Transgenic Research最新文献

To explore the effects of salt-tolerance gene accumulation on salt tolerance in transgenic plant, we used four types of plant expression vector (N27, N28, N29, and N30) carrying mtlD, mtlD + gutD, mtlD + gutD + BADH, mtlD + gutD + BADH + sacB genes respectively, to transform tobacco through Agrobacterium-mediated method. Transgenic lines were identified through polymerase chain reaction (PCR) detection. Transgenic lines and non-transgenic plant (CK) were subjected to 6‰ sodium chloride solution stress; then, fluorescence quantitative PCR (FQ-PCR) and salt tolerance indexes were used to assess characteristics. PCR showed the exogenous genes had been integrated into the tobacco genome. FQ-PCR showed under clean water treatment the target genes were expressed in all transgenic plants at the transcriptional level. The transcript abundances of target genes changed with the number of genes increased, and improved following salt stress. Comparative analyses of salt tolerance indexes showed height growth, biomass (except for N29), chlorophyll content, net photosynthetic rate, Fv/Fm, and PI of all transgenic plants and CK were lower under salt stress than under clean water treatment, to varying degrees. However, the descent ratio was smaller in transgenic plants. A comprehensive evaluation of multiple salt-tolerance indicators performed using the membership function method showed the average salt tolerance of each vector transgenic line was higher than that of CK, and salt tolerance was greater in transgenic polyvalent gene lines than in transgenic monovalent gene lines. The average salt tolerance was N29 > N28 > N30 > N27 > CK. This study provides a theoretical and practical reference for salt tolerance breeding in other plants.

The involvement of Loose Plant Architecture 1 (LPA1) in regulating plant growth and leaf angle has been previously demonstrated. However, the fundamental genetic background remains unidentified. To further understand the tissue expression profile of the NtLPA1 gene, an overexpression vector (pBI121-NtLPA1) was developed and employed to modify tobacco using the leaf disc method genetically. Validation confirmed the generation of transgenic tobacco plants with NtLPA1 overexpression. The findings indicated that increased NtLPA1 overexpression substantially decreased plant auxin sensitivity and modulated signal transduction and polar transport, significantly reducing leaf angle, diminished leaf area during early and late growth stages, and shortened root length. In summary, NtLPA1 augmented tobacco resistance to severe shin disease by modulating the expression of disease-associated genes PBZ1, PR1b, and the growth regulator auxin polar transport factor PIN1.

Drought, as an abiotic stressor, globally limits cereal productivity, leading to early aging of leaves and lower yields. The expression of the isopentenyl transferase (IPT) gene, which is involved in cytokinin (CK) biosynthesis, can delay drought-induced leaf senescence. In this study, the Agrobacterium Isopentenyl transferase (IPT) gene was introduced into two local hexaploid wheat cultivars, NR-421 and FSD-2008. The expression cassette was developed containing the IPT gene under transcriptional regulation of the stress-inducible promoter 'Dehydrin,' sourced from Hordeum vulgare. The gene expression cassette was assembled in pSB219M, a modified transformation vector for monocots, equipped with both an antibiotic (spectinomycin) and an herbicide selection marker (BASTA). Initial screening of transgenic plants involved BASTA selection (2 and 3 mg/L) and was subsequently confirmed through PCR analysis. The transformation efficiencies of NR-421 and FSD-2008 were 0.4% and 0.3%, respectively. The qRT-PCR analysis under stress conditions showed a 13.5-fold higher expression of the IPT gene in T₂ transgenic plants of NR-421 and a 5.8-fold higher expression in those of FSD-2008 than in non-transgenic controls. Under stress conditions, the wheat transgenic plants exhibited increased chlorophyll and relative water content. Additionally, for total soluble proteins, two transgenic lines from the NR-421 variety showed a significant increase, whereas no notable change was observed in the FSD-2008 transgenics. Moreover, the transgenic lines displayed increased plant height, higher fresh and dry biomass, and increased seed weight compared to the non-transgenic controls. These findings highlight that stress-inducible expression of the IPT gene in wheat leads to enhanced grain yield and subsequently improved drought tolerance.

Lignin is a crucial defense phytochemical against phytophagous insects. Cinnamoyl-CoA reductase (CCR) is a key enzyme in lignin biosynthesis. In this study, transgenic Populus davidiana × P. bolleana overexpressing the PdbCCR gene were generated via Agrobacterium-mediated transformation. Successful integration of PdbCCR into the poplar genome was confirmed by PCR amplification and quantitative reverse transcription PCR (qRT-PCR). The lignin content in the transgenic poplar leaves was significantly higher than that in the wild poplar, and after L. dispar larvae fed on the transgenic poplar, the CCR activity was clearly induced. The L. dispar larvae grew slowly after feeding on transgenic poplar and the laccase, cellulase and three detoxifying enzymes were induced compared with larvae after feeding on wild-type poplar. The bioassay further revealed that transgenic poplar plants overexpressing PdbCCR showed a high level of resistance to L. dispar larvae. These results confirmed that PdbCCR is a candidate gene for breeding insect resistant poplar.

Proto-oncogene KRAS, GTPase (KRAS) is one of the most intensively studied oncogenes in cancer research. Although several mouse models allow for regulated expression of mutant KRAS, selective isolation and analysis of transforming or tumor cells that produce the KRAS oncogene remains a challenge. In our study, we present a knock-in model of oncogenic variant KRAS^G12D that enables the "activation" of KRAS^G12D expression together with production of red fluorescent protein tdTomato. Both proteins are expressed from the endogenous Kras locus after recombination of a transcriptional stop box in the genomic DNA by the enzyme flippase (Flp). We have demonstrated the functionality of the allele termed RedRas (abbreviated Kras^RR) under in vitro conditions with mouse embryonic fibroblasts and organoids and in vivo in the lung and colon epithelium. After recombination with adenoviral vectors carrying the Flp gene, the Kras^RR allele itself triggers formation of lung adenomas. In the colon epithelium, it causes the progression of adenomas that are triggered by the loss of tumor suppressor adenomatous polyposis coli (APC). Importantly, cells in which recombination has successfully occurred can be visualized and isolated using the fluorescence emitted by tdTomato. Furthermore, we show that KRAS^G12D production enables intestinal organoid growth independent of epidermal growth factor (EGF) signaling and that the KRAS^G12D function is effectively suppressed by specific inhibitor MRTX1133.

The transcription factor Tbx20 is integral to heart development and plays a significant role in various cardiac diseases. Despite its established importance, the regulatory mechanisms and functional significance of Tbx20 remain incompletely understood. To elucidate these mechanisms, we initially conducted eQTL mapping to identify genetic loci associated with Tbx20 expression in heart tissue from BXD mice. Co-expression and enrichment analyses revealed pathways linked to Tbx20, including dilated cardiomyopathy, hypertrophic cardiomyopathy, and FoxO signaling. Additionally, protein-protein interaction studies identified essential cardiac proteins, such as Myl2 and Myl7, along with upstream regulators like Mef2c. To validate our bioinformatic findings, we performed quantitative reverse transcription polymerase chain reaction (qRT-PCR) to assess the relative mRNA expression levels of TBX20 and Mef2c in the heart tissues of BXD mice compared to their parental strains (B6 and D2). Our results demonstrated significant up-regulation of both TBX20 and Mef2c in the BXD group relative to the parental strains. Conversely, both genes were down-regulated in B6, D2, Control, and Treatment groups when compared to BXD mice. These findings confirm the predicted regulatory roles of TBX20 and Mef2c in cardiac development as suggested by our initial analyses.This study not only reinforces the critical role of Tbx20 in cardiac gene regulation but also highlights its potential as a therapeutic target for cardiovascular disorders. Further investigations into Tbx20 and its interactions will enhance our understanding of heart biology and contribute to the development of targeted therapies for heart diseases.

The environmental risk assessment (ERA) of genetically modified (GM) crops in Japan requires collecting data from a comparative study of a GM and non-GM control in an in-country confined field trial (CFT). This in-country CFT requirement is used to address concerns that differences in the local environmental conditions may lead to differences in growth and/or risks of GM crops. However, this requirement for in-country CFT has recently been exempted for certain GM maize and GM cotton traits, and instead CFT data from other countries are used to inform the ERA of these GM events. However, in-country CFTs continue to be required for GM B. napus. Our objective is to assess whether using B. napus as a host crop increases the potential for differences between GM B. napus and conventional B. napus that may have an impact on biodiversity occurring only under the Japanese environment. In this paper agronomic data was compiled from seven local CFTs of GM B. napus events to assess the potential for differences between GM and non-GM B. napus for three key areas; competitiveness, potential to produce harmful substances, and outcrossing. Considering these elements, the need for conducting CFTs locally for ERA of future GM B. napus traits is discussed. The assessment concluded that conducting CFT locally is not necessary for GM B. napus events if traits do not bring competitive advantage or produce harmful substances only under Japanese environment.

This study aimed to develop a reliable and efficient genetic transformation method for the ornamental Indian Lotus (Nelumbo nucifera Gaertn.) using the sonication-assisted Rhizobium radiobacter-mediated transformation technique. To conduct the transformation, shoot apical meristem explants were infected with Rhizobium radiobacter (synonym Agrobacterium tumefaciens) strain LBA 4404 containing a binary vector pBI121 that harbours the GUS reporter gene (uidA) and kanamycin resistance gene nptII for plant selection. To improve the transformation efficiency, we optimized parameters such as bacterial cell density, sonication duration, infection time, co-cultivation duration, acetosyringone concentration, cefotaxime, and kanamycin concentrations. Sonication treatment at 42 kHz for 90 s recorded the highest transformation efficiency. The selection of regenerated plantlets was performed on a kanamycin-supplemented selection medium. The putative transformants showed GUS expression in the leaves and petioles. The presence of the GUS gene was also confirmed in the putative transformants through PCR, with the appearance of the expected amplicon size of 520 bp. The presence of nptII was confirmed by PCR in the putatively transformed plants with an amplicon size of 530 bp. The maximum regeneration frequency obtained was 72.66%, and the highest transformation efficiency achieved was 9.0% in the Indian Lotus.

Bombyx mori nuclear polyhedrosis, caused by B. mori nucleopolyhedrovirus (BmNPV), threatens sericulture seriously. To explore strategies for controlling it, the UDP glycosyltransferase gene UGT41A3 (BmUGT41A3) was targeted. UGT is involved in exogenous substances detoxification and endogenous biomass regulation in insects. Early embryos of the BmNPV-sensitive variety 'HYB' were used to obtain the transgenic line HYB-UGT41A3, overexpressing BmUGT41A3 under the IE1 promoter. qPCR results revealed that, compared with the wild-type control 'HYB', BmUGT41A3 was upregulated during the individual developmental stages of HYB-UGT41A3 from silkworm eggs to fifth-instar larvae; peak expression was observed in the third-instar larvae, which presented the most significantly upregulated expression. Individual-tissues qPCR results revealed that BmUGT41A3 expression was highest in the hemocytes of HYB-UGT41A3, followed by the midgut, whereas expression in HYB was very low. Gradient feeding of BmNPV on HYB-UGT41A3 and control 'HYB' larvae on the first day of the second-instar stage. The results revealed that the LC₅₀ of HYB-UGT41A3 reached 4.040 × 10⁷ particles/mL, which was 20-fold greater than that of HYB. The decrease in the BmNPV load was more significant in HYB-UGT41A3 than in HYB at 48 h after viral inoculation. These results indicate BmUGT41A3 overexpression inhibits BmNPV proliferation and improve resistance to BmNPV in B. mori.

Eremopyrum triticeum is a typical spring ephemeral species, which in China mainly distributed in the desert regions of northern Xinjiang, and play an important role in the desert ecosystems. E. triticeum has several adaptive characteristics such as short growth rhythms, high photosynthetic efficiency, high seed production, drought and salt resistance. However, the molecular regulatory mechanism of E. triticeum in responses to abiotic stress resistance is still unknown. In this study, two NAC-like transcription factor-encoding genes, EtNAC1 and EtNAC2, were isolated from E. triticeum. The predicted EtNAC1 and EtNAC2 proteins possess a typical NAC DNA-binding domain at the N-terminal region. The qRT-PCR analysis showed that EtNAC1 and EtNAC2 were highly expressed in mature roots of E. triticeum, and were significantly up-regulated under drought, high salt and abscisic acid (ABA) stresses. Subcellular localization analysis in onion epidermal cells revealed that EtNAC1 and EtNAC2 were located in the nucleus. Expression of EtNAC1 and EtNAC2 in yeast cells improved the survival rate of yeast under low temperature, H₂O₂, high drought and salt stresses. Overexpression of EtNAC1 and EtNAC2 in Arabidopsis thaliana conferred enhanced tolerance to drought and salt stresses, increased ABA sensitivity, and transgenic plants showed higher proline (Pro) content, but lower malondialdehyde content, lower chlorophyll leaching, lower water loss rate and stomatal aperture (width/length) than WT plants. In conclusion, EtNAC1 and EtNAC2 play important roles in abiotic stress responses of E. triticeum, which might have significant potential in crop molecular breeding for abiotic stress tolerance.