Use Of Genetic Engineering Research Articles

The competence system of Streptococcus anginosus and its use for genetic engineering.

Streptococcus anginosus is considered a human commensal but improvements in species identification in recent years have highlighted its role as an emerging pathogen. However, our knowledge about the pathogenicity mechanisms in this species is scarce. One reason for this is the lack of published genetic manipulation techniques in the S.anginosus group. To establish a novel mutation technique we investigated the competence system of S.anginosus and created a Cre-recombinase-based mutation method that allows the generation of markerless gene deletions in S.anginosus. Insilico analysis of the competence system demonstrated that S.anginosus encodes homologues for the vast majority of genes that are known to be essential for the transformation of S.pneumoniae. Analysis of transformation kinetics confirmed that S.anginosus SK52 possesses an S.pneumoniae-like competence development with a rapid increase of competence after treatment with Competence Stimulating Peptide (CSP), reaching a maximum transformation efficiency of 0.24%±0.08%. The combination of CSP-induced transformation and the Cre-lox system allows the efficient and fast creation of markerless gene deletions and will facilitate the investigation of the pathogenicity of S.anginosus on a genetic level.

The current status and development of insect-resistant genetically engineered poplar in China

Poplar is one of the main afforestation tree species in China, and the use of a single, or only a few, clones with low genetic diversity in poplar plantations has led to increasing problems with insect pests. The use of genetic engineering to cultivate insect-resistant poplar varieties has become a hot topic. Over the past 20 years, there have been remarkable achievements in this area. To date, nearly 22 insect-resistant poplar varieties have been created and approved for small-scale field testing, environmental release, or pilot-scale production. Here, we comprehensively review the development of insect-resistant genetically modified (GM) poplars in China. This review mostly addresses issues surrounding the regulation and commercialization of Bt poplar in China, the various insecticidal genes used, the effects of transgenic poplars on insects, toxic protein expression, multigene transformation, the stability of insect resistance, and biosafety. The efficacy of GM poplars for pest control differed among different transgenic poplar clones, larval instars, and insect species. The Bt protein analysis revealed that the expression level of Cry3A was significantly higher than that of Cry1Ac. Temporal and spatial studies of Bt protein showed that its expression varied with the developmental stage and tissue. The inheritance and expression of the exogenous gene were reviewed in transgenic hybrid poplar progeny lines and grafted sections. Biosafety issues, in terms of transgene stability and the effects on soil microorganisms, natural enemies of insects, and arthropod communities are also discussed.

Floridians' Perceptions of GMOs: GMOs and Florida Citrus

This is a minor revision of an EDIS publication focusing on Floridians’ intent to purchase GMO citrus. It is intended to help Extension faculty understand public perceptions regarding the use of genetic engineering to combat citrus greening. Extension faculty can use this understanding of public perceptions as they develop and deliver programming for clientele. This 4-page fact sheet was written by Nicole M. W. Dodds, Laura M. Gorham, and Joy N. Rumble, and published by the Department of Agricultural Education and Communication, November 2017. AEC520/WC182: Floridians' Perceptions of GMOs: GMOs and Florida Citrus (ufl.edu)

Genetics and evolution of MIXTA genes regulating cotton lint fiber development.

Cotton, with cellulose-enriched mature fibers, is the largest source of natural textiles. Through a map-based cloning strategy, we isolated an industrially important lint fiber development gene (Li3 ) that encodes an MYB-MIXTA-like transcription factor (MML) on chromosome D12 (GhMML4_D12). Virus-induced gene silencing or decreasing the expression of the GhMML4_D12 gene in n2 NSM plants resulted in a significant reduction in epidermal cell prominence and lint fiber production. GhMML4_D12 is arranged in tandem with GhMML3, another MIXTA gene responsible for fuzz fiber development. These two very closely related MIXTA genes direct fiber initiation production in two specialized cell forms: lint and fuzz fibers. They may control the same metabolic pathways in different cell types. The MIXTAs expanded in Malvaceae during their evolution and produced a Malvaceae-specific family that regulates epidermal cell differentiation, different from the gene family that regulates leaf hair trichome development. Cotton has developed a unique transcriptional regulatory network for fiber development. Characterization of target genes regulating fiber production has provided insights into the molecular mechanisms underlying cotton fiber development and has allowed the use of genetic engineering to increase lint yield by inducing more epidermal cells to develop into lint rather than fuzz fibers.

Volatile terpenoids: multiple functions, biosynthesis, modulation and manipulation by genetic engineering.

Terpenoids play several physiological and ecological functions in plant life through direct and indirect plant defenses and also in human society because of their enormous applications in the pharmaceutical, food and cosmetics industries. Through the aid of genetic engineering its role can by magnified to broad spectrum by improving genetic ability of crop plants, enhancing the aroma quality of fruits and flowers and the production of pharmaceutical terpenoids contents in medicinal plants. Terpenoids are structurally diverse and the most abundant plant secondary metabolites, playing an important role in plant life through direct and indirect plant defenses, by attracting pollinators and through different interactions between the plants and their environment. Terpenoids are also significant because of their enormous applications in the pharmaceutical, food and cosmetics industries. Due to their broad distribution and functional versatility, efforts are being made to decode the biosynthetic pathways and comprehend the regulatory mechanisms of terpenoids. This review summarizes the recent advances in biosynthetic pathways, including the spatiotemporal, transcriptional and post-transcriptional regulatory mechanisms. Moreover, we discuss the multiple functions of the terpene synthase genes (TPS), their interaction with the surrounding environment and the use of genetic engineering for terpenoid production in model plants. Here, we also provide an overview of the significance of terpenoid metabolic engineering in crop protection, plant reproduction and plant metabolic engineering approaches for pharmaceutical terpenoids production and future scenarios in agriculture, which call for sustainable production platforms by improving different plant traits.

Meta-analysis of the effect of overexpression of CBF/DREB family genes on drought stress response

Transcription factors C-repeat/dehydration-responsive element binding proteins (CBF/DREB) play an important role in plant response to abiotic stresses. Over-expression of various CBF/DREB genes in diverse plants have been reported, but inconsistency of gene donor, recipient genus, parameters used in evaluating the transgenic plants have made it difficult to elucidate the complex physiological mechanisms by which DREBs impact drought tolerance. We performed a meta-analysis to categorize the response of plant parameters involved in drought tolerance in DREB-overexpressed plants and to evaluate experimental variables that affects transgenic plant performance. The results from across different studies indicated that 8 and 2 of 13 measured parameters exhibited a significant response by increasing or decreasing values by 25% or more in drought-stressed and in non-stressed, control plants, respectively. Among the examined moderating variables, promoters, type of media and stress, donor genus, and recipient genus significantly affected the extent of DREBs' influence. In plants subjected to drought stress, the overexpression of DREB genes derived from Arabidopsis led to greater increases in Fv/Fm (maximum quantum efficiency of photosystem II), relative water content, and proline content. These findings should guide future research on the function of the DREB genes in plant drought tolerance and the use of genetic engineering in drought tolerance breeding.

Transgenic Strategies for Enhancement of Nematode Resistance in Plants.

Plant parasitic nematodes (PPNs) are obligate biotrophic parasites causing serious damage and reduction in crop yields. Several economically important genera parasitize various crop plants. The root-knot, root lesion, and cyst nematodes are the three most economically damaging genera of PPNs on crops within the family Heteroderidae. It is very important to devise various management strategies against PPNs in economically important crop plants. Genetic engineering has proven a promising tool for the development of biotic and abiotic stress tolerance in crop plants. Additionally, the genetic engineering leading to transgenic plants harboring nematode resistance genes has demonstrated its significance in the field of plant nematology. Here, we have discussed the use of genetic engineering for the development of nematode resistance in plants. This review article also provides a detailed account of transgenic strategies for the resistance against PPNs. The strategies include natural resistance genes, cloning of proteinase inhibitor coding genes, anti-nematodal proteins and use of RNA interference to suppress nematode effectors. Furthermore, the manipulation of expression levels of genes induced and suppressed by nematodes has also been suggested as an innovative approach for inducing nematode resistance in plants. The information in this article will provide an array of possibilities to engineer resistance against PPNs in different crop plants.

The Absence of the Transcription Factor Yrr1p, Identified from Comparative Genome Profiling, Increased Vanillin Tolerance Due to Enhancements of ABC Transporters Expressing, rRNA Processing and Ribosome Biogenesis in Saccharomyces cerevisiae.

Enhancing the tolerance of Saccharomyces cerevisiae to inhibitors derived from lignocellulose is conducive to producing biofuel and chemicals using abundant lignocellulosic materials. Vanillin is a major type of phenolic inhibitor in lignocellulose hydrolysates for S. cerevisiae. In the present work, the factors beneficial to vanillin resistance in yeast were identified from the vanillin-resistant strain EMV-8, which was derived from strain NAN-27 by adaptive evolution. We found 450 SNPs and 44 genes with InDels in the vanillin-tolerant strain EMV-8 by comparing the genome sequences of EMV-8 and NAN-27. To investigate the effects of InDels, InDels were deleted in BY4741, respectively. We demonstrated that the deletion of YRR1 improved vanillin tolerance of strain. In the presence of 6 mM vanillin, deleting YRR1 increase the maximum specific growth rate and the vanillin consumption rate by 142 and 51%, respectively. The subsequent transcriptome analysis revealed that deleting YRR1 resulted in changed expression of over 200 genes in the presence of 5 mM vanillin. The most marked changes were the significant up-regulation of the dehydrogenase ADH7, several ATP-binding cassette (ABC) transporters, and dozens of genes involved in ribosome biogenesis and rRNA processing. Coincidently, the crude enzyme solution of BY4741(yrr1Δ) exhibited higher NADPH-dependent vanillin reduction activity than control. In addition, overexpressing the ABC transporter genes PDR5, YOR1, and SNQ2, as well as the RNA helicase gene DBP2, increased the vanillin tolerance of strain. Interestingly, unlike the marked changes we mentioned above, under vanillin-free conditions, there are only limited transcriptional differences between wildtype and yrr1Δ. This indicated that vanillin might act as an effector in Yrr1p-related regulatory processes. The new findings of the relationship between YRR1 and vanillin tolerance, as well as the contribution of rRNA processing and ribosome biogenesis to enhancing S. cerevisiae vanillin tolerance, provide novel targets for genetic engineering manipulation to improve microbes’ tolerance to lignocellulose hydrolysate.

Allergenicity Assessment of Transgenic Wheat Lines In Silico.

Agriculture biotechnology is a promising tool for developing varieties with enhanced quality and quantity. Transgenic proteins expressed by genetically modified (GM) food crops improve crop characteristics like nutritional value, taste, and texture, and endow plants with resistance against fungus, pests, and insects. Despite such potential benefits, there are concerns regarding possible adverse effects of GM crops on human health, animals and the environment. Among the proposed guidelines for GM food safety testing-the weight-of-evidence approach proposed by the Codex Alimentarius Commission (ALINORM 03/34A) is the most recent. Till date, several transgenic wheat lines have been developed and research is underway for further improvement. However, GM wheat is not being grown or consumed in any part of the world. In the present study, in silico tools were employed for safety testing of eight transgenes used for the development of transgenic wheat lines. Among the genes studied, none of them shared sequence homology with the reported allergens and may be safe for use in genetic engineering. In conclusion, gene selection for developing transgenic wheat lines should be done with utmost care to ensure its safety for feed and fodder.

Recent trends in bioethanol production from food processing byproducts.

The widespread use of corn starch and sugarcane as sources of sugar for the production of ethanol via fermentation may negatively impact the use of farmland for production of food. Thus, alternative sources of fermentable sugars, particularly from lignocellulosic sources, have been extensively investigated. Another source of fermentable sugars with substantial potential for ethanol production is the waste from the food growing and processing industry. Reviewed here is the use of waste from potato processing, molasses from processing of sugar beets into sugar, whey from cheese production, byproducts of rice and coffee bean processing, and other food processing wastes as sugar sources for fermentation to ethanol. Specific topics discussed include the organisms used for fermentation, strategies, such as co-culturing and cell immobilization, used to improve the fermentation process, and the use of genetic engineering to improve the performance of ethanol producing fermenters.

Plant Salt Stress: Adaptive Responses, Tolerance Mechanism and Bioengineering for Salt Tolerance

Salinity is an important abiotic environmental stress factor threatening agricultural productivity throughout the world. The detrimental effects of salinity stress are observed at cellular, organ and whole plant level at osmotic phase (early/short-term response) and ionic phase (late/long-term response). High salinity exerts its negative impact on major plant processes such as disrupting the osmotic and ionic equilibrium, protein synthesis, photosynthesis, energy, and lipid metabolism. To adapt and tolerate salt stress, plants have evolved physiological and biochemical mechanisms orchestrated by multiple biochemical pathways of ion homeostasis, osmolytes synthesis, ROS scavenging, and hormonal balance. At the molecular level, such adaptation involves activation of cascade(s) of gene modulations and synthesis of defense metabolites. In recent years, several candidate genes have been identified and employed to facilitate genetic engineering efforts to improve salt tolerance in crop plants. However, there is a further need of improvement for successful release of salt tolerant cultivars at the field level. In this article we present the physiological, biochemical and molecular signatures of plant responses to salinity, and outline their use in genetic engineering to improve salt stress tolerance.

КОНТРОЛЬ ГЕНЕТИЧНО МОДИФІКОВАНИХ РОСЛИН – ГАРАНТІЯ ПРОДОВОЛЬЧОЇ БЕЗПЕКИ

One of the problems in formation of mechanisms of development of environmentally safe environment is the proliferation of genetically modified organisms (GMOs). The use of genetic engineering in agriculture, food and agricultural industry have created the new opportunities for manufacturing the food and feed for meet the ever growing needs of the world's population. However, the opinions of scientists about the benefits and risks of genetic engineering is different. So today is particularly acute issue unpredictable consequences and danger for human health and the environment which are connected with GMO.The article present the results of determination of genetically modified organisms in plant material for the period 2013 – 2015 years, from the farms of different ownership forms. Analysis of the conducted research indicates on the circulation in farms of Lviv region transgenic plants.Found that the most common genetically modified plants were maize, soya and rape. The research of plant material on the presence of GMOs was performed by polymerase chain reaction in real time (PCR RF) by setting in them target sequences of promoter 35S virus of cauliflower mosaic (CaMV), promoter of FMV and (or) terminator NOS (T–NOS) T1 plasmid of Agrobacterium tumefaciens, Pat, EPSPS, Cry 3A, and determined species identification of positive samples. The use of PCR–RF allows quickly and qualitatively detect the GMO in the research samples. In 2013, the percentage of positive samples was 3.9% of the total, in 2014 – 2.3% and in 2015 – 3.3%. It is therefore necessary to monitor the vegetable raw and seed material on the presence of genetically modified organisms, to monitor the situation on GMO in Ukraine, because the problem of biosafety and the evaluation of the potential risks from their use is not studied.

Increased salt tolerance with overexpression of cation/proton antiporter 1 genes: a meta-analysis.

SummaryCation/proton antiporter 1 (CPA1) genes encode cellular Na+/H+ exchanger proteins, which act to adjust ionic balance. Overexpression of CPA1s can improve plant performance under salt stress. However, the diversified roles of the CPA1 family and the various parameters used in evaluating transgenic plants over‐expressing CPA1s make it challenging to assess the complex functions of CPA1s and their physiological mechanisms in salt tolerance. Using meta‐analysis, we determined how overexpression of CPA1s has influenced several plant characteristics involved in response and resilience to NaCl stress. We also evaluated experimental variables that favour or reduce CPA1 effects in transgenic plants. Viewed across studies, overexpression of CPA1s has increased the magnitude of 10 of the 19 plant characteristics examined, by 25% or more. Among the ten moderating variables, several had substantial impacts on the extent of CPA1 influence: type of culture media, donor and recipient type and genus, and gene family. Genes from monocotyledonous plants stimulated root K+, root K+/Na+, total chlorophyll, total dry weight and root length much more than genes from dicotyledonous species. Genes transformed to or from Arabidopsis have led to smaller CPA1‐induced increases in plant characteristics than genes transferred to or from other genera. Heterogeneous expression of CPA1s led to greater increases in leaf chlorophyll and root length than homologous expression. These findings should help guide future investigations into the function of CPA1s in plant salt tolerance and the use of genetic engineering for breeding of resistance.

Fighting Sharka in Peach: Current Limitations and Future Perspectives.

Sharka, caused by Plum Pox Virus (PPV), is by far the most important infectious disease of peach [P. persica (L.) Batsch] and other Prunus species. The progressive spread of the virus in many important growing areas throughout Europe poses serious issues to the economic sustainability of stone fruit crops, peach in particular. The adoption of internationally agreed-upon rules for diagnostic tests, strain-specific monitoring schemes and spatial–temporal modeling of virus spread, are all essential for a more effective sharka containment. The EU regulations on nursery activity should be modified based on the zone delimitation of PPV presence, limiting open-field production of propagation materials only to virus-free areas. Increasing the efficiency of preventive measures should be augmented by the short-term development of resistant cultivars. Putative sources of resistance/tolerance have been recently identified in peach germplasm, although the majority of novel resistant sources to PPV-M have been found in almond. However, the complexity of introgression from related-species imposes the search for alternative strategies. The use of genetic engineering, particularly RNA interference (RNAi)-based approaches, appears as one of the most promising perspectives to introduce a durable resistance to PPV in peach germplasm, notwithstanding the well-known difficulties of in vitro plant regeneration in this species. In this regard, rootstock transformation to induce RNAi-mediated systemic resistance would avoid the transformation of numerous commercial cultivars, and may alleviate consumer resistance to the use of GM plants.

The Current and Future Uses of Biotechnology in Animal Agriculture

Biotechnologies have been an integral part of improvements in animal genetics, nutrition and health over the past century. Many biotechnologies have become fundamental components of efficient livestock production systems. The genetic improvements that have been enabled by biotechnologies have dramatically decreased the environmental footprint of animal protein production in many parts of the world, and continued innovation is required to address the projected increase in demand for animal products in the future. Breeding programs increasingly utilize a combination of advanced reproductive technologies and genomic tools to accelerate the rate of genetic gain by manipulating components of the breeder’s equation. The use of these biotechnologies and breeding methods has met with little public opposition. In contrast, the use of modern biotechnologies, defined as those that employ the use of in vitro nucleic acid techniques, have been highly controversial, especially those involving the use of genetic engineering. This modern biotechnology distinction is somewhat arbitrary as there are a number of biotechnologies that involve the use of in vitro processes, and many result in genetic modifications that are indistinguishable from the naturally-occurring variation that is the driver of both traditional breeding programs and evolution. A number of useful traits including disease resistance and animal welfare traits have been successfully introduced into various livestock species using both genetic engineering and gene editing techniques. Ultimately these techniques complement the genetic improvement that can be accomplished using traditional selection techniques and, if judged acceptable, offer an opportunity to synergistically accelerate genetic improvement in food animal species.

Applications of genetically encoded photosensitizer miniSOG: from correlative light electron microscopy to immunophotosensitizing

Genetically encoded photosensitizers (PSs), e.g. ROS generating proteins, correspond to a novel class of PSs that are highly desirable for biological and medical applications since they can be used in combination with a variety of genetic engineering manipulations allowing for precise spatio-temporal control of ROS production within living cells and organisms. In contrast to the commonly used chemical PSs, they can be modified using genetic engineering approaches and targeted to particular cellular compartments and cell types. Mini Singlet Oxygen Generator (miniSOG), a small flavoprotein capable of singlet oxygen production upon blue light irradiation, was initially reported as a high contrast probe for correlative light electron microscopy (CLEM) without the need of exogenous ligands, probes or destructive permeabilizing detergents. Further miniSOG was successfully applied for chromophore-assisted light inactivation (CALI) of proteins, as well as for photo-induced cell ablation in tissue cultures and in Caenorhabditis elegans. Finally, a novel approach of immunophotosensitizing has been developed, exploiting the specificity of mini-antibodies or selective scaffold proteins and photo-induced cytotoxicity of miniSOG, which is particularly promising for selective non-invasive photodynamic therapy of cancer (PDT) due to the spatial selectivity and locality of destructive action compared to other methods of oncotherapy.

Напрями, досягнення та перспективи біотехнології у харчовій промисловості

The article provides analytical screening of biotechnological facilities in the food industry. Presents the basic areas of application of biotechnology research and development, described the feasibility and relevance of the use of genetic engineering, specialized strains of microorganisms and enzyme preparations in the production of food products with functional directivity highest level of quality and safety. Are formulated the most important requirements and selection criteria for the biotechnological component for the various areas of the food industry. Summarizes the scientific achievements of biotechnology and genetic engineering, which promote the development of innovation in creating products for a healthy diet, make it possible to realize the potential of of this direction.

Efficient production of cynomolgus monkeys with a toolbox of enhanced assisted reproductive technologies.

The efficiency of assisted reproductive technologies (ARTs) in nonhuman primates is low due to no screening criterions for selecting sperm, oocyte, and embryo as well as its surrogate mothers. Here we analyzed 15 pairs of pregnant and non-pregnant cynomolgus monkeys, each pair of which received embryos from one batch of fertilized oocytes, and found ratio of endometrial to myometrial thicknesses in abdominal ultrasonic transverse section of uterus is a reliable indicator for selection of recipients for embryo transfer. We performed 305 ovarian stimulations in 128 female cynomolgus monkeys and found that ovarian stimulation can be performed in a whole year and repeated up to six times in the same monkey without deteriorating fertilization potential of eggs until a poor response to stimulation happened. Fertilization can be efficiently achieved with both conventional and piezo-driven intracytoplasmic sperm injection procedures. In semen collection, semen quality is higher with the penile robe electrical stimulus method compared with the rectal probe method. Moreover, caesarean section is an effective strategy for increasing baby survival rates of multiple pregnancies. These findings provide a practical guidance for the efficient use of ARTs, facilitating their use in genetic engineering of macaque monkeys for basic and translational neuroscience research.

Contradictions teleology of Legal Policy of the Russian Federation in the field of genetic engineering

The article deals with the analysis of Russian legal policy in the sphere of genetic engineering. Over the past ten years, the risks of genetic engineering in the Russian society are mythologized. At the same time attempts were made to change the model of the legal regulation of genetic engineering legislation to tighten it. They worried the scientific community. At the same time the legislative initiatives to tighten the model of the legal regulation of genetic engineering become frequent. This situation worries the scientific community. The authors explore the goals which the Russian legislator aims in the process of legal regulation of genetic engineering, and relate them to the needs of the development of science. The authors identified two problems in the teleology of the Russian legal policy in the field of genetic engineering. Firstly, the concept of national biosafety has not been created. This concept is necessary for specifying not only negative but also positive risks of genetic engineering, that is, for the purposes of definition the use of genetic engineering. Second, the degree of influence of the scientific community to the social representations about the risks of genetic engineering is weak.

The ways to increase efficiency of soil bioremediation

Abstract The aim of this paper was to present possibilities of using different substrates to assist the bioremediation of soils contaminated with heavy metals, pesticides and other substances. Today's bioengineering offers many solutions that enable the effective conduct of biological remediation, including both biostimulation and bioaugmentation. For this purpose, they are used to enrich various organic substances, sorbents, microbiological and enzymatic preparations, chemical substances of natural origin or nanoparticles. The use of genetic engineering as a tool to obtain microorganisms and plants capable of efficient degradation of pollutants may cause the risks that entails the introduction of transgenic plants and microorganisms into the environment. In order to determine the efficacy and possible effects of the various bioremediation techniques, it is required to conduct many studies and projects on a larger scale than only in the laboratory. Furthermore, it should be emphasized that bioremediation involves interdisciplinary issues and therefore, there is a need to combine knowledge from different disciplines, such as: microbiology, biochemistry, ecology, environmental engineering and process engineering.