• The Plant Pathology Journal
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• v.36 no.5
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• pp.468-475
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• 2020

Malva vein clearing virus (MVCV) is a member of the Potyvirus species, and has a negative impact on the aesthetic development of Alcea rosea. It was first reported in Germany in 1957, but its complete genome sequence data are still scarce. In the present work, A. rosea leaves with vein-clearing and mosaic symptoms were sampled and analyzed with small RNA deep sequencing. By denovo assembly the raw sequences of virus-derived small interfering RNAs (vsiRs) and whole genome amplification of malva vein cleaning virus SX strain (MVCV-SX) by specific primers targeting identified contig gaps, the full-length genome sequences (9,645 nucleotides) of MVCV-SX were characterized, constituting of an open reading frame that is long enough to encode 3,096 amino acids. Phylogenetic analysis showed that MVCV-SX was clustered with euphorbia ringspot virus and yam mosaic virus. Further analyses of the vsiR profiles revealed that the most abundant MVCV-vsiRs were between 21 and 22 nucleotides in length and a strong bias was found for "A" and "U" at the 5′-terminal residue. The results of polarity assessment indicated that the amount of sense strand was almost equal to that of the antisense strand in MVCV-vsiRs, and the main hot-spot region in MVCV-SX genome was found at cylindrical inclusion. In conclusion, our findings could provide new insights into the RNA silencing-mediated host defence mechanism in A. rosea infected with MVCV-SX, and offer a basis for the prevention and treatment of this virus disease.

• Asian-Australasian Journal of Animal Sciences
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• v.29 no.9
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• pp.1363-1370
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• 2016

Rennet, a complex of enzymes found in the stomachs of ruminants, is an important component for cheese production. In our study, we described that yak chymosin gene recombinant Pichia pastoris strain could serve as a novel source for rennet production. Yaks total RNA was extracted from the abomasum of an unweaned yak. The yak preprochymosin, prochymosin, and chymosin genes from total RNA were isolated using gene specific primers based on cattle chymosin gene sequence respectively and analyzed their expression pattern byreal time-polymerase chain reaction. The result showed that the chymosin gene expression level of the sucking yaks was 11.45 times higher than one of adult yaks and yak chymosin belongs to Bovidae family in phylogenetic analysis. To express each, the preprochymosin, prochymosin, and chymosin genes were ligated into the expression vector $pPICZ{\alpha}A$, respectively, and were expressed in Pichia pastoris X33. The results showed that all the recombinant clones of P. pastoris containing the preprochymosin, prochymosin or chymosin genes could produce the active form of recombinant chymosin into the culture supernatant. Heterologous expressed prochymosin (14.55 Soxhlet unit/mL) had the highest enzyme activity of the three expressed chymosin enzymes. Therefore, we suggest that the yak chymosin gene recombinant Pichia pastoris strain could provide an alternative source of rennet production.

• Research in Plant Disease
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• v.23 no.1
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• pp.35-41
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• 2017

Pseudomonas syringae pv. actinidiae is the causative agent of bacterial canker of kiwifruit. The population of this pathogen is differentiated into three biovars, biovar 1, 2 and 3, according to their molecular characteristics. In this work, we determined biovars of P. syringae pv. actinidiae strains isolated in Korea since 1997 and stored in Department of Biology, Sunchon National University, Suncheon, Korea. The biovars of P. syringae pv. actinidiae strains were determined by PCR using biovar specific primers developed previously. Of 682 strains investigated, 288 strains belonged to biovar 2, while 394 strains were biovar 3. There were no P. syringae pv. actinidiae strains belonging to biovar 1 among the strains isolated in Korea. Sudden outbreak and spreading of bacterial canker caused by biovar 3 strain suggest that this strain has character of rapid transmission.

• Journal of Life Science
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• v.19 no.11
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• pp.1522-1528
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• 2009

A marine bacterium was isolated from brown seaweeds for its ability to degrade alginate. Analysis of 16S ribosomal DNA sequence revealed that the strain belongs to Streptomyces like strain ALG-5 which was reported previously. New alginate lyase gene of Streptomyces sp. M3 was cloned by using PCR with the specific primers designed from homologous nucleotide sequences. The consensus sequences of N-terminal YXRSELREM and C-terminal YFKAGXYXQ were conserved in the M3 alginate lyase amino acid sequences. The homology model for the M3 alginate lyase showed a characteristic structure of $\beta$-jelly roll fold main domain like alyPG from Corynebacterium sp. ALY-1. The homogenate of the recombinant E. coli with the alginate lyase gene showed more degrading activity for polyguluronate block than polymannuronate block. The results from the multiple alignments and the homology modeling elucidated in the M3 alginate lyase can be classified into family PL-7.

• Food Science and Biotechnology
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• v.17 no.4
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• pp.888-891
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• 2008

Twenty-three fungal strains were isolated from meju that had originated from the Sunchang province, the famous location for making fermented soybean foods in Korea. The restriction fragment length polymorphism (RFLP) of the internal transcribed spacer (ITS) region of the rDNA (ITS-RFLP) was applied to differentiate the isolated fungal strains. First, the ITS region by polymerase chain reaction (PCR) with specific primers was amplified and then cleaved the products with different restriction enzymes. Cleavage of the amplified fragments with the restriction enzymes AluI, HaeIII, HhaI, and TaqI revealed extensive polymorphisms. The ITS-RFLP results highly correlated with ITS sequence analysis. All of the 23 fungal strains were classified into 5 groups by ITS-RFLP analysis. Aspergillus oryzae was the major fungal strain isolated from Sunchang meju (12 out of 23), while Aspergillus fumigatus was the next most frequently isolated strain (7 out of 23). In contrast, it was found that Fusarium asiaticum, Aspergillus sydowii, and Arthrinium sp. were the minor fungal strains in meju.

• The Plant Pathology Journal
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• v.32 no.2
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• pp.136-144
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• 2016

Pseudomonas fluorescens pc78 is an effective biocontrol agent for soil-borne fungal diseases. We previously constructed a P43-gfp tagged biocontrol bacteria P. fluorescens pc78-48 to investigate bacterial traits in natural ecosystem and the environmental risk of genetically modified biocontrol bacteria in tomato rhizosphere. Fluctuation of culturable bacteria profile, microbial community structure, and potential horizontal gene transfer was investigated over time after the bacteria treatment to the tomato rhizosphere. Tagged gene transfer to other organisms such as tomato plants and bacteria cultured on various media was examined by polymerase chain reaction, using gene specific primers. Transfer of chromosomally integrated P43-gfp from pc78 to other organisms was not apparent. Population and colony types of culturable bacteria were not significantly affected by the introduction of P. fluorescens pc78 or pc78-48 into tomato rhizosphere. Additionally, terminal restriction fragment length polymorphism profiles were investigated to estimate the influence on the microbial community structure in tomato rhizosphere between non-treated and pc78-48-treated samples. Interestingly, rhizosphere soil treated with strain pc78-48 exhibited a significantly different bacterial community structure compared to that of non-treated rhizosphere soil. Our results suggest that biocontrol bacteria treatment influences microbial community in tomato rhizosphere, while the chromosomally modified biocontrol bacteria may not pose any specific environmental risk in terms of gene transfer.

• Korean Journal of Microbiology
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• v.44 no.3
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• pp.212-220
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• 2008

A Pn10 DNA probe was introduced as a Prevotella nigrescens ATCC $33563^T$-specific DNA probe. In that study, the specificity of the Pn10 was tested with only type or reference strains of 5 oral bacterial species. The purpose of this study is to evaluate the specificity of the Pn10 using the wild type strains of P. nigrescens and is to develop the P. nigrescens ATCC $33563^T$-specific PCR primers based on the nucleotide sequence of the Pn10. The specificity of the Pn10 DNA probe was determined by Southern blot analysis. The nucleotide sequence of Pn10 DNA probes was determined by chain termination method. The PCR primers were designed based on the nucleotide sequence of cloned DNA fragment. The data showed that Pn10 DNA probe were hybridized with the genomic DNAs from P. nigrescens ATCC $33563^T$ and KB6. The Pn10 homologous region, KB6-Pn10, of P. nigrescens KB6 was cloned by PCR and sequenced. The Pn10 and KB6-Pn10 DNA fragments were consisted of 1,875 bp and 1,873 bp, respectively. The percent identity of the two was 98.8% and the divergence of them was 0.6%. The two primer sets (Pn10-F-AC/ Pn10-R-AC and Pn10-F-A/ Pn10-R-A), designed base on the nucleotide sequences of Pn10 DNA probe, were specific to the P. nigrescens ATCC $33563^T$. The two PCR primer sets could detect as little as 4 pg of genomic DNA of P. nigrescens ATCC $33563^T$. These results indicate that the two PCR primer sets have proven useful for the identification of P. nigrescens ATCC $33563^T$, especially with regard to the maintenance of the strain.

• Korean Journal of Clinical Laboratory Science
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• v.49 no.4
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• pp.413-419
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• 2017

Four imipenem-resistant bacteria were isolated from the clinical specimens of a patient with pneumonia. To identify the isolates, we used the GN card of Vitek II system and performed a phylogenetic analysis based on 16S rRNA gene sequence. The isolates were identified as P. aeruginosa (2 strains), P. monteilii (1 strain), and P. putida (1 strain), and were tested for antibiotic resistance after determining the MIC of imipenem to be $${\geq_-}8{\mu}g/mL$$ using the AST-N225 card of Vitek II system. The imipenem-resistant genotypes were determined using PCR products amplified using specific ${\beta}-Lactamase$ gene primers. The MBL gene was identified in all four isolates. One strain of P. aeruginosa exhibited the VIM and SHV-1 type genes, while the other strain exhibited both VIM and OXA group II genes. According to the antimicrobial susceptibility test, the bacteria were more susceptible to amikacin than other antibiotics. DNA fingerprint analysis using ERIC-PCR to analyze the epidemiological relationship between strains estimated that both the P. aeruginosa isolates were similar, but exhibited different DNA band types. It is uncommon to find four strains of imipenem-resistant bacteria with different DNA band types in a single patient.

• Journal of Mushroom
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• v.13 no.1
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• pp.79-83
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• 2015

In this study, SCAR marker that differentiates Pleurotus eryngii strains with higher ${\beta}$-glucan from control strain was developed. Genomic DNAs of 9 control strains of Pleurotus eryngii and 9 Pleurotus eryngii strains with higher ${\beta}$-glucan were analyzed by bulked segregant analysis (BSA) using randomly amplified polymorphic DNA (RAPD). One-hundred twenty RAPD primers were screened on bulked DNA samples and a unique DNA fragment with the size of 91 bp was yielded by OP-R03 primer from the Pleurotus eryngii strains with higher ${\beta}$-glucan. A sequence characterized amplified region (SCAR) marker, designated as OP-R03-1-F and OP-R03-1-R, was designed on the basis of the determined sequence. The PCR analysis with the OP-R03-1 primer showed that this SCAR marker can clearly distinguish the Pleurotus eryngii strains with higher ${\beta}$-glucan from the control strains.

• Journal of Mushroom
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• v.12 no.3
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• pp.226-231
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• 2014

In this study, SCAR marker that differentiates Pleurotus eryngii strains adaptable to high-temperature from control strain was developed. Genomic DNAs of 7 control strains of Pleurotus eryngii and 7 Pleurotus eryngii strains adaptable to high-temperature were analyzed by bulked segregant analysis (BSA) using randomly amplified polymorphic DNA (RAPD). Onehundred twenty RAPD primers were screened on bulked DNA samples and a unique DNA fragment with the size of 385 bp was yielded by OP-A06 primer from the Pleurotus eryngii strains adaptable to high-temperature. A sequence characterized amplified region (SCAR) marker, designated as OP-A06-1-F and OP-A06-1-R, was designed on the basis of the determined sequence. The PCR analysis with the OP-A06-1 primer showed that this SCAR marker can clearly distinguish the Pleurotus eryngii strains adaptable to high-temperature from the control strains.