• Title/Summary/Keyword: Gene Screening

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Physiological and Proteome Responses of Korean F1 maize (Zea mays L.) Hybrids to Water-deficit Stress during Tassel Initiation (옥수수 영양생장기 한발 스트레스에 의한 광합성의 생리적 반응 및 프로테옴 변화 분석)

  • Bae, Hwan Hee;Kwon, Young-Sang;Son, Beom-Young;Kim, Jung-Tae;Go, Young Sam;Kim, Sun-Lim;Baek, Seong-Bum;Shin, Seonghyu;Kim, Sang Gon
    • KOREAN JOURNAL OF CROP SCIENCE
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    • v.64 no.4
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    • pp.422-431
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    • 2019
  • Severe droughts in spring have occurred frequently in Korea in recent years, exerting a critical impact on corn yield. Therefore, it is necessary to find physiological and/or molecular indicators of the response to drought stress in maize plants. In this study, we investigated the effects of water-deficit stress on two Korean elite F1 maize hybrids, Ilmichal and Gwangpyeongok, by withholding water for 10 days at tassel initiation. The water deficit drastically reduced the relative leaf water content, leaf number, leaf area, and stem length, leading to dry matter reduction. Moreover, it reduced the SPAD values and stomatal conductance of leaves in drought-stressed plants of both hybrids. Importantly, the number of leaves and SPAD value were non-destructive and easy to investigate in response to water-deficit stress, suggesting that they may be useful indicators for screening drought-tolerant genetic resources. We detected more than 100 spots that were differentially accumulated under drought stress. Of these spots, a total of 21 protein spots (≥1.5-fold) from drought-exposed maize leaves were successfully analyzed by MALDI-TOF-TOF mass spectrometry. Functional annotation using Gene Ontology analysis revealed that most of the identified proteins were involved in carbohydrate metabolism, stress response fatty acid catabolism, photosynthesis, energy metabolism, and transport. The protein expression levels were increased in both Ilmichal and Gwangpyeongok, except for triosephosphate isomerase, fructose-bisphosphate aldolase, and an uncharacterized protein. The lactoylglutathione lyase delta (3,5)-delta (2,4)-dienoyl-CoA isomerase was overexpressed in Gwangpyeongok only. The results obtained from this study suggest that the drought-specific genes may be useful as molecular markers for screening drought-tolerant maize genotypes.

Functional Screening for Cell Death Suppressors and Development of Multiple Stress-Tolerant Plants

  • Moon Hae-Jeong;Baek Dong-Won;Lee Ji-Young;Nam Jae-Sung;Yun Dae-Jin
    • Journal of Plant Biotechnology
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    • v.5 no.3
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    • pp.143-148
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    • 2003
  • Bax, a mammalian pro-apoptotic member of the Bcl-2 family induces cell death when expressed in yeast. To investigate whether Bax expression can induce cell death in plant, we produced transgenic Arabidopsis plants that contained murine Bax cDNA under control of a glucocorticoid-inducible promoter. Transgenic plants treated with dexamethasone, a strong synthetic glucocorticoid, induced Bax accumulation and cell death, suggesting that some elements of cell death mechanism by Bax may be conserved among various organisms. Therefore, we developed novel yeast genetic system, and cloned several Plant Bax Inhibitors (PBIs). Here, we report the function of two PBIs in detail. PBI1 is ascorbate peroxidase (sAPX). Fluorescence method of dihydrorhodamine123 oxidation revealed that expression of Bax in yeast cells generated reactive oxygen species (ROS), and which was greatly reduced by co-expression with sAPX. These results suggest that sAPX inhibits the generation of ROS by Bax, which in turn suppresses Baxinduced cell death in yeast. PBI2 encodes nucleoside diphosphate kinase (NDPK). ROS stress strongly induces the expression of the NDPK2 gene in Arabidopsis thaliana (AtNDPK2). Transgenic plants overexpressing AtNDPK2 have lower levels of ROS than wildtype plants. Mutants lacking AtNDPK2 had higher levels of ROS than wildtype. $H_2O_2$ treatment induced the phosphorylation of two endogenous proteins whose molecular weights suggested they are AtMPK3 and AtMPK6. In the absence of $H_2O_2$ treatment, phosphorylation of these proteins was slightly elevated in plants overexpressing AtNDPK2 but markedly decreased in the AtNDPK2 deletion mutant. Yeast two-hybrid and in vitro protein pull-down assays revealed that AtNDPK2 specifically interacts with AtMPK3 and AtMPK6. Furthermore, AtNDPK2 also enhances the MSP phosphorylation activity of AtMPK3 in vitro. Finally, constitutive overexpression of AtNDPK2 in Arabidopsis plants conferred an enhanced tolerance to multiple environmental stresses that elicit ROS accumulation in situ. Thus, AtNDPK2 appears to playa novel regulatory role in $H_2O_2$-mediated MAPK signaling in plants.

Characterization of Cellulase and Xylanase from Bacillus subtilis NC1 Isolated from Environmental Soil and Determination of Its Genes (Bacillus subtilis NC1 유래 cellulase와 xylanase의 특성 규명 및 효소 유전자의 규명)

  • Park, Chang-Su;Kang, Dae-Ook;Choi, Nack-Shick
    • Journal of Life Science
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    • v.22 no.7
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    • pp.912-919
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    • 2012
  • A Bacillus sp. strain producing celluase and xylanase was isolated from environmental soil with LB agar plate containing carboxymethylcellulose (CM-cellulose) and beechwood xylan stained with trypan blue as substrates, respectively. Based on the 16S rRNA gene sequence and API 50 CHL test, the strain was identified as B. subtilis and named B. subtilis NC1. The cellulase and xylanase from B. subtilis NC1 exhibited the highest activities for CM-cellulose and beechwood xylan as substrate, respectively, and both enzymes showed the maximum activity at pH 5.0 and $50^{\circ}C$. We cloned and sequenced the genes for cellulase and xylanase from genomic DNA of the B. subtilis NC1 by the shot-gun cloning method. The cloned cellulase and xylanase genes consisted of a 1,500 bp open reading frame (ORF) encoding a 499 amino acid protein with a calculated molecular mass of 55,251 Da and a 1,269 bp ORF encoding a 422 amino acid protein with a calculated molecular mass of 47,423 Da, respectively. The deduced amino acid sequences from the genes of cellulase and xylanase showed high identity with glycosyl hydrolases family (GH) 5 and 30, respectively.

A Possible Mechanism Related with Non-spinning Syndrome of Bombyx mori that Intimidates the Sericultural Industry in Northern Kyungbuk (경북 북부지역의 양잠산업에 피해를 주고 있는 누에(Bombyx mori) 미화용 기작에 관한 연구)

  • Kim, Yong-Kyun;Bae, Sang-Ki;Lee, Sun-Young;Ji, Dong-Jin;Kim, Jin;Hong, Yong-Pyo;Kim, Gil-Ho
    • Korean journal of applied entomology
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    • v.43 no.2
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    • pp.143-153
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    • 2004
  • Non-spinning syndrome of Bombyx mori has been serious issue in sericulture industry near Kyungbuk area. This study was focused on the analysis of the mechanism and on screening candidate chemicals inducing the anti-metamorphosis of the silkworms. Rearing temperatures or initial body weight of the final instar larvae did not affect a normal larval to pupal metamorphosis of B. mori. However, pyriproxyfen (a juvenile hormone (JH) agonist) induced follicle patency significantly even at its 10$\^$-8/ M concentration and inhibited metamorphosis of B. mori in both developmental time and dose dependent manners. Pyriproxyfen induced JH esterase (JHE) activity and downregulated expression of JH binding protein of 5. mori. These results suggests that pyriproxyfen induced JHE activity as a JH agonist and that the elevated JHE activity degraded endogenous JH and resulted in JHBP gene expression. Based on the fact that the JH agonist induced follicle patency and inhibited metamorphosis of B. mori, follicle patency bioassay suggested that three commercial pesticides including simazine, molinate or alachlor were proved to give potent JH agonistic effect on B. mori. Further direct exposure experiments to these candidates are required to determine the chemicals responsible for the non-spinning syndrome of 8. mori.

The Estrogenic Effects of Phthalates(DEHP, DBP) in Yeast Recombinant Assay (효모재조합 검색시험법을 이용한 DEHP, DBP의 에스트로젠 효과)

  • Jung, Ji-Youn
    • Journal of Food Hygiene and Safety
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    • v.22 no.3
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    • pp.218-222
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    • 2007
  • Di(2-ethylhexyl) phthalate (DEHP) and di-n-butyl phthalate (DBP) were screened for estrogenic activity using a recombinant yeast screening system consisted with estrogen receptors and ${\beta}-galactosidase$ as reporter gene. The chemicals showed estrogenic activity in ranges of $1{\times}10^{-10}\;to\;1{\times}10^{-7}M(DEHP)\;and\;of\;1{\times}10^{-9}M\;to\;1{\times}10^{-6}M(DBP)$ respectively. $17{\beta}-estradiol$, as a positive control of, showed maximal activity at $1{\times}10^{-9}M$. The concentration of half-maximal estrogenic activity was $1{\times}10^{-9}M$ for both chemicals. However, the concentration of maximal estrogenic activity was $1{\times}10^{-7}M$ for DEHP and $1{\times}10^{-8}M$ M for DBP. These results suggested that DBP was higher in relative potencies and more sensitive than DEHP. In conclusion, DEHP and DBP were both estrogenic, even though DBP was more reactive to estrogen receptor.

Screening of Natural Product Libraries for the Extension of Cell Life-span through Immune System (면역시스템을 통한 세포수명연장 천연물질 스크린)

  • Yoo, Bo-Kyung;Kwon, Kisang;Ko, Young Hwa;Kim, Hong Geun;Lee, Seokhyun;Park, Kwan-Ho;Choi, Ji-Young;Kwon, O-Yu
    • Journal of Life Science
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    • v.26 no.3
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    • pp.359-363
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    • 2016
  • We have screened four natural products against 640 single compounds, which shows more two folds gene expression for both endoplasmic reticulum aminopeptidase 1 (ERAP1) and FOXO-family transcription factor (FOXO1). The results were as follows. (±)-Car-3-ene-2,5-dione from Asarum sieboldii Miq. is C10H12O2 molecular formula and the 164 kDa molecular weight. Cinobufagin from Bufonis Venennum is C26H34O6 molecular formula and 442 kDa molecular weight. So far reported main biological function is Na+/K+-ATPase inhibition. Corilagin from Euphorbia pekinensis is C27H22O18 molecular formula and 634 kDa molecular weight. Carbonic anhydrase inhibition is well known its biological function. Corydaline from Corydalis turtschaninovii is C22H27NO4 molecular formula and 369 kDa molecular weight. The main biological function is acetylcholinesterase inhibition. In the short future, four types of natural products will be used in longevity experiments with insects. The results may give one of the clues for studying new drug development candidates of the longevity.

Functional Screening for Cell Death Suppressors and Development of Multiple Stress-Tolerant Plants

  • Moon, Hae-Jeong;Baek, Dong-Won;Lee, Ji-Young;Nam, Jae-Sung;Yun, Dae-Jin
    • Proceedings of the Korean Society of Plant Biotechnology Conference
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    • 2003.04a
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    • pp.65-71
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    • 2003
  • Bax, a mammalian pro-apoptotic member of the Bcl-2 family, induces cell death when expressed in yeast. To investigate whether Bax expression can induce cell death in plant, we produced transgenic Arabidopsis plants that contained murine Bax cDNA under control of a glucocorticoid-inducible promoter. Transgenic plants treated with dexamethasone, a strong synthetic glucocorticoid, induced Bax accumulation and cell death, suggesting that some elements of cell death mechanism by Bax may be conserved among various organisms. Therefore, we developed novel yeast genetic system, and cloned several Plant Bax Inhibitors (PBIs). Here, we report the function of two PBIs in detail. PBI1 is ascorbate peroxidase (sAPX). Fluorescence method of dihydrorho-damine 123 oxidation revealed that expression of Bax in yeast cells generated reactive oxygen species (ROS), and which was greatly reduced by co-expression with sAPX. These results suggest that sAPX inhibits the generation of ROS by Bax, which in turn suppresses Baxinduced cell death in yeast. PBI2 encodes nucleoside diphosphate kinase (NDPK). ROS stress strongly induces the expression of the NDPK2 gene in Arabidopsis thaliana (AtNDPK2). Transgenic plants overexpressing AtNDPK2 have lower levels of ROS than wildtype plants. Mutants lacking AtNDPK2 had higher levels of ROS than wildtype. $H_2O_2$ treatment induced the phosphorylation of two endogenous proteins whose molecular weights suggested they are AtMPK3 and AtMPK6. In the absence of $H_2O_2$ treatment, phosphorylation of these proteins was slightly elevated in plants overexpressing AtNDPK2 but markedly decreased in the AtNDPK2 deletion mutant. Yeast two-hybrid and in vitro protein pull-down assays revealed that AtNDPK2 specifically interacts with AtMPK3 and AtMPK6. Furthermore, AtNDPK2 also enhances the MBP phosphorylation activity of AtMPK3 in vitro. Finally, constitutive overexpression of AtNDPK2 in Arabidopsis plants conferred an enhanced tolerance to multiple environmental stresses that elicit ROS accumulation in situ. Thus, AtNDPK2 appears to play a novel regulatory role in $H_2O_2$-mediated MAPK signaling in plants.

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Autosomal-dominant Polycystic Kidney Disease in a Family of Scottish Fold Cats (스코티쉬 폴드 고양이 가족에 발생한 상염색체 우성 다낭성 신병)

  • Seo, Kyoung-Won;Kim, Sae-Um;Ahn, Jin-Ok;Coh, Ye-Rin;Han, Sung-Young;Youn, Hwa-Young
    • Journal of Veterinary Clinics
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    • v.27 no.6
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    • pp.726-728
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    • 2010
  • Autosomal-dominant polycystic kidney disease (AD-PKD) is common in Persian and Persian-related breeds, and is sporadically reported in Scottish Fold cats. A 5-year-old male Scottish Fold cat was diagnosed with polycystic kidney disease based on screening tests and abdominal ultrasonography and died 3.5 months after diagnosis. The cat had 14 kittens with three queens, including his female sibling, with an age range of 3 months to 8 years. Genetic testing to confirm the genetic transmission of AD-PKD which detects the mutated PKD1 gene was performed. Abdominal ultrasonography confirmed the presence of renal cysts. Nineteen cats were screened in the present study (13 males and 6 females), with an age range of 3 months to 8 years. The results of renal ultrasonography agreed with the genetic test results in the 19 cats in which both tests were performed and 8 cats were diagnosed as ADPKD based on these tests. AD-PKD has not been investigated in cats in South Korea. Moreover, this is the first report of AD-PKD in a family unit of Scottish Fold cats.

Selection of Representative Magnaporthe oryzae Isolates and Rice Resistant Gene Types for Screening of Blast-resistant Rice Cultivars (우리나라 벼 도열병균의 대표 균주 및 벼의 저항성 유전자형 선발)

  • Goh, Jaeduk;Kim, Byung-Ryun;Lee, Se-Won;Roh, Jae-Hwan;Shin, Dong-Bum;Jeung, Ji-Ung;Cho, Young-Chan;Han, Seong-Sook
    • Research in Plant Disease
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    • v.19 no.4
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    • pp.243-253
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    • 2013
  • Rice blast is one of the most serious disease threatening stable production of rice. Breeding of resistant cultivars has been used as the most effective and useful method to controll rice blast caused by Magnaporthe oryzae. To collect rice blast isolates in fields and test their pathogenicity on new cultivars are important for establishment of new resistant cultivars breeding program of rice. Pathotypes of Korean rice blast isolates have been categorized to Korean differential race system developed in 1985. However, it is little known about genetic background of Korean differential cultivars, so that it is hard to understand for relationship between each pathogen and each host plant at genetic level. In this study, we suggested necessity of a new differential system by analyzing pathogenic responses between 24 monogenic rice lines and 200 Korean rice blast isolates. In addition, we determined the nine representative resistant genes based on the resistance responses of the monogenic lines to rice blast isolates, indexed resistant responses of the monogenic lines to ten representative rice blast isolates and selected 30 Korean representative rice blast isolates proper to Korean system. We think the newly developed differential race system can be broadly used to select resistant cultivars to rice blast in Korea.

Recent Advances in Cancer Diagnosis: On an Overview of Diagnostic Cytopathologic Modalities and Ancillary Techniques (세포병리학적 기초에 의한 암진단의 발전: 진단방법과 보조기법)

  • Kim, Ki-Tai;Ham, Eui-Keun
    • The Korean Journal of Cytopathology
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    • v.7 no.1
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    • pp.1-11
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    • 1996
  • From the concepts of cellular pathology and of exfoliative cytology, as elucidated by Virchow and Papanicolaou respectively in the late 19th and early 20th century, have evolved the primary methods for the diagnosis of cancer today. From Papanicolaou's concept of exfoliative cytology developed fine needle aspiration biopsy in the early 1960's, this has become a major diagnostic procedure and has contributed to a significant reduction in open biopsies and, therefore, to medical cost-effectiveness immunobiochemical techniques provided us with a supplement to cancer diagnosis in the 1980's. The immunoperoxidase method, using monoclonal antibodies, is applied primarily as an ancillary measure to elucidate the nature of cancers The availability of specific monoclonal antibodies has greatly facilitated the identification of cell products or surface markers. For example, antibodies directed against intermediate filaments have proved to be of value in determining the histogenesis oi poorly differentiated neoplasms. Tumor markers may serve as biochemical indicators of the presence of a neoplasm. They can be detected In plasma and other body fluids. Their concentration can be applied as a diagnostic test, for monitoring the clinical course of known cancer, and as a screening measure to detect certain cancers in a population at risk. Flow cytometry is a useful tool for distinguishing several cell characteristics, such as the immunophenotype of leukemia-lymphoma cells, the DNA content of neoplastic cells, and cell proliferation rate. Molecular biologic techniques provided a giant step for the management of cancer patients encompassing diagnosis, prognostic evaluation, and therapy. Nucleic acid hybridization techniques are utilized as Southern, Northern, and dot blots and in situ hybridization. Molecular biology and its techniques may bring a blight new horizon for understanding cancer biology and in designing therapy on the basis of gene manipulation.

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