Analysis of the transcriptome of OGA-treated Arabidopsis leaf by SAGE method

Abstract

To analyze the transcriptome of OGA-treated Arabidopsis leaf, Serial analysis of gene expression (SAGE) method was utilized in this study. During the plant defense response against various pathogenic infection or stress, metabolic change and change of gene expression are supposed to be followed. Oligogalacturonic acid (OGA) is one of the well-characterized class of plant cell wall-derived signals and induce responses that may help the plant to resist disease and physiological changes. In this study, gene expression pattern of OGA-treated Arabidopsis, OGA was prepared and the transcriptome of the plant was analyzed. OGA was prepared by acid-hydrolysis of Polygalacturonic acid (PGA) and analyzed its size distribution by capillary electrophoresis. The OGAs known as effective size were obtained. Ethylene production induced by OGA-treated Arabidopsis was examined. In 500 ㎍/ml OGA treated Arabidopsis for 6-hours, ethylene accumulation was increased approximately 80% compared with untreated. To know the gene expression patterns in OGA-treated plants, a recently developed method, SAGE was used. From mRNAs of OGA-treated Arabidopsis, cDNA conversion was performed. After cDNA cut and linker ligation, ditags production, PCR amplification, and construction of ditag concatemers were sequentially performed. cDNA tag library was constructed and as an initial analysis of gene expression pattern, total 19 tag sequences were examined using FASTA program. Some known genes and ESTs including hsp90A and ACC oxidase homolog, which are known to be related to responses against various stresses. were obtained. and also unknown new ESTs were obtained.;Oligogalcturonic acid (OGA)는 잘 알려진 식물 세포벽에서 파생된 신호 물질의 하나로, 식물의 질병에 대한 저항 반응들을 유도하는 물질로 식물 세포에 여러 가지 생리적 변화를 일으킨다. 본 연구에서는 OGA에 의해 어떠한 유전적 발현 양상이 만들어지는지를 조사하기 위해 OGA를 제조하고, 제조된 OGA를 처리한 Arabidopsis잎에서의 transcriptome을 조사하였다. 산 가수분해 방법으로 Polygalacturonic acid를 분해시키고, 그 산물을 Capillary electrophoresis방법으로, 크기 분포를 조사하여 효과적인 크기인 oligo size를 다량 포함함을 확인하였다. 제조된 OGA를 Arabidopsis의 잎에 6시간 동안 500 μg/㎖의 OGA로 처리했을 때, 처리하지 않는 경우보다 에틸렌 축적이 80% 증가하였다. 또한 OGA 처리한 식물에서의 유전자 발현 상황을 보기 위하여 최근에 개발된 SAGE방법을 적용하였다. OGA처리한 Arabidopsis잎의 mRNA로부터 얻은 cDNA를 잘라 linker를 이은 후, ditag를 만들어 PCR로 증폭한 뒤 ditag concatemer를 얻는 과정을 순차적으로 진행하였다. 유전자 발현 상황을 분석하는 초기 작업으로, 제작된 cDNA tag library에서 20여개의 tag sequence를 얻어 FASTA program으로 조사하였다. 스트레스에 대한 반응과 관련 있다고 알례진 ACC oxidase homologue 및 hsp90A를 포함한 몇 가지 알려진 유전자와 그 역할이 알려지지 않은 새로운 EST들이 확인되었다.