• Journal of Plant Biotechnology
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• v.49 no.1
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• pp.30-38
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• 2022

Solanum brevicaule is one of the tuber-bearing wild Solanum species. Because of its resistance to several important pathogens infecting potatoes during cultivation, it can be used for potato breeding. However, the fact that S. brevicaule used in this study has an EBN value of two causes the sexual reproduction barriers between the species and cultivated potatoes. In this study, specific markers for discriminating S. brevicaule from other Solanum species were developed on the basis of the results of sequence alignments with the whole chloroplast genomes of S. brevicaule and seven other Solanum species. The chloroplast genome of S. brevicaule was completed by next-generation sequencing technology described in other recent studies. The total sequence length of the chloroplast genome of S. brevicaule is 155,531 bp. Its structure and gene composition are similar to those of other Solanum species. Phylogenetic analysis revealed that S. brevicaule was closely grouped with other Solanum species. BLASTN search showed that its genome sequence had 99.99% and 99.89% identity with those of S. spegazzinii (MH021562) and S. kurtzianum (MH021495), respectively. Sequence alignment identified 27 SNPs that were specific to S. brevicaule. Thus, three PCR-based CAPS markers specific to S. brevicaule were developed on the basis of these SNPs. This study will facilitate in further studies on evolutionary and breeding aspects in Solanum species.

• International Journal of Industrial Entomology and Biomaterials
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• v.12 no.2
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• pp.57-61
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• 2006

Suppressor of cytokine signaling (SOCS) is known to be as a negative feedback regulator in Janus kinase signal transducer and activator of transcription signaling. Highly conserved SOCS box domain was cloned from a Korean malaria vector, Anopheles sinensis. Sequence analysis indicates that it has identity to Anopheles gambiae (96%), Aedes aegypti (94%), Drosophila melanogaster (78%), Mus musculus (72%) and Homo sapiens (72%), respectively. Tissue specificity RT-PCR demonstrated that the expression level of AsSOCS transcript was high at abdomen, midgut, and ovary, whereas developmental expression patterns showed that the level of AsSOCS was high at egg, early pupae, and adult female. On the other hand, RT-PCR analysis after bacterial challenge showed that SOCS mRNA was strongly induced in larvae. In addition, it was also induced by various immune elicitors such as lipoteicoic acid, CpG-DNA, and laminarin. It seems that AsSOCS, repressor of JAK-STAT pathway, is highly conserved in mosquito, and may play an important role in mosquito innate immune response.

• Journal of Embryo Transfer
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• v.13 no.2
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• pp.159-172
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• 1998

사람 성장호르몬 수용체(hGHR) cDNA는 PCR방법에 의하여 fagment로서 보고되어진 바 있으나, liver cDNA로 부터 전장을 cloning한 보고는 없는 실정으로 본 연구에서는 기능을 가진 약 4.6kbp의 cDNA hGHR을 cloning 하는데 성공하였다. 먼저 cloning하기 위하여 human liver mRNA와 human breast cancer tissue로부터 회수한 mRNA를 RT-PCR방법에 의하여 human cDNA library와 cloning에 필요한 probe를 제작하였다. human library mRNA는 GT-PCR방법에 의하여 증폭하여 증폭되어진 산물은 λZAP Vector를 이용하여 cDNA library를 구축하였고,screeing을 위하여 임 보고 되어진 hGHR fragment native sequence를 기초로 N-terminal부분의 primer를 설계하여 950bp의 probe를 얻는데 성공하였다. 이 probe를 이용하여 준비된 human liver cDNA library로부터 2.5$\times$10 6개의 plaque로부터 6개의 positive clone을 획득하였고, 이들중 poly Asignal인 "AATAAA"를 포함하고 있는 가장 긴 약 3.8kbp의 clone을 sequencing한 결과 open reading frame을 포함하고 있었으나, 5'부분의 결손되어 있었다. 그리하여 이 부분은 human breast cancer tissue로 부터 회수한 mRNA를 RT-PCR에 의하여 증폭하였고, sequencing결과 이미 보고되어진 native hGHR와 비교한 결과 하나의 nucleotide가 silent mutation으로 판명되었다.한편 human liver cDNA library로부터 cloning한 3.8cp의 positive clone의 5'end의 결손된 부분에 silent mutation된 PCR 산물을 연결함으로써 native hGHR와 유사한 cDNA hGHR subcloning에 성공하였다. 이러한 cDNA hGHR의 clone이 function을 가지고 있는지를 검토하기 위하여 eukaryotic 발현 vector인 pCXN2에 의거 ligation한 후 chinese hamster ovary cell[CHO-KI]에 transfect를 실시하였다. Dexamethasone은 첨가하지 않고 hGH만의 존재하에서 이들 cell을 배양시키고 cell menbrane에서 발현 여부를 판정키 위하여 hGHR monocloual antibody를 사용하여 flow cytometery해석을 실시하는 한편 125I-hGH binding assay에 의하여 hGH binding activity를 측정하였다. 최종적으로 GH signal transduction의 target genedf으로 알려져 있는 serine protease inhibitor 2.1(Spi 2.1) gene의 promotor activity를 검토한 결과 hGHR을 transfect한 CHO Cell에 있어서 hGH의 농도에 의존적으로 증가되었다. 따라서 본 실험에서 cloning한 cDNA hGHR는 native hGHR와 같은 기능을 가지는 것으로 판명되었다.것으로 판명되었다.

• Research in Plant Disease
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• v.19 no.4
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• pp.313-318
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• 2013

In March, 2013, twenty symptomatic freesia plants (10 plants of cultivar Shiny Lemon and 10 plants of cultivar Shiny Gold), with striking virus-like symptoms were collected in Cheongju, Korea. The plants showed chlorotic, coalescing, interveinal, whitish, necrotic, mosaic, mottling or dark brown-to-purple necrotic spots on leaves. Freesia crude sap was directly analyzed by transmission electron microscopy, which potyvirus particles as well as long virus-like particles were detected. Total RNA extracts were analyzed for the infection of Freesia sneak virus (FreSV) by reverse transcription (RT)-PCR with primers specific to FreSV coat protein (CP) gene based on the sequences of FreSV isolates (GenBank No. GU071089, FJ807730 and DQ885455), showing 9 of 20 plants were infected. All 1305bp RT-PCR products were cloned and sequenced. Comparisons of nucleotide and deduced amino acid sequences using BLAST and bioinformatics tools resulted in 99 to 100% sequence identity with FreSV isolates FOV, Virginia, and Italy, confirming FreSV in 9 symptomatic freesia plants. Of 9 determined cDNAs of FreSV isolates, sequences of 5 cDNA clones were identical (GenBank No. AB811437) and sequences of 4 cDNA clones were identical (GenBank No. AB811792). To our knowledge, this is the first report of FreSV from Freesia spp. in Korea.

• Asian-Australasian Journal of Animal Sciences
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• v.31 no.1
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• pp.63-70
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• 2018

Objective: The aim of the study was to isolate gossypol-degrading bacteria and to assess its potential for gossypol degradation. Methods: Rumen liquid was collected from fistulated cows grazing the experimental pasture. Approximately 1 mL of the rumen liquid was spread onto basal medium plates containing 2 g/L gossypol as the only source of carbon and was then cultured at $39^{\circ}C$ to isolate gossypol-degrading bacteria. The isolated colonies were cultured for 6 h and then their size and shape observed by microscope and scanning electron microscope. The 16S rRNA gene of isolated colonies was sequenced and aligned using National Center for Biotechnology Information-Basic Local Alignment Search Tool. The various fermentation conditions, initial pH, incubation temperature, inoculum level and fermentationperiod were analyzed in cottonseed meal (CSM). The crude protein (CP), total gossypol (TG), and free gossypol (FG) were determined in CSM after fermentation with isolated strain at $39^{\circ}C$ for 72 h. Results: Screening results showed that a single bacterial isolate, named Rumen Bacillus Subtilis (RBS), could use gossypol as a carbon source. The bacterium was identified by 16S rDNA sequencing as being 98% homologous to the sequence of Bacillus subtilis strain GH38. The optimum fermentation conditions were found to be 72 h, $39^{\circ}C$, pH 6.5, moisture 50%, inoculum level $10^7cell/g$. In the optimum fermentation conditions, the FG and TG content in fermented CSM decreased 78.86% and 49% relative to the control. The content of CP and the essential amino acids of the fermented CSM increased respectively, compared with the control. Conclusion: The isolation of a gossypol-degrading bacterium from the cow rumen is of great importance for gossypol biodegradation and may be a valuable potential source for gossypol-degradation of CSM.

• Korean Journal of Plant Taxonomy
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• v.34 no.4
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• pp.335-358
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• 2004

To investigate molecular phylogenetic relationships and to test hypothesis of hybrid origin of Ranunculus cantoniensis (Ranunculaceae), the sequences of nrDNA and chloroplast DNA were analyzed for 8 taxa and 25 accessions including 5 accessions of outgroup. In the phylogenetic trees by analyses of maximum parsimony and maximum likelihood for ITS nrDNA sequences and combined data of psbA-trnH, rps16 and trnL sequences of cpDNA, R. cantoniensis was most closely related to R. chinensis, and then to R. taciroi and R. silerifolius. The molecular phylogenetic relationships were not congruent with the previous report that R. cantoniensis was most closely related to R. silerifolius. In the sequence analysis of ITS and psbA-trnH, rps16, trnL for R. cantoniensis and the related taxa, R. cantoniensis showed polymorphism. It supported that the polymorphism also was reported in chromosome number and karyotype of R. cantoniensis. Ranunculus cantoniensis shared the marker gene of R. chinensis and R. silerifolius in ITS, and one of R. silerifolius in cpDNA. These results supported the hypothesis that R. cantoniensis was caused by hybridization between R. chinensis and R. silerifolius based on chromosome number and karyotype, and also estimated that R. silerifolius might be of maternal origin and R. chinensis be paternal.

• Research in Plant Disease
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• v.30 no.1
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• pp.60-65
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• 2024

In July 2020, total RNA was extracted from passion fruit (Passiflora edulis) leaves showing virus symptoms such as chlorotic spots and vein banding in Haenam, South Korea. Cucumber mosaic virus (CMV)-HN2 was identified through reverse transcription polymerase chain reaction and sequencing analysis. To confirm the biological characteristics of the CMV infecting passion fruit, 10 indicator plants were inoculated with CMV-HN2, and the results showed a typical CMV symptoms. Phylogenetic analysis based on the amino acid of the coat protein (CP) of CMVs revealed that the CMV passion fruit isolates belonged to subgroup I, among which CMV-HN2 belonged to subgroup IA. Additionally, CMVs isolated from passion fruit in Korea have amino acid sequence variation between the subgroup. Among them, CMV-HN2 had four to eight amino acid differences in CP from other CMV isolates from passion fruit. These results confirm the presence of genetic diversity in the CPs of passion fruit CMV isolates.

• Research in Plant Disease
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• v.9 no.2
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• pp.89-93
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• 2003

An isolate of Tobacco mosaic virus (TMV) was isolated from potato cultivar ‘Chubak’ showing vein clearing and mild mosaic at Namhae in Korea. This isolate, TMV-St, was differentiated from other tobamoviruses based on biological properties, serological relationships and nucleotide sequence analyses of coat protein genes. TMV-St caused typical symptoms on four indicator plants as compared to the tobamovirus of TMV-U1, Pepper mild mottle virus (PMMoV), and Tomato mosaic virus (ToMV), which caused economic losses in Solanaceous vegetables, tomato, pepper, and eggplant. Remarkably, the TMV-St induced distinctly different symptom of systemic chlorotic spots on Chenophodium murale. On C. murale, Gomphorena globosa, and Nic-otiana rustica, the four viruses were classed by the virulence of systemic or local infections. In serological test TMV-St antiserum showed a precipitation line with each other tabamovirus. The CP gene of TMV-St contain 477 nucleotides, and the nucleotides sequence was the most similar to that of TMV-U1.

• Horticultural Science & Technology
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• v.35 no.3
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• pp.333-343
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• 2017

Bloomless cucumber fruits are commercially produced by grafting onto the pumpkin stocks (Cucurbita moschata) to restricted silicon ($SiO_2$) absorption. Inhibition of silicon absorption in bloomless stocks is conferred by a mutant allele of the CmLsi1 homologous to Lsi1 in rice. In this study, we characterized the Lsi1 homologs in pumpkin (C. moschata) and its cold-tolerant wild relative C. ficifolia ('Heukjong') in order to develop a DNA marker for selecting a bloomless trait and to establish the molecular basis for breeding bloomless stock cultivars of C. ficifolia. A Cleaved amplified polymorphic sequence (CAPS) marker (CM1-CAPS) was designed based on a non-sysnonymous single nucleotide polymorphism (SNP, C>T) of the CmLsi1 mutant-type allele, and its applicability for Marker-assisted selection (MAS) was confirmed by evaluating three bloom and five bloomless pumpkin stock cultivars. Quantitative RT-PCR of the CmLsi1 for these stock cultivers implied that expression level of the CmLsi1 gene does not appear to be associated with the bloom/bloomless trait and may differ depending on plant species and tissues. A full length cDNA of the Lsi1 homolog [named CfLsi1($B^+$)] of 'Heukjong' (C. ficifolia), was cloned and sequence comparison between CmLsi1($B^+$) and CfLsi1($B^+$) revealed that there exists total 24 SNPs, of which three were non-synonymous. Phylogenetic analysis of CfLsi1($B^+$) and Lsi1 homologs further revealed that CfLsi1($B^+$) is closesly related to Nodulin 26-like intrinsic proteins (NIPs) and most similar to CpNIP1 of C. pepo than C. moschata.

• Journal of Plant Biotechnology
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• v.49 no.3
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• pp.178-186
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• 2022

The tetraploid Solanum acaule is a wild potato species from Bolivia widely used for potato breeding because of its diverse attractive traits, including resistance to frost, late blight, potato virus X, potato virus Y, potato leafroll virus, potato spindle tuber viroid, and cyst nematode. However, the introgression of useful traits into cultivated potatoes via crossing has been limited by differences in endosperm balance number between species. Somatic fusion could be used to overcome sexual reproduction barriers and the development of molecular markers is essential to select proper fusion products. The chloroplast genome of S. acaule was sequenced using next-generation sequencing technology and specific markers for S. acaule were developed by comparing the obtained sequence with those of seven other Solanum species. The total length of the chloroplast genome is 155,570 bp, and 158 genes were annotated. Structure and gene content were very similar to other Solanum species and maximum likelihood phylogenetic analysis with 12 other species belonging to the Solanaceae family revealed that S. acaule is very closely related to other Solanum species. Sequence alignment with the chloroplast genome of seven other Solanum species revealed four InDels and 79 SNPs specific to S. acaule. Based on these InDel and SNP regions, one SCAR marker and one CAPS marker were developed to discriminate S. acaule from other Solanum species. These results will aid in exploring evolutionary aspects of Solanum species and accelerating potato breeding using S. acaule.