• 한국미생물·생명공학회지
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• 제49권3호
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• pp.305-315
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• 2021

The cotton leafworm, Spodoptera littoralis, is a major pest in Egypt and many countries worldwide, and causes heavy economic losses. As a result, management measures to control the spread of the worm are required. S. littoralis nucleopolyhedrovirus (SpliNPV) is one of the most promising bioagents for the efficient control of insect pests. In this study, a chitinase gene (chitA) of a 1.8 kb DNA fragment was cloned and fully characterized from SpliNPV-EG1, an Egyptian isolate. A sequence of 601 amino acids was deduced when the gene was completely sequenced with a predicted molecular mass of 67 kDa for the preprotein. Transcriptional analyses using reverse transcription polymerase chain reaction (RT-PCR) revealed that chitA transcripts were detected first at 12 h post infection (hpi) and remained detectable until 168 hpi, suggesting their transcriptional regulation from a putative late promoter motif. In addition, quantitative analysis using quantitative RT-PCR showed a steady increase of 7.86-fold at 12 hpi in chitA transcription levels, which increased up to 71.4-fold at 120 hpi. An approximately 50 kDa protein fragment with chitinolytic activity was purified from ChitA-induced bacterial culture and detected by western blotting with an anti-recombinant SpliNPV chitinase antibody. Moreover, purification of the expressed ChitA recombinant protein showed in vitro growth inhibition of two different fungi species, Fusarium solani and F. oxysporum, confirming that the enzyme assembly and activity was correct. The results supported the potential role and application of the SpliNPV-ChitA protein as a synergistic agent in agricultural fungal and pest control programs.

• Journal of Microbiology and Biotechnology
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• 제29권6호
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• pp.923-932
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• 2019

Current strategies of strain improvement processes are mainly focused on enhancing the synthetic pathways of the products. However, excessive metabolic flux often creates metabolic imbalances, which lead to growth retardation and ultimately limit the yield of the product. To solve this problem, we applied a dynamic regulation strategy to produce $\text\tiny{L}$-phenylalanine ($\text\tiny{L}$-Phe) in Escherichia coli. First, we constructed a series of Phe-induced promoters that exhibited different strengths through modification of the promoter region of tyrP. Then, two engineered promoters were separately introduced into a Phe-producing strain xllp1 to dynamically control the expression level of one pathway enzyme AroK. Batch fermentation results of the strain xllp3 showed that the titer of Phe reached 61.3 g/l at 48 h, representing a titer of 1.36-fold of the strain xllp1 (45.0 g/l). Moreover, the $\text\tiny{L}$-Phe yields on glucose of xllp3 (0.22 g/g) were also greatly improved, with an increase of 1.22-fold in comparison with the xllp1 (0.18 g/g). In summary, we successfully improved the titer of Phe by using dynamic regulation of one key enzyme and this strategy can be applied for improving the performance of strains producing other aromatic amino acids and derived compounds.

• Journal of Ginseng Research
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• 제43권4호
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• pp.645-653
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• 2019

Background: Cytochrome P450 enzymes catalyze a wide range of reactions in plant metabolism. Besides their physiological functions on primary and secondary metabolites, P450s are also involved in herbicide detoxification via hydroxylation or dealkylation. Ginseng as a perennial plant offers more sustainable solutions to herbicide resistance. Methods: Tissue-specific gene expression and differentially modulated transcripts were monitored by quantitative real-time polymerase chain reaction. As a tool to evaluate the function of PgCYP736A12, the 35S promoter was used to overexpress the gene in Arabidopsis. Protein localization was visualized using confocal microscopy by tagging the fluorescent protein. Tolerance to herbicides was analyzed by growing seeds and seedlings on Murashige and Skoog medium containing chlorotoluron. Results: The expression of PgCYP736A12 was three-fold more in leaves compared with other tissues from two-year-old ginseng plants. Transcript levels were similarly upregulated by treatment with abscisic acid, hydrogen peroxide, and NaCl, the highest being with salicylic acid. Jasmonic acid treatment did not alter the mRNA levels of PgCYP736A12. Transgenic lines displayed slightly reduced plant height and were able to tolerate the herbicide chlorotoluron. Reduced stem elongation might be correlated with increased expression of genes involved in bioconversion of gibberellin to inactive forms. PgCYP736A12 protein localized to the cytoplasm and nucleus. Conclusion: PgCYP736A12 does not respond to the well-known secondary metabolite elicitor jasmonic acid, which suggests that it may not function in ginsenoside biosynthesis. Heterologous overexpression of PgCYP736A12 reveals that this gene is actually involved in herbicide metabolism.

• 한국어병학회지
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• 제34권1호
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• pp.23-29
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• 2021

Interferon regulatory factors (IRFs) are a family of transcription factors essential to the control of antiviral immune response, cell growth, differentiation and apoptosis. IRF10 of zebrafish (Danio rerio) was negative regulation of the interferonΦ1 and 3 response in vitro. In this study, we analyze the induction of in vivo immune response activation from the IRF10 gene of zebrafish and the protective effect against VHSV. As the results, the group inoculated with IRF10 expression vectors, there was no expression of IFNΦ1, suggestion that IRF10 may function as a negative regulator of IRF3, which binds to the IFNΦ1 promoter. And other types of interferon genes (IFNΦ2-4) are thought to have been activated, inducing to the expression of pro-inflammatory cytokine and Mx genes. As the results of challenge test performed at 14 days after inoculation of the expression vectors, the maximum survival rate [50% (1㎍ DNA) and 42.5% (10㎍ DNA)] for IRF10 group were recorded. Meanwhile, the survival rates of pcDNA3.1 and PBS as the control groups were 10% and 15%, respectively. This study suggests that the possibility that activation of IRF10 molecule could be exploited as a VHS control method.

• Journal of Animal Science and Technology
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• 제63권1호
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• pp.16-24
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• 2021

The suppressive effect of monensin as an ionophore-feed additive on enteric methane (CH4) emission and renewable methanogenesis were evaluated. To clarify the suppressive effect of monensin a respiratory trial with head cage was performed using Holstein-Friesian steers. Steers were offered high concentrate diets (80% concentrate and 20% hay) ad libitum with or without monensin, galacto-oligosaccharides (GOS) or L-cysteine. Steers that received monensin containing diet had significantly (p < 0.01) lower enteric CH4 emissions as well as those that received GOS containing diet (p < 0.05) compared to steers fed control diets. Thermophilic digesters at 55℃ that received manure from steers fed on monensin diets had a delay in the initial CH4 production. Monensin is a strong inhibitor of enteric methanogenesis, but has a negative impact on biogas energy production at short retention times. Effects of the activity of coprophagous insects on CH4 and nitrous oxide (N2O) emissions from cattle dung pats were assessed in anaerobic in vitro continuous gas quantification system modified to aerobic quantification device. The CH4 emission from dungs with adults of Caccobius jessoensis Harold (dung beetle) and the larvae of the fly Neomyia cornicina (Fabricius) were compared with that from control dung without insect. The cumulative CH4 emission rate from dung with dung insects decreased at 42.2% in dung beetles and 77.8% in fly larvae compared to that from control dung without insects. However, the cumulative N2O emission rate increased 23.4% in dung beetles even though it reduced 88.6% in fly larvae compared to dung without coprophagous insects. It was suggested that the antibacterial efficacy of ionophores supplemented as a growth promoter still continued even in the digested slurry, consequently, possible environmental contamination with the antibiotics might be active to put the negative impact to land ecosystem involved in greenhouse gas mitigation when the digested slurry was applied to the fields as liquid manure.

• Genomics & Informatics
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• 제20권2호
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• pp.19.1-19.15
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• 2022

Alfalfa (Medicago sativa) is an important food and feed crop which rich in mineral sources. The WUSCHEL-related homeobox (WOX) gene family plays important roles in plant development and identification of putative gene families, their structure, and potential functions is a primary step for not only understanding the genetic mechanisms behind various biological process but also for genetic improvement. A variety of computational tools, including MAFFT, HMMER, hidden Markov models, Pfam, SMART, MEGA, ProtTest, BLASTn, and BRAD, among others, were used. We identified 34 MsWOX genes based on a systematic analysis of the alfalfa plant genome spread in eight chromosomes. This is an expansion of the gene family which we attribute to observed chromosomal duplications. Sequence alignment analysis revealed 61 conserved proteins containing a homeodomain. Phylogenetic study sung reveal five evolutionary clades with 15 motif distributions. Gene structure analysis reveals various exon, intron, and untranslated structures which are consistent in genes from similar clades. Functional analysis prediction of promoter regions reveals various transcription binding sites containing key growth, development, and stress-responsive transcription factor families such as MYB, ERF, AP2, and NAC which are spread across the genes. Most of the genes are predicted to be in the nucleus. Also, there are duplication events in some genes which explain the expansion of the family. The present research provides a clue on the potential roles of MsWOX family genes that will be useful for further understanding their functional roles in alfalfa plants.

• Journal of Microbiology and Biotechnology
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• 제33권3호
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• pp.329-338
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• 2023

Enterohemorrhagic Escherichia coli (EHEC) is a foodborne pathogen that produces attaching and effacing lesions on the large intestine and causes hemorrhagic colitis. It is primarily transmitted through the consumption of contaminated meat or fresh produce. Similar to other bacterial pathogens, antibiotic resistance is of concern for EHEC. Furthermore, since the production of Shiga toxin by this pathogen is enhanced after antibiotic treatment, alternative agents that control EHEC are necessary. This study aimed to discover alternative treatments that target virulence factors and reduce EHEC toxicity. The locus of enterocyte effacement (LEE) is essential for EHEC attachment to host cells and virulence, and most of the LEE genes are positively regulated by the transcriptional regulator, Ler. GrlA protein, a transcriptional activator of ler, is thus a potential target for virulence inhibitors of EHEC. To identify the GrlA inhibitors, an in vivo high-throughput screening (HTS) system consisting of a GrlA-expressing plasmid and a reporter plasmid was constructed. Since the reporter luminescence gene was fused to the ler promoter, the bioluminescence would decrease if inhibitors affected the GrlA. By screening 8,201 compounds from the Korea Chemical Bank, we identified a novel GrlA inhibitor named Grlactin [3-[(2,4-dichlorophenoxy)methyl]-4-(3-methylbut-2-en-1-yl)-4,5-dihydro-1,2,4-oxadiazol-5-one], which suppresses the expression of LEE genes. Grlactin significantly diminished the adhesion of EHEC strain EDL933 to human epithelial cells without inhibiting bacterial growth. These findings suggest that the developed screening system was effective at identifying GrlA inhibitors, and Grlactin has potential for use as a novel anti-adhesion agent for EHEC while reducing the incidence of resistance.

• Genomics & Informatics
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• 제20권4호
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• pp.45.1-45.7
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• 2022

Food security will be affected by climate change worldwide, particularly in the developing world, where the most important food products originate from plants. Plants are often exposed to environmental stresses that may affect their growth, development, yield, and food quality. Auxin is a hormone that plays a critical role in improving plants' tolerance of environmental conditions. Auxin controls the expression of many stress-responsive genes in plants by interacting with specific cis-regulatory elements called auxin-responsive elements (AuxREs). In this work, we performed an in silico prediction of AuxREs in promoters of five auxin-responsive genes in Zea mays. We applied a data fusion approach based on the combined use of Dempster-Shafer evidence theory and fuzzy sets. Auxin has a direct impact on cell membrane proteins. The short-term auxin response may be represented by the regulation of transmembrane gene expression. The detection of an AuxRE in the promoter of prolyl oligopeptidase (POP) in Z. mays and the 3-fold overexpression of this gene under auxin treatment for 30 min indicated the role of POP in maize auxin response. POP is regulated by auxin to perform stress adaptation. In addition, the detection of two AuxRE TGTCTC motifs in the upstream sequence of the bx1 gene suggests that bx1 can be regulated by auxin. Auxin may also be involved in the regulation of dehydration-responsive element-binding and some members of the protein kinase superfamily.

• Animal Bioscience
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• 제37권6호
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• pp.1041-1052
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• 2024

Objective: Bacillus subtilis, a kind of probiotic with broad-spectrum antibacterial function, was commonly used in livestock and poultry production. Recent research suggested that Bacillus subtilis may have antioxidant properties and improve immune response. This study aimed to verify the probiotic function of Bacillus subtilis in the production of broiler chickens. Methods: A total of 324 (1-day-old) Arbor Acres broilers were selected and randomly divided into three groups: basal diet group (Ctr Group), basal diet + antibiotic growth promoter group (Ctr + AGP) and basal diet + 0.5% Bacillus subtilis preparation group (Ctr + Bac). The experiment lasted for 42 days. Muscle, serum and liver samples were collected at 42 days for determination. Results: The results showed that Bacillus subtilis could decrease malondialdehyde content in the serum and liver (p<0.05) and increase superoxide dismutase 1 mRNA expression (p<0.01) and total superoxide dismutase (p<0.05) in the liver. In addition, compared with AGP supplementation, Bacillus subtilis supplementation increased interleukin-10 (IL-10) and decreased tumor necrosis factor-α and IL-1β level in the serum (p<0.05). At 45 minutes after slaughter Ctr + Bac presented a higher a^* value of breast muscle than Ctr Group (p<0.05), while significant change in leg muscle was not identified. Moreover, there was no difference in weight, shear force, cooking loss and drip loss of breast and leg muscle between treatments. Conclusion: Our results demonstrate that Bacillus subtilis in diet can enhance antioxidant capacity and optimize immune response of broilers.

• Journal of Microbiology
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• 제44권1호
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• pp.64-71
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• 2006

To investigate the effects of the nucleotide sequences in Shine-Dalgarno (SD) and the spacer region (SD-ATG) on bovine growth hormone (bGH) gene expression, the expression vectors under the control of the T7 promoter (pT7-7 vector) were constructed using bGH derivatives (bGH1 & bGH14) which have different 5'-coding regions and were induced in E. coli BL21 (DE3). Oligonucleotides containing random SD sequences and a spacer region were chemically synthesized and the distance between the SD region and the initiation codon were fixed to nine bases in length. The oligonucleotides were annealed and fused to the bGH1 and bGH14 cDNA, respectively. When the bGH gene was induced with IPTG in E. coli BL21(DE3), some clones containing only bGH14 cDNA produced considerable levels of bGH in the range of $6.9\%\;to\;8.5\%$ of total cell proteins by SDS-PAGE and Western blot. Otherwise, the bGH was not detected in any clones with bGH1 cDNA. Accordingly, the nucleotide sequences of SD and the spacer region affect on bGH expression indicates that the sequences sufficiently destabilize the mRNA secondary structure of the bGH14 gene. When the free energy was calculated from the transcription initiation site to the +51 nucleotide of bGH cDNA using a program of nucleic acid folding and hybridization prediction, the constructs with values below -26.3 kcal/mole (toward minus direction) were not expressed. The constructs with the original sequence of bGH cDNA also did not show any expression, regardless of the free energy values. Thus, the disruption of the mRNA secondary structure may be a major factor regulating bGH expression in the translation initiation process. Accordingly, the first stem-loop among two secondary structures present in the 5'-end region of the bGH gene should be disrupted for the effective expression of bGH.