GFP Fusion Research Articles

Creation of a functional unc-11/PICALM GFP knock-in by CRISPR.

unc-11 is the only C. elegans ortholog of mammalian PICALM/AP180, paralogs that play an important role in Clathrin-Mediated Endocytosis (CME) and the recycling of a subset of SNAREs, the vesicle-associated membrane proteins (VAMPs). In this publication we report the creation of a new unc-11 allele that is endogenously-tagged with GFP just upstream of the stop codon. Moreover, we demonstrate that the UNC-11::GFP fusion protein functions like wild type with an expression pattern similar to UNC-11 antibody staining described previously.

Simple and sensitive detection of deoxyribonucleic acid using a RecA-GFP fusion protein-DNA filament as probe.

A simple, rapid and highly sensitive method for detection of double-stranded DNA (dsDNA) was developed using a novel fluorescence probe composed of a RecA-GFP fusion protein that had specific recognition of ssDNA complexes (RecA-GFP-DNA filament). The RecA-GFP fusion protein not only had strong fluorescence, but could also occur by homologous recombination. In the presence of the target dsDNA, the complementary ssDNA of the RecA-GFP-DNA filaments invaded one end of the dsDNA chain. In addition, the other end of the ssDNA dissociated the RecA-GFP filaments. An assay of the probe showed a linear relationship with dsDNA concentration in the range 1-11 nM, with a correlation coefficient of 0.9923. The limit of detection for dsDNA was determined experimentally to be 0.3 nM (3δ). Compared with conventional methods, this method has the advantages of simple operation, high specificity, and high sensitivity.

Design and evaluation of small interfering RNAs for the treatment of Severe Acute Respiratory Syndrome-Coronavirus-2

SARS-CoV-2 is the virus responsible for the COVID-19 pandemic. As the 2019 coronavirus disease continues to spread, it will be useful to have as many effective treatment options as possible. This research has the potential to create a siRNA treatment that is safe, effective, and practical in design and administration; 192 siRNAs were designed to target conserved regions of the SARS-CoV-2 genome. The first aim of this study is to confirm, via sequence analysis, that these target sites have remained highly conserved over the course of the pandemic. Multiple sequence alignments were generated for the first half of 30,312 full SARS-CoV-2 genomes, which were averaged and compared with the Wuhan-Hu-1 reference genome. Most target sites maintained a very high level of conservation, suggesting that potential repressor siRNAs could be effective in a majority of infected individuals. To evaluate the efficacy of the 192 test siRNAs, we cloned sections of the SARS-CoV-2 RNA genome into GFP fusion genes. Some of these constructs were transfected in different conditions to set up a screening assay based on GFP expression. Preliminary data on the setup of this GFP reporter assay show that the M, N, E, ORF8, and ORF10 constructs produced a good GFP signal, whereas the S, F1, F2 and F3 constructs did not produce a sufficiently strong GFP signal to detect above background. In a preliminary experiment, we evaluated siRNAs targeting the M, N, and E open reading frames and found some to be efficacious. Future directions for this project include generating alignments of the second half of the SARS-CoV-2 genome for a complete sequence conservation estimate, and cell metabolism assays for supplementing visual observations of siRNA toxicity, optimization of GFP readout, and screening of all designed siRNAs.

Cprp-An Unusual, Repetitive Protein Which Impacts Pleuromutilin Biosynthesis in the Basidiomycete Clitopilus passeckerianus.

Interrogation of an EST database for Clitopilus passeckerianus identified a putative homolog to the unusual stress response gene from yeast; ddr48, as being upregulated under pleuromutilin production conditions. Silencing of this gene, named cprp, produced a population of transformants which demonstrated significantly reduced pleuromutilin production. Attempts to complement a Saccharomyces cerevisiae ddr48 mutant strain (strain Y16748) with cprp were hampered by the lack of a clearly identifiable mutant phenotype, but interestingly, overexpression of either ddr48 or cprp in S. cerevisiae Y16748 led to a conspicuous and comparable reduction in growth rate. This observation, combined with the known role of DDR48 proteins from a range of fungal species in nutrient starvation and stress responses, raises the possibility that this family of proteins plays a role in triggering oligotrophic growth. Localization studies via the production of a Cprp:GFP fusion protein in C. passeckerianus showed clear localization adjacent to the hyphal septa and, to a lesser extent, cell walls, which is consistent with the identification of DDR48 as a cell wall-associated protein in various yeast species. To our knowledge this is the first study demonstrating that a DDR48-like protein plays a role in the regulation of a secondary metabolite, and represents the first DDR48-like protein from a basidiomycete. Potential homologs can be identified across much of the Dikarya, suggesting that this unusual protein may play a central role in regulating both primary and secondary metabolism in fungi.

Adenovirus Terminal Protein Contains a Bipartite Nuclear Localisation Signal Essential for Its Import into the Nucleus.

Adenoviruses contain dsDNA covalently linked to a terminal protein (TP) at the 5′end. TP plays a pivotal role in replication and long-lasting infectivity. TP has been reported to contain a nuclear localisation signal (NLS) that facilitates its import into the nucleus. We studied the potential NLS motifs within TP using molecular and cellular biology techniques to identify the motifs needed for optimum nuclear import. We used confocal imaging microscopy to monitor the localisation and nuclear association of GFP fusion proteins. We identified two nuclear localisation signals, PV(R)6VP and MRRRR, that are essential for fully efficient TP nuclear entry in transfected cells. To study TP–host interactions further, we expressed TP in Escherichia coli (E. coli). Nuclear uptake of purified protein was determined in digitonin-permeabilised cells. The data confirmed that nuclear uptake of TP requires active transport using energy and shuttling factors. This mechanism of nuclear transport was confirmed when expressed TP was microinjected into living cells. Finally, we uncovered the nature of TP binding to host nuclear shuttling proteins, revealing selective binding to Imp β, and a complex of Imp α/β but not Imp α alone. TP translocation to the nucleus could be inhibited using selective inhibitors of importins. Our results show that the bipartite NLS is required for fully efficient TP entry into the nucleus and suggest that this translocation can be carried out by binding to Imp β or Imp α/β. This work forms the biochemical foundation for future work determining the involvement of TP in nuclear delivery of adenovirus DNA.

Membrane localized thaumatin-like protein from tea (CsTLP) enhanced seed yield and the plant survival under drought stress in Arabidopsis thaliana

Thaumatin-like proteins (TLPs) are pathogenesis-related (PR5) proteins, which are induced in response to various biotic and abiotic stresses. The present work was carried out to clone TLP of Camellia sinensis (CsTLP) and to evaluate the response of transgenic lines of Arabidopsis constitutively expressing CsTLP under drought conditions. Data showed that transgenic lines exhibited lower relative electrolyte leakage and higher water retention capacity as compared to the wild-type (WT) plants under drought stress. In addition, results with confocal microscopy showed CsTLP + GFP fusion protein to be localized in the cell membrane which moved to the intercellular spaces under prolonged drought stress. Expression of CsTLP enhanced seed yield and the plant survival in transgenic lines as compared to the WT plants under drought stress. Results suggested the importance of CsTLP in improving drought tolerance in Arabidopsis.

Fluorescence-detection size-exclusion chromatography utilizing nanobody technology for expression screening of membrane proteins

GFP fusion-based fluorescence-detection size-exclusion chromatography (FSEC) has been widely employed for membrane protein expression screening. However, fused GFP itself may occasionally affect the expression and/or stability of the targeted membrane protein, leading to both false-positive and false-negative results in expression screening. Furthermore, GFP fusion technology is not well suited for some membrane proteins, depending on their membrane topology. Here, we developed an FSEC assay utilizing nanobody (Nb) technology, named FSEC-Nb, in which targeted membrane proteins are fused to a small peptide tag and recombinantly expressed. The whole-cell extracts are solubilized, mixed with anti-peptide Nb fused to GFP for FSEC analysis. FSEC-Nb enables the evaluation of the expression, monodispersity and thermostability of membrane proteins without the need for purification but does not require direct GFP fusion to targeted proteins. Our results show FSEC-Nb as a powerful tool for expression screening of membrane proteins for structural and functional studies.

The Doublesex/Mab-3 domain transcription factor DMD-10 regulates ASH-dependent behavioral responses.

The Doublesex/Mab-3 Domain transcription factor DMD-10 is expressed in several cell types in C. elegans, including in the nervous system. We sought to investigate whether DMD-10 is required for normal neuronal function using behavioral assays. We found that mutation of dmd-10 did not broadly affect behavior. dmd-10 mutants were normal in several behavioral assays including a body bends assay for locomotion, egg laying, chemotaxis and response to gentle touch to the body. dmd-10 mutants did have defects in nose-touch responsiveness, which requires the glutamate receptor GLR-1. However, using quantitative fluorescence microscopy to measure levels of a GLR-1::GFP fusion protein in the ventral nerve cord, we found no evidence supporting a difference in the number of GLR-1 synapses or in the amount of GLR-1 present in dmd-10 mutants. dmd-10 mutants did have decreased responsiveness to high osmolarity, which, along with nose-touch, is sensed by the polymodal sensory neuron ASH. Furthermore, mutation of dmd-10 impaired behavioral response to optogenetic activation of ASH, suggesting that dmd-10 promotes neuronal signaling in ASH downstream of sensory receptor activation. Together our results suggest that DMD-10 is important in regulating the frequency of multiple ASH-dependent behavioral responses.

The faulty SOS response of Pseudomonas putida KT2440 stems from an inefficient RecA-LexA interplay.

Despite its environmental robustness Pseudomonas putida strain KT2440 is very sensitive to DNA damage and displays poor homologous recombination efficiencies. To gain an insight into this deficiency isogenic ∆recA and ∆lexA1 derivatives of prophage-free strain P. putida EM173 were generated and responses of the recA and lexA1 promoters to DNA damage tested with GFP reporter technology. Basal expression of recA and lexA1 of P. putida were high in the absence of DNA damage and only moderately induced by norfloxacin. A similar behaviour was observed when equivalent GFP fusions to the recA and lexA promoters of E. coli were placed in P. putida EM173. In contrast, all SOS promoters were subject to strong repression in E. coli, which was released only when cells were treated with the antibiotic. Replacement of P. putida's native LexA1 and RecA by E. coli homologues did not improve the responsiveness of the indigenous functions to DNA damage. Taken together, it seems that P. putida fails to mount a strong SOS response due to the inefficacy of the crucial RecA-LexA interplay largely tractable to the weakness of the corresponding promoters and the inability of the repressor to shut them down entirely in the absence of DNA damage.

Identification, Gene Structure, and Expression of BnMicEmUP: A Gene Upregulated in Embryogenic Brassica napus Microspores.

Microspores of Brassica napus can be diverted from normal pollen development into embryogenesis by treating them with a mild heat shock. As microspore embryogenesis closely resembles zygotic embryogenesis, it is used as model for studying the molecular mechanisms controlling embryo formation. A previous study comparing the transcriptomes of three-day-old sorted embryogenic and pollen-like (non-embryogenic) microspores identified a gene homologous to AT1G74730 of unknown function that was upregulated 8-fold in the embryogenic cells. In the current study, the gene was isolated and sequenced from B. napus and named BnMicEmUP (B. napus microspore embryogenesis upregulated gene). Four forms of BnMicEmUP mRNA and three forms of genomic DNA were identified. BnMicEmUP2,3 was upregulated more than 7-fold by day 3 in embryogenic microspore cultures compared to non-induced cultures. BnMicEmUP1,4 was highly expressed in leaves. Transient expression studies of BnMicEmUP3::GFP fusion protein in Nicotiana benthamiana and in stable Arabidopsis transgenics showed that it accumulates in chloroplasts. The features of the BnMicEmUP protein, which include a chloroplast targeting region, a basic region, and a large region containing 11 complete leucine-rich repeats, suggest that it is similar to a bZIP PEND (plastid envelope DNA-binding protein) protein, a DNA binding protein found in the inner envelope membrane of developing chloroplasts. Here, we report that the BnMicEmUP3 overexpression in Arabidopsis increases the sensitivity of seedlings to exogenous abscisic acid (ABA). The BnMicEmUP proteins appear to be transcription factors that are localized in plastids and are involved in plant responses to biotic and abiotic environmental stresses; as well as the results obtained from this study can be used to improve crop yield.

Comparison between two isoforms of glycerol-3-phosphate acyltransferase in microalga Myrmecia incisa: Subcellular localization and role in triacylglycerol synthesis

Microalgae represent a rich and naturally occurring source of triacylglycerol (TAG), and the microalgae-derived TAG has enormous potential in bioenergy, bio-based materials and other agricultural innovations. Glycerol-3-phosphate acyltransferase (GPAT) is a key enzyme that catalyzes the biosynthesis of TAG. Previously our lab has reported the first GPAT in the oleaginous alga Myrmecia incisa, and in the present study, a new GPAT was further identified from the same alga, which was designated as MiGPAT2. A full-length cDNA of MiGPAT2 consisting of a 1374-bp ORF, a 153-bp 5′-UTR and a 271-bp 3′-UTR was cloned. The putative protein composed of 457 amino acids, possessing the HX4D motif that acts as the activity center of GPAT catalysis. Emphasis was put on the comparison between two MiGPATs regarding subcellular localization and TAG synthesis function. A polyclonal and monoclonal antibody were separately prepared for MiGPAT1 and MiGPAT2, and the following immunogold labeling showed they were localized on chloroplast and endoplasmic reticulum (ER), respectively. This was further confirmed by the GFP fusion studies. Both MiGPATs were expressed heterologously in a GPAT-deficient yeast mutant, and it was found that MiGPAT2 played key roles in the de novo TAG biosynthesis, whereas MiGPAT1 was more involved in the phospholipid formation, contributing to TAG production via an indirect way. Findings of the present study expanded our knowledge on functional and evolutionary relationships of GPAT members in M. incisa, suggesting the possibility of genetic manipulation of GPAT in this alga for enhanced TAG production.

The ten amino acids of the oxygen-evolving enhancer of tobacco is sufficient as the peptide residues for protein transport to the chloroplast thylakoid

Key messageThe thylakoid transit peptide of tobacco oxygen-evolving enhancer protein contains a minimal ten amino acid sequences for thylakoid lumen transports. This ten amino acids do not contain twin-arginine, which is required for typical chloroplast lumen translocation.Chloroplasts are intracellular organelles responsible for photosynthesis to produce organic carbon for all organisms. Numerous proteins must be transported from the cytosol to chloroplasts to support photosynthesis. This transport is facilitated by chloroplast transit peptides (TPs). Four chloroplast thylakoid lumen TPs were isolated from Nicotiana tabacum and were functionally analyzed as thylakoid lumen TPs. Typical chloroplast stroma-transit peptides and thylakoid lumen transit peptides (tTPs) are found in N. tabacum transit peptides (NtTPs) and the functions of these peptides are confirmed with TP–GFP fusion proteins under fluorescence microscopy and chloroplast fractionation, followed by Western blot analysis. During the functional analysis of tTPs, we uncovered the minimum 10 amino acid sequence is sufficient for thylakoid lumen transport. These ten amino acids can efficiently translocate GFP protein, even if they do not contain the twin-arginine residues required for the twin-arginine translocation (Tat) pathway, which is a typical thylakoid lumen transport. Further, thylakoid lumen transporting processes through the Tat pathway was examined by analyzing tTP sequence functions and we demonstrate that the importance of hydrophobic core for the tTP cleavage and target protein translocation.

Functional diversity of three tandem C-terminal carbohydrate-binding modules of a β-mannanase

Carbohydrate active enzymes, such as those involved in plant cell wall and storage polysaccharide biosynthesis and deconstruction, often contain repeating noncatalytic carbohydrate-binding modules (CBMs) to compensate for low-affinity binding typical of protein–carbohydrate interactions. The bacterium Saccharophagus degradans produces an endo-β-mannanase of glycoside hydrolase family 5 subfamily 8 with three phylogenetically distinct family 10 CBMs located C-terminally from the catalytic domain (SdGH5_8-CBM10x3). However, the functional roles and cooperativity of these CBM domains in polysaccharide binding are not clear. To learn more, we studied the full-length enzyme, three stepwise CBM family 10 (CBM10) truncations, and GFP fusions of the individual CBM10s and all three domains together by pull-down assays, affinity gel electrophoresis, and activity assays. Only the C-terminal CBM10-3 was found to bind strongly to microcrystalline cellulose (dissociation constant, Kd = 1.48 μM). CBM10-3 and CBM10-2 bound galactomannan with similar affinity (Kd = 0.2–0.4 mg/ml), but CBM10-1 had 20-fold lower affinity for this substrate. CBM10 truncations barely affected specific activity on carob galactomannan and konjac glucomannan. Full-length SdGH5_8-CBM10x3 was twofold more active on the highly galactose-decorated viscous guar gum galactomannan and crystalline ivory nut mannan at high enzyme concentrations, but the specific activity was fourfold to ninefold reduced at low enzyme and substrate concentrations compared with the enzyme lacking CBM10-2 and CBM10-3. Comparison of activity and binding data for the different enzyme forms indicates unproductive and productive polysaccharide binding to occur. We conclude that the C-terminal-most CBM10-3 secures firm binding, with contribution from CBM10-2, which with CBM10-1 also provides spatial flexibility.

Hsp104 contributes to freeze-thaw tolerance by maintaining proteasomal activity in a spore clone isolated from Shirakami kodama yeast.

The supply of oven-fresh bakery products to consumers has been improved by frozen dough technology; however, freeze-thaw stress decreases the activity of yeast cells. To breed better baker's yeasts for frozen dough, it is important to understand the factors affecting freeze-thaw stress tolerance in baker's yeast. We analyzed the stress response in IB1411, a spore clone from Saccharomyces cerevisiae Shirakami kodama yeast, with an exceptionally high tolerance to freeze-thaw stress. Genes encoding trehalose-6-phosphate synthase (TPS1), catalase (CTT1), and disaggregase (HSP104) were highly expressed in IB1411 cells even under conditions of non-stress. The expression of Hsp104 protein was also higher in IB1411 cells even under non-stress conditions. Deletion of HSP104 (hsp104Δ) in IB1411 cells reduced the activity of the ubiquitin-proteasome system (UPS). By monitoring the accumulation of aggregated proteins using the ΔssCPY*-GFP fusion protein under freeze-thaw stress or treatment with proteasomal inhibitor, we found that IB1411 cells resolved aggregated proteins faster than the hsp104Δ strain. Thus, Hsp104 seems to contribute to freeze-thaw tolerance by maintaining UPS activity via the disaggregation of aggregated proteins. Lastly, we found that the IB1411 cells maintained high leavening ability in frozen dough as compared with the parental strain, Shirakami kodama yeast, and thus will be useful for making bread.

Identification of a cross-talk between EGFR and Wnt/beta-catenin signaling pathways in HepG2 liver cancer cells

EGFRis a transmembrane receptor tyrosine kinase involved in regulating cell proliferation, differentiation and survival. EGFR is actively pursued as a therapeutic target because its aberrant expression or activity has been reported in several cancers. Several studies have reported the nuclear localization of the EGFR in various cell types, however, its exact nuclear functions are not clear yet. In this study, we have generated GFP fusion constructs of EGFR and its mutants to analyze their subcellular localizationin normal and cancer cells and impact of its sub-cellular location on its various activities using immunoblotting, confocal microscopy, reporter assays, loss-of-function EGFR mutants, and EGFR specific small molecule inhibitors. We show that EGFR is involved in modulating TCF dependent β-catenin transcriptional activity in HepG2 cells in a similar fashion as IGF1R tyrosine kinase. Moreover, we show that cytoplasmic and nuclear functions are two independent activities of EGFR.

Thylakoid Transit Peptide Is Related to the Expression and Localization of NdhB Subunits in Soybean

The chloroplast NAD(P)H dehydrogenase (NDH) complex, as one of the most important photosynthesis protein complexes in thylakoid membrane, is involved in photosystem I (PSI) cyclic electron transport (CEF). Under abiotic environmental stress, the photosynthetic apparatus is susceptible to the damage caused by the strong light illumination. However, the enhancement of NDHdependent CEF could facilitate the alleviation of the damage to the photosynthetic apparatus. The NdhB subunit encoded by chloroplast genome is one of most important subunits of NDH complex and consists of 510 amino acids. Here, according to cloning ndhB from Melrose (cultivated soybean), ACC547 (wild salt-tolerant soybean), S113-6 and S111-9 (hybrid descendant), based on the comparison and analysis of the sequences of NdhB subunits, we found that there is a novel thylakoid transit peptide of NdhB subunit in S111-9. In addition, crosslink immunoprecipitation, immunogold labeling and co-expression of GFP fusion protein indicated that the novel thylakoid transit peptide is favorable to the expression and localization of NdhB subunit in chloroplast. Therefore, we suggest that this novel thylakoid transit peptide plays the same role as chaperonin and contributes to facilitating the expression and localization of NdhB subunit.

Genes encoding lipid II flippase MurJ and peptidoglycan hydrolases are required for chloroplast division in the moss Physcomitrella patens.

Homologous genes for the peptidoglycan precursor flippase MurJ,and peptidoglycan hydrolases: lytic transglycosylase MltB, and DD-carboxypeptidase VanY are required for chloroplast division in the moss Physcomitrella patens. The moss Physcomitrella patens is used as a model plant to study plastid peptidoglycan biosynthesis. In bacteria, MurJ flippase transports peptidoglycan precursors from the cytoplasm to the periplasm. In this study, we identified a MurJ homolog (PpMurJ) in the P. patens genome. Bacteria employ peptidoglycan degradation and recycling pathways for cell division. We also searched the P. patens genome for genes homologous to bacterial peptidoglycan hydrolases and identified genes homologous for the lytic transglycosylase mltB, N-acetylglucosaminidase nagZ, and LD-carboxypeptidase ldcA in addition to a putative DD-carboxypeptidase vanY reported previously. Moreover, we found a ß-lactamase-like gene (Pplactamase). GFP fusion proteins with either PpMltB or PpVanY were detected in the chloroplasts, whereas fusion proteins with PpNagZ, PpLdcA, or Pplactamase localized in the cytoplasm. Experiments seeking PpMurJ-GFP fusion proteins failed. PpMurJ gene disruption in P. patens resulted in the appearance of macrochloroplasts in protonemal cells. Compared with the numbers of chloroplasts in wild-type plants (38.9 ± 4.9), PpMltB knockout and PpVanY knockout had lower numbers of chloroplasts (14.3 ± 6.7 and 28.1 ± 5.9, respectively). No differences in chloroplast numbers were observed after PpNagZ, PpLdcA, or Pplactamase single-knockout. Chloroplast numbers in PpMltB/PpVanY double-knockout cells were similar to those in PpMltB single-knockout cells. Zymogram analysis of the recombinant PpMltB protein revealed its peptidoglycan hydrolase activity. Our results imply that PpMurJ, PpMltB and PpVanY play a critical role in chloroplast division in the moss P. patens.

Photocaged Nanobodies Delivered into Cells for Light Activation of Biological Processes

AbstractWe have recently reported photocaged nanobodies, termed photobodies, that can be light‐activated in their antigen binding properties due to the removal of the photolabile protection group. We aimed to establish the delivery and activation of photobodies inside mammalian cells, because intracellular protein manipulation and detection by using antibodies and antibody fragments like nanobodies is highly attractive, however, the spatial and temporal control of their application in cells is extremely limited. AntiGFP photobodies with ortho‐nitrobenzyltyrosine in the paratope region were delivered into HeLa cells using the bead‐loading technique. Light‐activation was carried out with spatial control in single cells to trigger binding to a GFP tag fused to endogenously expressed target proteins. By means of its fused nuclear localization sequence, an activated photobody dragged the GFP fusion protein into the nucleus, the kinetics of which could be monitored using live cell microscopy. These results show the potential of photobodies for intracellular applications.

Cloning and salt tolerance analysis of the PnHB7 transcription factor in Populus nigra L

HD-Zip transcription factors play an important role in regulating plants’ response to salt stress, but there have been few studies of HD-Zip transcription factors in poplar and their role in salt-stress responses. In this study, the PnHB7 was cloned from Populus nigra L., a bioinformatics analysis was conducted, and a genetic transformation vector was constructed. The results show that PnHB7 was a transcription factor belonging to the HD-Zip family I subfamily, located on chromosome 14, with a length of 717 bp, encoding 238 amino acids, a molecular formula of C1185H1861N329O387S8. Multiple cis-acting elements related to stress response are contained in the upstream promoter region. Subcellular localization showed that the PnHB7:GFP fusion protein was located in the nucleus. Under a 0.4 % NaCl treatment, the growth and physiological indexes of overexpressed tobacco (Nicotiana tabacum L.) were better than those of wild-type tobacco, and salt tolerance was higher. The stress responses of tobacco seedlings transformed by 35S:pro-PnHB7:GUS showed that PnHB7 responded to abscisic acid, ethylene, methyl jasmonate, and NaCl stress. The transcriptome sequencing results of overexpressed tobacco showed that hormone-related protein genes (abscisic acid, gibberellin, ethylene, salicylic acid), oxidase genes (oxygenase, peroxidase, polyphenol oxidase) and transcription factor protein genes were significantly upregulated, and most were located in the signal pathway of ABA transduction. It is speculated that PnHB7 plays an important role in the ABA regulation pathway.

Fish IKKα from Japanese eel (Anguilla japonica) can activate NF-κB, AP1, and type I IFN signaling pathways

Inhibitor of nuclear factor kappa-B kinase subunit alpha (IKKα) plays a pivotal role in the activation of nuclear factor kappa-B (NF-κB) pathway in response to pathogens infections in mammals, but the information about IKKα in the regulation of immune responses is still limited in teleost fishes. In the present study, the full-length cDNA of an IKKα homologue, AjIKKα, was cloned by 5′ and 3′ SMART RACE from Japanese eel, and its characteristics of expression in response to various PAMPs and A. hydrophila infection were investigated both in vivo and in vitro using quantitative real-time polymerase chain reaction (qRT-PCR). In addition, the subcellular localization of AjIKKα GFP fusion protein and the induction of AjIKKα in the activation of NF-κB, type I IFN and AP1 performed using Dual-Glo luciferase assay system were also detected. Sequence comparison analysis revealed that AjIKKα has typical conserved domains, including an N-terminal kinase domain, an ubiquitin-like domain, a scaffold dimerization domain, and a C-terminal NEMO-binding domain. The predicted three-dimensional structure of AjIKKα is similar to that of human IKKα. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis revealed a broad expression for AjIKKα in a wide range of tissues, with the highest expression in the liver, followed by the intestine, gills, and spleen, and with a lower expression in the muscle and heart. The AjIKKα expressions in the liver and kidney were significantly induced following injection with the viral mimic poly I:C and Aeromonas hydrophila infection, whereas the bacterial mimic LPS down-regulated the expression of AjIKKα in the spleen. In vitro, the AjIKKα transcripts of Japanese eel liver cells were significantly enhanced by the treatment of LPS, poly I:C, CpG-DNA, and PGN or the stimulation of different concentration of Aeromonas hydrophila (1 × 106 cfu/mL, 1 × 107 cfu/mL, and 1 × 108 cfu/mL). Luciferase assays demonstrated that AjIKKα expression could significantly induce NF-κB, AP-1 and type I IFN promoter activation in a dose-dependent manner. Additionally, subcellular localization studies showed that AjIKKα was evenly distributed in the cytoplasm in the natural state, but AjIKKα was found to aggregate into spots in the cytoplasm after the stimulation of LPS and poly I:C. These results collectively indicated that AjIKKα plays an important role in innate immunity of host against antibacterial and antiviral infection likely via the activation of NF-κB, AP1and type I IFN signaling pathway.