고추 탄저병균의 포자 발아 단계 발현 유전자 동정 Identification of Genes Expressed during Conidial Germination of the Pepper Anthracnose Pathogen, Colletotrichum acutatum원문보기
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, histoneprotein, 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 고추탄저병균의 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.
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 고추탄저병균의 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 cont...
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.
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.
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문제 정의
In this study, we describe a pilot-scale cDNA sequencing that provides the first opportunity for inspection of the gene expression during conidial germination of pepper anthracnose fungus C. acutatum. We have produced 983 ESTs from conidial germination stage cDNA library, resulting 464 genes.
The main objectives of this project were to identify C. acutatum genes expressed during early development of conidia and to investigate their expression profile (abundance) by sequence analysis. The data presented here represent a preliminary survey.
이론/모형
coli transformation by electroporation, the library was directly ligated into the plasmid vector, pBlueScriptIISK+, which eliminates the need to use in vivo excision to create phagemids. A 96-well format plasmid DNA isolation method was established for these experiments. Several methods were evaluated including the Wizard system (Promega, USA), Polyfiltronics filter plate (Whatman, USA) and Qiagen miniprep kit (Qiagen, USA) for high-throughput DNA isolation.
Partial nucleotide sequences of the cDNA inserts were determined using the dideoxy chain termination method. Purified plasmid DNA was sequenced using Applied Biosystems (ABI) Big Dye terminator kits (Perkin-Elmer) and the ABI model 3700 DNA sequencers in the NICEM (National Instrumentation Center for Environmental Management) of Seoul National University.
성능/효과
Several methods were evaluated including the Wizard system (Promega, USA), Polyfiltronics filter plate (Whatman, USA) and Qiagen miniprep kit (Qiagen, USA) for high-throughput DNA isolation. It was concluded that using the alkaline lysis method employing the Polyfiltronics filter plate for purification yielded DNA of acceptable quality/quantity in a cost-effective way. This method is now applied widely to isolate plasmid DNA or BAC DNA in a high-throughput manner.
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