인삼 모상근 프로테옴 데이터 분석 : 인삼 EST database와의 통합 분석에 의한 단백질 동정

권경훈,김승일,김경욱,김은아,조건,김진영,김영환,양덕춘,허철구,유종신,박영목,Kwon, Kyung-Hoon,Kim, Seung-Il,Kim, Kyung-Wook,Kim, Eun-A,Cho, Kun,Kim, Jin-Young,Kim, Young-Hwan,Yang, Deok-Chun,Hur, Cheol-Goo,Yoo, Jong-Shin,Park, Young-M 한국식물생명공학회 2002 식물생명공학회지 Vol.29 No.3

인삼 모상근의 프로테옴 분석에 의해 얻은 질량분석 스펙트럼 데이터는 MALDI/TOF/MS에서 얻는 질량 스펙트럼과 ESI/Q-TOF/MS에서 얻는 탄뎀 질량 스펙트럼으로 구분된다. 질량 스펙트럼은 단백질이 효소에 의해 분해된 펩타이드들의 분자량 정보를 제공하며, 탄뎀 질량 스펙트럼에서는 아미노산 단위로 분해된 절편 단백질의 분자량으로부터 아미노산 서열을 결과로 얻는다. 펩타이드의 아미노산 서열을 BLAST로 검색하면 유사한 단백질을 GenBank에서 검색할 수 있다. 이러한 단백질 동정 방법은 완전한 유전체 서열이 알려진 생물체의 경우 높은 정확도로 단백질을 동정할 수 있으나, 그렇지 않은 경우는 유사한 단백질이 데이터베이스에 존재하지 않아 분석이 용이하지 않다. 본 연구에서는 질량 스펙트럼 및 절편 단백질의 아미노산 서열을 EST (expressed sequence tag) 서열과 비교하여 프로테옴 데이터와 일치하는 EST 서열을 찾아내고 이를 BLAST검색에 의해 단백질 동정에 활용하였다. ESI/Q-TOF/MS 에서 얻은 아미노산 서열은 길이는 짧지만 데이터의 신뢰도가 높으므로 EST 서열과의 연관 관계를 밝힘으로써 단백질에 대한 정보를 보완할 수 있었다. ESI/Q-TOF/MS에서 얻은 펩타이드의 아미노산 서열을 EST 서열과 비교한 결과 90%의 아미노산 서열이 EST DB에서 발견되었다. NCBI의 nr 데이터베이스에서 아미노산 서열을 검색하여 찾은 단백질이 68%임에 비하여, 인삼 EST 서열에 의한 검색이 22% 더 많은 결과를 얻었다. MALDI/TOF/MS의 질량 스펙트럼에서 nr 데이터베이스로 검색한 결과와 인삼 EST 데이터베이스를 검색한 결과가 일치하는 경우는 47개 중 9개인 19%에 불과하여, 탄뎀 질량 분석으로 아미노산 서열을 얻지 않고, 단지 질량 스펙트럼으로부터 단백질을 동정하는 방법으로는 단백질 동정의 정확한 결과를 기대하기 어려움을 확인하였다. For the hairy root of Panax ginseng, we have got mass spectrums from MALDI/TOF/MS analysis and Tandem mass spectrums from ESI/Q-TOF/MS analysis. While mass spectrum provides the molecular weights of peptide fragments digested by protease such as trypsin, tandem mass spectrum produces amino acid sequence of digested peptides. Each amino acid sequences can be a query sequence in BLAST search to identify proteins. For the specimens of animals or plants of which genome sequences were known, we can easily identify expressed proteins from mass spectrums with high accuracy. However, for the other specimens such as ginseng, it is difficult to identify proteins with accuracy since all the protein sequences are not available yet. Here we compared the mass spectrums and the peptide amino acid sequences with ginseng expressed sequence tag (EST) DB. The matched EST sequence was used as a query in BLAST search for protein identification. They could offer the correct protein information by the sequence alignment with EST sequences. 90% of peptide sequences of ESI/Q-TOF/MS are matched with EST sequences. Comparing 68% matches of the same sequences with the nr database of NCBI, we got more matches by 22% from ginseng EST sequence search. In case of peptide mass fingerprinting from MALDI/TOF/MS, only about 19% (9 proteins of 47 spots) among peptide matches from nr DB were correlated with ginseng EST DB. From these results, we suggest that amino acid sequencing using tandem mass spectrum analysis may be necessary for protein identification in ginseng proteome analysis.

Identification of Genes Induced by Venturia nashicola in Indigenous Korean Pear ‘Hwangsilri’

신일섭,배경미,남가영,강병철,천재안,조강희,김세희,최형석,김현란,황해성,이희재 한국원예학회 2012 Horticulture, Environment, and Biotechnology Vol.53 No.6

Indigenous Korean pear ‘Hwangsilri’ leaves were inoculated with Venturia nashicola to build expressed sequence tags (ESTs) database as resources for transcripts induced in the inoculated leaves. After performing subtractive suppression hybridization using cDNA of the inoculated and uninoculated leaves harvested at 1, 48, and 96 hours after inoculation, 159 (1 hour), 384 (48 hours), and 110 clones (96 hours) were selected and sequenced. BLASTX searches each cDNA library against Genbank revealed 15, 50, and 22 unique sequences at 1, 48, and 96hours after inoculation, respectively. Most highly represented ESTs at 1 hour after inoculation were photosynthesis-and senescence-related genes such as light harvesting chlorophyll a/b binding protein, ribulose-bishosphate carboxylase/oxyganase, and senescence-associated protein. Cytochrome P450-like TATA box binding proteins and manganese superoxide dismutase associated with the defense response, biotic and abiotic stresses were differentially expressed at 1 hour after inoculation. Although more than 50% of ESTs at 1 and 48 hours after inoculation were also associated with photosynthesis- and carbon fixation-related genes, defense-related genes were annotated as 14 clones at 48 hours after inoculation. Six ESTs associated with pathogen-defense and biotic and abiotic stresses were expressed only at 48 hours after inoculation. At 96 hours after inoculation, seven ESTs were involved in defense-response and biotic and abiotic stresses, and three out of the seven ESTs such as cyclophilin, F-box family, and leucine-rich repeat receptor-like kinase (LRR-RLK) were uniquely expressed. Quantitative real-time polymerase chain reaction analysis revealed that the transcripts for aldo-keto reductase (AKR) and LRR-RLK were highly expressed in the incompatible interaction. AKR gene was more highly expressed in resistant cultivar ‘Hwangsilri’ and moderately susceptible one ‘Gamcheonbae’ than in other cultivars. LRR-RLK showed differentially higher expression pattern in inoculated than in uninoculated leaves of susceptible cultivars including ‘Gamcheonbae’ and ‘Hwangkeumbae’. The ARK and LRRRLK genes were found to be associated with plant defense mechanism. However, more detailed further study using transformants introgressed these genes is required to understand how these genes are expressed and regulated by infection with V. nashicola.

Spliced leader sequences detected in EST data of the dinoflagellates Cochlodinium polykrikoides and Prorocentrum minimum

Ruoyu Guo,기장서 한국조류학회I 2011 ALGAE Vol.26 No.3

Spliced leader (SL) trans-splicing is a mRNA processing mechanism in dinoflagellate nuclear genes. Although studies have identified a short, conserved dinoflagellate SL (dinoSL) sequence (22-nt) in their nuclear-encoded transcripts,whether the majority of nuclear-coded transcripts in dinoflagellates have the dinoSL sequence remains doubtful. In this study, we investigated dinoSL-containing gene transcripts using 454 pyrosequencing data (Cochlodinium polykrikoides,93 K sequence reads, 31 Mb; Prorocentrum minimum, 773 K sequence reads, 291 Mb). After making comparisons and performing local BLAST searches, we identified dinoSL for one C. polykrikoides gene transcript and eight P. minimum gene transcripts. This showed transcripts containing the dinoSL sequence were markedly fewer in number than the total expressed sequence tag (EST) transcripts. In addition, we found no direct evidence to prove that most dinoflagellate nuclear-coded transcripts have this dinoSL sequence.

Spliced leader sequences detected in EST data of the dinoflagellates Cochlodinium polykrikoides and Prorocentrum minimum

Guo, Ruoyu,Ki, Jang-Seu The Korean Society of Phycology 2011 ALGAE Vol.26 No.3

Spliced leader (SL) trans-splicing is a mRNA processing mechanism in dinoflagellate nuclear genes. Although studies have identified a short, conserved dinoflagellate SL (dinoSL) sequence (22-nt) in their nuclear-encoded transcripts, whether the majority of nuclear-coded transcripts in dinoflagellates have the dinoSL sequence remains doubtful. In this study, we investigated dinoSL-containing gene transcripts using 454 pyrosequencing data (Cochlodinium polykrikoides, 93 K sequence reads, 31 Mb; Prorocentrum minimum, 773 K sequence reads, 291 Mb). After making comparisons and performing local BLAST searches, we identified dinoSL for one C. polykrikoides gene transcript and eight P. minimum gene transcripts. This showed transcripts containing the dinoSL sequence were markedly fewer in number than the total expressed sequence tag (EST) transcripts. In addition, we found no direct evidence to prove that most dinoflagellate nuclear-coded transcripts have this dinoSL sequence.

Analysis and Identification of Expressed Sequence Tags in Hairy Root Induced from Korea Ginseng (Panax ginseng C.A. Meyer)

Deok Chun Yang 한국약용작물학회 2004 韓國藥用作物學會誌 Vol.12 No.2

Hairy roots were induced from Korean ginseng (Panax ginseng C. A. Meyer) root explants and studied for their gene expression. A total of 3,000 ESTs (expressed sequence tags) from ginseng hairy root were determined and about 2,700 ESTs have a length of readable sequence, which result in 1,352 unique ESTs sequences. The 879 ESTs showed significant similarities to known nucleotide or amino acid sequences in other plant species, which were divided into eleven categories depending upon gene unction. The remaining 473 sequences showed no significant matches, which are likely to be transcripts or to be matched to other organisms. The results indicated that the analysis of the ginseng hairy root ESTs by partial sequencing of random cDNA clone may be an efficient approach to isolate genes that are functional in ginseng root in a large scale. Our extensive EST analysis of genes expressed in ginseng hairy rppt not only contributes to the understanding of the dynamics of genome expression patterns in root organ but also adds data to the repertoire of all genomic genes.

Analysis and Identification of Expressed Sequence Tags in Hairy Root Induced from Korean Ginseng (Panax ginseng C. A. Meyer)

Yang, Deok-Chun,In, Jun-Gyo The Korean Society of Medicinal Crop Science 2004 韓國藥用作物學會誌 Vol.12 No.2

Hairy roots were induced from Korean ginseng (Panax ginseng C. A. Meyer) root explants and studied for their gene expression. A total of 3,000 ESTs (expressed sequence tags) from ginseng hairy root were determined and about 2,700 ESTs have a length of readable sequence, which result in 1,352 unique ESTs sequences. The 879 ESTs showed significant similarities to known nucleotide or amino acid sequences in other plant species, which were divided into eleven categories depending upon gene function. The remaining 473 sequences showed no significant matches, which are likely to be transcripts or to be matched to other organisms. The results indicated that the analysis of the ginseng hairy root ESTs by partial sequencing of random cDNA clones may be an efficient approach to isolate genes that are functional in ginseng root in a large scale. Our extensive EST analysis of genes expressed in ginseng hairy root not only contributes to the understanding of the dynamics of genome expression patterns in root organ but also adds data to the repertoire of all genomic genes.

Analysis and Identification of Expressed Sequence Tags in Hairy Root Induced from Korean Ginseng (Panax ginseng C. A. Meyer)

Deok-Chun Yang,Jun-Gyo In 韓國藥用作物學會 2004 한국약용작물학회지 Vol.12 No.2

Hairy roots were induced from Korean ginseng (Panax ginseng C. A. Meyer) root explants and studied for their gene expression. A total of 3,000 ESTs (expressed sequence tags) from ginseng hairy root were determined and about 2,700 ESTs have a length of readable sequence, which result in 1,352 unique ESTs sequences. The 879 ESTs showed significant similarities to known nucleotide or amino acid sequences in other plant species, which were divided into eleven categories depending upon gene function. The remaining 473 sequences showed no significant matches, which are likely to be transcripts or to be matched to other organisms. The results indicated that the analysis of the ginseng hairy root ESTs by partial sequencing of random cDNA clones may be an efficient approach to isolate genes that are functional in ginseng root in a large scale. Our extensive EST analysis of genes expressed in ginseng hairy root not only contributes to the understanding of the dynamics of genome expression patterns in root organ but also adds data to the repertoire of all genomic genes.

Analysis of 176 Expressed Sequence Tags Generated from cDNA Clones of Hot Pepper by Single-pass Sequencing

Hong, Sung Tae,Chung, Jae Eun,An, Gyn Heung,Kim, Seong Ryong 한국식물학회 1998 Journal of Plant Biology Vol.41 No.2

As a part of the project to identify novel genes from the hot pepper (Capsicum annuum L. cv. Happy Dry), we have constructed several cDNA libraries and 176 randomly selected cDNA clones were partially sequenced. This expressed sequence tag (EST) analysis identified 95 clones (54.0%) that had a significant homology to a known protein sequence in the NCBI database. Of these clones, eighteen of them are related to genes not from the plant kingdom, indicating that 10.2% of the ESTs were newly identified in plants. Functional categorization of these clones revealed that the genes involved in metabolic pathways such as glycolysis and photosynthesis are most abundant, and genes in translational apparatus ranked next in abundance. Expression patterns of four ESTs were examined by RNA blot analysis. The CAN14 clone, which has a 58% identity to the potato patatin protein over a 117 amino acid overlap, was highly expressed in anther tissue but not in fruit tissues. The CFR2 clone, which is 88% identical to apospory-associated protein, showed relatively higher expression levels in seedlings and roots compared to other tissues. The transcript of the CFR11 clone, which shows a homology to γ-thionin, was abundant in every organ that was examined except roots. The CFR 29 clone which is 92% identical to the putative osmoprotectant from tomato root showed a root-preferential expression pattern.

Identification of Genes Expressed during Conidial Germination of the Pepper Anthracnose Pathogen, Colletotrichum acutatum

Jeong-Hwan Kim(김정환),Jong-Hwan Lee(이종환),Woobong Choi(최우봉) 한국생명과학회 2013 생명과학회지 Vol.23 No.1

고추 탄저병균의 포자 발아 단계에서 발현되는 유전자를 파악하기 위해 포자 발아단계cDNA library를 제작하고, 임의로 선택된 cDNA clone들에 대한 EST sequencing을 실시하였다. 총 983개 EST를 확보하여 contig assembly를 실시한 결과, 197개 contigs와 267개 singletons으로 조합되어, 최종적으로 464개의 유전자를 동정하였다. 464개 유전자 서열에서 유추한 아미노산 서열을 이용한 상동유전자 검색을 통해 절반의 유전자가 GenBank에 기존 등록된 유전자와 유의성 있는 유사성을 보였다. 가장 높은 빈도로 발현된 유전자는 elongation factor, histone protein, ATP synthease, 14-3-3 protein, clock controlled protein을 암호화하는 유전자들이었다. 그리고 고추 탄저병균의 세포 발달과정에 관여 하는것으로 추정되는 GTP-binding protein, MAP kinase, transaldolase, ABC transporter 유전자들도 검출되었다. 또한 고추 탄저병균의 병원성에 영향을 미치는 것으로 파악되는 ATP citrate lyase, CAP20, manganese-superoxide dismutase 유전자들도 검출되어, EST sequencing 을 통한 세포 발달 단계 발현 유전자 탐색이 효과적임을 알 수 있었다. Genes expressed during conidial germination of the pepper anthracnose fungus Colletotrichum acutatum were identified by sequencing the 5’ end of unidirectional cDNA clones prepared from the conidial germination stage. A total of 983 expressed sequence tags (ESTs) corresponding to 464 genes, 197 contigs and 267 singletons, were generated. The deduced protein sequences from half of the 464 genes showed significant matches (e value less than 10-5) to proteins in public databases. The genes with known homologs were assigned to known functional categories. The most abundantly expressed genes belonged to those encoding the elongation factor, histone protein, ATP synthease, 14-3-3 protein, and clock controlled protein. A number of genes encoding proteins such as the GTP-binding protein, MAP kinase, transaldolase, and ABC transporter were detected. These genes are thought to be involved in the development of fungal cells. A putative pathogenicity function could be assigned for the genes of ATP citrate lyase, CAP20 and manganese-superoxide dismutase.

Expressed Sequence Tag Analysis of Antarctic Hairgrass Deschampsia antarctica from King George Island, Antarctica

Lee, Hyoungseok,Cho, Hyunhee,Kim, Ilchan,Yim, Jounghan,Lee, Hongkum,Lee, Yookyung Korean Society for Molecular Biology 2008 Molecules and cells Vol.25 No.2

Deschampsia antarctica is the only monocot that thrives in the tough conditions of the Antarctic region. It is an invaluable resource for the identification of genes associated with tolerance to various environmental pressures. In order to identify genes that are differentially regulated between greenhouse-grown and Antarctic field-grown plants, we initiated a detailed gene expression analysis. Antarctic plants were collected and greenhouse plants served as controls. Two different cDNA libraries were constructed with these plants. A total of 2,112 cDNA clones was sequenced and grouped into 1,199 unigene clusters consisting of 243 consensus and 956 singleton sequences. Using similarity searches against several public databases, we constructed a functional classification of the ESTs into categories such as genes related to responses to stimuli, as well as photosynthesis and metabolism. Real-time PCR analysis of various stress responsive genes revealed different patterns of regulation in the different environments, suggesting that these genes are involved in responses to specific environmental factors.