Transgenic Research最新文献

The Agrobacterium tumefaciens mediated transformation is the prevailing methodology for plant genetic manipulation; however, A. tumefaciens overgrowth is a common constraint in the process. Exploring auxotrophic A. tumefaciens could reduce overgrowth and enhance plant transformation efficiency. The ILVC gene, which encodes the ketol-acid isomeroreductase, is critical for Valine (Val) and isoleucine (Ile) biosynthesis in some microorganisms. However, its function in A. tumefaciens is unclear. To ascertain the function of ILVC and generate an auxotrophic A. tumefaciens, this study employed an allelic exchange to disrupt the ILVC in A. tumefaciens strain GV3101. This resulted in the loss of ketol-acid isomeroreductase activity and the prevention of Val and Ile biosynthesis, creating a dual-auxotrophic GV3101_∆ILVC. Transient expression assays in Nicotiana benthamiana transformation demonstrated that the GV3101_∆ILVC was capable of T-DNA transfer. Moreover, stable genetic transformation analysis in N. benthamiana indicated that the introduction of GV3101_∆ILVC led to a reduction in overgrowth within infected plant tissues. Additionally, an enhancement in transformation efficiency was observed with the prolongation of the co-cultivation time of the explant-infected strain. This study revealed the function of ILVC and explored a dual-auxotrophic A. tumefaciens for Val and Ile, potentially broadening the utilization of auxotrophic strains in plant genetic transformation.

Brucellosis, caused by Brucella species, is a global threat to livestock farming, resulting in economic losses and socio-economic challenges, particularly in rural areas. Despite its impact, no licensed human vaccines are available. Animal vaccination remains the most cost-effective control method, but traditional vaccine production is expensive. Edible vaccines, using plants as bioreactors to produce immunogenic antigens, offer a low-cost alternative by eliminating complex purification processes. This study developed a transgenic plant by expressing the Brucella abortus outer membrane protein OMP25 in tobacco plants. OMP25, a conserved transmembrane protein with high immunogenicity, was cloned into a Gateway pDONR vector via a Boundary Pairing reaction and transferred to a binary destination vector via a Left-Right reaction. The destination vector was introduced into Agrobacterium tumefaciens and subsequently used for Agrobacterium-mediated transformation of tobacco plants. Transgenic plants were selected on media containing kanamycin, and the expression of the transgene was verified through the fluorescence of green fluorescent protein. Microcallus formation and shoot development on selective media confirmed kanamycin resistance and the successful integration of the transgene. After phenotypic selection, genomic DNA was extracted from transgenic plants and analyzed by PCR (Polymerase Chain Reaction) using primers specific to the OMP25 gene. Positive PCR results validated the successful integration of the OMP25 gene into the plant genome. Gene expression was further confirmed at the RNA level through real-time quantitative PCR (qRT-PCR) and at the protein level via Western blot analysis. Future studies will evaluate immune responses in animal models. This approach demonstrates the potential for low-cost, effective vaccines to combat brucellosis, addressing critical economic and public health challenges.

Appropriate flowering time is important for rice regional adaptation and optimum rice production, but little is known about the omics of heading date in rice. Here, we studied omics including transcriptome, proteome and transcriptional factors to identify regulatory genes related to flowering time. A total of 1402 differentially expressed genes (DEGs, 721 up-regulated and 681 down-regulated) were detected in wild and mutant. These transcripts are classified according to biological processes, cellular components, and molecular functions. Among these differentially expressed genes, many transcription factor genes demonstrated multiple regulatory pathways involved in flowering time. Gene expression analysis showed that Os03g0122600 (OsMADS50), Os08g0105000 (Ehd3), Os06g0275000 (Hd1) were expressed higher and Os06g0199500 (OsHAL3), Os06g0498800 (OsMFT1), Os08g0105000 (Ehd3), Os06g0157700 (Hd3a), and Os02g0731700 (Ghd2), were expressed lower in wild compared to mutant, which are the key genes that regulate the flowering in rice. In addition, Ghd7 interacted with Os10g30860 and Os12g08260 using yeast two-hybrid assay. We identified 28 potential Ghd7 transcriptional regulators using the transcription factor-centered yeast one hybrid (TF-Centered Y1H) assay. Taken together, this study developed a new set of genomic resources to identify and characterize genes, proteins, and motifs associated with flowering time.

Folate (vitamin B9) is an essential nutrient that plays a crucial role in various bodily functions. Its deficiency can lead to health issues, such as megaloblastic anemia, neural tube defects in the developing fetus and increased risk of cardiovascular diseases. Consequently, there is interest in increasing folate content in food crops by classical and molecular breeding. Since folate is a water-soluble vitamin that is sensitive to heat, we chose to manipulate its synthesis pathway in lettuce, which is often used for salads and sandwiches, without heat treatment, which makes it a good vehicle for making folate available to the population via biofortification. Transgenic lettuce plants were generated to express the GTP-cyclohydrolase I and aminodeoxychorismate synthase coding sequences from Arabidopsis thaliana. Plants were cultivated under greenhouse conditions and two field trials were carried out. Results have shown that transgenic lines presented up to 3.4 times more folate than the non-transgenic lettuce plants and 1.9 times more folate than spinach, considered one of the plants richest in folates. Advanced generations of homozygous plants were cultivated under field conditions for two years and the results showed that folate-biofortification was stable. A folate-biofortified lettuce serving would provide about 36 to 64% of the recommended daily intake. This technology is a foundation to produce folate-biofortified commercial varieties that can help to reduce hidden hunger, decreasing the number of cases of fetus malformations and other diseases.

With the continuous expansion of the planting area of genetically modified (GM) crops, the demand for efficient and comprehensive monitoring systems is becoming increasingly urgent. To establish a method suitable for large-scale monitoring of genetically modified rapeseed planting, beehives were strategically deployed at specific locations around genetically modified rapeseed fields, and the TaqMan quantitative PCR (qPCR) method was used to detect and analyze the genetically modified components in the rapeseed pollen collected by bees. The results demonstrated that the average Ct values for the CaMV35S promoter, Bar gene, NPTII gene, and HPT gene in the pollen of each hive were 27.91, 29.58, 31.49, and 31.97, respectively. The average ΔCt values for these four genes in hive pollen from 100 to 200 m were - 0.35, 1.66, 2.58, and 5.06, respectively, which were significantly lower than those from 300 to 1100 m (2.85, 4.01, 6.66, and 5.63). The results of this study have demonstrated the feasibility of using pollen collected by bees for large-scale detection of genetically modified rapeseed plants. This early warning model for GM crop spread based on bee pollination provides an efficient and practical solution for monitoring and managing genetically modified crops.

One of the nutrients that is necessary for plant growth and development is potassium (K⁺). The uneven production and distribution of global potassium resources significantly challenge crop yields and quality. A moderate increase in the potassium content within plants can enhance both crop yield and quality. This study identifies the Shaker K⁺ channel NKT3A within the model crop, tobacco. The yeast heterologous expression system demonstrated its capability for K⁺ inward transportation. GUS staining and RT-qPCR analyses of the constructed promoter materials revealed NKT3A's activity during the tobacco seedling stage. Expression levels are higher in the leaf and stems, with low potassium levels inducing upregulation of its expression, also observed in roots. Gene editing technology was employed to construct overexpression and knockout mutants, with subsequent measurement of their phenotypes. Results indicate that NKT3A expression enhances facilitates potassium absorption and transport in tobacco seedlings under low potassium conditions. For the first time, this article identifies the Shaker potassium channel gene NKT3A, which functions as an inward rectifier K⁺ channel in tobacco. It elucidates the gene's role in regulating potassium distribution under low potassium conditions, thereby deepening our understanding of plant responses in such environments and offering a potential target for enhancing crop potassium use efficiency.

Prostatic acid phosphatase (PAP) is a specific protein that is highly expressed in prostate cancer. In this study, we constructed two recombinant PAP fusion genes: PAP fused to the immunoglobulin G (IgG) Fc fragment (designated PAP-Fc) and PAP-Fc fused to the endoplasmic reticulum retention sequence KDEL (designated PAP-FcK). Transgenic Nicotiana tabacum plants expressing these recombinant macromolecular proteins (MPs) were generated using Agrobacterium-mediated transformation, and the presence of both genes was confirmed through genomic PCR. Western blot analysis validated the expression of PAP-Fc and PAP-FcK MPs, which were successfully purified via protein A affinity chromatography. Size-exclusion high-performance liquid chromatography revealed dimeric peaks for PAP-Fc (PAP-Fc^P) and PAP-FcK (PAP-FcK^P). Bio-transmission electron microscopy demonstrated 'Y'-shaped protein particles resembling antibody structures. Moreover, PAP-Fc^P and PAP-FcK^P exhibited a high association rate with human FcγR and FcRn. Vaccination of mice with both PAP-Fc^P and PAP-FcK^P resulted in increased total IgG against PAP and enhanced activation of CD4⁺ T cells, comparable to mice immunized with PAP, which served as a positive control. These findings indicate that both plant-derived MPs can effectively induce adaptive immunity, positioning them as promising candidates for prostate cancer vaccines. Overall, plants expressing PAP-Fc and PAP-FcK represent a viable production system for antigenic macromolecule-based prostate cancer vaccines.

Gene knockout using CRISPR-Cas9 is often employed in research aimed at elucidating gene functions in fish. However, CRISPR-Cas9 sometimes introduces unintended alterations, known as off-target mutations. These mutations can reduce the robustness of data during phenotypic analysis. In this study, we focused on the culture temperature, which is known to significantly influence mutagenesis, and examined whether low-temperature culture after introducing CRISPR-Cas9 into early embryos of medaka and zebrafish suppresses off-target mutations. Continuous incubation of medaka at 16 °C significantly reduced off-target mutation rates compared to those at 28 °C; the drawback is that it decreased the survival rate of medaka embryos. Therefore, low-temperature incubation was limited to early development in both zebrafish and medaka, and then the temperature was increased to 28 °C. Under these conditions, the mutation rates of the three off-target regions in medaka (Off-D, Off-P, and Off-A) significantly decreased, whereas those of the three target regions (DJ-1, p4hb, and avt) were unaffected. Similarly, the mutation rate of the zebrafish target region (ywhaqa) remained high, whereas the off-target (Off-Y1) mutation rate significantly reduced. Furthermore, this method effectively suppressed the germ line transmission of off-target mutations in medaka. This approach is effective to obtain more reliable data from the G0 generation of medaka and zebrafish and may reduce the screening effort required to remove individuals with off-target mutations in the F1 generation.