• Proceedings of the Korean Society of Plant Pathology Conference
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• 2003.10a
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• pp.90.1-90
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• 2003

Colonization of Pseudomonas chlororaphis O6, a nonpathogenic rhizobacterium, on the roots induced systemic resistance in cucumber plants against tai-get leaf spot, a foliar disease caused by Corynespora cassiicola. A cDNA library was constructed using mRNA extracted from the cucumber leaves 12 h after inoculation with C. cassiicola, which roots had been previously treated with O6. To identify the genes involved in the O6-mediated induced systemic resistance (ISR), we employed a subtractive hybridization method using mRNAs extracted from C cassiicola-inoculated cucumber leaves with and without previous O6 treatment on the plant roots. Differential screening of the cDNA library led to the isolation of 5 distinct genesencoding a GTP-binding protein, a putative senescence-associated protein, a galactinol synthase, a hypersensitive-induced reaction protein, and a putative aquaporin. Expressions of these genes are not induced by O6 colonization alone. Before challenge inoculation, no increase in the gene transcriptions could be detected in previously O6-treated and untreated plants but, upon subsequent inoculation with the pathogenic fungus, transcription levels in O6-treated plants rose significantly faster and stronger than in untreated plants. Therefore, the O6-mediated ISR may be associated with an enhanced capacity for the rapid and effective activation of cellular defense responses which becomes apparent only after challenge inoculation on the distal, untreated plant parts, as suggested by Conrath et al. (2002). This work was supported by a grant R11-2001-092-02006-0 from the Korea Science and Engineering Foundation through the Agricultural Plant Stress Research Center at Chonnam National University.

• Journal of Plant Biotechnology
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• v.42 no.1
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• pp.25-33
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• 2015

A PyrcpPR-10 gene with differentially expressed was isolated by using the suppression subtractive hybridization assay between '93-3-98' (highly resistant against scab caused by Venturia nashicola) and 'Sweat Skin'(highly susceptible) and analyzed the expression pattern according to organs and cultivars. The full length of PyrcpPR-10 was cloned as 743bp with 480bp's ORP, and was determined to encode a protein of 159 amino acid residues. On analyzing PyrcpPR-10 gene sequence compared with resistant and susceptible cultivars, 'Hwangsilri' (resistant), 'Gamcheonbae' (moderately resistant), 'Wonhwang' (moderately susceptible), 'Niitaka' (highly susceptible), and 'Sweat Skin' (highly susceptible) had identical gene sequence but 'Bartlett' (highly resistant) showed partly different sequences. The deduced amino acid sequence showed 64 ~ 98% homology and had the GXGGXG motif to known amino acid of other plants PR-10 by the BLAST X analysis. Among several organs or tissues, petal was showed highest expression level of PyrcpPR-10 gene followed by leaf, floral axis, bud, and bark. The expression level of PyrcpPR-10 gene was dramatically increased at 24 hr after inoculation in all cultivars and also up-regulated in accordance with resistant degree of cultivars. While resistant cultivars ('Bartlett', '93-3-98', and 'Hwangsilri') induced relatively high expression level of PyrcpPR-10 gene, susceptible cultivars ('Niitaka', and 'Sweat Skin') showed low expression level. PyrcpPR-10 gene is assumed that it is directly connected with defense mechanisms to pear scab.

• Journal of Life Science
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• v.14 no.4
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• pp.650-656
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• 2004

Oryza grandiglumis (CCDD, 2n=48), one of the wild rice species, has been known to possess fungal-,bacterial-, and insect-resistance against sheath blight, rice blast, bacterial leaf blight and brown plant hopper (Nilaparvata lugens). To rapidly isolate differentially expressed genes responding to fungal and wounding stress, wounding and yeast extract were treated to O. grandiglumis for 24 hrs. Suppression subtractive hybridization (SSH) method was used to obtain differentially expressed genes from yeast extract and wounding treated plants. Seven hundreds and seventy six clones were obtained by subcloning PCR product, and colony array and screening were carried out using radio-isotope labeled cDNA probes prepared from the wounding and yeast extract treated plants. One hundred and fifteen colonies were confirmed as true positive ones. Average insert size of the clones were ranged from 400 bp to 700 bp and all the inserts were sequenced. To decide the identity of those clones, sequences were analyzed by sequence homology via GenBank database. The homology search result showed that 68 clones were matched to the genes with known function; 16 were related to primary metabolism, 5 to plant retrotransposons, 5 to defense related metallothionein-like genes. In addition to that, others were matched to various genes with known function in amino acid synthesis and processing, membrane transport, and signal transduction, so on. In northern blot analysis, induced expressions of ogwfi-161, ogwfi-646, ogwfi-663, and ogwfi-695 by wounding and yeast extract treatments were confirmed. The result indicates that SSH method is very efficient for rapid screening of differentially expressed genes.

• Asian-Australasian Journal of Animal Sciences
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• v.21 no.4
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• pp.489-493
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• 2008

In 2004, Jorgensen and coworkers proposed the MUC4 gene as a candidate gene of enterotoxigenic Escherichia coli (ETEC) F4ab/ac receptor in piglets and a mutation of $G{\rightarrow}C$ in intron 7 of MUC4 was identified to be associated with the ETEC F4ab/ac adhesion phenotypes. In this study, we used 310 piglets of three breeds (Landrace, Large White and Chinese Songliao Black) to analyze the relationship between this mutation and the F4ab/ac adhesion phenotype. The results show that the genotypes at this site and the ETEC F4ab/ac adhesion phenotypes were not completely consistent, although they are very strongly associated. Among the individuals with genotype CC, which was identified as a resistant genotype to F4ab/ac adhesion, only 72.1% (124/172) were non-adhesive to ETEC F4ab and 77.9% (134/172) were non-adhesive to ETEC F4ac infections. This suggests that this mutation may not be the causative mutation for ETEC F4ab/ac adhesion, rather, the actual causative mutation may be in another gene closely linked to MUC4, or at aother site within the MUC4 gene. Our results also suggest that the receptors of F4ab and F4ac may be determined by two different but closely linked loci. In order to screen other genes related to F4ab/ac adhesion in piglets, the mRNA profiles from six full sib piglets, of which three were adhesive to ETEC F4ab/ac and three non-adhesive, were analyzed by suppression subtractive hybridization (SSH). One up-regulated gene, Ep-CAM, was selected for further analysis based on its role in the intestinal epithelial cells adhesion. Using real-time RT-PCR, we found that the Ep-CAM gene was significantly up-regulated in the piglets adhesive to F4ab/ac. It was mapped to SSC3q11-q14 by radiation hybrid mapping.

• Korean Journal of Ichthyology
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• v.23 no.1
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• pp.21-29
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• 2011

Expression analysis of development-related genes was conducted using differential screening of 6-month-old [18M(-), 6M-18M] specific and 18-month-old [6M(-), 18M-6M] specific subtracted cDNA libraries constructed by subtractive hybridization using skeletal muscle of 6- and 18-month-old dark-banded rockfish Sebastes inermis. A total 202 cDNA clones displaying different expression levels in each stage were obtained; among them, 32 clones showing up-regulation were finally selected for further expression analysis. We sequenced the clones and analyzed individual sequences. Genes expressed specifically in 6-month-old skeletal muscle were identified as myosin, adenylate kinase, calsequestrin, dystrobrevin beta, and diphosphate kinase-Z1. Genes showing strong expression in 18-month-old rockfish were identified as desmin, TGFBR2 (transforming growth factor-beta receptor), muscle-type creatine kinase, and cathepsin D. Expression of these genes was checked further in 6-18-30-42 month-old dark-banded rock fish. Rapid reduction of expression was observed in dystrobrevin beta and diphosphate kinase. However, expression of creatine kinase (muscle type) and cathepsin D increased as dark-banded rockfish grew, and remained even after 18 months. The results reported here demonstrate that genes related to muscles contract are expressed at an early stage of development, and genes controlling energy in muscles are predominantly expressed at a late developmental stage.

• Asian-Australasian Journal of Animal Sciences
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• v.21 no.1
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• pp.19-24
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• 2008

The purpose of this study was to detect expression of genes related to egg production in Taiwan Country chickens by suppression subtractive hybridization. Liver samples of mRNA extraction from two Taiwan Country chicken strains (L2 and B), originated from the same population but with very distinct egg production rates after long-term selection for egg and meat production respectively. Two-way subtraction was performed. The hepatic cDNA from the low egg production chickens (B) was subtracted from the hepatic cDNA from the high egg production strain (L2). The reversed subtraction (L2 from B) was also performed. The resulting differentially expressed gene fragments were cloned and sequenced. We sequenced 288 clones from the forward subtraction and 96 clones from the reverse subtraction. These genes were subjected to further screening to confirm the differential expression between the two genetic breeds of chickens. The apolipoprotein B (apoB) was expressed to a greater extent in the liver of the L2 than in the B line chickens. The 5-aminoimidazole- 4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase (PURH) was expressed to a greater extent in the liver of the B than in the L2 strain chickens. We demonstrated that both apoB and PURH were more highly expressed in the liver than that in other tissues (muscle, ovary, and oviduct) in laying Taiwan Country chickens. Taken together, these data suggest that after the selection for egg production, expression of apoB and PURH genes were also changed. Whether the changed expression of these genes is directly related to egg production is not known, but these two genes may be useful markers for egg laying performance in Taiwan Country chickens.

• 대한생식의학회:학술대회논문집
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• 2001.03a
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• pp.195-205
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• 2001

태반 영양배엽 (trophoblast)은 포유동물의 발생과정 중 가장 먼저 분화되는 세포로서, 자궁환경내에서 배아가 착상, 발생, 및 분화하기 위해서 반드시 필요한 태반을 형성하는 색심적인 세포이다. 영양배엽 세포의 분화과정중의 결함은 배아의 사산이나 임신질환 등의 치명적 결과를 초래한다. 하지만, 영양배엽 세포의 분화를 조절하는 분자생물학적인 메카니즘은 아직 규명되지 않고 있다. 영양배엽 세포의 분화를 조절하는 경로를 규경하기 위한 선결과제는 분화된 영양배엽 세포에서만 발현하는 많은 유전자들이 밝혀져야만 한다. 본 연구팀은 최근에 분화된 영양배엽 세포에서만 발현하는 두 종류의 새로운 유전자들을 찾았다. 한 종류는 homeobox를 보유하고 있는 조절 유전자 Psx이고, 다른 한 종류는 임신호르몬인 태반 프로락틴 라이크 단백질 유전자 PLP-C${\beta}$이다. 본 연구과제의 목표는 이들 유전자의 기능과 조절 메카니즘을 규명함으로써, 영양배엽 세포의 분화를 조절하는 조절경로를 밝히는 것이다. 이를 위하여 다음과 같은 일련의 연구를 수행할 것이다. 1) Psx 유전자가 분화된 영양배엽 세포에서만 발현케 하는 조절 메카니즘을 규명하기 위해 functional assays, in vitro footprinting, gel mobility shift assays, 생쥐형질전화, UV crosslinking, Southwestern blot 등의 방법을 통해 Psx 유전자의 cis-acting 요인과 trans-acting factor를 밝혀 분석한다. 2) 영양배엽 세포의 분화조절 경로를 규명하기 위해 random oligonuclotide library screening, DD-PCR, subtractive screening 등의 방법을 이용하여 Psx 유전자에 의해 조절되는 하부유전자를 밝힌다. 3) Psx 유전자를 knock-out시켜 영양배엽 세포가 발달 및 분화하는데 미치는 역할을 밝힌다. 4) Yeast two-hybrid screening방법을 이용하여 태반 프로락틴 유전자의 수용체를 찾아 이들의 신호전달 기전을 밝힌다. 제1차년 연구결과로서, mouse와 rat으로부터 각각 Psx 유전자의 genomic DNA를 클로닝하여, 유전자 구조를 비교한 결과, mouse Psx (mPsx2)는 4개의 exons으로 이루어져 있는 반면에, rat Psx (Psx3)는 3개의 exons으로 구성되어 있었다. 즉, rPsx3는 mPsx2의 exon1이 없었다. Notrhern blot과 in situ hybridization 분석에 의해 mouse와 rat에서 Psx 유전자가 다르게 발현 조절되는 현상을 밝혔다. 실제로 mPsx2와 rPsx3의 5'-flanking지역을 클로닝하여 염기서열 분석 결과 전혀 homology를 찾을 수 없었다. 또한, 이들 각각 promoter의 activity를 luciferase reporter를 이용하여 조사한 결과 Rcho-1 trophoblast cells에서 각기 다른 activity를 보여 주는 것을 발견하였다. Psx 유전자의 transcription start sites는 Primer extension에 의해 밝혔다. 또한 Psx2 유전자를 knock-out 시키기 위해 targeting vector를 Osdupde1에 제작하였다. 본 과제를 시작할 때 새로운 프로락틴 유전자 하나를 클로닝하여 이 유전자를 PLP-I라고 이름을 붙였다. 이 후 이 유전자 (PLP-I)는 PLP-C${\beta}$라고 이름을 붙이게 되었다. Mouse PLP-C${\beta}$ 유전자의 counterpart를 rat에서 찾아 염기서열을 비교한 결과 mouse와 rat에서 PLP-C${\beta}$유전자의 homology는 약 79% (amino acid level)였다. 본 연구과정을 통해 또 하나의 새로운 PLP-C subfamily member를 mouse로부터 클로닝 하였고, 이 유전자를 PLP-C${\gamma}$라 하였다. PLP-C${\beta}$와 PLP-C${\gamma}$의 발현 유형은 Northern blot과 in 냐셔 hybridization 분석에 의해 태반의 제한된 spongitrophoblast와 trophoblast giant cells에서만 발현하는 것을 밝혔다. 놀랍게도 이들 두 새로운 유전자는 alternative splicing에 의해 두 종류의 isoform이 있음을 밝혔다. PLP family member 유전자로서 splicing에 의한 isoforms을 보여 주는 유전자로는 PLP-C${\beta}$와 PLP-C${\gamma}$가 최초이다. 이들 isoform mRNAs의 발현 유형은 RT-PCR 방법을 이용하여 규명하였다. 또 하나의 새로운 발견은 PLP-C${\beta}$와 PLP-C${\gamma}$가 독특한 유전자 구조를 갖고 있었다. 즉, PLP-C${\beta}$는 exon3의 alternative splicing에 의해 5개 혹은 6개의 exons을 갖는 two isoforms이 생긴다. 반면에 PLP-C${\gamma}$는 exon2가 alternative splcing이 되면서 7개의 exons을 갖거나 6개의 exons을 갖는 isoforms을 만든다. 그리고, PLP-C${\gamma}$의 promoter activity를 trophoblast Rcho-l${\gamma}$ 세포주를 이용하여 PLP-C${\gamma}$ 의 1.5 kb 5'-flanking 지역이 trophoblast-specific promoter activity를 갖고 있음을 밝혔다. PLP-C${\gamma}$ 유전자의 transcription start site는 Primer extension에 의해 밝혔다. 제 1차 년도의 연구결과를 토대로, 2차년에서는 다음단계의 연구를 수행하고자 한다. 즉, 1) mPsx2와 rPsx3의 promoter를 비교분석 함으로서 mouse와 rat에서 Psx 유전자가 다르게 조절되는 메카니즘 규명, 2) Psx와 PLP-C 유전자의 promoter에 있는 cis-acting elements 탐색, 3) Psx2와 Psx3의 단백질을 이용하여 이들이 binding하는 target sequence 규명, 4) 제작한 Psx2 targeting vector를 이용하여 ES cells에서 Psx2 유전자 knock-out, 5) Psx 유전자를 과발현시키는 세포주를 만들고 Psx에 의해 조절되는 유전자 탐색, 6) 새로 밝히 PLP-C members 유전자들의 조절기전을 Rcho-1 세포주를 이용하여 여러 거지 성장인자와 다른 호르몬에 대한 반응을 탐색, 7) Psx와 PLP-C${\gamma}$ 유전자의 chromosomal mapping 등을 밝힐 것이다.

• Journal of The Korean Society of Inherited Metabolic disease
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• v.5 no.1
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• pp.65-75
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• 2005

Purpose: To understand genetic differences and similarities between mucolipidosis and control. Methods: Using the fibroblast of the mucolipidosis II and control, forward and reverse subtracted libraries were constructed. Among these clones, we investigated mutations in the GNPTA (MGC4170) gene, which codes for the ${\alpha}/{\beta}$ subunits of phosphotransferase, and in the GNPTAG gene, which codes for the ${\gamma}$ subunits in 5 Korean patients with mucolipidosis type II or IIIA. Result: Several differentially expressed cDNAs were cloned and their sequences were determined. Mutation analysis of the interested gene, GNPTA was performed and we identified 7 mutations in the GNPTA gene, but none in the GNPTAG gene. The mutations in type II patients included p.Q104X(c.310C>T), p.R1189X(c.3565C>T), p.S1058X(c.3173C>G), p.W894X(c.2681G>A) and p.H1158fsX15(c.3474_3475delTA), all of which are non-sense or frame shift mutations. However, a splicing site mutation, IVS13+1G>A (c.2715+1G>A) was detected along with a non-sense or a frame shift mutation (p.R1189X or p.E858fsX3(c.2574_2575delGA)) in two mucolipidosis type IIIA patients. Conclusion: This report shows that mutations in the GNPTA gene coding for the ${\alpha}{\beta}$subunits of phosphotransferase, and not mutations in the GNPTAG gene, account for most of mutations found in Korean patients with mucolipidosis type II or IIIA.