GENETIC ENGINEERING AND BIOSAFETY JOURNAL
Year:2019 | Volume:7 | Issue:2 |Papers Count:13

LEC2 as a transcriptional factor attaches to seed-specific promoters and induces the expression of downstream genes. Analysis of permanent gene transfer in plants is time-consuming and has long process. Using this transcription factor along with the agroinfiltration method can reduce significantly the time of seed specific promoter evaluation. In this research, seed specificity of FAD2-1 promoter, isolated from safflower plants, was studied by GUS expression analysis in tobacco leaves. Fad2-1 promoter sequence analysis indicated that this promoter is containing essential elements for seed-specific function. For analyzing FAD2-1 promoter function, we constructed two gene cassettes: pFAD2-GUS (GUS gene under the control of FAD2-1 promoter) and pBI-LEC2 (LEC2 gene under the control of CaMV35S promoter). These cassettes were cloned in pBI121 vector and transferred to Agrobacteriun tumefacience EHA105, separately. For seed-specific function analysis of FAD2-1 promoter in tobacco leaves, the overnight culture of Agrobacteriums were adjusted to OD600=0. 6, mixed equally and then used to infiltrate into tobacco leaves. GUS assay analysis of infiltrated leaves indicated the specific expression of the reporter gene (GUS). Therefore, FAD2-1 promoter is suggested as a seed specific promoter. Our results suggest that LEC2 along with agroinfiltration method can use as a rapid and confident tool for verifying seed specific expression of any seed-specific promoters.

Light is an important source of energy for the photosynthetic organisms. Photoreceptor proteins are able to perception of the specific wavelengths of light and initiate a signal transduction pathway. Volvox carteri is a simple multicellular green alga with many features that is recommended as a lower eukaryote model organism for studying the development of photoreception. In this research, the effect of UV-B radiation (0. 056 mw. cm-2) was studied on gene expression of 13 photoreceptors using RNA-seq data. These photoreceptors are required for light-monitoring and adaptation of physiological activities to environmental changes. According to our results, under low intensity of UV-B radiation, the photoreceptors were differentially expressed neither in reproductive cells nor in somatic cells as compared to their corresponding control groups. However, comparing the transcriptome of somatic cells with reproductive cells revealed that Phot, CRYp, and ChR1-2, HKR1-4 and Vop (VR1) photoreceptors exhibited a cell-type specific expression pattern while photoreceptors such as UVR8, CRYd1-2 and CRYa were differentially expressed. However, it seems that due to dominantly transcript accumulations of target genes of UV-B response pathway in somatic cells, likely UV-B may indirectly affect gene transcription in this organism. The transcriptional profile pattern between two cell-types, both for photoreceptors and for target genes of UV-B response pathway, shows a different nature and differentiation between reproductive and somatic cells.

The Cel B enzyme from Thermobifidia fusca, a CBHII, belongs to the family of cellulase B, which is highly resistant to heat. Due to the low level of production of this enzyme in this host, it cannot be used at industrial scale. For expression of cellobihydrolase in yeast, cel B gene in pSZ plasmid was amplified by PCR and then was cloned in to pPICZα A vector. Recombinant construct contains cel B gene transformed to E. coli Jm and recombinant were bacteria selected on LB medium with μ g/ml Belleomycin. Recombinant plasmid was linearized and then transformed in Gs and KM H yeast strains by electroporation method. Finally, the best concentration of methanol and the best day of expression of enzyme were determined by DNS method. The results in yeast indicated, the most CBH activity in GS and KM H strains and on PC substrate in C for h was and U ( mol/min)/ml, respectively. Only sample from high yield colony of GS strain for CBH expression was confirmed in SDS-PAGE and then the best methanol concentration and day for enzyme expression with same strain was obtained in th days after induction with three percent methanol. In this study, we did not optimize the codons of cel B gene for expression in P. pastoris. Thus, its decreased expression level and cellobiohydrolase activity may be due to codon usage in Pichia.

Gene expression in bacteria have already been used to study the protein of viruses and to produce specific antibodies. In this research, potato samples were collected from eight provinces of Iran and were tested for Potato virus S, as one of the most destructive viruses of potato fields, by DAS-ELISA and RT-PCR. Dominant isolate of PVS was selected according to coat protein (CP) gene sequences following phylogenetic analysis. Full length CP gene was amplified, cloned and sequenced, then was digested from pJET1. 2/blunt vector, ligated into the expression vector pET-28a and the construct (pET-28a: PVS: CP) was transformed into Escherichia coli BL21 strain. Gene expression was optimized by induction with 0. 5, 1 and 2 mM final concentrations of IPTG for 3, 4 and 6 h and was verified by SDS-PAGE and Western blotting. Based on the nucleotide analysis, the Iranian isolates of PVS were divided into three groups that one group with 22 members known as the dominant group. The expression of recombinant CP of about 34 kDa was proved by western blotting. Induction by 1 mM IPTG for 4 h proved to be the most efficient method of expression. This is the first report of the expression of PVS CP gene, which is important for the preparation of anti-PVS antibody.

Cucumber Mosaic Virus (CMV) is one of the most important viruses infecting Cucurbitaceae and has a wide host range. However, presence of mixed infections with other viruses increases symptoms and damages of viruses. The present study aimed to investigate the mixed infection of CMV with Potyvirus in plants collected from various plant families including Cucurbitaceae with cucumber and Cucurbit, Solanaceae with tomato, pepper and Solanum nigrum and Malvaceae with Malva. sp and Althea sp. Determining in which hosts Potyviruses are mix infected with CMV and in which plants this infection is negligible, is important. Thus, 300 plant samples suspected to be infected were collected and preserved from above-mentioned families from Northwestern of Iran for laboratory and greenhouse experiments with symptoms of Mosaic, deformity and mottling, shrub dwarfism, yellowing and other symptoms of viral plant diseases including (shoe – string in tomato) etc. from each host, 25 samples related to various isolates were scrutinized. In order to conduct primary research on the presence of CMV, systemic and local host of CMV were applied in greenhouse and a pair of CMV coat protein-specific primers were utilized in RT-PCR. Then, detecting mixed infection with Potyviruses was conducted using RT-PCR with universal primers of Potyvirus genus NIb2F/NIb3 in samples whose infection to CMV was proved. The most of mix infections were observed in tomato and cucurbit and the least of mix infections were observed in Malva, pepper and cucumber.

Diamondback moth (DBM), Plutella xylostella (L. ) is one of the most destructive insect pests of brassicaceous crops in Iran and throughout the world. Chemical pesticides play a key role in managing DBM, however, the adverse effects of chemical insecticides and the significant resistance of the pest to the broad range of synthetic insecticides has led to increased attention to the use of safe and eco-friendly components. Therefore, in this research we examined pathogenicity of two Metarhizium anisopliae isolates (AM111 & AH112) and synthesized Metarhizium anisopliae AM111@Fe3O4 against the 3rd instar larva of diamondback moth under laboratory conditions. Toxicity comparisons of nanoformulated fungus and non-formulated one demonstrated a significant decrease in the LC50 value of Metarhizium anisopliae AM111@Fe3O4 estimated as 2. 5 × 104 conidia/ ml that exhibited nano-formulated fungus was more efficient against the pest. Mean comparisons of pure and formulated fungus repellencies showed that nano-fungus significantly was more repellent against the third instar larvae of DBM, so mean repellency values of nonformulated fungus and nano-fungus on 3rd instar larvae of DBM were equivalent to 69 and 74. 66%, respectively. Also, in enzymatic study, protease Pr1 activity comparisons in optimal pH (=11) exhibited significant difference between control and fungal treatment and the most level of Pr1 activity was observed in fungal treatment. Pr1 activity levels in nanofungus and non-formulated fungus treatments were equivalent to 2. 994 and 0. 051 mg/ml, respectively. Our results showed that nano-fungus was more effective on P. xylostella relative to pure-fungus and Metarhizium anisopliae AM111@Fe3O4 can be used as an efficient new component in integrated P. xylostella management.

Ocimum basilicum L. (2n=48) is a medicinal plant that belongs to Lamiaceae family. Eugenol is one of the most important components of basil essential oil which is utilized as an anti-bacterial and anti-fungal material in various industries. Phenylpropanoids such as methyl eugenol possess anti-oxidant properties against free radicals and act as protective compounds in plant responses to the biotic and abiotic stresses. Methyl eugenol O-methyl transferase (EOMTs), as a key enzyme of phenylpropanoids biosynthesis pathway, converts eugenol into methyl eugenol by methylation. In this study, we isolated EOMTs gene promoter from the basil plant and cloned in pTG19-T vector for sequencing. Then, this sequence was characterized and analyzed by bioinformatics softwares. We aimed to investigate the possible gene expression regulation of EOMTs gene under different environmental conditions and recognition different regulatory motifs of its promoter. Analysis of EOMTs gene promoter (1209 bp) indicated that this sequence included some important regulatory elements which could respond to high temperature and drought stress such as MYB, MYC, HSE, and WRKY. Transient expression of the GUS gene under ObEOMTs promoter control in Nicotiana tabacum leaves showed that this promoter including core elements was able to direct the basal expression of this gene. It is expected that this promoter will be capable of changing the expression levels of EOMTs gene in reaction to different environmental stresses because there are stress-responsive regulatory sites in ObEOMTs gene promoter.

The ability of endophyte bacteria Pseudomonas flourescens FY32 in growth improving and increasing the resistance of rapeseed to salinity stress, through proteomic approach was investigated. This research was arranged in a factorial experiment on the basis of completely randomized design with four replications in hydroponic culture system. First factor was inoculation with bacteria and non-inoculation and the second factor was salinity stress in two levels (0 and 300 NaCl mM). The results showed that the effects of salinity stress, bacteria inoculation and canola cultivars were significant for dry weight, shoot and root (P< 0. 01). The highest weight was related to the control and the lowest to severe stress. Inoculated plants had also the highest dry weight comparing to non-inoculated plants that indicates the positive effect of bacterial inoculation on canola in reducing the negative effects of salinity stress. Protein pattern of leaf tissue was determined by 2 dimensional electrophoresis. After scanning, second dimensional gels were analyzed through PD-quest software, then quality and quantity of protein expression in different treatment was compared. A total of 15 spots were significantly changed. Among them, four spots were related to oxidative stress, four spots were involved in metabolism pathway and seven spots were related to photosynthesis. Based on the comparison between bacteria and control gels under both stressed and non-stressed environments, the bacteria increased the tolerance of the plant under salinity stress.

In order to investigate the haplotype and allelic diversity of 22 rice (Oryza sativa L. ) genotypes, a factorial experiment was conducted based on RCBD with three replications under normal (no stress) and drought stress (-5 bar) conditions at seedling stage. The 16 primer pairs of SSR markers related to drought tolerance were used for genotyping. The studied traits were included root diameter, root dry weight, root number, root length, stem dry weight, stem length, genotypic score and total biomass. Analysis of variance for studied traits showed that there was a significant genetic variation among genotypes. 16 used SSR loci produced 49 alleles. The number of alleles per locus generated by each primer pair varied from 2 to 6 alleles with an average of 3. The polymorphic information content (PIC) values ranged from 0. 207 to 0. 678 with an average of 0. 462. RM8030 and RM3302 microsatellite markers were useful for discriminating between tolerant and susceptible genotypes and therefore may be useful for marker-assisted selection. 15 haplotypes were identified among these genotypes using Bala haplotype as reference for haplotype diversity. Genotypes of haplotype group 1 including Salari, CT6516-24-3-2, IR77298-14-1-2 and IR50 genotypes can be useful for breeding programs to drought tolerance.

Gene transfer technology is based on the use of animals and birds as bioreactors in the production of high-quality pharmaceutical, industrial and recombinant proteins, as well as the use of animal models for specific diseases and the investigation of the effects of therapeutic genes in them. Different methods have been developed for gene transfer in eukaryotic cells, including physical, chemical and viral methods. Technological advances and the ever-growing knowledge of molecular virology and virus-host cell relationships have improved the safety of viral vectors that are now used to study cellular gene function, to correct genetic defects (gene therapy), to express therapeutic proteins, to vaccinate against infectious agents and tumors, to produce experimental animal models, and for other purposes. By increasing the researchers' knowledge about the life cycle of viruses and due to their natural ability of viruses to transmit and integrate into the host genome, viruses have become one of the most powerful tools for transferring the gene. Viral vectors have been used in various forms for gene transfer and the production of transgenic animals, including the direct injection of recombinant viruses into the target tissue or the treatment of the stem cells with the recombinant virus and the transfer of recombinant cells to the target tissue or treatment of embryonic cells in the early stages of the fetus. In this study, we attempt to refer some of the most methods for gene transfer using viral vectors, their advantages and limitations in gene transfer.

A major part of the gene-expression products has composed by non-coding protein-binding ribonucleotide sequences including short and long RNA molecules ranged in ten to hundreds which more than 200 nucleotides are called “ long non-coding protein RNA, or lncRNA” . LncRNAs act important roles in some cellular processes by their special molecular structure. Purposes of this study were to evaluate the function of regulator LncRNAs as a major component in intracellular processes and regulating gene expression at different epigenetic levels, transcription, and post-transcription. We also aimed to evaluate the function of LncRNAs in development of the central nervous system, increase or decrease of their expression in some disease like cancer and their roles in some animal diseases. Many modern technologies and advanced bioinformatics methods have been developed to identify LncRNA molecules. Here, we review the current state of knowledge of the lncRNA field, discussing what is known about the genomic contexts, biological functions, and mechanisms of action of lncRNAs. Although there are several online databases to facilitate researches and share scientific information related to these molecules. Therefore, it is expected that studying on this particular type of RNAs to find out their functional and regulatory roles is increasing; and there is still a great deal of space for developing researches in human and animal diseases. There are hopes that more perspectives to find out therapies for many types of diseases, especially those with no definitive treatment. In addition, we wish to obtain significant advances in genetics and livestock breeding by finding new functional and regulatory roles of LncRNAs in animal productive traits.

Despite the increasing development of genetic engineering technology and the use of its products (transgenic products) around the world, in some areas, including Iran, the production of transgenic products continues to be challenged. Despite the scholarly explanations of the problem by the experts and the issuance of a solid fatwa by the high Shiite jurisprudents, the void that is clearly seen is the lack of precise explanation of the jurisprudential ruling on the production of these products on the basis of religious evidence. However, due to the central role of Sharia in determining the laws of society, this issue is of particular importance. Therefore, the present research with a descriptive-analytic approach is an attempt to study the jurisprudential ruling on the production of transgenic products based on Imamieh jurisprudence. In this regard, due to the necessity of subjectology, first, experts' viewpoints regarding the transboundary issue were examined and then the jurisprudential section and the jurisprudence of the subject were distinguished. The findings indicate that the health of these products is accepted globally and it is certain that its rejection in some societies, including the Iranian circles, is not based on scientific evidence. Because its certainty has not been proven in the department of jurisprudence, given the lack of valid reason for transgenic humor, its initial verdict is Permission. Nevertheless, given the climate in Iran and the challenges, the use of biotechnology and the production of genetically modified organisms to prevent the domination of foreigners and importations is imperative to be obligatory from the introduction of the obligatory.

Plant parasitic nematodes are amongst the most economically important groups of pathogens. The use of resistant cultivar, crop rotation, chemical control, antagonistic organisms and biocontrol agents are the principal methods for management of the nematodes. Natural nematode resistance genes present in gene pools of crop species and their relatives have been used with the aim of transferring such traits into economically important plants where effective resistance is lacking. Biotechnology contributes to this process via marker-assisted selection to identify the best nematode resistance genes, and increasingly in providing new knowledge of target genes, and the potential to exploit this knowledge using transgenic technology. Thus recent advances make it possible to exploit specific aspects of nematode-host plant interactions to design control strategies that include enabling plants to prevent nematode invasion, migration through tissues and reducing feeding ability or nematode fecundity. Application of RNAi, new biotechnology-based chemical nematicides and some other methods are amongst the modern strategies of control. New traits would be added to existing crop genotypes with the best conventional or natural nematode resistance to increase the effectiveness and durability of the nematode resistance trait. Biotech trait expression could also be limited to roots to minimize expression in harvested parts.