Application Of Genetic Engineering Research Articles

Phytoremediation of Mercury Contamination: Bibliometric Analysis

Mercury is classified as one of the world’s most toxic and dangerous pollutants as it tends to bioaccumulate and biomagnify within the trophic chain and is persistent. Various approaches are available to remediate Hg-affected sites including phytoremediation, which includes the use of plants to clean up contaminated environments. The phytoremediation of mercury contamination is attracting increasing attention because of its advantages: it is environmentally friendly, inexpensive, simple, and can improve soil fertility. In this report, VOSviewer (version 1.6.1) and Bibliometrix (version 4.16) software were used to analyze 457 and 697 documents published from 2000 to 2023, retrieved from the databases WoS and Scopus, respectively. China, India, the United States, and Spain were the top four most productive countries. The largest topic area was environmental sciences, and the Chinese Academy of Sciences was the organization that contributed the most to the overall number of publications. The keywords with the highest frequency excluding phytoremediation and mercury in WoS were heavy metals, accumulation, cadmium, soils, and phytoextraction. In Scopus, the most frequent keywords were bioremediation, heavy metals, soil pollution, bioaccumulation, biodegradation, and environmental. From the above analysis, we concluded that future research should focus on (1) finding native plants, (2) genetic engineering applications, (3) increasing remediation ability through assisted phytoremediation, and (4) the detoxification mechanism of mercury. This study provides insights into trending themes and serves as a reference for future research.

Mutations of methionine 444 interacting with T1Cu-coordinating amino acids affect the structure and function of multicopper oxidase CopA.

Manganese is an essential trace element for humans, animals, and plants, but excessive amounts of manganese can cause serious harm to organisms. The biological manganese oxidation process mainly oxidizes Mn(II) through the secretion of unique manganese oxidase by manganese-oxidizing bacteria. The T1 Cu site of multicopper oxidase is the main site for substrate oxidation, and its role is to transfer electrons to TNC, where dioxygen reduction occurs. In this study, methionine (Met) No. 444 interacting with the T1Cu-coordinating amino acid in the multicopper oxidase CopA from Brevibacillus panacihumi MK-8 was mutated to phenylalanine (Phe) and leucine (Leu) by the enzyme. Based on the analysis of enzymatic properties and the structural model, the mutant protein M444F with 4.58 times the catalytic efficiency of the original protein CopA and the mutant protein M444L with 1.67 times the catalytic efficiency of the original protein CopA were obtained. The study showed that the manganese removal rate of the manganese-oxidizing engineered bacterium Rosetta-pET-copAM444L cultured for 7 days was 88.87%, which was 10.77% higher than that of the original engineered bacterium. Overall, this study provides a possibility for the application of genetic engineering in the field of biological manganese removal.

An Efficient CRISPR/Cas Cooperative Shearing Platform for Clinical Diagnostics Applications.

The CRISPR/Cas system is a powerful genome editing tool and possesses widespread applications in molecular diagnostics, therapeutics and genetic engineering. But easy folding of the target sequences causes remarkable deterioration of the recognition and shear efficiency in the case of single Cas-CRISPR RNA (crRNA) duplex. Here, we develop a CRISPR/Cas cooperative shearing (CRISPR-CS) system. Compared with traditional CRISPR/Cas system, two CRISPR/Cas-crRNA duplexes simultaneously recognize different sites in the target sequence, increasing recognition possibility and shearing efficiency. Cooperative shearing cuts more methylene blue-ssDNA reporters on the electrode, enabling unamplified nucleic acid electrochemical assay in less than 5 minutes with a detection limit of 9.5×10-20 M, 2 to 9 orders of magnitude lower than those of other electrochemical assays. The CRISPR-CS platform detects monkeypox, human papilloma virus and amyotrophic lateral sclerosis with an accuracy up to 98.1%, demonstrating the potential application of the efficient cooperative shearing.

An Efficient CRISPR/Cas Cooperative Shearing Platform for Clinical Diagnostics Applications

The CRISPR/Cas system is a powerful genome editing tool and possesses widespread applications in molecular diagnostics, therapeutics and genetic engineering. But easy folding of the target sequences causes remarkable deterioration of the recognition and shear efficiency in the case of single Cas‐CRISPR RNA (crRNA) duplex. Here, we develop a CRISPR/Cas cooperative shearing (CRISPR‐CS) system. Compared with traditional CRISPR/Cas system, two CRISPR/Cas‐crRNA duplexes simultaneously recognize different sites in the target sequence, increasing recognition possibility and shearing efficiency. Cooperative shearing cuts more methylene blue‐ssDNA reporters on the electrode, enabling unamplified nucleic acid electrochemical assay in less than 5 minutes with a detection limit of 9.5×10−20 M, 2 to 9 orders of magnitude lower than those of other electrochemical assays. The CRISPR‐CS platform detects monkeypox, human papilloma virus and amyotrophic lateral sclerosis with an accuracy up to 98.1%, demonstrating the potential application of the efficient cooperative shearing.

Analysis of codon usage patterns in complete plastomes of four medicinal Polygonatum species (Asparagaceae).

Polygonati Rhizoma and Polygonati odorati Rhizoma, known as "Huangjing" and "Yuzhu" in China, are medicinal Polygonatum species resources with top-grade medical and edible properties. The chloroplast (cp) genome has been used to study species diversity, evolution, and breeding of species for applications in genetic engineering. Codon usage bias (CUB), a common and complex natural phenomenon, is essential for studies of codon optimization of exogenous genes, genetic engineering, and molecular evolution. However, the CUB of medicinal Polygonatum species chloroplast genomes has not been systematically studied. In our study, a detailed analysis of CUB was performed in the medicinal Polygonatum species chloroplast genomes. We investigated the codon bias of 204 plastid protein-coding genes (PCGs) in 4 medicinal Polygonatum species using CodonW and CUSP online software. Through the analysis of the codon bias index, we found that the medicinal Polygonatum species chloroplast genomes had weak codon usage bias. In addition, our results also showed a high preference for AT bases in medicinal Polygonatum species chloroplast genomes, and the preference to use AT-ending codons was observed in these species chloroplast genomes. The neutrality plot, ENC plot, PR2-Bias plot, and correspondence analysis showed that compared with mutation pressure, natural selection was the most important factor of CUB. Based on the comparative analysis of high-frequency codons and high expression codons, we also determined the 10-11 optimal codons of investigative medicinal Polygonatum species. Furthermore, the result of RSCU-based cluster analysis showed that the genetic relationship between different medicinal Polygonatum species could be well reflected. This study provided an essential understanding of CUB and evolution in the medicinal Polygonatum species chloroplast genomes.

Microbial degradation of contaminants of emerging concern: metabolic, genetic and omics insights for enhanced bioremediation.

The perpetual release of natural/synthetic pollutants into the environment poses major risks to ecological balance and human health. Amongst these, contaminants of emerging concern (CECs) are characterized by their recent introduction/detection in various niches, thereby causing significant hazards and necessitating their removal. Pharmaceuticals, plasticizers, cyanotoxins and emerging pesticides are major groups of CECs that are highly toxic and found to occur in various compartments of the biosphere. The sources of these compounds can be multipartite including industrial discharge, improper disposal, excretion of unmetabolized residues, eutrophication etc., while their fate and persistence are determined by factors such as physico-chemical properties, environmental conditions, biodegradability and hydrological factors. The resultant exposure of these compounds to microbiota has imposed a selection pressure and resulted in evolution of metabolic pathways for their biotransformation and/or utilization as sole source of carbon and energy. Such microbial degradation phenotype can be exploited to clean-up CECs from the environment, offering a cost-effective and eco-friendly alternative to abiotic methods of removal, thereby mitigating their toxicity. However, efficient bioprocess development for bioremediation strategies requires extensive understanding of individual components such as pathway gene clusters, proteins/enzymes, metabolites and associated regulatory mechanisms. "Omics" and "Meta-omics" techniques aid in providing crucial insights into the complex interactions and functions of these components as well as microbial community, enabling more effective and targeted bioremediation. Aside from natural isolates, metabolic engineering approaches employ the application of genetic engineering to enhance metabolic diversity and degradation rates. The integration of omics data will further aid in developing systemic-level bioremediation and metabolic engineering strategies, thereby optimising the clean-up process. This review describes bacterial catabolic pathways, genetics, and application of omics and metabolic engineering for bioremediation of four major groups of CECs: pharmaceuticals, plasticizers, cyanotoxins, and emerging pesticides.

Identification and functional characterization of bidirectional gene pairs and their intergenic regions in cotton

BackgroundIn research to improve the quality of transgenic crops, it is often necessary to introduce multiple functionally related genes into recipient plants simultaneously to improve crop genetic traits effectively. Compared with unidirectional promoters, bidirectional promoters simultaneously regulate the expression of multiple genes and improve the efficiency of biotechnology. Therefore, in this study, bidirectional gene pairs were systematically analyzed in Gossypium hirsutum TM-1, and the structure, function and evolutionary relationships of the bidirectional genes were analyzed. The endogenous bidirectional promoters of cotton were mined, and their specific regulatory elements and biological functions were explored to provide useful promoter resources and a theoretical basis for cultivating new cotton germplasms with excellent fiber quality.ResultsUsing an improved search model, a total of 1,383 bidirectional transcript pairs were identified in the Gossypium hirsutum TM-1 genome, and their gene structure and functional annotations were systematically analyzed. Thirty bidirectional intergenic sequences were randomly screened for promoter activity analysis via a transient expression system, and 25 intergenic sequences were found to have bidirectional promoter activity. Comparative analysis of the bidirectional gene profiles of the four cotton subspecies revealed that these subspecies presented abundant bidirectional gene pairs with high homology and that the bidirectional genes in the cotton subspecies were more similar in terms of their molecular functions, cellular components and biological processes. In addition, parallel analysis of bidirectional genes in dicotyledons and monocotyledons revealed that abundant bidirectional gene pairs exist in different species. Although the total number of orthologous bidirectional genes was similar, there was a significant difference in the number of orthologous bidirectional gene pairs between dicotyledons and monocotyledons. This evolutionary analysis of the function and structure of homologous bidirectional gene pairs in different varieties and different subspecies of the same species revealed potential pathways by which these gene pairs originated, which may be necessary for the evolution of a new species.ConclusionIn this study, many bidirectional gene pairs in Gossypium hirsutum TM-1 were identified using computer programming, and systematic analysis was conducted to explore their functions and evolutionary relationships. In addition, the promoter activity of the bidirectional intergenic sequences was verified. The combination of computer programming screening, experimental validation and other methods is expected to provide preferred bidirectional promoters for transgenic breeding work via multigene cotransformation methods, and this information is valuable for genetic engineering research and applications.

Regulation of Oil Biosynthesis and Genetic Improvement in Plants: Advances and Prospects.

Triglycerides are the main storage form of oil in plant seeds. Both fatty acids and triglycerides possess important functions in the process of plant growth and development. To improve the seed oil content and improve its fatty acid composition, this paper analyzed the research progress on the oil regulation and synthesis metabolism process of plant seeds and summarized the strategies for the improvement of plant seed oil: (a) To regulate carbon distribution by inhibiting the expression of genes encoding key enzymes, allocating carbon sources into the protein synthesis pathway, and enhancing the expression of key genes encoding key enzymes, leading carbon sources into the synthesis pathway of fatty acids; (b) To intervene in lipid synthesis by promoting the biosynthesis of fatty acids and improving the expression level of key genes encoding enzymes in the triacylglycerol (TAG) assembly process; (c) To improve seed oil quality by altering the plant fatty acid composition and regulating the gene expression of fatty acid desaturase, as well as introducing an exogenous synthesis pathway of long chain polyunsaturated fatty acids; (d) To regulate the expression of transcription factors for lipid synthesis metabolism to increase the seed oil content. In addition, this article reviews the key enzymes involved in the biosynthesis of plant fatty acids, the synthesis of triacylglycerol, and the regulation process. It also summarizes the regulatory roles of transcription factors such as WRI, LEC, and Dof on the key enzymes during the synthesis process. This review holds significant implications for research on the genetic engineering applications in plant seed lipid metabolism.

Genome editing using type I-E CRISPR-Cas3 in mice and rat zygotes

The type I CRISPR system has recently emerged as a promising tool, especially for large-scale genomic modification, but its application to generate model animals by editing zygotes had not been established. In this study, we demonstrate genome editing in zygotes using the type I-E CRISPR-Cas3 system, which efficiently generates deletions of several thousand base pairs at targeted loci in mice with 40%-70% editing efficiency without off-target mutations. To overcome the difficulties associated with detecting the variable deletions, we used a newly long-read sequencing-based multiplex genotyping approach. Demonstrating remarkable versatility, our Cas3-based technique was successfully extended to rats as well as mice, even by zygote electroporation methods. Knockin for SNP exchange and genomic replacement with a donor plasmid were also achieved in mice. This pioneering work with the type I CRISPR zygote editing system offers increased flexibility and broader applications in genetic engineering across different species.

Agrobacterium tumefaciens: Biology and application in genetic engineering

Agrobacterium tumefaciens is a rod-shaped soil bacterium renowned for its unique ability to transfer tumour-inducing plasmid (Ti plasmid) segments to plant cells. This mechanism has been extensively exploited in plant genetic engineering. This review delves into the intricate biological interactions between A. tumefaciens and plant cells, including the critical steps of bacterial attachment, activation of virulence (Vir) genes, generation and transport of the T-complex, and integration of T-DNA into the plant chromosome. Furthermore, the review examines the engineering of A. tumefaciens as a transformation tool, focusing on the modifications of Ti plasmids to create binary and co-integrative vector systems, which have significantly improved the efficiency and versatility of transformation protocols. The paper also highlights applications of Agrobacterium-mediated transformation in the production of edible vaccines. By providing a detailed examination of the biological, technical, and practical aspects of Agrobacterium-mediated transformation, this review aims to offer insights into optimizing this technique for various plant biotechnology applications. Ultimately, understanding and improving Agrobacterium-mediated transformation is crucial for advancing plant biotechnology.

CRISPR Tools for Engineering Prokaryotic Systems: Recent Advances and New Applications.

In the past decades, the broad selection of CRISPR-Cas systems has revolutionized biotechnology by enabling multimodal genetic manipulation in diverse organisms. Rooted in a molecular engineering perspective, we recapitulate the different CRISPR components and how they can be designed for specific genetic engineering applications. We first introduce the repertoire of Cas proteins and tethered effectors used to program new biological functions through gene editing and gene regulation. We review current guide RNA (gRNA) design strategies and computational tools and how CRISPR-based genetic circuits can be constructed through regulated gRNA expression. Then, we present recent advances in CRISPR-based biosensing, bioproduction, and biotherapeutics across in vitro and in vivo prokaryotic systems. Finally, we discuss forthcoming applications in prokaryotic CRISPR technology that will transform synthetic biology principles in the near future.

Development of super-infective ternary vector systems for enhancing the Agrobacterium-mediated plant transformation and genome editing efficiency.

Agrobacterium-mediated transformation remains a cornerstone of plant biology, fueling advancements in molecular genetics, new genomic techniques (NGTs), and the biotech industry. However, recalcitrant crops and technical hurdles persist as bottlenecks. The goal was to develop super-infective ternary vector systems that integrate a novel salicylic acid-degrading enzyme, GABA, and ethylene-degrading enzymes, targeting the transformation of crops by neutralizing plant defense system on Agrobacterium. Firstly, both the effect and activity of introducing enzymes were validated in EHA105, an important Agrobacterium strain. Our study demonstrates that all ternary vector (Tv) system variants significantly enhance reporter expression in transient assays with Nicotiana benthamiana and Cannabis sativa. Specifically, incorporating a constitutive virG mutation with novel enzyme combinations increased GFP and RUBY expression in C. sativa by >5-fold and 13-fold, respectively. The Tv system, combined with a geminivirus replicon, markedly boosted GUS gene expression in tomato, enhancing genome editing efficiency. Notably, compared to controls, Tv-VS demonstrated up to 18-fold and 4.5-fold increases in genome editing efficiency in C. sativa and tomato, respectively. Additionally, stable transformation rates in tomato and Arabidopsis improved significantly, with Tv-VS showing a remarkable 2.5-fold increase in transformation efficiency compared to control strains. The research marks notable progress in Agrobacterium-mediated plant transformation. The innovative ternary vectors overcome plant defense mechanisms, enabling genetic manipulation in previously challenging plant species. This development is anticipated to broaden the applications of plant genetic engineering, contributing to advancements in crop genome editing.

3'-UTR Sequence of Exosomal NANOGP8 DNA as an Extracellular Vesicle-Localization Signal.

Extracellular vesicles (EVs) are garnering attention as a safe and efficient biomolecule delivery system. EVs intrinsically play a crucial role in intercellular communication and pathophysiology by transporting functionally active DNA molecules. The internalized DNA pleiotropically affects the recipient cells. Considering these salient features, an intentional incorporation of specific DNA gene cassettes into EVs and their subsequent delivery to the target cells has potential applications in genetic engineering. Moreover, efficient ways to insert the DNA into EVs during their biogenesis is valuable. Our current research is a step in the development of this technology. As such, cancer cells are known to secrete exosomes containing increased amounts of double-stranded DNA than normal cells. The clonal analysis in our previously published data revealed that exosomes released from various cancer cells contained a significantly larger population of NANOGP8 DNA with a 22-base pair insertion in the 3'-untranslated region (UTR) compared to those secreted by normal cells. This finding led us to hypothesize that the 22-base pair insertion may act as a signal to facilitate the incorporation of NANOGP8 DNA into the exosomes. To test this hypothesis, we compared the EV localization of an Enhanced Green Fluorescent Protein (EGFP) gene fused with the NANOGP8 3'-UTR, with and without the 22-base pair insertion. The quantitative PCR analysis showed a significantly higher EGFP DNA accumulation in exosomes released from cells transfected with the gene cassette containing the 3'-UTR with the 22-base pair insertion. The discovery of a DNA localization signal in exosomal DNA's 3'-UTR could pave the way for the development of an EV-based DNA delivery system. This technology will open new possibilities in genetic engineering and innovative therapies using nucleic acid medicine.

β‐Galactosidase: Insights into source variability, genetic engineering, immobilisation and diverse applications in food, industry and medicine

β‐Galactosidases are crucial enzymes that hydrolyse oligosaccharides and polysaccharides with terminal β‐1,4‐glycosidic bonds. Though the traditional application of β‐Galactosidases has been to catalyse the breakdown of lactose in dairy products, its application extends beyond the production of lactose‐free products since variants capable of facilitating lactose condensation and exhibiting galactosyl transferase activity are extensively utilised for the synthesis of prebiotic galacto‐oligosaccharides. This review analyses β‐Galactosidase in multiple aspects, including sources, classification, characterisation, immobilisation, genetic engineering and applications in terms of whey treatment, biofuel production, production of lactose‐free dietary product, synthesis of galacto‐oligosaccharides and the early detection of cellular senescence and tumours.

Molecular Insight into the Structural Dynamics of Holliday Junctions Modulated by Integration Host Factor.

The integration host factor (IHF) in Escherichia coli is a nucleoid-associated protein with multifaceted roles that encompass DNA packaging, viral DNA integration, and recombination. IHF binds to double-stranded DNA featuring a 13-base pair (bp) consensus sequence with high affinity, causing a substantial bend of approximately 160° upon binding. Although wild-type IHF (WtIHF) is principally involved in DNA bending to facilitate foreign DNA integration into the host genome, its engineered counterpart, single-chain IHF (ScIHF), was specifically designed for genetic engineering and biotechnological applications. Our study delves into the interactions of both IHF variants with Holliday junctions (HJs), pivotal intermediates in DNA repair, and homologous recombination. HJs are dynamic structures capable of adopting open or stacked conformations, with the open conformation facilitating processes such as branch migration and strand exchange. Using microscale thermophoresis, we quantitatively assessed the binding of IHF to four-way DNA junctions that harbor specific binding sequences H' and H1. Our findings demonstrate that both IHF variants exhibit a strong affinity for HJs, signifying a structure-based recognition mechanism. Circular dichroism (CD) experiments unveiled the impact of the protein on the junction's conformation. Furthermore, single-molecule Förster resonance energy transfer (smFRET) confirmed the influence of IHF on the junction's dynamicity. Intriguingly, our results revealed that WtIHF and ScIHF binding shifts the population toward the open conformation of the junction and stabilizes it in that state. In summary, our findings underscore the robust affinity of the IHF for HJs and its capacity to stabilize the open conformation of these junctions.

In vitro matured oocytes have a higher developmental potential than in vivo matured oocytes after hormonal ovarian stimulation in Callithrix jacchus

BackgroundThe common marmoset, Callithrix jacchus, is an invaluable model in biomedical research. Its use includes genetic engineering applications, which require manipulations of oocytes and production of embryos in vitro. To maximize the recovery of oocytes suitable for embryo production and to fulfil the requirements of the 3R principles to the highest degree possible, optimization of ovarian stimulation protocols is crucial. Here, we compared the efficacy of two hormonal ovarian stimulation approaches: 1) stimulation of follicular growth with hFSH followed by triggering of oocyte maturation with hCG (FSH + hCG) and 2) stimulation with hFSH only (FSH-priming).MethodsIn total, 14 female marmosets were used as oocyte donors in this study. Each animal underwent up to four surgical interventions, with the first three performed as ovum pick-up (OPU) procedures and the last one being an ovariohysterectomy (OvH). In total, 20 experiments were carried out with FSH + hCG stimulation and 18 with FSH-priming. Efficacy of each stimulation protocol was assessed through in vitro maturation (IVM), in vitro fertilization (IVF) and embryo production rates.ResultsEach study group consisted of two subgroups: the in vivo matured oocytes and the oocytes that underwent IVM. Surprisingly, in the absence of hCG triggering some of the oocytes recovered were at the MII stage, moreover, their number was not significantly lower compared to FSH + hCG stimulation (2.8 vs. 3.9, respectively (ns)). While the IVM and IVF rates did not differ between the two stimulation groups, the IVF rates of in vivo matured oocytes were significantly lower compared to in vitro matured ones in both FSH-priming and FSH + hCG groups. In total, 1.7 eight-cell embryos/experiment (OPU) and 2.1 eight-cell embryos/experiment (OvH) were obtained after FSH + hCG stimulation vs. 1.8 eight-cell embryos/experiment (OPU) and 5.0 eight-cell embryos/experiment (OvH) following FSH-priming. These numbers include embryos obtained from both in vivo and in vitro matured oocytes.ConclusionA significantly lower developmental competence of the in vivo matured oocytes renders triggering of the in vivo maturation with hCG as a part of the currently used FSH-stimulation protocol unnecessary. In actual numbers, between 1 and 7 blastocysts were obtained following each FSH-priming. In the absence of further studies, FSH-priming appears superior to FSH + hCG stimulation in the common marmoset under current experimental settings.

Engineering Gut Symbionts: A Way to Promote Bee Growth?

Bees play a crucial role as pollinators, contributing significantly to ecosystems. However, the honeybee population faces challenges such as global warming, pesticide use, and pathogenic microorganisms. Promoting bee growth using several approaches is therefore crucial for maintaining their roles. To this end, the bacterial microbiota is well-known for its native role in supporting bee growth in several respects. Maximizing the capabilities of these microorganisms holds the theoretical potential to promote the growth of bees. Recent advancements have made it feasible to achieve this enhancement through the application of genetic engineering. In this review, we present the roles of gut symbionts in promoting bee growth and collectively summarize the engineering approaches that would be needed for future applications. Particularly, as the engineering of bee gut symbionts has not been advanced, the dominant gut symbiotic bacteria Snodgrassella alvi and Gilliamella apicola are the main focus of the paper, along with other dominant species. Moreover, we propose engineering strategies that will allow for the improvement in bee growth with listed gene targets for modification to further encourage the use of engineered gut symbionts to promote bee growth.

Post-harvest Biotechnology or Genetic Engineering Solutions: Extending Shelf Life and Reducing Food Waste

Post-harvest losses and food waste have become critical challenges in the global food supply chain, contributing to economic losses, environmental degradation, and food insecurity. This article explores the innovative applications of post-harvest biotechnology and genetic engineering as promising solutions to address these issues by extending the shelf life of perishable products and minimizing food waste. Advancements in genetic engineering techniques have paved the way for the development of crops with enhanced resistance to pests, diseases, and environmental stresses. Additionally, the manipulation of genes associated with ripening and senescence has allowed scientists to engineer fruits and vegetables with extended shelf life. These genetically modified organisms (GMOs) exhibit improved post-harvest characteristics, providing a longer window for transportation, storage, and consumption. Biotechnological interventions also include the use of biocontrol agents and beneficial microorganisms to suppress post-harvest pathogens, thereby reducing spoilage and decay. The development of bio-preservatives, such as antimicrobial peptides and natural compounds, offers an eco-friendly alternative to traditional chemical preservatives, contributing to both food safety and sustainability. Furthermore, the integration of smart packaging technologies with genetic modifications enhances the monitoring and control of environmental conditions during storage and transportation. Intelligent packaging materials equipped with sensors can detect changes in temperature, humidity, and gas composition, enabling real-time adjustments to prolong the freshness of perishable goods.

Advancements in Genetic Engineering for Enhanced Traits in Horticulture Crops: A Comprehensive Review

Genetic engineering has emerged as a powerful tool in the field of horticulture to enhance the traits of crops, leading to improved yield, quality, and resistance to biotic and abiotic stresses. This comprehensive review explores the recent advancements in genetic engineering techniques applied to horticulture crops, providing a detailed overview of the innovative strategies employed to manipulate plant genomes. The review begins by discussing the evolution of genetic engineering in horticulture, highlighting key milestones and breakthroughs that have paved the way for current advancements. It covers a range of techniques such as CRISPR-Cas9, RNA interference, and synthetic biology, shedding light on their applications in modifying specific genes responsible for desired traits. The main focus of the review is on the enhancement of key horticultural traits, including disease resistance, pest resistance, abiotic stress tolerance, and post-harvest attributes. Examples from various horticultural crops, such as fruits, vegetables, and ornamental plants, illustrate the success stories and potential applications of genetic engineering in each category. The ethical and regulatory aspects of genetic engineering in horticulture are also explored, addressing concerns related to environmental impact, biodiversity, and consumer acceptance. The review emphasizes the importance of responsible and sustainable practices in the application of genetic engineering to ensure the long-term benefits without adverse consequences. Furthermore, the review delves into emerging trends and future prospects in the field, including the potential of genome editing for precision breeding, the use of omics technologies for targeted trait improvements, and the integration of genetic engineering with other breeding techniques. It also discusses the challenges and opportunities associated with the global adoption of genetically modified horticulture crops.

Discovery of novel replication proteins for large plasmids in cyanobacteria and their potential applications in genetic engineering.

Numerous cyanobacteria capable of oxygenic photosynthesis possess multiple large plasmids exceeding 100 kbp in size. These plasmids are believed to have distinct replication and distribution mechanisms, as they coexist within cells without causing incompatibilities between plasmids. However, information on plasmid replication proteins (Rep) in cyanobacteria is limited. Synechocystis sp. PCC 6803 hosts four large plasmids, pSYSM, pSYSX, pSYSA, and pSYSG, but Rep proteins for these plasmids, except for CyRepA1 on pSYSA, are unknown. Using Autonomous Replication sequencing (AR-seq), we identified two potential Rep genes in Synechocystis 6803, slr6031 and slr6090, both located on pSYSX. The corresponding Rep candidates, Slr6031 and Slr6090, share structural similarities with Rep-associated proteins of other bacteria and homologs were also identified in various cyanobacteria. We observed autonomous replication activity for Slr6031 and Slr6090 in Synechococcus elongatus PCC 7942 by fusing their genes with a construct expressing GFP and introducing them via transformation. The slr6031/slr6090-containing plasmids exhibited lower copy numbers and instability in Synechococcus 7942 cells compared to the expression vector pYS. While recombination occurred in the case of slr6090, the engineered plasmid with slr6031 coexisted with plasmids encoding CyRepA1 or Slr6090 in Synechococcus 7942 cells, indicating the compatibility of Slr6031 and Slr6090 with CyRepA1. Based on these results, we designated Slr6031 and Slr6090 as CyRepX1 (Cyanobacterial Rep-related protein encoded on pSYSX) and CyRepX2, respectively, demonstrating that pSYSX is a plasmid with "two Reps in one plasmid." Furthermore, we determined the copy number and stability of plasmids with cyanobacterial Reps in Synechococcus 7942 and Synechocystis 6803 to elucidate their potential applications. The novel properties of CyRepX1 and 2, as revealed by this study, hold promise for the development of innovative genetic engineering tools in cyanobacteria.