Introduction Wall-associated kinases (WAKs) are pivotal in linking plant cell walls to intracellular signaling networks, thereby playing essential roles in plant growth, development, and stress responses. Methods The bioinformatics analysis was employed to identify WAK genes in tobacco. The expression levels of NtWAK genes were assessed by qRT-PCR. The subcellular localization of WAK proteins was observed in tobacco cells and Arabidopsis protoplasts. Kinase activity of the WAK proteins was evaluated through in vitro assays. Results We conducted a comprehensive genome-wide identification and analysis of the WAK gene family in tobacco ( Nicotiana tabacum ). A total of 44 WAK genes were identified in the tobacco genome, which were further classified into three distinct groups. Phylogenetic analysis comparing tobacco WAKs (NtWAKs) with Arabidopsis WAKs (AtWAKs) revealed species-specific expansion of these genes. The WAK proteins within each group displayed similar gene structures and conserved motif distributions. Promoter region analysis indicated that cis-elements of NtWAK genes are primarily involved in regulating plant growth and development, phytohormone signaling, and stress responses. Expression profiling under NaCl, PEG, and ABA treatments suggested that certain NtWAK genes may play key roles in modulating responses to abiotic stress. Three-dimensional structural predictions and subcellular localization analysis showed that NtWAK proteins from the three subgroups exhibit high cytoplasmic similarity and are primarily located to the plasma membrane. Kinase activity assay confirmed that they possess phosphorylation activity. Discussion This study represents the first genome-wide analysis of the WAK gene family in N. tabacum , laying the groundwork for future functional investigations.
Long non-coding RNAs (lncRNAs) regulate many biological processes in plants, including defense against pathogens and herbivores. Recently, many small ORFs embedded in lncRNAs have been identified to encode biologically functional peptides (small ORF-encoded peptides [SEPs]) in many species. However, it is unknown whether lncRNAs mediate defense against herbivore attack and whether there are novel functional SEPs for these lncRNAs. By sequencing Spodoptera litura-treated leaves at six time-points in Nicotiana tabacum, 22,436 lncRNAs were identified, of which 787 were differentially expressed. Using a comprehensive mass spectrometry (MS) pipeline, 302 novel SEPs derived from 115 tobacco lncRNAs were identified. Moreover, 61 SEPs showed differential expression after S. litura attack. Importantly, several of these peptides were characterized through 3D structure prediction, subcellular localization validation by laser confocal microscopy, and western blotting. Subsequent bioinformatic analysis revealed some specific chemical and physical properties of these novel SEPs, which probably represent the largest number of SEPs identified in plants to date. Our study not only identifies potential lncRNA regulators of plant response to herbivore attack but also serves as a valuable resource for the functional characterization of SEP-encoding lncRNAs.
Endophytes, symbiotically with their host plants, are frequently found in the roots of Panax ginseng. To explore ginsenosides biotransformation by endophytic fungi, 26 such fungi were isolated from ginseng roots sourced from four distinct planting regions, and their glycosyl hydrolysis activities were measured. Of these endophytic, 20 isolates exhibited glycosidase enzymes capable of cleaving glycosidic bonds of glucose, arabinofuranose and arabinopyranose. Notably, the glycosidase BglNh gene was isolated and cloned from Nectria haematococca, one of the endophytic fungi. Enzymatic assessments unveiled that BglNh specifically catalyzes the hydrolysis of glucose residues located external to the C-3 position of protopanaxadiol ginsenosides under optimal reaction conditions (pH 6.0 and temperature 35 °C). Our findings demonstrate the efficient conversion of major ginsenosides Rb1, Rb2 and Rc to minor ginsenosides Gyp17, CO and CMc1, highlighting the potential of these fungi for ginsenoside biotransformation and bioactive compound production.
The plant hormone jasmonic acid (JA) has an important role in many aspects of plant defense response and developmental process. JA triggers interaction between the F-box protein COI1 and the transcriptional repressors of the JAZ family that leads the later to proteasomal degradation. The Jas-motif of JAZs is critical for mediating the COI1 and JAZs interaction in the presence of JA. Here, by using the protoplast transient gene expression system we reported that the Jas-motif of JAZ1 was necessary and sufficient to target a foreign reporter protein for COI1-facilitated degradation. We fused the Jas-motif to the SHY2 transcriptional repressor of auxin signaling pathway to create a chimeric protein JaSHY. Interestingly, JaSHY retained the transcriptional repressor function while become degradable by the JA coreceptor COI1 in a JA-dependent fashion. Moreover, the JA-induced and COI1-facilitated degradation of JaSHY led to activation of a synthetic auxin-responsive promoter activity. These results showed that the modular components of JA signal transduction pathway can be artificially redirected to regulate auxin signaling pathway and control auxin-responsive gene expression. Our work provides a general strategy for using synthetic biology approaches to explore and design cell signaling networks to generate new cellular functions in plant systems.
The aim of the present study was to explore the underlying mechanism of microRNA‑338‑3p (miR‑338‑3p) in lung cancer cell (A549) invasion and proliferation. A microarray assay showed that miR‑338‑3p upregulated 216 and downregulated 147 genes in A549 cells, and the differentially expressed genes were enriched for several signaling pathways, including AKT and β‑catenin signaling pathways. Western blotting results showed that phosphorylated (p)‑AKT at Ser473 and at Thr308 and p‑β‑catenin at Ser552 were downregulated. Inhibiting AKT signaling pathway decreased β‑catenin signaling pathway. Overexpression of β‑catenin promoted the invasion of A549 cells. In addition, miR‑338‑3p showed inhibitory effect on the primary tumor developed from KrasG12D mice. Together, the data of the present study support that miR‑338‑3p inhibits lung cancer cell invasion and proliferation by downregulating AKT/β‑catenin signaling pathway. The present findings supported the potential use of miR‑338‑3p in the treatment of lung cancer.
To prepare the monoclonal antibodies (mAbs) against human carbohydrate antigen 19-9 (CA19-9) and establish a double-antibody sandwich chemiluminescent immunoassay (CLIA) detection system for CA19-9 in the human serum.BALB/c mice were immunized with human CA19-9 antigen. The mAbs were obtained by hybridoma technique. The purity, titer, specificity and pairing of the mAbs were characterized and the sandwich CLIA system was established. The system was evaluated in its accuracy, limit of detection, linearity and repetitiveness after optimization of coating buffer, coating concentration and pipetting mode, and the serum sample was tested with it.Four mAbs named #1-1, #2-1, #3-1 and #4-1 were obtained against human CA19-9. The titers of the anti-CA19-9 mAbs were above 10(-8). The mAbs had nearly no cross-reaction with CA125, CA15-3 and CA72-4. The double-antibody sandwich CLIA system was established by #3-1 mAb and #2-1 mAb-HRP. After optimization, its property was detection range of 0-1 000 U/mL, limit detection of 2.0 U/mL, linear correlation coefficient of 0.9999. The results which were contrasted with Roche test showed that: Kappa>0.75, r(correlation coefficient)>0.9.The mAbs against human CA9-9 have been prepared and a sandwich CLIA system for detecting CA19-9 in the human serum has been established successfully.
Objective Construction of monoclonal antibody cell lines against human free prostate specific antigen (f-PSA), and establish double antibody sandwich CLIA for human f-PSA. Methods Two hybridoma cell lines secreting anti-f-PSA mAb were obtained by hybridoma technique. The cell lines were expanded by spinner bottles and purified by affinity purification, from which the antibodies were tested for antibody titers, specificity, epitopes by ELISA and affinity by surface plasmon resonance. Establishment, analysis and performance evaluation of a double mAbs sandwich CLIA system using standard curve quantitative method. In total 426 (130 gray area) clinical specimens were used for system comparison between our assay and Roche f-PSA assay. Results anti- f-PSA(f-10-1, f-14-1) mAbs were obtained. The CLIA system with detection range of 0.1-30 ng/mL, sensitivity of 0.05 ng/mL, relative detection deviation of ±5% and not cross-react with the tumor marker AFP, CEA and c-PSA. The correlation coefficient of our reagent with Roche's was 0.99. The positive coincidence rate, negative coincidence rate and total coincidence rate of gray area specimens were all higher than 90%. Conclusion mAbs against human f-PSA were successfully prepared, and the double mAbs sandwich CLIA for specific quantitative detection of human f-PSA was established.
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