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ACS Central Science May 2024In this study, an innovative approach is presented in the field of engineered plant living materials (EPLMs), leveraging a sophisticated interplay between synthetic...
In this study, an innovative approach is presented in the field of engineered plant living materials (EPLMs), leveraging a sophisticated interplay between synthetic biology and engineering. We detail a 3D bioprinting technique for the precise spatial patterning and genetic transformation of the tobacco BY-2 cell line within custom-engineered granular hydrogel scaffolds. Our methodology involves the integration of biocompatible hydrogel microparticles (HMPs) primed for 3D bioprinting with capable of plant cell transfection, serving as the backbone for the simultaneous growth and transformation of tobacco BY-2 cells. This system facilitates the concurrent growth and genetic modification of tobacco BY-2 cells within our specially designed scaffolds. These scaffolds enable the cells to develop into predefined patterns while remaining conducive to the uptake of exogenous DNA. We showcase the versatility of this technology by fabricating EPLMs with unique structural and functional properties, exemplified by EPLMs exhibiting distinct pigmentation patterns. These patterns are achieved through the integration of the betalain biosynthetic pathway into tobacco BY-2 cells. Overall, our study represents a groundbreaking shift in the convergence of materials science and plant synthetic biology, offering promising avenues for the evolution of sustainable, adaptive, and responsive living material systems.
PubMed: 38799669
DOI: 10.1021/acscentsci.4c00338 -
Plant Signaling & Behavior Dec 2024The purpose of this study was to analyze the role of transcription factor in , proving that the transcription factor was related to the plant phenotypes of - cv....
The purpose of this study was to analyze the role of transcription factor in , proving that the transcription factor was related to the plant phenotypes of - cv. 'GuangYaoDa1' and it could be used in molecular-assisted breeding. 'GuangYaoDa1' was used as the material and its DNA was the template to clone DsWRKY6, the transgenic line was constructed by agrobacterium tumefaciens‑mediated transformation. Transgenic was cultivated to study phenotype and physiological and biochemical indexes. Phenotypic observation showed that transgenic had a faster growth rate while compared with the control group, they had longer lengths of main stem, lateral branches of cauline leaves, and root, but a lower number of cauline leaves and lateral branches of cauline leaves. And it also showed that their flowering and fruiting periods were advanced. The results of physiological and biochemical indexes showed that the relative expressions of increased and the abscisic acid content significantly increased in transgenic compared with the control group. According to the above results, could regulate the advancing of flowering and fruiting periods caused by the improvement of abscisic acid content, and expression of the transcription factor might be the cause of the upright growth of 'GuangYaoDa1'.
Topics: Arabidopsis; Plants, Genetically Modified; Cloning, Molecular; Plant Proteins; Transcription Factors; Gene Expression Regulation, Plant; Fabaceae; Phenotype; Abscisic Acid; Genes, Plant
PubMed: 38743594
DOI: 10.1080/15592324.2024.2349868 -
PloS One 2024Sunflower is one of the four major oil crops in the world. 'Zaoaidatou' (ZADT), the main variety of oil sunflower in the northwest of China, has a short growth cycle,...
Sunflower is one of the four major oil crops in the world. 'Zaoaidatou' (ZADT), the main variety of oil sunflower in the northwest of China, has a short growth cycle, high yield, and high resistance to abiotic stress. However, the ability to tolerate adervesity is limited. Therefore, in this study, we used the retention line of backbone parent ZADT as material to establish its tissue culture and genetic transformation system for new variety cultivating to enhance resistance and yields by molecular breeding. The combination of 0.05 mg/L IAA and 2 mg/L KT in MS was more suitable for direct induction of adventitious buds with cotyledon nodes and the addition of 0.9 mg/L IBA to MS was for adventitious rooting. On this basis, an efficient Agrobacterium tumefaciens-mediated genetic transformation system for ZADT was developed by the screening of kanamycin and optimization of transformation conditions. The rate of positive seedlings reached 8.0%, as determined by polymerase chain reaction (PCR), under the condition of 45 mg/L kanamycin, bacterial density of OD600 0.8, infection time of 30 min, and co-cultivation of three days. These efficient regeneration and genetic transformation platforms are very useful for accelerating the molecular breeding process on sunflower.
Topics: Helianthus; Transformation, Genetic; Agrobacterium tumefaciens; Plants, Genetically Modified; Tissue Culture Techniques; Plant Roots; Plant Breeding; Crops, Agricultural
PubMed: 38722945
DOI: 10.1371/journal.pone.0298299 -
Open Life Sciences 2024Allene oxide synthase (AOS) is a key enzyme involved in the jasmonic acid (JA) synthesis pathway in plants. To explore its function on the regulatory mechanism of JA...
Allene oxide synthase (AOS) is a key enzyme involved in the jasmonic acid (JA) synthesis pathway in plants. To explore its function on the regulatory mechanism of JA synthesis, we screened and identified two genes and in qingke. Both HvnAOS1 and HvnAOS2 contained conserved heme-binding motif, which is most closely related to AtsAOS2, indicating controlled dehydration of fatty acid hydroperoxides to allene oxides. Molecular docking simulations identified the key amino acid sites that were important for heme binding and interaction with 13()-HPOT, respectively. The expression pattern also indicated that and were highly induced by JA, abscisic acid, and salicylic acid. Subcellular localization of and using transient expression of showed the green fluorescent protein signal in the cell cytoplasm of the . leaves. Overexpression of and in mutant restored male fertility and plant resistance to , indicating that and can restore the functions of in mutant.
PubMed: 38681731
DOI: 10.1515/biol-2022-0855 -
Life (Basel, Switzerland) Apr 2024Quorum sensing (QS) controls the virulence of This study aims to determine the anti-QS activity of aspirin alone and in combination with chitosan to reach maximum...
BACKGROUND
Quorum sensing (QS) controls the virulence of This study aims to determine the anti-QS activity of aspirin alone and in combination with chitosan to reach maximum inhibition. We tested ten virulent (. ) isolates and screened for N-acyl homoserine lactone (AHL) production using as a biosensor. isolates were treated with sub-minimum inhibitory concentrations (MICs) of aspirin and chitosan-aspirin. We used broth microdilution and checkerboard titration methods to determine the MICs and the synergistic effect of these two compounds, respectively. Real-time polymerase chain reaction (PCR) was used to estimate the anti-QS activity of the aspirin-chitosan combination on the expression of and genes.
RESULTS
Aspirin decreased the motility and production of AHLs, pyocyanin, and biofilm. Chitosan potentiated the inhibitory effect of aspirin. The chitosan-aspirin combination inhibited and gene expression in PAO1 (ATCC 15692) by 7.12- and 0.92-fold, respectively. In clinical isolates, the expression of and was decreased by 1.76 × 10- and 1.63 × 10-fold, respectively. Molecular docking analysis revealed that aspirin could fit into the active sites of the QS synthases and with a high binding affinity, causing conformational changes that resulted in their inhibition.
CONCLUSIONS
The chitosan-aspirin combination provides new insights into treating virulent and resistant
PubMed: 38672752
DOI: 10.3390/life14040481 -
Frontiers in Microbiology 2024It is essential to consider a practical antibody test to successfully implement marker vaccines and validate vaccination efficacy against classical swine fever virus...
BACKGROUND
It is essential to consider a practical antibody test to successfully implement marker vaccines and validate vaccination efficacy against classical swine fever virus (CSFV). The test should include a serological antibody assay, combined with a tool for differentiating infected from vaccinated animals (DIVA). The immunochromatographic test strip (ICS) has been exclusively designed for detecting CSFV E2 antibodies while lacking in detecting E antibodies, which can be employed and satisfy DIVA strategy. This study developed a novel ICS for detecting CSFV E2/E dual-antibody. The effectiveness of ICS in evaluating the DIVA capability of two novel chimeric pestivirus vaccine candidates was assessed.
METHODS
Recombinant E2 or E protein was transiently expressed in the plant using . ICS was subsequently assembled, and goat anti-rabbit IgG and recombinant CSFV E2 or E protein were plated onto the nitrocellulose membrane as control and test lines, respectively. The sensitivity and specificity of ICS were evaluated using sera with different neutralizing antibody titers or positive for antibodies against CSFV and other pestiviruses. The coincidence rates for detecting E2 and E antibodies between ICS and commercial enzyme-linked immunosorbent assay (ELISA) kits were also computed. ICS performance for DIVA capability was evaluated using sera from pigs vaccinated with conventional vaccine or chimeric vaccine candidates.
RESULTS
E2 and E proteins were successfully expressed in -produced recombinant proteins. ICS demonstrated high sensitivity in identifying CSFV E2 and E antibodies, even at the low neutralizing antibody titers. No cross-reactivity with antibodies from other pestiviruses was confirmed using ICS. There were high agreement rates of 93.0 and 96.5% between ICS and two commercial ELISA kits for E2 antibody testing. ICS also achieved strong coincidence rates of 92.9 and 89.3% with two ELISA kits for E antibody detection. ICS confirmed the absence of CSFV E-specific antibodies in sera from pigs vaccinated with chimeric vaccine candidates.
CONCLUSION
E2 and E proteins derived from the plant showed great potential and can be used to engineer a CSFV E2/E dual-antibody ICS. The ICS was also highly sensitive and specific for detecting CSFV E2 and E antibodies. Significantly, ICS can fulfill the DIVA concept by incorporating chimeric vaccine candidates.
PubMed: 38666258
DOI: 10.3389/fmicb.2024.1383976 -
Heliyon Apr 2024The study aimed to enhance quercetin production in radish by optimizing -mediated transformation. This protocol involved infecting radish seed embryo axis with EHA105...
The study aimed to enhance quercetin production in radish by optimizing -mediated transformation. This protocol involved infecting radish seed embryo axis with EHA105 strain carrying the . Radish seeds were infected with the suspension (0.8 OD) for 30 min, followed by sonication for 60 s and vacuum infiltration for 90 s at 100 mm Hg. A 3-day co-cultivation in Murashige and Skoog medium with 150 μM acetosyringone yielded a transformation efficiency of 59.6% and a transgenic callus induction rate of 32.3%. Transgenic plant and callus lines were confirmed by GUS histochemical assay, PCR, and qRT-PCR. The transgenic lines showed an increased expression of flavonoid pathway genes ( and ) and antioxidant genes ( and ) compared to WT plants. Overexpression of in transgenic callus increased enzyme activity of phenylalanine ammonia lyase, catalase, and ascorbate peroxidase. In half-strength MS medium with 116.8 mM sucrose, the highest growth index (7.63) was achieved after 20 days. In overexpressed callus lines, phenolic content (357.31 mg g dry weight), flavonoid content (463 mg g dry weight), and quercetin content (48.24 mg g dry weight) increased significantly by 9.41-fold. Micro-wounding, sonication, and vacuum infiltration improved transformation in radishes. These high-quercetin-content transgenic callus lines hold promise as valuable sources of flavonoids.
PubMed: 38660267
DOI: 10.1016/j.heliyon.2024.e27053 -
PloS One 2024Most legumes are able to develop a root nodule symbiosis in association with proteobacteria collectively called rhizobia. Among them, the tropical species Aeschynomene...
Setting up Agrobacterium tumefaciens-mediated transformation of the tropical legume Aeschynomene evenia, a powerful tool for studying gene function in Nod Factor-independent symbiosis.
Most legumes are able to develop a root nodule symbiosis in association with proteobacteria collectively called rhizobia. Among them, the tropical species Aeschynomene evenia has the remarkable property of being nodulated by photosynthetic Rhizobia without the intervention of Nod Factors (NodF). Thereby, A. evenia has emerged as a working model for investigating the NodF-independent symbiosis. Despite the availability of numerous resources and tools to study the molecular basis of this atypical symbiosis, the lack of a transformation system based on Agrobacterium tumefaciens significantly limits the range of functional approaches. In this report, we present the development of a stable genetic transformation procedure for A. evenia. We first assessed its regeneration capability and found that a combination of two growth regulators, NAA (= Naphthalene Acetic Acid) and BAP (= 6-BenzylAminoPurine) allows the induction of budding calli from epicotyls, hypocotyls and cotyledons with a high efficiency in media containing 0,5 μM NAA (up to 100% of calli with continuous stem proliferation). To optimize the generation of transgenic lines, we employed A. tumefaciens strain EHA105 harboring a binary vector carrying the hygromycin resistance gene and the mCherry fluorescent marker. Epicotyls and hypocotyls were used as the starting material for this process. We have found that one growth medium containing a combination of NAA (0,5 μM) and BAP (2,2 μM) was sufficient to induce callogenesis and A. tumefaciens strain EHA105 was sufficiently virulent to yield a high number of transformed calli. This simple and efficient method constitutes a valuable tool that will greatly facilitate the functional studies in NodF-independent symbiosis.
Topics: Fabaceae; Agrobacterium tumefaciens; Symbiosis; Phenotype; Vegetables; Transformation, Genetic; Plants, Genetically Modified
PubMed: 38625963
DOI: 10.1371/journal.pone.0297547 -
Bio-protocol Apr 2024Citrus fruits encompass a diverse family, including oranges, mandarins, grapefruits, limes, kumquats, lemons, and others. In citrus, -mediated genetic transformation of...
Citrus fruits encompass a diverse family, including oranges, mandarins, grapefruits, limes, kumquats, lemons, and others. In citrus, -mediated genetic transformation of Hongkong kumquat ( Swingle) has been widely employed for gene function analysis. However, the perennial nature of woody plants results in the generation of transgenic fruits taking several years. Here, we show the procedures of -mediated transient transformation and live-cell imaging in kumquat ( Swingle) fruit, using the actin filament marker GFP-Lifeact as an example. Fluorescence detection, western blot analysis, and live-cell imaging with confocal microscopy demonstrate the high transformation efficiency and an extended expression window of this system. Overall, -mediated transient transformation of kumquat fruits provides a rapid and effective method for studying gene function in fruit, enabling the effective observation of diverse cellular processes in fruit biology.
PubMed: 38618180
DOI: 10.21769/BioProtoc.4968 -
Plants (Basel, Switzerland) Mar 2024Transient protein expression is a versatile tool with diverse applications and can be used in soybeans to study gene function, obtain mutants, and produce proteins for...
Transient protein expression is a versatile tool with diverse applications and can be used in soybeans to study gene function, obtain mutants, and produce proteins for commercial use. However, soybeans are considered recalcitrant for agroinfiltration. Subsequent studies on soybeans have demonstrated a green fluorescent protein (GFP) expression in seedpods, but not in leaves, using syringe agroinfiltration. To evaluate agroinfiltration-based transient protein expression levels in plant cells, we used the transient expression vector pTKB3 harboring the gene. Using , vacuum agroinfiltration of the leaves and needle agroinfiltration of the seedlings of different soybean varieties were performed. GFP was transiently expressed in all of the samples. However, the Enrei and Williams 82 varieties presented better results than the other varieties in the leaf tissue, with results confirmed by immunoblot analysis, demonstrating that both varieties are good candidates for molecular biological studies. GFP expression in the seedlings was less extensive than that in the leaves, which may be due to the tissue characteristics, with Enrei showing the best results. Based on this observation, we conclude that the Tsukuba system is an effective tool that can be used for different tissues and soybean varieties.
PubMed: 38592852
DOI: 10.3390/plants13060858