느티만가닥버섯의 ITS (internal transcribed spacer) 영역의 2차구조 분석

우주리(Ju-Ri Woo),윤혁준(Hyeokjun Yoon),유영현(Young-Hyun You),이창윤(Chang-Yun Lee),공원식(Won-Sik Kong),김종국(Jong-Guk Kim) 한국생명과학회 2013 생명과학회지 Vol.23 No.10

본 연구에서는 H. marmoreus 3-10균주와 H. marmoreus 1-1균주의 ribosomal DNA (rDNA) cluster의 분석이 수행되었다. Small subunit (SSU)와 intergenic spacer 2 (IGS 2)는 부분적으로 염기서열이 결정되었고, internal transcribed spacer 1 (ITS 1), 5.8S, internal transcribed spacer 2 (ITS 2), large subunit (LSU), intergenic spacer 1 (IGS 1), 5S는 완전하게 염기서열이 결정 되었다. 팽이버섯 H. marmoreus 3-10균주와 H. marmoreus 1-1균주의 rDNA cluster는 총 7,049 bp로 결정되었다. SSU은 1,796 bp, ITS1은 229 bp, 5.8S은 153 bp, ITS2는 223 bp, LSU은 3,348 bp, IGS1은 390 bp, IGS2은 900 bp로 염기서열이 분석되었다. 결정된 rDNA cluster의 총 7,049 bp 중에서 17 bp가 다름이 확인되었고, 각각 SSU (2 bp), ITS (3 bp), LSU (9 bp), IGS (3 bp)에서 차이를 확인하였다. ITS regions의 2차 구조 결과 5개의 stem-loop가 있음이 드러났다. 흥미롭게도, 이들 stem-loop 사이에서 stem-loopⅤ에서 한 개의 상이한 염기가 다른 2차 구조를 나타냄을 확인하였다. The ribosomal DNA (rDNA) clusters of Hypsizygus marmoreus 3-10 and H. marmoreus 1-1 were analyzed in this study. The small subunit (SSU) and intergenic spacer 2 (IGS 2) was partially sequenced. The internal transcribed spacer 1 (ITS 1), 5.8S, internal transcribed spacer 2 (ITS 2), large subunit (LSU), intergenic spacer 1 (IGS 1), and 5S were completely sequenced. The rDNA clusters of H. marmoreus 3-10 and H. marmoreus 1-1 were 7,049 bp in length. The sequence of SSU rDNA, which corresponded to 18S rDNA, was 1,796 bp in length, and the sequence of LSU rDNA, which corresponded to 28S rDNA, was 3,348 bp in length. The ITS region that variable region and IGS region that nontranscribed spacer was 462 bp and 1,290 bp in length. The sequence of 5.8S rDNA and 5S rDNA was 153 bp and 43 bp in length, respectively. The 17 bp of the rDNA cluster in the H. marmoreus 3-10 strain was different to that in the H. marmoreus 1-1 strain, with 2 bp in the SSU, 3 bp in the ITS, 9 bp in the LSU, and 3 bp in the IGS. The analysis of the secondary structure revealed that the ITS regions of H. marmoreus 3-10 and H. marmoreus 1-1 have five stem-loop structures. Interestingly, among these structures, one different nucleotide sequence resulted in a different secondary structure in stem-loop V.

Flammulina velutipe의 국내 균주와 외래 균주 간의 ITS region을 이용한 계통학적 유연관계 분석

Gwang-Rip Hwang(황광립),Ju-Ri Woo(우주리),Hyeokjun Yoon(윤혁준),Chang-Yun Lee(이창윤),Sang-Han Lee(이상한),Won-Sik Kong(공원식),Jong-Guk Kim(김종국) 한국생명과학회 2012 생명과학회지 Vol.22 No.1

본 연구는 팽이버섯(Flammulina velutipes)의 국내 균주와 외래 균주간의 유전적 유연관계를 조사하기 위하여 수행되었으며, 계통수 측정 결과 3개 그룹으로 나눠짐을 확인하였다. 20개 F. velutipes 균주들의 ITS region 염기서열을 확보하였으며, 이들 서열을 바탕으로 multiple alignment를 보았으며, Neighbor-joining method를 이용하여 계통수를 작성하였으며, 2개 그룹으로 나눠짐을 확인하였다. 또한 F. velutipes 4154 균주의 rDNA-cluster를 최초로 분석한 결과, 총 10,974 bp임을 밝혔다. SSU는 1,806 bp, ITS region은 553 bp의 염기배열이 결정되었다. ITS 1 부분은 245 bp이고 ITS 2는 308 bp였으며, LSU에 해당되는 염기서열은 3,402 bp, IGS 1은 1,400 bp, 5S는 83 bp, IGS 2는 3,571 bp임을 확인하였다. This study was carried out to investigate the genetic relationship of Flammulina velutipes with other species. The ribosomal DNA cluster containing 4 rRNA genes from F. velutipes 4154 were sequenced. The length of the rDNA cluster sequence was estimated at 7,403 bp long and consisted of 1,806 bp of SSU rDNA, 245 bp of ITS 1 region, 159 bp of 5.8S rDNA, 308 bp of ITS 2 region, 3,402 bp of LSU rDNA, 1,400 bp of IGS 1 region, and 83 bp of 5S rDNA. The F. velutipes 4154 genes were contained in the rDNA cluster of F. velutipes in the order of SSU rDNA - ITS 1 - 5.8S rDNA - ITS 2 - LSU rDNA - IGS 1 - 5S rDNA. The phylogenetic relationships of 20 strains of Tricholomataceae and Physalacriaceae were analyzed by conducting distance analysis using the Neighbor-joining (NJ) method. The 20 strains used in this study were divided into three groups and the strains of the genus Flammulina were related very closely to strains of Physalacria bambusae.

Plant Growth-Promoting Activity of Endophytic Fungi Isolated from the Roots of Native Plants in Dokdo Islands

유영현(Young-Hyun You),윤혁준(Hyeokjun Yoon),우주리(Ju-Ri Woo),서영교(Yeonggyo Seo),김미애(Miae Kim),추연식(Yeon-Sik Choo),김종국(Jong-Guk Kim) 한국생명과학회 2011 생명과학회지 Vol.21 No.11

독도에 자생하고 있는 6 종류의 식물뿌리로부터 내생균을 분리하였다. 동도에서 갯제비쑥, 명아주, 까마중과서도에서 도깨비쇠고비, 술패랭이 그리고 번행초를 연구재료로 사용하였다. 총 32 종의 내생균을 분리하였고, universal primers ITS1과 ITS4를 사용하여 ITS 영역을 PCR로 증폭하여 동정하였다. 염기서열 분석결과, 6 종류의 식물의 뿌리에서 모두 32종류의 다양한 내생균류를 분리 및 동정 할 수 있었다. 독도 자생식물인 갯제비쑥 4종, 명아주 8종, 까마중 3종, 도깨비쇠고비 3종, 술패랭이 3종, 번행초 11종의 내생균이 분리되었다. 내생균들의 배양여액은 식물생장촉진활성을 확인하기 위하여 난장이벼의 유묘에 처리되었다. 그 결과, 명아주에서 분리된 Ca-5-2-2 균주가 우수하게 식물생장촉진활성을 나타내었다. 그리고 독도에 자생하는 6종류의 식물에서 Penicillium sp.와 Fusarium sp. 그리고 Aspergillus sp.의 내생균이 가장 많이 존재함을 알 수 있었다. Endophytic fungal strains were isolated from the roots of six species plants in the Dokdo islands. Native plant samples, such as Artemisia japonica, Chenopodium album and Solanum nigrum were isolated from Dongdo, and those such as Cyrtomium falcatum, Dianthus longicalyx and Tetragonia tetragonoides were isolated from Seodo. In total, thirty two fungal strains were isolated from these native plants. To identify the fungal strains, polymerase chain reaction (PCR) amplification of internal transcribed spacer (ITS: containing ITS1, 5.8s and ITS2 region) regions was done with universal primers ITS1 and ITS4. Endophytic fungi of four species were isolated from A. japonica, eight species from C. album, three species from S. nigrum, three species from C. falcatum, three species from D. longicalyx and eleven species from T. tetragonoides. Culture filtrates (CF) of isolated endophytic fungi were used to treat-waito-c rice seedlings to test plant growth-promoting activity. As a result of bioassay, Ca-5-2-2 strain isolated from C. album expressed highest plant growth-promotion activity. Of all the endophytic fungi isolated, Penicillium sp., Fusarium sp. and Aspergillus sp. were the most abundantly distributed fungal strains in the six plants used in this study.

보문: 고창갯벌의 염생식물 뿌리로부터 분리된 내생진균의 다양성

유영현 ( Young Hyun You ),윤혁준 ( Hyeok Jun Yoon ),우주리 ( Ju Ri Woo ),서영교 ( Yeong Gyo Seo ),김미애 ( Mi Ae Kim ),이경민 ( Gyeong Min Lee ),김종국 ( Jong Guk Kim ) 한국균학회 2012 韓國菌學會誌 Vol.40 No.2

염생식물인 칠면초, 갈대, 갯질경, 해홍나물을 고창갯벌로부터 채집하였으며, 39점의 내생진균을 염 습지에 자생하고 있는 염생식물의 뿌리로부터 분리하였다. 내생진균들은 ITS1, 5.8s와 ITS2를 포함하는 ITS-rDNA 영역에 의해 분석되었다. 다양한 내생진균은 Eurotiales (36%), Pleosporales (26%), Hypocreales (18%), Incertae sedis (8%), Glomerellales (5%), Sordariales (2%), Xylariales (2%), Capnodiales (3%)등 8종류 목에 속하는 것으로 확인되었다. 39점의 내생진균은 속(genus)단계에서 Alternaria, Aspergillus, Chaetomium, Cladosporium, Colletotrichum, Coniothyrium, Dothideomycete, Fusarium, Gibberella, Macrophoma, Penicillium, Pestalotiopsis, Phaeosphaeria, Phoma, Pleosporales, Pseudozyma, Talaromyces, 그리고 Termitomyces에 포함되는 것으로 분석되었다. 그리고 내생진균은 Eurotiales목의 Penicillium속이 가장 많이 분포하고 있는 것으로 분석되었다. Halophyte samples, such as Suaeda japonica, Phragmites australis, Limonium tetragonum, Suaeda maritima were collected from Go-chang salt marsh. Thirty-nine endophytic fungal strains were isolated from the roots of halophytes naturally growing in salt marsh. All endophytic fungal strains isolated were analyzed by internal transcribed spacer (ITS) containing ITS1, 5.8 s and ITS2 region. Endophytic fungal strains belong to eight orders, i.e., Eurotiales (36%), Pleosporales (26%), Hypocreales (18%), Incertae sedis (8%), Glomerellales (5%), Sordariales (2%), Xylariales (2%), and Capnodiales (3%). On genus level, they were composed of Alternaria, Aspergillus, Chaetomium, Cladosporium, Colletotrichum, Coniothyrium, Dothideomycete, Fusarium, Gibberella, Macrophoma, Penicillium, Pestalotiopsis, Phaeosphaeria, Phoma, Pleosporales, Pseudozyma, Talaromyces, and Termitomyces. Of them, Penicillium (26%), Fusarium (13%) of Eurotiales and Hypocreales were predominant.

독도의 자생식물 뿌리에서 분리한 내생진균의 다양성과 생장촉진활성

Young-Hyun You(유영현),Hyeokjun Yoon(윤혁준),Gil Seong Lee(이길성),Ju-Ri Woo(우주리),Jae-Ho Shin(신재호),In-Jung Lee(이인중),Soon-Ok Rim(임순옥),Yeon-Sik Choo(추연식),Jong-Guk Kim(김종국) 한국생명과학회 2011 생명과학회지 Vol.21 No.7

독도에 자생하고 있는 식물의 뿌리로부터 내생진균의 분리를 시도하여, 동도로 부터 참억새, 돌피, 쇠무릎과 서도에서 갯별꽃 및 율무쑥등을 연구재료로 사용하였다. 총 21종의 내생진균을 분리하였고, universal primers ITS-1과 ITS-4를 사용하여 ITS 영역을 PCR로 증폭하여 동정하였다. 염기서열 분석결과, 동도에 자생하는 참억새에서는 Penicillium 속 균주가 75%, Aspergillus 균주가 25%, 분리되었고, 돌피에서는 Penicillium 속 균주가 55%, Aspergillus 속 균주가 30%, Zygorhynchus 속 균주가 15%가 분리되었으며, 쇠무릎에서는 Penicillium 속 균주가 50%, Aspergillus 속 균주가 12%, Gibberella 속 균주가 13%, Talaromyces 속 균주가 9%, Umbelopsis 속 균주가 8% 분리되었다. 서도에 자생하는 갯별꽃에서는 Penicillium 속 균주가 76%, Pestalotiopsis 속 균주가 24% 분리되었고, 율무쑥에서는 Penicillium 속 균주가 81%, Mucor 속 균주가 19% 분리되었다. Bioassay결과, 돌피에서 분리된 Ec-3-1균주가 식물생장촉진활성을 나타내었다. 그리고 독도의 5종류 식물에서 Penicillium 속의 내생진균이 가장 많이 존재함을 알 수 있었다. Endophytic fungi were isolated from the roots of plants growing naturally on the island of Dokdo. Plant samples, such as Miscanthus sinensis, Achyranthus japonica and Echinochloa crusgali were isolated from Dongdo, and those such as Honkenya peploides and Artemsia koidzumii were isolated from Seodo. Twenty one strains of endophytic fungi were isolated from these plants. To identify the strains, PCR (polymerase chain reaction) amplification of the partial ITS (Internal Transcribed Spacer) regions was done with universal primers ITS-1 and ITS-4 to determine the nucleotide sequence of the ITS regions. Of the strains isolated from Miscanthus sinensis, 75% were Penicillium sp. and 25% were Aspergillus sp. Fifty five percent of strains isolated from Achyranthus japonica were Penicillium sp., 30% were Aspergillus sp. and 15% were Zygorhynchus sp. Strains isolated from Echinochloa crusgali were Penicillium sp. (50%), Aspergillus sp. (12%), Giberella sp. (13%), Talaromyces sp. (9%) and Umbelopsis sp. (8%). Of the strains isolated from Honkenya peploides, 76% were Penicillium sp. and 24% were Pestalotiopsis sp. Strains isolated from Artemisia koidzumii were Penicillium sp. (81%) and Mucor sp. (19%). As a result of bioassay, Ec-3-1 strain isolated from Echinochloa crusgalli showed plant growth-promotion activity. Of all the endophytic fungi isolated, Penicillium sp. was the most abundantly distributed fungal strain in all plants used in this study.

느티만가닥버섯의 신품종에 대한 endo-, exo-cellular 효소 활성도의 비교

Chang-Yun Lee(이창윤),Ho-Sung Song(송호성),Hyeon-Su Ro(노현수),Ju-Ri Woo(우주리),Young-Hyun You(유영현),Jong-Guk Kim(김종국) 한국생명과학회 2012 생명과학회지 Vol.22 No.6

본 연구는 새롭게 개발된 느티만가닥버섯의 6개 품종에 대한 형태적, 생리적 특성을 조사하고 endo-, exo-cellular 효소 활성을 측정하기 위해서 수행되었다. 국내 야생종인 Hm3-10과 일본 재배종인 Hm1-1과의 단핵균사 교배를 통하여 343개의 교배 균주를 획득하여 재배를 실시하여 58개 균주를 1차 선발하고 2차로 6개 균주를 선발하였다. 6개 선발 균주를 대상으로 배양 일수 별로 재배를 실시한 결과 배양일수가 80일 이상에서는 재배일수가 19~20일로 단축되어 최적 배양일수를 80일로 결정하였다. 80일 배양일수에서 각 품종별 형태적 특성을 검증한 결과 Hm15-3, Hm15-4, Hm17-5의 3균주가 재배에 적합한 균주로 판명되었다. 각 균주의 endo-cellular 효소 활성도를 측정한 결과, α-amylase의 효소 활성도가 73.9~102,2 unit/㎎ protein으로 가장 높았으며, chitinase 의 효소활성도가 8.1~13.1 unit/㎎ protein으로 측정되었다. Exo-cellular효소 활성도를 측정한 결과, α-amylase의 효소활성도가 5,292~1,184 unit/㎎ protein으로 가장 높았으며, CMCase와 Xylanase의 효소 활성도가 각각 1,140~245 unit/㎎ protein, 94~575 unit/㎎ protein으로 측정되었다. 그러나 β-glucosidase와 chitinase의 활성도는 비교적 낮은 활성도를 나타내었다. This study was carried out to investigate the morphological and physiological characteristics of six new cultivars of Hypsizygus marmoreus (Hm) and measure endo-, exo-cellular enzyme-specific activity. The domestic wild stain (Hm3-10) and commercial strain in Japan (Hm1-1) were mated by crossing monokaryon mycelia. We gained 58 strains from one of 400 crosses through the 1<SUP>st</SUP> cultivation experiment, and selected six strains from one of 58 strains through the 2<SUP>nd</SUP> cultivation experiment. When six of the selected new strains were grown during several spawn culture periods (60, 70, 80, 90, and 100 days), a spawn culture period of more 80 days was considered to be excellent as being shorter than 19~20 days. Therefore, we determined the period of spawn culture as 80 days. Three strains such as Hm15-3, Hm15-4, and Hm17-5 showed an excellent result. When endo-cellular enzyme activity measured eight strains, we obtained a result of that specific activity of α-amylase at the highest as 73.9~102.2 unit/㎎ protein, and chitinase is lower than α-amylase at 8.1~13.1 unit/㎎ protein. When exo-cellular enzyme activity measured eight strains, we determined the result of that specific activity of α-amylase is the highest at 5,292~1,184 unit/㎎ protein, and CMCase and xylanase were 1,140~245 unit/㎎ protein, 94~575 unit/㎎ protein, compared to each other. However, the enzyme activity of β-glucosidase and chitinase is low.

단보 : 송이버섯과 공생하는 소나무 세근으로부터 분리된 내생균의 다양성

유영현 ( Young Hyun You ),윤혁준 ( Hyeok Jun Yoon ),우주리 ( Ju Ri Woo ),임순옥 ( Soon Ok Rim ),이진형 ( Jin Hyung Lee ),공원식 ( Won Sik Kong ),김종국 ( Jong Guk Kim ) 한국균학회 2011 韓國菌學會誌 Vol.39 No.3

Endophytic fungi were isolated from the Pinus densiflora rootlet colonized by ectomycorrhizal fungus Tricholoma matsutake. Eighteen species of endophytic fungi were identified by analyzing rDNA-ITS sequence. As the result of the rDNA-ITS analysis, ascomycota of 15 species and Mucoromycotina of 3 species were isolated. Of all the endophytic fungi isolated, Penicillium sp. was confirmed as the highest frequency.

유전자조작, 균주분리 갯메꽃 뿌리로부터 분리된 내생진균의 식물생장촉진활성

유영현 ( Young Hyun You ),윤혁준 ( Hyeok Jun Yoon ),우주리 ( Ju Ri Woo ),서영교 ( Yeong Gyo Seo ),신재호 ( Jae Ho Shin ),추연식 ( Yeon Sik Choo ),이인중 ( In Jung Lee ),김종국 ( Jong Guk Kim ) 한국미생물생명공학회 2011 한국미생물·생명공학회지 Vol.39 No.4

Eight endophytic fungal strains were isolated from the roots of Calystegia soldanella from the western coast of South Korea. The culture filtrate of the eight endophytic fungi were applied to waito-c rice seedlings in order to verify potential plant growth promotion activities. The results of bioassay indicated that the Cs-9-7 fungal strain possessed the highest plant growht promotion activity. Fungal culture filtrates were analyzed to verify secondary metabolites using gas chromatography and mass spectroscopy with selected ion monitoring (GC/MS-SIM). The culture filtrate of the Cs-9-7 fungal strain was confirmed to contain gibberellins GA3 (1.229 ng/mL), GA4 (3.535 ng/mL), GA7 (1.408 ng/mL) and GA12 (0.378 ng/mL). Polymerase chain reactions (PCR) were performed so as to determine the internal transcribed spacer (ITS) regions for the identification of isolated strains with universal primers ITS-1 and ITS-4. The Cs-9-7 fungal strain, isolated from the root of C. soldanella, has been named Aspergillus tubingensis Cs-9-7.

보문 : 순천만에 자생하는 염생식물의 뿌리로부터 분리된 내생진균의 유전적 다양성

서영교 ( Yeong Gyo Seo ),김미애 ( Mi Ae Kim ),유영현 ( Young Hyun You ),윤혁준 ( Hyeok Jun Yoon ),우주리 ( Ju Ri Woo ),이경민 ( Gyeong Min Lee ),김종국 ( Jong Guk Kim ) 한국균학회 2012 韓國菌學會誌 Vol.40 No.1

독도의 자생식물의 근권에서 분리한 원핵 미생물의 다양성 분석

김예은(Ye-Eun Kim),윤혁준(Hyeokjun Yoon),유영현(Young-Hyun You),김현(Hyun Kim),서영교(Yeonggyo Seo),김미애(Miae Kim),우주리(Ju-Ri Woo),남윤종(Yoon-Jong Nam),이리나(Khalmuratova Irina),이경민(Gyeong-Min Lee),송진하(Jin-Ha Song),진영주(Y 한국생명과학회 2014 생명과학회지 Vol.24 No.4

본 연구에서는 독도에 자생하는 3종의 해안식물 해국(Aster sphathulifolius), 땅채송화(Sedum oryzifolium), 갯까치 수영(Lysimachia mauritiana)을 연구 재료로 사용하였다. 각 식물에서 근권세균을 각 9주씩 분리하고, 동정하여 이들 세균의 다양성을 조사하고 근권 토양의 세균 집단 조성을 분석하였다. 16S rDNA의 염기서열 분석 결과, 계통학적으로 총 4문 19종의 근권세균이 분리, 동정, 확인되었다. 또한 본 실험을 통하여 독도 자생식물 근권 균주의 최적 생육 환경이 확인되었다. S. oryzifolium는 Rhodobacterales목, Micrococcales목, Corynebacteriales목, Flavobacteriales목, Oceanospirillales목, Bacillales목의 세균 균주가 확인되었으며, 자생식물 L. mauritiana에서는 Micrococcales목, Flavobacteriales목, Rhizobiales목, Oceanospirillales목, Rhodobacterales목, Bacillales목의 세균균주가 확인되었다. 자생식물 A. sphathulifolius에서는 Flavobacteriales목, Rhizobiales목, Corynebacteriales목, Micrococcales목, Alteromonadales목 및 Bacillales목의 균주가 분리, 동정, 확인되었다. 본 연구를 바탕으로 3종의 자생식물에서 방선균문(Actinobacteria)과 프로테오박테리아문(Proteobacteria)에 속하는 근권세균이 많이 존재함을 확인할 수 있었다. Three plant species, Aster sphathulifolius, Sedum oryzifolium, and Lysimachia mauritiana, native to the Dokdo Islands in South Korea, were examined for rhizosphere microorganisms by using 16S rDNA sequences. Nine species of rhizosphere microorganisms were isolated from the three native plant species, respectively. Phylogenetic analysis showed that the microorganisms could be classified into 19 species belonging to four phyla (Actinobacteria, Bacteroidetes, Firmicutes and Proteobacteria), and the characteristics of the microbes were confirmed. Rhizosphere microorganisms from the six orders (Bacillales, Corynebacteriales, Flavobacteriales, Micrococcales, Oceanospirillales, and Rhodobacterales) were isolated from S. oryzifolium. From L. mauritiana, microbes belonging to the seven orders (Bacillales, Flavobacteriales, Micrococcales, Oceanospirillales, Rhizobiales, and Rhodobacterales) were isolated. From A. sphathulifolius, the six orders of rhizosphere microorganisms (Alteromonadales, Bacillales, Corynebacteriales, Flavobacteriales, Micrococcales, and Rhizobiales) were isolated. These data showed that Actinobacteria and Proteobacteria were the dominant phyla for the rhizosphere of all three plants. To confirm the bacterial diversity in rhizospheres, Shannon’s diversity index (H’) was used at the genus level. In these data, the rhizosphere from S. oryzifolium and L. mauritiana had more diverse bacteria compared to that from A. sphathulifolius.