients with thyroid tumors and 106 healthy individuals were evaluated in a case-control study. Polymorphic (726 G / A) rs1130233 genotypes of Akt1 gene were determined by Tetra-ARMS PCR and compared and analyzed. Significant difference was not observed between the distribution of these genotypes in the patient and control groups. There was a significant relationship between genotype type and tumor size (P> 0.05). These findings suggest that rs1130233 polymorphism (726 G / A) has no effect on thyroid cancer susceptibility in northwest of Iran but may contribute to the relative risk of developing thyroid tumors.

Late flowering in lettuce produces additional leaves and maintains the edible quality of its product. Control of flowering time in plants is vital for plant fitness and for agricultural purposes and is an important factor in successful reproduction and seed crop yield and depends on the environmental and internal parameters of the plant and it is controlled by the regulatory network of genes. One of the problems in the field of crops and horticulture is the mismatch of flowering time with favorable environmental conditions. by increase in population, it is necessary to produce efficient plants with higher biomass. In the flowering regulatory network, Flowering Locus C (FLC), a MADS box transcription factor, prevents flowering. The autonomous pathway accelerates the onset of flowering independently of daylight by suppressing FLC. The MSI4/FVE gene is a key gene in the autonomous pathway. It is involved in controlling cell proliferation and early differentiation, organ growth and plays a key role in accelerating flowering by reducing the expression of FLC gene via epigenetic changes. in this study, using the RNA Silencing technique, MSI4/FVE gene expression was repressed at post transcriptional level. To this end, first, the tissue culture of lettuce in MS culture medium was optimized using BAP, Kinetin and NAA hormones for direct regeneration from cotyledon and leaf explants.  The MS medium containing 0.1 mg/l NAA and 0.01 mg/l BAP has the highest regeneration rate for cotyledon explants. Then, for construction of silencing hairpin construct, a fragment of Arabidopsis thaliana MSI4 gene was amplified by PCR and then cloned in sense and in antisense directions into pFGC5941 vector by two consecutive cloning. The hairpin structure in this recombinant vector was then transferred to the lettuce plant by Agrobacterium. The obtained transgenic plants were tested by PCR to confirm the presence of the transgene and the study on the flowering phenotype of these transgenic plants showed that the silencing of MSI4 in lettuce caused the plant to flower late (about 1.5 months delay) and produce additional leaves.

Hairy root induction by Agrobacterium rhizogenes is a reliable method to increase P. oleracea medicinal compounds. The hairy roots in addition to high growth rates are genetically and biochemically stable. Purslane (Portulaca oleracea L.) is one of the valuable medicinal plants and has important secondary metabolites such as dopamine and noradrenaline. In this study, hairy roots of Purslane were produced by using Agrobacterium rhizogenes strain A4 and different concentrations of macro elements (0.5x, 1x, 1.5x, 2x and 2.5x concentrations of MS base medium) were investigated. For confirming hairy roots transformation, the presence of the rolB gene in the hairy roots was performed by PCR and using rolB genechr('39')s specific primers. The results show that maximum hairy root induction was observed by using 0.5x KNO3 (80 percentage). The highest phenolic content was obtained by using 0.5x KNO3 (4.52 mg/g DW) and also maximum flavonoid content, (18.2 and 17.02 mg/g DW respectively) was belonged to 0.5x and 1x KNO3. Molecular confirmation of transgenic hairy roots was done with PCR reaction.

Thyme (Thymus vulgaris L.) is xerophytes plant that can tolerat against drought and water loss stress easily. Most important secondary metabolites in Thyme are Thymol that have medicinal, antioxidant and antimicrobial properties. No research has carried out so far on the effects of cold stress on the synthesis of Thymol pathway genes and on the physiological and biochemical characteristics. This study was designed to investigate the effect of low temperature stress (4 °C) on the expression Thymol pathway genes such as TPS2 and TPS5 genes using RT-PCR in the time period of 0, 3, 12, 24 and 48 hours after treatment. To evaluate the levels of relative expression of these genes for the synthesis of Thymol (MVA, MEP) t-test was used. TPS2 gene has highest expression within the 3 hours after treatment (3.46 times) compared to the control.

Soybean (Glycine Max (L.) Merr), like those maize, wheat, rice and cotton crops, is an important agricultural crop traded in the global commodity markets. the production of soybean has been curtailed by numerous harsh environmental stresses, especially drought, a water deficit state of soil. The isotopic dimethyl labeling was applied to study the proteomic changes associated with the drought responses of two contrasting soybean cultivars. A total of 874 non-redundant and repeatable peptides were identified among 17787 peptides in tolerant cultivar (these peptides changed significantly to compare with sensitive cultivar) by using MS/MS analysis which corresponding to 260 of protein groups. Among these 260 protein groups, there were 142 groups being up-regulated in the drought-tolerant cultivar under drought treatment and in contrast, there were 118 groups being down-regulated by drought-tolerant cultivar. InterPro protein domain enrichment and Gene Ontology were performed for functional analysis. According to the results most of these proteins are related to photosynthesis activity, energy metabolism, ROS-scavenging activity, channel and transporter activity, gene expression and translation regulation, Carbohydrate derivative metabolic process, Organic acid and substance metabolic process and sugar alcohols catabolic process.

In this paper, a new method for the problem of predicting the compound molecule properties in the lead optimization step in drug design is presented. In the lead optimization step, the amount of available biological data on small molecule compounds is low. In recent years, this challenge has been considered and transfer learning and deep learning techniques have been used to solve it. For this purpose, similar data sets have been used as auxiliary data to learn a reliable model. In this method, compound feature extraction plays an essential role in transferring knowledge from similar (auxiliary) data sets to the target data set. In this paper, the effect of using Edge weighted Graph Convolutional Network (EGCN) is assessed which able to consider the feature vector of the compound bond as well as the atom feature vector. To evaluate the method, we have applied the proposed approach on BACE and HIV datasets. The obtained results show that the proposed method is able to extract more efficient knowledge from similar data sets to transfer to the target data set.

Protease cascade in insect hemolymph is important in proper response against invading microorganisms and parasitoids. Serine proteases lead to the induction of antimicrobial peptide syntheses and phenoloxidases. In the current study, the effects of the least concentration of the three specific inhibitors of serine proteases including AEBSF.HCl [4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride], TLCK [tosyllysine chloromethyl ketone hydrochloride] and TPCK [tosyl phenylalanyl chloromethyl ketone] were evaluated on the activities of trypsin, chymotrypsin, elastase and phenoloxidase of Cydalima perspectalis larvae. The 5 µM concentration of AEBSF.HCl lead to the significant decrease of trypsin, chymotrypsin and phenoloxidase activities at the time intervals of 1, 15, 30- and 60-min post-injection while the activities reached to the value of control after 120 and 180 min. The inhibitor showed no significant effect on elastase activity. The 5 µM concentration of TLCK also decreased the activity of trypsin at the time intervals of 1, 15, 30- and 60-min while the activity of phenoloxidase decreased at all-time intervals except for 180 min post-injection which the least value was obtain after 15 min of injection. TPCK decreased the activity of chymotrypsin at all-time intervals following injection except for 180 min in which the least activity was recorded after 1 min. This inhibitor decreased the activity of phenoloxidase at the time intervals of 1, 15 and 30 min after injection while no significant differences were observed at other time intervals. These results demonstrate the important role of serine proteases in induction of phenoloxidase within C.perspectalis larvae. Inhibitors used in this study can prevent effective immunity of insects by stopping the serine protease process in insect hemolymph and increase the sensitivity of larvae against pathogen. In other words, the use of these compounds improves the effective control of pests through microbial control.

Increasing the world population and subsequently increasing demand for food has led to the use of new biotechnology methods to produce food. Transgenic plants are one of the major biotechnological achievements in agriculture that have gained part of the world food market in recent years and the cultivation area of these plants is constantly increasing. Human needs to increase agricultural production and food security, the use of biotechnology has made it inevitable and on the other hand, the potential dangers of producing these products have raised concerns for human health and consumer rights. One of the concerns expressed by people is transferring unknown genes to plants from other countries and importing these products into the country without using GMO label, that these products may have an impact on human health and other living organisms. Therefore, first step is identifying and labeling transgenic products. In this study, 30 samples of domestic and foreign rice were randomly purchased from Shiraz market. DNA was extracted using the standard CTAB method and the polymerase chain reaction for SPS gene was performed to prove the presence of rice genome. The PCR for EPSPS gene, 35S promoter and NOS terminator were performed to identify transgenic rice. The results of PCR using specific primers showed that none of the rice samples purchased from Shiraz market were transgenic.

Because of the small size of viroids (~246- 436 nucleotides), lack of protein-coding capacity, and causing some sever diseases, the virulence mechanism of viroids in host is being one of the interesting aspects of their study. Direct and indirect interaction between viroids and host factors in various studies has been proved; resulting to change in proteome and host transcripts and finally disease in plants. Latest presented model for these changes is the RNA silencing mechanism that has been proved for both viroid families. This review highlights the latest finding on pathogenicity mechanisms of viroids.

Agrobacterium-mediated transformation is one of the most effective methods for producing the transgenic soybean plants. The efficiency of soybean transformation with Agrobacterium depends on the various factors. The aim of this study was investigating the possibility of human interferon gamma production in soybean. For this purpose, some effective factors in soybean transformation such as Agrobacterium concentration (OD600nm = 0.2, 0.4, 0.6, 0.8 and 1), inoculation time (10, 20, 40 and 60 minutes), co-cultivation time (1, 2, 3, 4 and 5 days) and different levels of acetosyringone (0, 50, 100, 150, 200 and 250 μM) were evaluated. The results showed that the highest percentage of transformation was observed in the OD600nm= 0.8 and the lowest percentage of transformation was obtained at the OD600nm= 0.2. Also, the highest percentage of transformation was obtained in the 40 minutes of inoculation and 4 days of co-cultivation and the lowest percentage of transformation was observed in the 10 minutes of inoculation and 1 day of co-cultivation. The presence of acetosyringone had the increasing effect on the soybean transformation efficiency, and the highest percentage of transformation was obtained at the 150 until 200 μM of acetosyringone. Finally, the presence of human interferon gamma gene in the probable transgenic plants was confirmed by the PCR technique and specific primers. The optimal conditions that obtained in this study can be used to transfer target genes to this soybean cultivar.

Accumulation of unfolded and mis-folded proteins cause ER stress. In response to ER stress, the unfolded protein response (UPR) is triggered by ER transmembrane sensor. UPR, a set of integrated signaling pathways, designed to restore ER homeostasis.  Two bZIP transcriptional factors regulate the UPR pathway in Marchantia Polymorpha: bZIP14 and bZIP7. In this study, bzip14 mutant lines were generated using CRISPR/Cas9. In this order, the bZIP14 DNA sequence was identified by Blastp in Marchantia.info, a database website for M.  polymorpha. Exons are implied to find out the proper sgRNAs as candidates in http://crispor.tefor.net/. Among all suggested sgRNAs, three of them were selected as candidates. sgRNAs were introduced into pMpGE013, a specific CRISPR/Cas9 vector in M. polymorpha. After transformation to agrobacteria GWB303+pSOUP, it was transformed into the plants. DNA was extracted from plants regenerated in selective media containing hygromycin, amplified using PCR by specific primers, and sent to sequencing. Eight out of 11 lines that were sequenced properly showed deletion and insertion in the DNA sequence. One mutant line was chosen for phenotypic assay and a survival test in the media containing tunicamycin, ER stress inducer. Our result showed that there are two isoforms of bZIP17 and bZIP28 in Arabidopsis thaliana, as homologs with bZIP14 in M. polymorpha. Also, a significant difference (p-value ≤ 0.05) was observed between bzip14 mutant and wild type under ER stress conditions.

Thymus daenensis Celak. is a perennial plant belonging to the family Lamiaceae that contains various monoterpenes such as thymol and carvacrol. In order to investigate the effect of salinity stress on growth, the content of thymol and carvacrol compounds and the expression of genes involved in the biosynthetic pathway of these compounds, an experiment was conducted in the research greenhouse in the form of a randomized complete block design with three replications. The results showed that salinity stress had a significant effect on the studied traits at a 1% probability level. Salinity stress at the level of 150 mM NaCl caused a significant reduction in plant height, fresh and dry weight and increasing of total phenol, flavonoids contents, PAL enzyme activity. The content of thymol and carvacrol in salinity stress significantly increased. Study of the genes expression involved in the biosynthesis of these compounds indicated that the genes expression of DXR, terpenes synthase, α-terpinene synthase and ү-terpinene synthase increased in comparison to control. These increments of increase in gene expression of DXR compared with other studied genes were much higher. Therefore, it can be concluded that the increase of secondary metabolites in Thymus daenensis, thymol and carvacrol, in salinity stress conditions due to increased expression of DXR key gene. It is expected to increase these metabolites in the future with its engineering.

Tissue culture is one of the most critical steps in genetic engineering and other breeding programs of plants. In this study, the effect of some factors in regeneration of four rice cultivars, including: Hashemi, Kazemi, Tarom and Shafagh has been studied. In the first experiment the effect of 2,4-D at 4 levels (1.5, 2, 2.5 and 3 mg/l) combination with Kin at three levels (0, 0.4 and 0.8 mg/l) on callus induction and plant regeneration in rice cultivars were studied. After two months calli were transferred to MS medium supplemented with 30g sucrose containing 2.5 mg/l BAP. Analysis of variance showed that Shafagh and Kazemi give the highest and lowest callus respectively. The best callus was obtained in medium supplemented with 0.8 mg/l Kin and 2.5 mg/l of 2,4-D. Analysis of variance showed that plant growth regulators used in callus induction medium, significantly affected the regeneration of plants. Cultivar had a significant effect on the regeneration rate. The highest plant regeneration and number of shoots per explant were obtained from callus derived from medium containing 0.8 mg/l Kin and 1.5 mg/l of 2,4-D. The Kazemi gave the highest percentage (74 %) and Tarom gave the lowest percentage (10%). Among the three strains used for transformation of rice, transgenic callus was obtained only with strain EHA105.