• The Plant Pathology Journal
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• 제29권4호
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• pp.465-470
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• 2013

In January 2012, spreading type petunia cv. Wave Pink plants showing an abnormal growth habit of sprouting unusual multiple plantlets from the lateral buds were collected from a greenhouse in Gwacheon, Gyeonggi Province, Korea. The presence of phytoplasma was investigated using PCR with the primer pairs P1/P6, and R16F1/R1 for nested-PCR. In the nested PCR, 1,096 bp PCR products were obtained, and through sequencing 12 Pet-Stol isolates were identified. Comparison of the nucleotide sequences of 16S rRNA gene of the 12 Pet-Stol isolates with other phytoplasmas belonging to aster yellows or Stolbur showed that Pet-Stol isolates were members of Stolbur. The presence of phytoplasma in petunia was also confirmed by microscopic observation of the pathogens. In this study, Stolbur phytoplasma was identified from spreading type petunia cultivars by sequence analysis of 16S rRNA gene of phytoplasma and microscopic observation of phytoplasma bodies. This is the first report of Stolbur phytoplasma in commercial Petunia hybrida cultivars.

• 한국수산과학회지
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• 제29권6호
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• pp.770-778
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• 1996

A rapid and economical method of simultaneous extraction of DNA and RNA from seaweeds has been developed by the use of lithium chloride. Lithium chloride facilitates the softening of cell walls resulting in a decrease in both compressive and tensile modulus of elasticity. The DNA was characterized by high molecular weight larger than 27 kb and a relative lack of carbohydrate and protein contamination. The DNA and RNA extracted by the method from many seaweeds were of sufficient quality to be used as a template for per amplification with a plant intergenic gene primer set, for RAPD analysis with arbitrary primers, and for differential display with arbitrary primers in the morphologically distinct regions of the matured Porphyra thallus. The cDNA polymorphism indicated that the reproductive tissue types (male, female, patch) had a relatively high degree of similarity; the vegetative tissue types (dividing, non-dividing) also showed a similar pattern with respect to each other. Holdfast tissue had very low similarity with the other tissues, but appeared most similar to vegetative non-dividing tissue type.

• 한국수산과학회지
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• 제50권4호
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• pp.447-452
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• 2017

In this study, we tried to identify a sea anemone collected from the coast of Gijang, Busan. The anemone was morphologically similar to species belonging to the genus Anthopleura, but its morphological characteristics did not allow for confirmed identification to species level. Multiple genes from mitochondrial cytochrome oxidase III, 12S and 16S rRNA, and nuclear 18S and 28S rRNA, were amplified for multilocus sequence typing (MLST) analysis using genomic DNA extracted from the sampled anemone and a different primer set. Based on the MLST analysis, the anemone obtained in this study was identified as Anthopleura artemisia. Also, the sequence of internal transcribed spacer-2 was most closely related to A. artemisia, indicating that this single region might be useful for anemone identification. This study shows significance of molecular identification for sea anemones, and will be helpful in studies of sea anemone identification using genotyping-by-sequencing.

• 대한바이러스학회지
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• 제27권1호
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• pp.49-57
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• 1997

We developed a sensitive, nested reverse transcription-polymerase chain reaction (RT-PCR) to detect Hantaan, Seoul, Belgrade, Puumala and Sin Nombre viruses in animal tissues. Total RNA was extracted from blood, lung or kidney samples of experimentally-infected hamsters by using the guanidine isothiocyanate buffer-acid phenol-chloroform method. Genus-reactive outer primers were derived from the consensus region of the G1 gene sequences of several hantaviruses. Serotype-specific primers were selected within the region amplified by the outer primers. To examine the sensitivity and specificity of the test, we diluted known quantities of Hantaan, Seoul, Belgrade, Puumala and Sin Nombre viruses in human or hamster immune sera before performing the nested RT-PCR. We could detect as little as 1 pfu of virus, even in the presence of high-titer neutralizing antibodies, and the serotype-specific primers amplified only homologous serotype viruses. RT-PCR with these primers demonstrated virus in the blood of experimentally-infected hamsters as early as four days to as late as 30 days after infection. A comparison of a standard immunofluorescent antibody screening test (IFAT) to nested RT-PCR with RNA extracted from lung or kidney tissues of the hamsters, demonstrated that RT-PCR to be more sensitive for identifying viruses in these tissues.

• Mycobiology
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• 제41권1호
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• pp.37-41
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• 2013

Laccases (EC 1.10.3.2) are copper-containing polyphenol oxidases found in white-rot fungi. Here, we report the cloning and analysis of the nucleotide sequence of a new laccase gene, fvlac7, based on the genomic sequence of Flammulina velutipes. A primer set was designed from the putative mRNA that was aligned to the genomic DNA of F. velutipes. A cDNA fragment approximately 1.6-kb long was then amplified by reverse transcriptase-PCR using total RNA, which was subsequently cloned and sequenced. The cDNA sequence of fvlac7 was then compared to that of the genomic DNA, and 16 introns were found in the genomic DNA sequence. The fvlac7 protein, which consists of 538 amino acids, showed only 42~51% identity with 12 different mushroom species containing two laccases of F. velutipes, suggesting the fvlac7 is a novel laccase gene. The first 25 amino acids of Fvlac7 correspond to a predicted signal sequence, four copper-binding sites, and four N-glycosylation sites. Fvlac7 cDNA was heterologously overexpressed in an Escherichia coli system with an approximate expected molecular weight of 60 kDa.

• Archives of Pharmacal Research
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• 제28권5호
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• pp.561-565
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• 2005

Bacterial arylsulfate sulfotransferase (ASST) catalyzes the transfer of sulfate group from a phenyl sulfate ester to a phenolic acceptor. The promoter region and the transcripti on start sites of Enterobacter amnigenus astA have been determined by primer extension analysis. Northern blot analysis resolved two mRNA species with lengths of 3.3 and 2.0 kb, which correspond to the distances between the transcriptional initiation sites and the two inverted repeat sequences (IRSs). By length, the 3.3 kb RNA could comprise the three-gene (astA with dsbA and dsbB) operon. ASST has three highly conserved cysteine residues. Reducing and non-reducing SDS-PAGE and activity staining showed that disulfide bond is needed for the activity of the enzyme. To identify the cysteine residues responsible for the disulfide bond formation, a series of Cys to Ser mutants has been constructed and the enzymatic activity was measured. Based on the results, we assumed that the first cysteine (Cys349) might be involved in disulfide bond mainly with the second cysteine (Cys445) and result in active conformation.

• The Plant Pathology Journal
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• 제27권1호
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• pp.93-97
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• 2011

For quarantine purpose, we selected five plant RNA viruses including Cucumber vein yellowing virus (CVYV), Cucurbit yellow stunting disorder virus (CYSDV), Potato aucuba mosaic virus (PAMV), Potato yellow dwarf virus (PYDV), and Tomato chlorosis virus (ToCV), which are not reported in Korea and cause serious economic losses to the family Cucurbitaceae or Solanaceae. To detect those viruses, we employed RT-PCR technique with specific oligonucleotide primer pairs and tested their detection efficiency for each virus. To design RT-PCR primers, coat protein was used for CVYV, CYSDV, and ToCV whereas RNA polymerase and nucleocapsid regions were used for PAMV and PYDV, respectively. The development of an RT-PCR based method proved a useful tool for rapid detection and identification of quarantine virus infections.

• 한국임상수의학회지
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• 제19권3호
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• pp.295-298
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• 2002

본 연구에서는 개의 Helicobacter 균속 감염을 비침습적 방법으로 진단하기 위해 분변을 시료로한 PCR 검사법을 확립하고 그 효능을 검정하고자 하였다. 분변 PCR 분석은 분변에서 채취된 DNA에서 Helicobacter 균속의 16S RNA의 특이적인 영역에 반응하는 primer를 이용해 수행하였다. 분변 PCR분석법에 의한 결과와 위조직 검사 법 결과를 비교해 본 결과 분변 PCR분석법은 높은 특이도 (100%)와 민감도(96%)를 나타내었다. 도한 확립된 분볍 PCR분석법을 이용해 국내 애완결들의 위내 Helicobacter 균속 감염율을 조사한 결과 감염율이 72.1%로 나타났다. 이상의 결과 본 연구에서 확립한 분변 PCR 분석법은 개의 Helicobacter 균 감염을 진단할 수 있는 새로운 비침습적 진단방법으로 이용될 수 있으리라 사료된다.

• 한국자원식물학회지
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• 제21권3호
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• pp.210-215
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• 2008

Gene-expression analysis is increasingly important in biological research, with real-time reverse PCR (RTPCR) becoming the method of choice for high-throughput and accurate expression profiling of selected genes. However, this technique requires important preliminary work for standardizing and optimizing the many parameters involved in the analysis. Plant stress studies are more and more based on gene expression. The analysis of gene expression requires sensitive and reproducible measurements for specific mRNA sequence. Several genes are regulated in response to abitoic stresses, such as salinity, and their gene products function in stress response and tolerance. The design of the primers and TaqMan probes for real-time PCR assays were carried out using the Primer $Express^{TM}$ software 3.0. The PCR efficiency was estimated through the linear regression of the dilution curve. To understand the expression pattern of various genes under salt stressed condition, we have developed a unique public resource of 9 stress-related genes in poplar. In this study, real-time RT-PCR was used to quantify the transcript level of 10 genes (9 stress-related genes and 1 house keeping gene) that could play a role in adaptation of Populus davidiana. Real-time RT-PCR analyses exhibited different expression ratios of related genes. The data obtained showed that determination of mRNA levels could constitute a new approach to study the stress response of P. davidiana after adaptation during growth in salinity condition.

• The Plant Pathology Journal
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• 제35권1호
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• pp.51-62
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• 2019

A great variable response was observed when PP-3 and PP-J encumbered with 116 populations of root knot nematode (RKN) at two different temperatures ($25{\pm}2^{\circ}C$ and $30{\pm}2^{\circ}C$) and concentrations ($10^4$ and $10^5$ spores/ml). The PCR reaction amplified intergenic region between cytochrome oxidase subunit II gene (COII) and large subunit of rRNA gene (lrRNA) of the mitochondrial genome of different RKN species. The primer C2F3 and 1108 identified M. incognita with the highest frequency (52.6%) followed by M. javanica (36.8%) and M. arenaria (10.5%). The sizes of PCR products were 1.7 kb for M. incognita and M. javanica populations while populations of M. arenaria produced 1.1 kb fragment. The digestion with Hinf I yielded three different fragment length patterns on 1.5 % agarose gel. From current research it is concluded that intra-Meloidogyne genetic variability exist in RKN populations which have better encumbrance with P. penetrans.