Search Results (230)

Passion fruit (Passiflora edulis Sims), belonging to the Passifloraceae family, is an economically important plant in tropical and subtropical regions. The advances in functional genomics research of passion fruit have been significantly hindered by its recalcitrance to regeneration and stable transformation. This study establishes the first efficient Agrobacterium rhizogenes-mediated hairy root transformation system for passion fruit. Utilizing the eGFP marker gene, transformation efficiencies of 11.3% were initially achieved with strains K599, MSU440, and C58C1, with K599 proving most effective. Key transformation parameters were systematically optimized to achieve the following: OD₆₀₀ = 0.6, infection duration 30 min, acetosyringone concentration 100 μM, and a dark co-cultivation period of 2 days. The system’s utility was further enhanced by incorporating the red visual marker RUBY, enabling direct, instrument-free identification of transgenic roots via betaxanthin accumulation. Finally, this system was applied for functional analysis using PeMYB123, which may be involved in proanthocyanidin accumulation. Overexpression of PeMYB123 produced a higher content of proanthocyanidin in hairy roots. Additionally, the PeANR gene involved in the proanthocyanidin pathway was strongly activated in the transgenic hairy roots. This rapid and efficient visually simplified hairy root transformation system provides a powerful tool for functional gene studies in passion fruit. Full article

Weeds present a pervasive challenge in agricultural fields. The integration of herbicide-resistant crops with chemical weed management offers an effective solution for sustainable weed control while reducing labor inputs, particularly in large-scale intensive farming systems. Consequently, the development of herbicide-resistant cultivars has emerged as an urgent priority. In this study, we found that the G311E mutant gene of Arabidopsis MATE (multidrug and toxic compound extrusion) family transporter DTX6, designated DTX6m, confers robust resistance to bipyridyl herbicides paraquat and diquat in rice. DTX6m-overexpression lines exhibited marked resistance to these two herbicides, tolerating diquat concentrations up to 5 g/L, which is five-fold higher than the recommended field application dosage. Agronomic assessments demonstrated that grain yields of DTX6m-overexpressing plants were statistically equivalent to those of wild-type plants. Moreover, the plants displayed beneficial phenotypic changes, such as accelerated flowering and a slight reduction in height. Seed morphometric analysis indicated that in comparison with the wild-type control, DTX6m-transgenic lines exhibited altered grain dimensions while maintaining consistent 1000-grain weight. Nutritional assays further demonstrated that DTX6m increased the levels of free amino acids in seeds, while normal protein and starch contents were retained. Collectively, these results establish that DTX6m effectively boosts rice resistance to paraquat and diquat, validating DTX6m as a candidate gene for engineering plant herbicide resistance and also implying a potential role for DTX6m in amino acid homeostasis in plants. Full article

Cadmium (Cd) contamination in agricultural soils severely threatens rice production and food safety. To address this issue, this study developed transgenic rice lines co-expressing three Vitis vinifera genes: the ABCC transporter Vvmrp1 and metallothioneins Vvmt1 and Vvmt2. AlphaFold computational modeling confirmed the conserved ABCC-type transporter domain in VvMRP1. Under hydroponic conditions, transgenic rice showed remarkable Cd tolerance, surviving 30 mM Cd (lethal to wildtype, WT) without growth penalties, and exhibited 62.5% survival at 1 mM Cd vs. complete wild-type mortality. Field-relevant Cd exposure (1 mM) reduced Cd accumulation to 35.8% in roots, 83% in stems, and 76.8% in grains compared to WT. Mechanistic analyses revealed that Vvmrp1 mediates cellular Cd efflux while Vvmt1 and 2 chelate free Cd ions, synergistically inhibiting Cd translocation. Transgenic plants also maintained better Fe, P, and Mg homeostasis under Cd stress. This study pioneers the co-expression of a transporter with metallothioneins in rice, demonstrating their complementary roles in Cd detoxification without pleiotropic effects from endogenous gene modification. The findings provide an effective genetic strategy for cultivating low-Cd rice in contaminated soils, offering significant implications for food safety and sustainable agriculture. Full article

Lycopene, a natural pigment, is valuable for human health because of its strong antioxidant capacity. However, studies on the involvement of tomato miR394 in the regulation of lycopene have not been reported. The aim of this study was to reveal the molecular mechanism by which miR394 regulates lycopene synthesis by targeting ζ-carotene dehydrogenase (ZDS). The miR394-silenced transgenic tomato plants were constructed by short tandem target mimicry (STTM) technology, and the association between lycopene content and antioxidant capacity was analyzed by combining qRT-PCR, UV spectrophotometry, and a free radical scavenging assay. The targeting relationship between miR394 and ZDS was verified using a subcellular localization assay. The results showed that the silencing of miR394 significantly upregulated the expression of the ZDS gene and promoted lycopene accumulation. The antioxidant enzyme activities of STTM394 transgenic plants were significantly enhanced, and the free radical scavenging ability was obviously improved. Subcellular localization experiments confirmed that miR394 directly inhibited the chloroplast expression of ZDS. In conclusion, this study reveals for the first time that the miR394-ZDS module enhances the antioxidant capacity by regulating lycopene metabolism, which provides a new target for themolecular breeding of highly nutritious tomatoes. Full article

Conditional transgenic systems and multi-copy target transgenes were used to produce transient fluorescent protein expression in adult Drosophila melanogaster, with the goal of developing an in vivo assay of protein turnover. Free-moving flies were assayed at multiple time points using video, and decay in fluorescence was used to calculate protein half-life. Additional experiments involved image capture of anesthetized flies. The half-life of eGFP was increased by the proteasome inhibitor bortezomib, both in vivo and in vitro, indicating proteasomal degradation of eGFP. The accumulation of eGFP in vivo was decreased by the protein synthesis inhibitor cycloheximide, without affecting half-life. The half-lives of several fluorescent proteins were determined, using both tissue-general and tissue-specific expression, in flies of both sexes and varying ages. Typical half-life values varied by fluorescent protein. DsRED showed a greater half-life than eGFP, and little if any degradation was detected for mCherry. Half-life also varied by tissue, with greater eGFP half-life observed in muscle relative to other tissues. Increased half-life with age was detected for DsRED but not for eGFP. Limited effects were observed for sex and female mating status. Taken together, the data indicate the in vivo assays are promising tools for the study of protein degradation regulated by protein sequence, subcellular compartment, tissue and small molecules. Full article

There is currently a worldwide trend towards deregulating the use of genome-edited plants. Virus-induced genome editing (VIGE) is a novel technique that utilizes viral vectors to transiently deliver clustered regularly interspaced short palindromic repeat (CRISPR) components into plant cells. It potentially allows us to obtain transgene-free events in any plant species in a single generation without in vitro tissue culture. This technology has great potential for agriculture and is already being applied to more than 14 plant species using more than 20 viruses. The main limitations of VIGE include insufficient vector capacity, unstable expression of CRISPR-associated (Cas) protein, plant immune reaction, host specificity, and reduced viral activity in meristem. Various solutions to these problems have been proposed, such as fusion of mobile elements, RNAi suppressors, novel miniature Cas proteins, and seed-borne viruses, but the final goal has not yet been achieved. In this review, the mechanism underlying the ability of different classes of plant viruses to transiently edit genomes is explained. It not only focuses on the latest achievements in virus-induced editing of crops but also provides suggestions for improving the technology. This review may serve as a source of new ideas for those planning to develop new approaches in VIGE. Full article

Hexaploid wheat has a large genome, making it difficult for transgenes to produce phenotypes due to gene redundancy and tight linkage among genes. Multiple gene copies typically necessitate multiple targeting events during gene editing, followed by several generations of self-crossing to achieve homozygous genotypes. The high cost of transgenesis in wheat is another issue, which hinders the easy availability of gene-edited materials in wheat. In this study, we developed a comprehensive approach to improve wheat gene editing efficiency. First, we established a protoplast-based system to evaluate the relative efficiency of gene editing targets, which enabled the rapid and effective selection of optimal sgRNAs. We then compared two transformation strategies: biolistic bombardment and Agrobacterium-mediated transformation for generating edited wheat lines. Although biolistic bombardment showed higher initial editing efficiency, Agrobacterium-mediated transformation proved more effective for obtaining homozygous mutants. Notably, we discovered that deploying the same sgRNA through different vectors enhanced editing efficiency, whereas overlapping but distinct sgRNAs exhibited interference effects. Finally, we optimized the VITF-edit (virus-induced transgene free editing) technique using BSMV delivery to establish a relatively simple and easily applied wheat gene editing method for general laboratories. Full article

Background: Mucin-1 (MUC1) is a glycoprotein that is hypoglycosylated and overexpressed in most adenocarcinomas, making it a promising target for cancer vaccines. Our group previously demonstrated that C3 (OPSS)-liposomes enhance antigen uptake by antigen-presenting cells (APCs) via the complement C3 pathway and, when combined with toll-like receptor (TLR) agonists, reduce tumor growth in murine cancer models. Methods: In the present study, we evaluate the immunogenicity of MUC1 peptide vaccines encapsulated in C3-liposomes, with and without TLR agonists, using MUC1-tolerant transgenic mice challenged with Lewis lung carcinoma (LLC.MUC1) cells. To assess vaccine effectiveness, tumor volumes were measured, and flow cytometry and ELISA and ELISPOT assays were used to assess the immune response. Results: Both male and female C57BL/6 transgenic mice vaccinated with MUC1 C3-liposomes developed significantly smaller tumors than those vaccinated with free MUC1 peptide or PBS. Notably, a sex-dependent response emerged in mice vaccinated with MUC1 C3-liposomes with TLR agonists (TLR4, TLR7/8, and TLR9); male mice exhibited greater tumor suppression than females. Flow cytometry analysis revealed that female mice had significantly higher levels of CD11b⁺, LY6C⁺, and LY6G⁺ MDSC cells, suggesting a potential mechanism for the sex difference. Additionally, MUC1 C3-liposome vaccination elicited robust adaptive immune responses, including significantly higher levels of IFN-γ-producing T cells and MUC1-specific IgG antibodies compared to non-encapsulated MUC1 or TLR adjuvant-only formulations. Conclusions: These findings underscore the potential of C3-liposome-based antigen vaccines to enhance anti-tumor immunity and highlight the impact of sex differences in vaccine efficacy. Full article

Excessive amounts of free-oxygen radicals produced during inflammation induce oxidative stress and lead to cell damage, thus delaying the transition of inflammation into the proliferation in the wound healing process. Oxidative stress on skin cells also plays an important role in the pathogenesis of inflammatory skin diseases. The aim of the planned in vitro studies was to assess the mechanisms of regenerative action and protection of cells against oxidative stress of three oil emulsions from transgenic (GMO) flax varieties M, B, and MB and a linseed emulsion from traditional NIKE linseed oil. Antioxidant and gene-protective properties were identified for the tested oil emulsions in a healthy cell model and in an in vitro model of cells under oxidative stress. The wound-healing regenerative potential of these linseed emulsions was also assessed in the proliferation, cell cycle, migration, and apoptosis and necrosis assays. The conducted research presented that the tested transgenic oil emulsions are safe for human skin because they do not induce the proliferation of skin cancer cells and, at the same time, induce the migration processes of normal human skin cells. Additionally, their use increases the ability to eliminate damaged cells. Transgenic linseed oils provide a gene-protective effect and an increased antioxidant effect, resulting in increased protection of skin cells against oxidative stress, which plays an important role in the pathogenesis of atopic dermatitis and psoriasis. Linen emulsion B has the best regenerative and protective properties against human epidermis cancer, which is probably due to the presence of an increased amount of stigmasterol in its composition along with the appropriate content of polyphenol compounds, as well as an increased amount of oleic and linoleic acids. Full article

Moniliophthora perniciosa is one of the main pathogens affecting cocoa, and controlling it generally involves planting resistant genotypes followed by phytosanitary pruning. The identification of plant genes related to defense mechanisms is crucial to unravel the molecular basis of plant–pathogen interactions. Among the candidate genes, BiP stands out as a molecular chaperone located in the endoplasmic reticulum that facilitates protein folding and is induced under stress conditions, such as pathogen attacks. In this study, the SoyBiPD gene was expressed in Solanum lycopersicum plants and the plants were challenged with M. perniciosa. The control plants exhibited severe symptoms of witches’ broom disease, whereas the transgenic lines showed no or mild symptoms. Gel-free proteomics revealed significant changes in the protein profile associated with BiP overexpression. Inoculated transgenic plants had a higher abundance of resistance-related proteins, such as PR2, PR3, and PR10, along with increased activity of antioxidant enzymes, including superoxide dismutase (SOD), catalase (CAT), guaiacol peroxidase, and fungal cell wall-degrading enzymes (glucanases). Additionally, transgenic plants accumulated less H₂O₂, indicating more efficient control of reactive oxygen species (ROS). The interaction network analysis highlighted the activation of defense-associated signaling and metabolic pathways, conferring a state of defensive readiness even in the absence of pathogens. These results demonstrate that BiP overexpression increases the abundance of defense proteins, enhances antioxidant capacity, and confers greater tolerance to biotic stress. This study demonstrates the biotechnological potential of the BiP gene for genetic engineering crops with increased resistance to economically important diseases, such as witches’ broom in cocoa. Full article

Alzheimer’s disease (AD) is marked by impaired cognitive functions, particularly in learning and memory, owing to complex and diverse mechanisms. Methionine restriction (MR) has been found to exert a mitigating effect on brain oxidative stress to improve AD. However, the bidirectional crosstalk between the gut and brain through which MR enhances learning and memory in AD, as well as the effects of fecal microbiota transplantation (FMT) from MR mice on AD mice, remains underexplored. In this study, APP/PS1 double transgenic AD mice were used and an FMT experiment was conducted. 16S rRNA gene sequencing, targeted metabolomics, and microbial metabolite short-chain fatty acids (SCFAs) of feces samples were analyzed. The results showed that MR reversed the reduction in SCFAs induced by AD, and further activated the free fatty acid receptors, FFAR2 and FFAR3, as well as the transport protein MCT1, thereby signaling to the brain to mitigate inflammation and enhance the learning and memory capabilities. Furthermore, the FMT experiment from methionine-restricted diet mouse donors showed that mice receiving FMT ameliorated Alzheimer’s learning and memory ability through SCFAs. This study offers novel non-pharmaceutical intervention strategies for AD prevention. Full article

Background/Objectives: Endothelial peroxisome proliferator-activated receptor gamma (PPARγ) regulates adipose tissue by facilitating lipid uptake into white adipocytes, but the role of endothelial lipid transport in systemic energy balance remains unclear. Ghrelin conveys nutritional information through the central nervous system and increases adiposity, while deficiency in its receptor, growth hormone secretagogue-receptor (GHSR), suppresses adiposity on a high-fat diet. This study aims to examine the effect of ghrelin/GHSR signaling in the endothelium on lipid metabolism. Methods: We compared the effects of ghrelin on adiposity and lipid uptake into adipocytes in wild-type and GHSR-null mice. Transgenic mice expressing GHSR selectively in endothelial cells were also generated and compared with global GHSR-null and wild-type mice. The impact of ghrelin on lipid uptake-related genes was assessed in cultured endothelial cells. Results: Ghrelin increased adiposity and triglyceride clearance in wild-type but not in GHSR-null mice. GHSR-null mice showed higher serum triglyceride after olive oil gavage and lower white adipose tissue (WAT) weight on a high-fat diet, suggesting impaired lipid uptake. Restoring GHSR expression in endothelial cells increased lipoprotein lipase activity, lipid uptake into WAT, and WAT weight. Ghrelin enhanced free fatty acid uptake and the expression of lipid uptake genes in cultured endothelial cells, whereas these effects were absent in GHSR-null mice-derived endothelial cells. Knockdown of PPARγ revealed that ghrelin/GHSR signaling in endothelial cells promoted lipid uptake via endothelial PPARγ. Conclusions: Endothelial GHSR is key for regulating lipid metabolism via PPARγ in response to ghrelin and for the role of endothelium in regulating white adipocyte metabolism. Targeting endothelial ghrelin signaling may be a promising therapeutic approach for managing excessive adiposity and associated metabolic disorders. Full article

Fruit firmness is crucial for storability, making cultivating varieties with higher firmness a key target in tomato breeding. In recent years, tomato varieties primarily rely on hybridizing ripening mutants to produce F₁ hybrids to enhance firmness. However, the undesirable traits introduced by these mutants often lead to a decline in the quality of the varieties. CRISPR/Cas9 has emerged as a crucial tool in accelerating plant breeding and improving specific target traits as technology iterates. In this study, we used a CRISPR/Cas9 system to simultaneously knock out two genes, FIS1 and PL, which negatively regulate firmness in tomato. We generated single and double gene knockout mutants utilizing the tomato genetic transformation system. The fruit firmness of all knockout mutants exhibited a significant enhancement, with the most pronounced improvement observed in the double mutant. Furthermore, we assessed other quality-related traits of the mutants; our results indicated that the fruit quality characteristics of the gene-edited lines remained statistically comparable to those of the wild type. This approach enabled us to create transgenic-free mutants with diverse genotypes across fewer generations, facilitating rapid improvements in tomato firmness. This study offers significant insights into molecular design breeding strategies for tomato. Full article

The creation of the CRISPR–Cas system has provided unprecedented opportunities in plant genome research and crop genetic improvement. In recent years, this system has been continuously improved to meet human needs through the expansion and modification of Cas proteins, the diversification of targeting locations, and the optimization of CRISPR vectors. In this review, we systematically describe the Class II Cas proteins that have been used in plants, deactivated Cas9 (dCas9) and its role in transcriptional regulation, precision editing systems, Cas9 protein variants, as well as methods and examples of CRISPR–Cas systems targeting various regions with different breadths. In addition, we outline the optimization plans based on CRISPR constructs that can overcome the pleiotropy of genes or accelerate the generation of transgene-free plants and the applications of CRISPR systems in plant breeding practices. Finally, we discuss the theory and development of “CRISPR plus”, and the integrated application of existing systems in more species. Full article

The cerebellum, a key target of ethanol’s toxic effects, is associated with ataxia following alcohol consumption. However, the impact of ethanol on Purkinje cell (PC) mitochondria remains unclear. To investigate how ethanol administration affects mitochondrial dynamics in cerebellar Purkinje cells, we employed a transgenic mouse model expressing mitochondria-targeted yellow fluorescent protein in Purkinje cells (PC-mito-eYFP). Both male and female PC-mito-eYFP mice received an intraperitoneal injection of ethanol or vehicle. One hour after ethanol administration, the animals were perfusion fixed or their cerebellum tissue or isolated mitochondria were collected. Cerebellum sections were analyzed using confocal microscopy to assess changes in mitochondrial length distribution. In vivo superoxide levels were measured using dihydroethidium (DHE), and mitochondrial NAD levels were determined by high-performance liquid chromatography (HPLC). Our findings revealed a sex-dependent response to ethanol administration in mitochondrial size distribution. While male Purkinje cell mitochondria exhibited no significant changes in size, female mitochondria became more fragmented after one hour of ethanol administration. This coincided with elevated phosphorylation of the fission protein Drp1 and increased superoxide production, as measured by DHE fluorescence intensity. Similarly, mitochondrial NAD levels were significantly reduced in female mice, but no changes were observed in males. Our results demonstrate that ethanol induced mitochondrial fragmentation through increased free radical levels, due to reduced NAD and increased p-Drp1, in PC cells of the female cerebellum. Full article