• International Journal of Industrial Entomology and Biomaterials
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• v.6 no.1
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• pp.49-55
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• 2003

We describe the generation of transgenic silkworm that carrying the chimeric fibroin light chain (L-chain) gene. Previously, we have cloned the complete fibroin L-chain gene from the silkworm Baekok-Jam, Bombyx mori, and the complete fibroin gene from the oak silkworm, Antheraea yamamai. The 444 bp repetitive sequence of A. yamamai fibroin gene was inserted into the exon 6 of B. mori fibroin L-chain gene to produce chimeric fibroin L-chain gene. The chimeric fibroin L-chain gene was cloned into the polyhedrin gene site of Autographa californica nuclear polyhedrosis virus (AcNPV) to yield a recombinant baculovirus as a fibroin gene targeting vector, One-day-old fifth instar female silkworm larvae were injected with the recombinant baculovirus and then mated with normal male moths. Genomic DNA from their progenies was extracted and screened for the desired targeting event by using PCR and Southern blot analysis. The analysis showed that the chimeric fibroin gene had intergrated into the L-chain gene on the genome by homologous recombination and was transmitted through generations. The transgenic silkworm carrying the chimeric fibroin gene were approximately 43.2% in $F_2$ generation, and the silkworms synthesized the fusion protein in cocoons layer.

• Asian-Australasian Journal of Animal Sciences
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• v.26 no.8
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• pp.1144-1151
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• 2013

In this study, protein domains with cellulase activity in goat rumen microbes were investigated using metagenomic and bioinformatic analyses. After the complete genome of goat rumen microbes was obtained using a shotgun sequencing method, 217,892,109 pair reads were filtered, including only those with 70% identity, 100-bp matches, and thresholds below $E^{-10}$ using METAIDBA. These filtered contigs were assembled and annotated using blastN against the NCBI nucleotide database. As a result, a microbial community structure with 1431 species was analyzed, among which Prevotella ruminicola 23 bacteria and Butyrivibrio proteoclasticus B316 were the dominant groups. In parallel, 201 sequences related with cellulase activities (EC.3.2.1.4) were obtained through blast searches using the enzyme.dat file provided by the NCBI database. After translating the nucleotide sequence into a protein sequence using Interproscan, 28 protein domains with cellulase activity were identified using the HMMER package with threshold E values below $10^{-5}$. Cellulase activity protein domain profiling showed that the major protein domains such as lipase GDSL, cellulase, and Glyco hydro 10 were present in bacterial species with strong cellulase activities. Furthermore, correlation plots clearly displayed the strong positive correlation between some protein domain groups, which was indicative of microbial adaption in the goat rumen based on feeding habits. This is the first metagenomic analysis of cellulase activity protein domains using bioinformatics from the goat rumen.

• Journal of Microbiology and Biotechnology
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• v.30 no.7
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• pp.974-981
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• 2020

Sequence type 410 (ST410) of Escherichia coli is an extraintestinal pathogen associated with multi drug resistance. In this study, we aimed to investigate the horizontal propagation pathway of a high-risk clone of E. coli ST410 that produces Klebsiella pneumoniae carbapenemase (KPC). blaKPC-encoding E. coli and K. pneumoniae isolates were evaluated, and complete sequencing and comparative analysis of blaKPC-encoding plasmids from E. coli and K. pneumoniae, antimicrobial susceptibility tests, polymerase chain reaction, multilocus sequence typing, and conjugal transfer of plasmids were performed. Whole-genome sequencing was performed for plasmids mediating KPC-2 production in E. coli and K. pneumoniae clinical isolates. Strains E. coli CPEc171209 and K. pneumoniae CPKp171210 were identified as ST410 and ST307, respectively. CPEc171209 harbored five plasmids belonging to serotype O8:H21, which is in the antimicrobial-resistant clade C4/H24. The CPKp171210 isolate harbored three plasmids. Both strains harbored various additional antimicrobial resistance genes. The IncX3 plasmid pECBHS_9_5 harbored blaKPC-2 within a truncated Tn4401a transposon, which also contains blaSHV-182 with duplicated conjugative elements. This plasmid displayed 100% identity with the IncX3 plasmid pKPBHS_10_3 from the K. pneumoniae CPKp171210 ST307 strain. The genes responsible for the conjugal transfer of the IncX3 plasmid included tra/trb clusters and pil genes coding the type IV pilus. ST410 can be transmitted between patients, posing an elevated risk in clinical settings. The emergence of a KPC-producing E. coli strain (ST410) is concerning because the blaKPC-2-bearing plasmids may carry treatment resistance across species barriers. Transgenic translocation occurs among carbapenem-resistant bacteria, which may spread rapidly via horizontal migration.

• The Plant Pathology Journal
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• v.30 no.2
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• pp.183-187
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• 2014

Lettuce mosaic virus (LMV) causes disease of plants in the family Asteraceae, especially lettuce crops. LMV isolates have previously been clustered in three main groups, LMV-Yar, LMV-Greek and LMV-RoW. The first two groups, LMV-Yar and LMV-Greek, have similar characteristics such as no seed-borne transmission and non-resistance-breaking. The latter one, LMV-RoW, comprising a large percentage of the LMV isolates contains two large subgroups, LMV-Common and LMV-Most. To date, however, no Korean LMV isolate has been classified and characterized. In this study, LMV-Muju, the Korean LMV isolate, was isolated from lettuce showing pale green and mottle symptoms, and its complete genome sequence was determined. Classification method of LMV isolates based on nucleotide sequence divergence of the NIb-CP junction showed that LMV-Muju was categorized as LMV-Common. LMV-Muju was more similar to LMV-O (LMV-Common subgroup) than to LMV-E (LMV-RoW group but not LMV-Common subgroup) even in the amino acid domains of HC-Pro associated with pathogenicity, and in the CI and VPg regions related to ability to overcome resistance. Taken together, LMV-Muju belongs to the LMV-Common subgroup, and is expected to be a seed-borne, non-resistance-breaking isolate. According to our analysis, all other LMV isolates not previously assigned to a subgroup were also included in the LMV-RoW group.

• The Plant Pathology Journal
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• v.36 no.1
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• pp.87-97
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• 2020

The development of reverse transcription-polymerase chain reaction using degenerate primers against conserved regions of most potyviral genomes enabled sampling of the potyvirome. However, these assays usually involve sampling potential host plants, but identifying infected plants when they are asymptomatic is challenging, and many plants, especially wild ones, contain inhibitors to DNA amplification. We used an alternative approach which utilized aphid vectors and indicator plants to identify potyviruses capable of infecting common bean (Phaseolus vulgaris). Aphids were collected from a range of asymptomatic leguminous weeds and trees in Iran, and transferred to bean seedlings under controlled conditions. Bean plants were tested serologically for potyvirus infections four-weeks postinoculation. The serological assay and symptomatology together indicated the presence of one potyvirus, and symptomology alone implied the presence of an unidentified virus. The partial genome of the potyvirus, encompassing the complete coat protein gene, was amplified using generic potyvirus primers. Sequence analysis of the amplicon confirmed the presence of an isolate of Wisteria vein mosaic virus (WVMV), a virus species not previously identified from Western Asia. Phylogenetic analyses of available WVMV sequences categorized them into five groups: East Asian-1 to 3, North American and World. The Iranian isolate clustered with those in the World group. Multiple sequence alignment indicated the presence of some genogroup-specific amino acid substitutions among the isolates studied. Chinese isolates were sister groups of other isolates and showed higher nucleotide distances as compared with the others, suggesting a possible Eastern-Asian origin of WVMV, the main region where Wisteria might have originated.

• Parasites, Hosts and Diseases
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• v.56 no.3
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• pp.295-300
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• 2018

Human sparganosis was diagnosed by morphological and genetic analyses in Korea. The complete mitochondrial genomes of Spirometra erinaceieuropaei and S. decipiens isolated in Korea have been recorded. Present study was performed to provide information to diagnose the etiologic agent of sparganosis by multiplex PCR using mitochondrial genome sequences of S. erinaceieuropaei and S. decipiens. In an effort to examine the differential diagnosis of spirometrid tapeworms, multiplex PCR assays were performed on plerocercoid larvae of S. erinaceieuropaei and S. decipiens. The PCR products obtained using species-specific primers were positively detected in all PCR assays on mixture of S. erinaceieuropaei and S. decipiens DNA. S. erinaceieuropaei-specific bands (239 bp and 401 bp) were obtained from all PCR assays using a mixture of S. erinaceieuropaei-specific primers (Se/Sd-1800F and Se-2018R; Se/Sd-7955F and Se-8356R) and S. erinaceieuropaei template DNA. S. decipiens-specific bands (540 bp and 644 bp) were also detected in all PCR assays containing mixtures of S. decipiens-specific primers (Se/Sd-1800F and Sd-2317R; Se/Sd-7955F and Sd-8567R) and S. decipiens template DNA. Sequence analyses on these species-specific bands revealed 100% sequence identity with homologous regions of the mtDNA sequences of S. erinaceieuropaei and S. decipiens. The multiplex PCR assay was useful for differential diagnosis of human sparganosis by detecting different sizes in species-specific bands.

• Korean Journal of Veterinary Service
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• v.34 no.2
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• pp.103-109
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• 2011

Asia1/Shamir that has been recommended by World Reference Laboratory for foot-and-mouth disease (FMD) is used as a vaccine strain, and is being prepared in many countries including Korea. Although it is assumed that vaccine strain Asia1/Shamir has a wide antigenicity, sufficient molecular biological analysis has not been accomplished yet. Complete genome sequence analysis showed that the region with the most severe variations was 1D region of structural protein-coding sequence; particularly amino acid 141~157 residues in 1D region RGD sites for binding to susceptible cells. In addition, five amino acids in 1D region were identified as characteristic sites that are different from other known Asia1 viruses. Asia1/Shamir strain was shown to be genetically similar to group VI that had occurred in the Middle East, but showed low level of genetic similarity to the group V viruses that had occurred in the Southeast Asia and China. It is considered that, if these viruses, group I and II including group V are introduced into Korea, care would be paid in case of inoculating the vaccine strain Shamir available in Korea.

• Research in Plant Disease
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• v.28 no.2
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• pp.98-107
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• 2022

The incidence of Tomato brown rugose fruit virus (ToBRFV) and biological and molecular characterization of the Palestinian isolates of ToBRFV are described in this study. Symptomatic leaf samples obtained from Solanum lycopersicum L. (tomatoes) and Nicotiana tabacum L. (cultivated tobacco) plants were tested for tobamoviruses infection by reverse transcription polymerase chain reaction. Tomato leaf samples collected from Tulkarm and Qalqilia are infected with ToBRFV-PAL with an infection rate of 76% and 72.5%, respectively. Leaf samples collected from Jenin and Nablus were found to be mixed infected with ToBRFV-PAL and Tobacco mosaic virus (TMV) (100%). Sequence analysis of the ToBRFV-PAL genome showed that the net average nucleotide divergence between ToBRFV/F48-PAL strain and the Israeli and Turkish strains was 0.0026398±0.0006638 (±standard error of mean), while it was 0.0033066±0.0007433 between ToBRFV/F42-PAL and these two isolates. In the phylogenetic tree constructed with the complete genomic sequence, all the ToBRFV isolates were clustered together and formed a sister branch with the TMV. The sequenced Palestinian isolates of ToBRFV-PAL shared the highest nucleotide identity with the Israeli ToBRFV isolate suggesting that the virus was introduced to Palestine from Israel. The findings of this study enhance our understanding of the biological and molecular characteristics of ToBRFV which would help in the management of the disease.

• The Plant Pathology Journal
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• v.22 no.3
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• pp.265-270
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• 2006

Cultivated hot pepper crops showing severe mosaic symptom were found in Korea in 2004 and their causal agent was identified as Cucumber mosaic virus (CMV). These pepper crops was resistant to the virus in the filled, and they belonged to pathotype 0 (P0) resistant pepper. Resistance screening of selected pepper plants showed that a pepper isolate of CMV was the P0 resistance-breaking virus. This P0 resistance-breaking isolate of CMV, named as Ca-P1, was isolated from leaves of the virus-infected Capsicum annuum cv. Manidda that showed systemic severe mosaic symptom. Ca-P1-CMV could induce systemic mosaic symptoms on P0-susceptible (P0-S) and P0-resistant (P0-R) cultivars whereas an ordinary strain (Fny-CMV) could not infect P0-R. This result suggests that Ca-P1-CMV can overcome P0 resistant pepper cultivars. To analyze its genome sequence, the complete nucleotide sequence of RNA3 of Ca-P1-CMV was determined from the infectious full-length cDNA clone of the virus. RNA3 of Ca-P1-CMV consisted of 2,219 nucleotides. Overall sequence homology of RNA3-encoded two viral proteins (movement protein and coat protein) revealed high similarity (75.2-97.2%) with the known CMV strains. By sequence analysis with known representative strains of CMV, Ca-P1-CMV belongs to a typical member of CMV subgroup IB. The resistance and resistance-breaking mechanisms of pepper and counterpart CMV, respectively, remain to be investigated, which will enrich the genetic resources and accelerate CMV-resistant pepper breeding programs.

• Molecules and Cells
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• v.23 no.2
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• pp.145-153
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• 2007

Elucidation of the roles of circadian associated factors requires a better understanding of the molecular mechanisms of circadian rhythms, control of flowering time through photoperiodic pathways, and photosensory signal transduction. In Arabidopsis, the APRR1 quintet, APRRs 1, 3, 5, 7, and 9, are known as central oscillator genes. Other plants may share the molecular mechanism underlying the circadian rhythm. To identify and characterize these circadian response genes in Brassica crops whose genome was triplicated after divergence from Arabidopsis, we identified B. rapa BAC clones containing these genes by BLAST analysis of B. rapa BAC end sequences against the five corresponding Arabidopsis regions. Subsequent fingerprinting, Southern hybridization, and PCR allowed identification of five BAC clones, one for each of the five circadian-related genes. By draft shotgun sequencing of the BAC clones, we identified the complete gene sequences and cloned the five expressed B. rapa circadian-associated gene members, BrPRRs 1, 3, 5, 7, and 9. Phylogenetic analysis revealed that each BrPRR was orthologous to the corresponding APRR at the sequence level. Northern hybridization revealed that the five genes were transcribed at distinct points in the 24 hour period, and Southern hybridization revealed that they are present in 2, 1, 2, 2, and 1 copies, respectively in the B. rapa genome, which was triplicated and then diploidized during the last 15 million years.