• Animal Bioscience
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• 제35권9호
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• pp.1327-1339
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• 2022

Objective: Fat deposition in poultry is an important factor in production performance and meat quality research. miRNAs also play important roles in regulating adipocyte differentiation process. This study was to investigate the expression patterns of miRNAs in duck adipocytes after differentiation and explore the role of miR-214 in regulating carnitine palmitoyltransferases 2 (CPT2) gene expression during duck adipocyte differentiation. Methods: Successful systems for the isolation, culture, and induction of duck primary fat cells was developed in the experiment. Using Illumina next-generation sequencing, the miRNAs libraries of duck adipocytes were established. miRanda was used to predict differentially expressed (DE) miRNAs and their target genes. The expression patterns of miR-214 and CPT2 during the differentiation were verified by quantitative real-time polymerase chain reaction and western blot. Luciferase reporter assays were used to explore the specific regions of CPT2 targeted by miR-214. We used a miR-214 over-expression strategy in vitro to further investigate its effect on differentiation process and CPT2 gene transcription. Results: There were 481 miRNAs identified in duck adipocytes, included 57 DE miRNA candidates. And the 1,046 targets genes of DE miRNAs were mainly involved in p53 signaling, FoxO signaling, and fatty acid metabolism pathways. miR-214 and CPT2 showed contrasting expression patterns before and after differentiation, and they were selected for further research. The expression of miR-214 was decreased during the first 3 days of duck adipocytes differentiation, and then increased, while the expression of CPT2 increased both in the transcriptional and protein level. The luciferase assay suggested that miR-214 targets the 3'untranslated region of CPT2. Overexpression of miR-214 not only promoted the formation of lipid droplets but also decreased the protein abundance of CPT2. Conclusion: Current study reports the expression profile of miRNAs in duck adipocytes differentiated for 4 days. And miR-214 has been proved to have the regulator potential for fat deposition in duck.

• Animal Bioscience
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• 제37권4호
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• pp.609-621
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• 2024

Objective: Hair follicle stem cells (HFSCs) differentiation is a critical physiological progress in skin hair follicle (HF) formation. Goat HFSCs differentiation is one of the essential processes of superior-quality brush hair (SQBH) synthesis. However, knowledge regarding the functions and roles of miR-133a-3p and miR-145-5p in differentiated goat HFSCs is limited. Methods: To examine the significance of chi-miR-133a-3p and chi-miR-145-5p in differentiated HFSCs, overexpression and knockdown experiments of miR-133a-3p and miR-145-5p (Mimics and Inhibitors) separately or combined were performed. NANOG, SOX9, and stem cell differentiated markers (β-catenin, C-myc, Keratin 6 [KRT6]) expression levels were detected and analyzed by using real-time quantitative polymerase chain reaction, western blotting, and immunofluorescence assays in differentiated goat HFSCs. Results: miR-133a-3p and miR-145-5p inhibit NANOG (a gene recognized in keeping and maintaining the totipotency of embryonic stem cells) expression and promote SOX9 (an important stem cell transcription factor) expression in differentiated stem cells. Functional studies showed that miR-133a-3p and miR-145-5p individually or together overexpression can facilitate goat HFSCs differentiation, whereas suppressing miR-133a-3p and miR-145-5p or both inhibiting can inhibit goat HFSCs differentiation. Conclusion: These findings could more completely explain the modulatory function of miR-133a-3p and miR-145-5p in goat HFSCs growth, which also provide more understandings for further investigating goat hair follicle development.

• Journal of Plant Biotechnology
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• 제39권2호
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• pp.106-113
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• 2012

Glycolysis is responsible for the conversion of glucose into pyruvate and for supplying reducing power and several metabolites. Fructose-1,6-bisphosphate aldolase (AtFBA1), a central enzyme in the glycolysis pathway, was isolated by functional complementation of the salt-sensitive phenotype of a calcineurin (CaN)-deficient yeast mutant. Under high salinity conditions, aldolase activity and the concentration of NADH were compromised. However, expression of AtFBA1 maintained aldolase activity and the NADH level in yeast cells. AtFBA1 shares a high degree of sequence identity with known class I type aldolases, and its expression was negatively regulated by stress conditions including NaCl. The fusion protein GFP-AtFBA1 was localized in the cytosol of Arabidopsis protoplasts. The seed germination and root elongation of AtFBA1 knock-out plants exhibited sensitivity to ABA and salt stress. These results indicate that AtFBA1 expression and aldolase activity is important for stress tolerance in yeast and plants.

• Journal of Microbiology and Biotechnology
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• 제17권11호
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• pp.1885-1889
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• 2007

The tissue blot immunobinding assay (TBIA) is widely used for the detection and localization of plant viruses in various plant tissues. The basic experimental procedures of TBIA sampling and blotting were simplified using commercially available micropipette tips. This method was termed the ring-blot immunobinding assay (R-BIA), as the blot on the membrane forms a ring shape. The detection efficacy of R-BIA was tested for two chili pepper viruses, pepper mild mottle tobamovirus (PMMoV) and pepper mottle potyvirus (PepMoV), following the optimized serological procedures of TBIA (length of the incubation period and BSA concentration, and primary and secondary antibodies). Sensitivity of the R-BIA was about 1 ng/ml of purified PMMoV in pepper leaf sap from a healthy pepper plant. R-BIA also showed high specificity in the detection of PMMoV and PepMoV. Moreover, the modified sampling and blotting procedures were simpler and more reliable than other TBIA methods (such as whole-leaf blotting and crushed-leaf blotting), suggesting that the R-BIA may be used for medium- to large-scale detection of plant viruses in laboratories with minimal facilities.

• The Plant Pathology Journal
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• 제23권4호
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• pp.314-317
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• 2007

A soft stem rot disease was observed on Chamaecereus silvestrii (Korean name: Sanchui), a scion of graft-cactus, in major growing areas of Suwon (National Horticulture Research Institute), Anseong, Eumseong, Cheonan, Daegu, and Goyang, Korea during 2000 and 2001. Typical symptoms were soft rots characterized by moist and watery decay of the whole cactus stem, which initiated as small water-soaked lesions and enlarged rapidly to the entire stem. The causal organism isolated from the infected stems was identified as Pectobacterium carotovorum subsp. carotovorum (Erwinia carotovora subsp. carotovora) based on its physiological and biochemical characteristics and confirmed by the cellular fatty acid composition and Biolog analyses. Artificial inoculation of the bacterium produced the same soft rot symptoms on the cactus stems, from which the same bacterium was isolated and identified. This is the first report of the P. carotovorum subsp. carotovorum in the graft-cactus C. silvestrii in Korea.

• 한국자원식물학회:학술대회논문집
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• 한국자원식물학회 2019년도 춘계학술대회
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• pp.61-61
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• 2019

In any crop breeding program Selection and use of genetically diverse genotypes to develop cultivars with a broad genetic base is important. Molecular markers play a major role in selecting diverse genotypes. Molecular breeding programs of the crop can be made more efficient by use of molecular markers. The present study was done with an aim of analyzing genetic diversity and the population structure in 24 accessions of sunflower (Helianthus annus L.) from Kenya genetic diversity using 35 EST-SSR and gSSR primers.Out of the 35 markers 3 were not polymorphic as they indicated Polymorphic Information content( PIC) of value 0.00 and so the data analysis was done using 32 markers . The 32 set of markers used produced 29 alleles ranging from 2 to 7with a mean of 3.0 alleles per locus.The average value of polymorphic information contents(PIC) were 0.3 .Genetic diversity analysis using these markers revealed 3 major clusters. This result could be useful for designing strategies to make elite hybrid and inbreeding of crossing block for breeding and future molecular breeding programs to make elite variety.

• 한국육종학회지
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• 제43권2호
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• pp.103-114
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• 2011

In recent years, genomic resources and information have accumulated at an ever increasing pace, in many plant species, through whole genome sequencing, large scale analysis of transcriptomes, DNA markers and functional studies of individual genes. Well-characterized species within key plant taxa, co-called "model systems", have played a pivotal role in nucleating the accumulation of genomic information and databases, thereby providing the basis for comparative genomic studies. In addition, recent advances to "Next Generation" sequencing technologies have propelled a new wave of genomics, enabling rapid, low cost analysis of numerous genomes, and the accumulation of genetic diversity data for large numbers of accessions within individual species. The resulting wealth of genomic information provides an opportunity to discern evolutionary processes that have impacted genome structure and the function of genes, using the tools of comparative analysis. Comparative genomics provides a platform to translate information from model species to crops, and to relate knowledge of genome function among crop species. Ultimately, the resulting knowledge will accelerate the development of more efficient breeding strategies through the identification of trait-associated orthologous genes and next generation functional gene-based markers.

• BMB Reports
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• 제40권1호
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• pp.22-28
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• 2007

MyoD, expressed in skeletal muscle lineages of vertebrate embryo, is one of muscle-specific basic helix-loop-helix (bHLH) transcription factors, which plays a key role in the determination and differentiation of all skeletal muscle lineages. In this study, a cDNA of grass carp MyoD was cloned and characterized from total RNA of grass carp embryos by RT-PCR. The full-length cDNA of grass carp MyoD is 1597 bp. The cDNA sequence analysis reveals an open reading frame of 825 bp coding for a protein of 275 amino acids, which includes a bHLH domain composed of basic domain (1-84th amino acids) and HLH domain (98-142th amino acids), without signal peptide. Then the MyoD cDNA of grass carp was cloned to yeast expression vector pPICZ$\alpha$A and transformed into P. pastoris GS115 strain, the recombinant MyoD protein with a molecular weight of about 31KD was obtained after inducing for 2d with 0.5% methanol in pH 8.0 BMGY medium, and the maximum yield was about 250 mg/L in shaking-flask fermentation. The results were expected to benefit for further studies on the crystal structure and physiological function of fish MyoD.

• International Journal of Industrial Entomology and Biomaterials
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• 제39권1호
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• pp.14-21
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• 2019

Genome editing by CRISPR/Cas9, a third-generation gene scissor in molecular breeding at the genome level, is attracting much attention as one of the breeding techniques of the future. In this study, genetic and phenotypic analysis was used to examine the responsiveness of the Bakokjam variety of the silkworm Bombyx mori to molecular breeding using CRISPR/Cas9 in editing the white egg 2 (w-2) gene. The nucleotide sequence of the w-2 gene was analyzed and three different guide RNAs (gRNA) were prepared. The synthesized gRNA was combined with Cas9 protein and then analyzed by T7 endonuclease I after introduction into the Bm-N silkworm cell line. To edit the silkworm gene, W1N and W2P gRNA and Cas9 complexes were microinjected into silkworm embryos. Based on the results of microinjection, the hatching rate was 16-24% and the incidence of mutation was 33-37%. The gene mutation was verified in the heterozygous F1 generation, but no phenotypic change was observed. In F2 homozygotes generated by F1 self-crosses, a mutant phenotype was observed. These results suggest that silkworm molecular breeding using the CRISPR/Cas9 system is possible and will be a very effective way to shorten the time required than the traditional breeding process.

• Asian-Australasian Journal of Animal Sciences
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• 제19권7호
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• pp.931-937
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• 2006

The full-length cDNA of the porcine SMPX gene was obtained by the rapid amplification of cDNA ends (RACE). The nucleotide sequences and the predicted protein sequences share high sequence identity with both human and mouse. The promoter of SMPX was sequenced and then analyzed to find the promoter binding sites. The reverse transcriptase-polymerase chain reaction (RT-PCR) revealed that SMPX has a high level of expression in heart and skeletal muscle, a very low expression in lung and spleen and no expression in liver, kidney, fat and brain. Moreover, SMPX has a differential expression level in skeletal muscle, the expression in 65-day embryos being higher than other stages. The porcine SMPX was mapped to SSCXp24 by using a somatic cell hybrid panel (SCHP) and was found closely linked to SW1903 using the radiation hybrid panel IMpRH. An A/G single nucleotide polymorphism (PCR-RFLP) in the 3'-untranslated region (3'-UTR) was detected in eight breeds. The analysis of allele frequency distribution showed that introduced pig breeds (Duroc and Large White) have a higher frequency of allele A while in the Chinese indigenous pig breeds (Qingping pig, Lantang pig, YushanBlack pig, Large Black-White pig, Small Meishan) have a higher frequencies of allele G. The association analysis using an experimental population (188 pigs), which included two cross-bred groups and three pure-blood groups, suggested that the SNP genotype was associated with intramuscular fat content.