• Animal Bioscience
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• v.34 no.10
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• pp.1590-1599
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• 2021

Objective: This study investigates the expression patterns of toll-like receptors (TLRs) and intracellular mediators in horse muscle cells after exercise, and the relationship between TLRS expression in stressed horse muscle cells and immune cell migration toward them. Methods: The expression patterns of the TLRs (TLR2, TLR4, and TLR8) and downstream signaling pathway-related genes (myeloid differentiation primary response 88 [MYD88]; activating transcription factor 3 [ATF3]) are examined in horse tissues, and horse peripheral blood mononuclear cells (PBMCs), polymorphonuclear cells (PMNs) and muscles in response to exercise, using the quantitative reverse transcription-polymerase chain reaction (qPCR). Expressions of chemokine receptor genes, i.e., C-X-C motif chemokine receptor 2 (CXCR2) and C-C motif chemokine receptor 5 (CCR5), are studied in PBMCs and PMNs. A horse muscle cell line is developed by transfecting SV-T antigen into fetal muscle cells, followed by examination of muscle-specific genes. Horse muscle cells are treated with stressors, i.e., cortisol, hydrogen peroxide (H₂O₂), and heat, to mimic stress conditions in vitro, and the expression of TLR4 and TLR8 are examined in stressed muscle cells, in addition to migration activity of PBMCs toward stressed muscle cells. Results: The qPCR revealed that TLR4 message was expressed in cerebrum, cerebellum, thymus, lung, liver, kidney, and muscle, whereas TLR8 expressed in thymus, lung, and kidney, while TLR2 expressed in thymus, lung, and kidney. Expressions of TLRs, i.e., TLR4 and TLR8, and mediators, i.e., MYD88 and ATF3, were upregulated in muscle, PBMCs and PMNs in response to exercise. Expressions of CXCR2 and CCR5 were also upregulated in PBMCs and PMNs after exercise. In the muscle cell line, TLR4 and TLR8 expressions were upregulated when cells were treated with stressors such as cortisol, H₂O₂, and heat. Migration of PBMCs toward stressed muscle cells was increased by exercise and oxidative stresses, and combinations of these. Treatment with methylsulfonylmethane (MSM), an antioxidant on stressed muscle cells, reduced migration of PBMCs toward stressed muscle cells. Conclusion: In this study, we have successfully cultured horse skeletal muscle cells, isolated horse PBMCs, and established an in vitro system for studying stress-related gene expressions and function. Expression of TLR4, TLR8, CXCR2, and CCR5 in horse muscle cells was higher in response to stressors such as cortisol, H₂O₂, and heat, or combinations of these. In addition, migration of PBMCs toward muscle cells was increased when muscle cells were under stress, but inhibition of reactive oxygen species by MSM modulated migratory activity of PBMCs to stressed muscle cells. Further study is necessary to investigate the biological function(s) of the TLR gene family in horse muscle cells.

• Animal Bioscience
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• v.34 no.8
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• pp.1392-1402
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• 2021

Objective: The growth rate of pigs is related to differentiation and proliferation of muscle cells, which are regulated by growth factors and expression of growth-related genes. Thus, the objective of this study was to establish optimal culture conditions for Jeju black pig (JBP) muscle cells and determine the relationship of various factors involved in muscle growth with the proliferation of JBP muscle cells. Methods: Muscles were taken from the femur skeletal muscle of JBP embryos. After isolation of the muscle cells, cells were cultured in a 6-well plate under four different culture conditions to optimize culture conditions for JBP muscle cells. To analyze proliferation rate of JBP muscle cells, these muscle cells were seeded into 6-well plates at a density of 1.5×10⁵ cells per well and cultured for 3 days. Western blot and quantitative real-time polymerase chain reaction were applied to verify the myogenic differentiation 1 (MyoD) expression and growth-related gene expression in JBP muscle cells, respectively. Results: We established a muscle cell line from JBP embryos and optimized its culture conditions. These muscle cells were positive for MyoD, but not for paired box 7. The proliferation rate of these muscle cells was significantly higher in a culture medium containing bFGF and epidermal growth factor + basic fibroblast growth factor (EGF+bFGF) than that without a growth factor or containing EGF alone. Treatment with EGF and bFGF significantly induced the expression of MyoD protein, an important transcription factor in muscle cells. Moreover, we checked the changes of expression of growth-related genes in JBP muscle cells by presence or absence of growth factors. Expression level of collagen type XXI alpha 1 gene was changed only when EGF and bFGF were added together to culture media for JBP muscle cells. Conclusion: Concurrent use of EGF and bFGF increased the expression of MyoD protein, thus regulating the proliferation of JBP muscle cells and the expression of growth-related genes.

• Animal Bioscience
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• v.36 no.4
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• pp.570-583
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• 2023

Objective: Fibroblast growth factors (FGFs) play critical roles in embryo development, and immune responses to infectious diseases. In this study, to investigate the roles of FGFs, we performed genome-wide identification, expression, and functional analyses of FGF family members in chickens. Methods: Chicken FGFs genes were identified and analyzed by using bioinformatics approach. Expression profiles and Hierarchical cluster analysis of the FGFs genes in different chicken tissues were obtained from the genome-wide RNA-seq. Results: A total of 20 FGF genes were identified in the chicken genome, which were classified into seven distinct groups (A-F) in the phylogenetic tree. Gene structure analysis revealed that members of the same clade had the same or similar exon-intron structure. Chromosome mapping suggested that FGF genes were widely dispersed across the chicken genome and were located on chromosomes 1, 4-6, 9-10, 13, 15, 28, and Z. In addition, the interactions among FGF proteins and between FGFs and mitogen-activated protein kinase (MAPK) proteins are limited, indicating that the remaining functions of FGF proteins should be further investigated in chickens. Kyoto encyclopedia of genes and genomes pathway analysis showed that FGF gene interacts with MAPK genes and are involved in stimulating signaling pathway and regulating immune responses. Furthermore, this study identified 15 differentially expressed genes (DEG) in 21 different growth stages during early chicken embryo development. RNA-sequencing data identified the DEG of FGFs on 1- and 3-days post infection in two indigenous Ri chicken lines infected with the highly pathogenic avian influenza virus H5N1 (HPAIV). Finally, all the genes examined through quantitative real-time polymerase chain reaction and RNA-Seq analyses showed similar responses to HPAIV infection in indigenous Ri chicken lines (R² = 0.92-0.95, p<0.01). Conclusion: This study provides significant insights into the potential functions of FGFs in chickens, including the regulation of MAPK signaling pathways and the immune response of chickens to HPAIV infections.

• Animal Bioscience
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• v.36 no.10
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• pp.1517-1529
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• 2023

Objective: The objective of this study was to investigate the phylogenetic and expression analysis of the angiopoietin-like (ANGPTL) gene family and their role in lipid metabolism in pigs. Methods: In this study, the amino acid sequence analysis, phylogenetic analysis, and chromosome adjacent gene analysis were performed to identify the ANGPTL gene family in pigs. According to the body weight data from 60 Jinhua pigs, different tissues of 6 pigs with average body weight were used to determine the expression profile of ANGPTL1-8. The ileum, subcutaneous fat, and liver of 8 pigs with distinct fatness were selected to analyze the gene expression of ANGPTL3, ANGPTL4, and ANGPTL8. Results: The sequence length of ANGPTLs in pigs was between 1,186 and 1,991 bp, and the pig ANGPTL family members shared common features with human homologous genes, including the high similarity of the amino acid sequence and chromosome flanking genes. Amino acid sequence analysis showed that ANGPTL1-7 had a highly conserved domain except for ANGPTL8. Phylogenetic analysis showed that each ANGPTL homologous gene shared a common origin. Quantitative reverse-transcription polymerase chain reaction analysis showed that ANGPTL family members had different expression patterns in different tissues. ANGPTL3 and ANGPTL8 were mainly expressed in the liver, while ANGPTL4 was expressed in many other tissues, such as the intestine and subcutaneous fat. The expression levels of ANGPTL3 in the liver and ANGPTL4 in the liver, intestine and subcutaneous fat of Jinhua pigs with low propensity for adipogenesis were significantly higher than those of high propensity for adipogenesis. Conclusion: These results increase our knowledge about the biological role of the ANGPTL family in this important economic species, it will also help to better understand the role of ANGPTL3, ANGPTL4, and ANGPTL8 in lipid metabolism of pigs, and provide innovative ideas for developing strategies to improve meat quality of pigs.

• Animal Bioscience
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• v.35 no.10
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• pp.1512-1523
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• 2022

Objective: The growth of pigs involves multiple regulatory mechanisms, and modern molecular breeding techniques can be used to understand the skeletal muscle growth and development to promote the selection process of pigs. This study aims to explore candidate lncRNAs and mRNAs related to skeletal muscle growth and development among Duroc pigs with different average daily gain (ADG). Methods: A total of 8 pigs were selected and divided into two groups: H group (high-ADG) and L group (low-ADG). And followed by whole transcriptome sequencing to identify differentially expressed (DE) lncRNAs and mRNAs. Results: In RNA-seq, 703 DE mRNAs (263 up-regulated and 440 down-regulated) and 74 DE lncRNAs (45 up-regulated and 29 down-regulated) were identified. In addition, 1,418 Transcription factors (TFs) were found. Compared with mRNAs, lncRNAs had fewer exons, shorter transcript length and open reading frame length. DE mRNAs and DE lncRNAs can form 417 lncRNA-mRNA pairs (antisense, cis and trans). DE mRNAs and target genes of lncRNAs were enriched in cellular processes, biological regulation, and regulation of biological processes. In addition, quantitative trait locus (QTL) analysis was used to detect the functions of DE mRNAs and lncRNAs, the most of DE mRNAs and target genes of lncRNAs were enriched in QTLs related to growth traits and skeletal muscle development. In single-nucleotide polymorphism/insertion-deletion (SNP/INDEL) analysis, 1,081,182 SNP and 131,721 INDEL were found, and transition was more than transversion. Over 60% of percentage were skipped exon events among alternative splicing events. Conclusion: The results showed that different ADG among Duroc pigs with the same diet maybe due to the DE mRNAs and DE lncRNAs related to skeletal muscle growth and development.

• Proceedings of the Plant Resources Society of Korea Conference
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• 2020.08a
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• pp.33-33
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• 2020

Watermelon (Citrullus lanatus) is a non-seasonal, economically important, cucurbit cultivated throughout the world with Asia as a continent contributing the most. As part of the effort in diversifying watermelon genetic resources in the already cultivated group, this study was devoted to providing baseline data on morphological quality traits and health-beneficial phytonutrients of watermelon germplasm collections, thereby promoting watermelon research and cultivation programs. To this end, we reported morphological traits, citrulline, and arginine levels of watermelon genetic resources obtained from the gene bank of Agrobiodiversity Center, Republic of Korea, and discussed the relationship between each other. Diverse characteristics were observed among many of the traits. But, most of the genetic resources (>90%) were either red or pink-fleshed. Korean origin fruits contained intermediate levels of soluble solid content (SSC) while The USA, Russian, Tajikistan, Turkmenistan, Taiwan, and Uruguay originated had generally the highest levels of soluble solids. The citrulline and arginine contents using HPLC method were ranged from 6.9 to 52.1 mg/g (average, 27.3 mg/g) and 1.8 to 21.3 mg/g (average, 9.8 mg/g), respectively. The citrulline content determined using Citrulline Assay Kit was ranged from 6.5 to 42.8 mg/g (average, 27.0 mg/g). Resources with high citrulline and arginine levels contained low SSC. Whereas, red- and pink-colored flesh samples had less citrulline compared to yellow and orange. In addition to the profiling of morphological characters and phytonutrients, molecular marker characterization and identification of sources of resistance to diseases and pests are recommended for a more complete diversity analysis of watermelon genetic resources.

• KOREAN JOURNAL OF CROP SCIENCE
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• v.68 no.3
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• pp.147-166
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• 2023

Seed color is controlled by several genes and is a key trait in determining the metabolite content and biological activities of legume genotypes. In this study, 296 adzuki bean accessions, including 159 grey, 99 red, and 38 white adzuki beans, were grown in Korea. Variations in total phenolic content (TPC), total saponin content (TSC), DPPH^• scavenging activity, ABTS^•+ scavenging activity, and ferric reducing antioxidant power (FRAP) were assessed and were reported to be in the ranges of 1.52-8.24 mg GAE/g, 14.36-114.22 mg DE/g, 0.23-12.84 mg AAE/g, 1.05-17.66 mg TE/g, and 0.59-13.14 mg AAE/g, respectively, with a wide variation across adzuki beans. Except for DPPH^• scavenging activity, the average values declined in the order gray > red > white adzuki beans, each demonstrating a significant variation (p < 0.05). White adzuki beans, which showed low metabolite content and antioxidant activity, were clearly separated from the gray and red genotypes using principal component and hierarchical cluster analyses. Moreover, TPC, TSC, and antioxidant activities were strongly correlated, regardless of seed color. Overall, the diversity of the TPC, TSC, and antioxidant activity in a broad population of adzuki bean genotypes was determined. Furthermore, this study found that seed color variation in adzuki beans had a significant effect on the metabolite content and antioxidant activity. Superior accessions with high levels of TPC, TSC, and antioxidant activity were also discovered and could be used for functional plant breeding and human consumption. The findings of this study may be useful for understanding the relationship between seed coat color and metabolite concentration in adzuki beans, paving the way for molecular-level analyses.

• Animal Bioscience
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• v.37 no.5
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• pp.929-943
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• 2024

Objective: The present study was executed to explore the molecular mechanism of fibroblast growth factor 10 (FGF10) gene in bovine adipogenesis. Methods: The bovine FGF10 gene was overexpressed through Ad-FGF10 or inhibited through siFGF10 and their negative control (NC) in bovine adipocytes, and the multiplicity of infection, transfection efficiency, interference efficiency were evaluated through quantitative real-time polymerase chain reaction, western blotting and fluorescence microscopy. The lipid droplets, triglycerides (TG) content and the expression levels of adipogenic marker genes were measured during preadipocytes differentiation. The differentially expressed genes were explored through deep RNA sequencing. Results: The highest mRNA level was found in omasum, subcutaneous fat, and intramuscular fat. Moreover, the highest mRNA level was found in adipocytes at day 4 of differentiation. The results of red-oil o staining showed that overexpression (Ad-FGF10) of the FGF10 gene significantly (p<0.05) reduced the lipid droplets and TG content, and their down-regulation (siFGF10) increased the measurement of lipid droplets and TG in differentiated bovine adipocytes. Furthermore, the overexpression of the FGF10 gene down regulated the mRNA levels of adipogenic marker genes such as CCAAT enhancer binding protein alpha (C/EBPα), fatty acid binding protein (FABP4), peroxisome proliferator-activated receptor-γ (PPARγ), lipoprotein lipase (LPL), and Fas cell surface death receptor (FAS), similarly, down-regulation of the FGF10 gene enriched the mRNA levels of C/EBPα, PPARγ, FABP4, and LPL genes (p<0.01). Additionally, the protein levels of PPARγ and FABP4 were reduced (p<0.05) in adipocytes infected with Ad-FGF10 gene and enriched in adipocytes transfected with siFGF10. Moreover, a total of 1,774 differentially expressed genes (DEGs) including 157 up regulated and 1,617 down regulated genes were explored in adipocytes infected with Ad-FGF10 or Ad-NC through deep RNA-sequencing. The top Kyoto encyclopedia of genes and genomes pathways regulated through DEGs were the PPAR signaling pathway, cell cycle, base excision repair, DNA replication, apoptosis, and regulation of lipolysis in adipocytes. Conclusion: Therefore, we can conclude that the FGF10 gene is a negative regulator of bovine adipogenesis and could be used as a candidate gene in marker-assisted selection.

• 한국균학회소식:학술대회논문집
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• 2015.05a
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• pp.53-53
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• 2015

Agrobacterium tumefaciens-mediated transformation(ATMT) of Flammulina velutipes was used to produce a diverse number of transformants to discover the functions of gene that is vital for its variation color, spore pattern and cellulolytic activity. Futhermore, the transformant pool will be used as a good genetic resource for studying gene functions. Agrobacterium-mediated transformation was conducted in order to generate intentional mutants of F. velutipes strain KACC42777. Then Agrobacterium tumefaciens AGL-1 harboring pBGgHg was transformed into F. velutipes. This method is use to determine the functional gene of F. velutipes. Inverse PCR was used to insert T-DNA into the tagged chromosomal DNA segments and conducting sequence analysis of the F. velutipes. But this experiment had trouble in diverse morphological mutants because of dikaryotic nature of mushroom. It needed to make monokaryotic fruiting varients which introduced genes of compatible mating types. In this study, next generation sequencing data was generated from 28 strains of Flammulina velutipes with different phenotypes using Illumina Hiseq platform. Filtered short reads were initially aligned to the reference genome (KACC42780) to construct a SNP matrix. And then we built a phylogenetic tree based on the validated SNPs. The inferred tree represented that white- and brown- fruitbody forming strains were generally separated although three brown strains, 4103, 4028, and 4195, were grouped with white ones. This topological relationship was consistently reappeared even when we used randomly selected SNPs. Group I containing 4062, 4148, and 4195 strains and group II containing 4188, 4190, and 4194 strains formed early-divergent lineages with robust nodal supports, suggesting that they are independent groups from the members in main clades. To elucidate the distinction between white-fruitbody forming strains isolated from Korea and Japan, phylogenetic analysis was performed using their SNP data with group I members as outgroup. However, no significant genetic variation was noticed in this study. A total of 28 strains of Flammulina velutipes were analyzed to identify the genomic regions responsible for producing white-fruiting body. NGS data was yielded by using Illumina Hiseq platform. Short reads were filtered by quality score and read length were mapped on the reference genome (KACC42780). Between the white- and brown fruitbody forming strains. There is a high possibility that SNPs can be detected among the white strains as homozygous because white phenotype is recessive in F. velutipes. Thus, we constructed SNP matrix within 8 white strains. SNPs discovered between mono3 and mono19, the parental monokaryotic strains of 4210 strain (white), were excluded from the candidate. If the genotypes of SNPs detected between white and brown strains were identical with those in mono3 and mono19 strains, they were included in candidate as a priority. As a result, if more than 5 candidates SNPs were localized in single gene, we regarded as they are possibly related to the white color. In F. velutipes genome, chr01, chr04, chr07,chr11 regions were identified to be associated with white fruitbody forming. White and Brown Fruitbody strains can be used as an identification marker for F. veluipes. We can develop some molecular markers to identify colored strains and discriminate national white varieties against Japanese ones.

• Korean Journal of Agricultural Science
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• v.25 no.1
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• pp.79-88
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• 1998

This study was performed in order to apply to effective breeding of Korean native cattle on the molecular genetic level obtained from PCR and nucleotide sequencing analysis of BoLA DRB3 exon2 that has important roles in host immune defence. Genomic DNA used in this study was prepared from the blood of Korean native cattle in Korean Native Cattle Improvement Center of National Livestock Cooperation. The results obtained from this study are summarized as follows: 1. Genomic DNA extracted from the blood of Korean native cattle was subjected to electrophoresis on 1.5% agarose gel. Major band was bigger than 12.2kb, indicating that genomic DNA was well prepared for PCR. Amplified products of 284bp fragments was obtained the amplification of BoLA DRB3 exon2 gene by PCR. 2. Cloning of BoLA DRB3 exon2 of Korean native cattle with pCR2.1 vector was conformed by 300bp fragment from recombinent plasmid that restricted with enzyme digestion of EcoRI. 3. Homology of BoLA DRB3 exon2 alleles of parent was 82.0% between sire's alleles and 90.1% between dam's alleles. 4. In pedigree analysis using BoLA DRB3 exon2 gene, sequencing result of BoLA DRB3 exon2 genes showed inheritance by Mendelian mode through the parents to their offspring. 5. Taking together those experimental results, pedigree was confirmed on the basis of sequencing for the alleles of parents and offspring. This knowledge by the molecular biological approach could be served for the improvement of Korean native cattle.