보고서 정보
주관연구기관 |
국립농업과학원 National Institute of Agricultural Sciences |
보고서유형 | 최종보고서 |
발행국가 | 대한민국 |
언어 |
한국어
|
발행년월 | 2016-02 |
과제시작연도 |
2015 |
주관부처 |
농촌진흥청 Rural Development Administration(RDA) |
등록번호 |
TRKO201600003177 |
과제고유번호 |
1395040743 |
사업명 |
농업기초기반연구 |
DB 구축일자 |
2016-06-25
|
DOI |
https://doi.org/10.23000/TRKO201600003177 |
초록
▼
Ⅳ. 연구개발결과
프라이머 PhRTF-PhRTR 조합을 이용하여 PCR을 실시한 결과, 국화역병균, 균핵병균, 흑반병균, 시들음병균 등 6종의 국화에서 발병되는 병원균에서는 DNA가 증폭되지 않지만 국화흰녹병균의 DNA만 증폭되는 것을 확인하였기에 본과제에서 선발한 PCR 프라이머는 국화흰녹병균만 특이적으로 검정할 수 있어 국화흰녹병의 감염여부를 알수있다. 병원균 접종 후 저항성과 감수성 품종간에 유전자발현 차이를 보이는 유전자를 발굴하기 위해 RT-qPCR 방법을 이용하였고 현재까지는 한 개의 유전자가 저항성과 감수성 품종간
Ⅳ. 연구개발결과
프라이머 PhRTF-PhRTR 조합을 이용하여 PCR을 실시한 결과, 국화역병균, 균핵병균, 흑반병균, 시들음병균 등 6종의 국화에서 발병되는 병원균에서는 DNA가 증폭되지 않지만 국화흰녹병균의 DNA만 증폭되는 것을 확인하였기에 본과제에서 선발한 PCR 프라이머는 국화흰녹병균만 특이적으로 검정할 수 있어 국화흰녹병의 감염여부를 알수있다. 병원균 접종 후 저항성과 감수성 품종간에 유전자발현 차이를 보이는 유전자를 발굴하기 위해 RT-qPCR 방법을 이용하였고 현재까지는 한 개의 유전자가 저항성과 감수성 품종간에 차이를 확인하였다.
충남 서천지역에서 발병한 이병주를 이용하여 국화재배지에 감염 시도하여 국화흰녹병균을 접종하였다. 발병된 국화 식물체로부터 균개를 분리하고, genomic DNA와 total RNA를 분리하였다. 분리된 DNA와 RNA로써 NGS 기기에 적합한 library를 제조하고 유전체, 전사체 염기서열을 결정하였다. 유전체 염기서열결정 결과 최종적으로 87개 contig가 얻어졌으며 7,589개 유전자가 예측되었다. 국화흰녹병균의 유전체 크기는 66 Mbp로 추정되었다.
Abstract
▼
<제1세부과제>
In South Korea, cut flower chrysanthemum has a large share in the flower industry and, compared to 2001, the export of cut flower chrysanthemum also increased by 1.9 times in 2011. However, Korean growers have faced with difficulty in keeping competitiveness in world chrysanthemum cut fl
<제1세부과제>
In South Korea, cut flower chrysanthemum has a large share in the flower industry and, compared to 2001, the export of cut flower chrysanthemum also increased by 1.9 times in 2011. However, Korean growers have faced with difficulty in keeping competitiveness in world chrysanthemum cut flower market due to increased production cost and selling price competition. In order to overcome these problems, it is needed to develop cultivars that consumers demand and to produce cut flower chrysanthemum with high quality. Diseases and insects can cause severe quality problem of cut flower chrysanthemum. The most serious damage is caused by Chrysanthemum white rust. The best option to minimize damage caused by white rust is to develop cultivars resistant to chrysanthemum white rust, but it will take so long time to develop a resistant cultivar. Therefore, an alternative way can be development of technique for early detection of infection by chrysanthemum white rust, which can minimize damage of cut flower chrysanthemum. We preferentially established the system for production of white rust susceptible chrysanthemum plants because it is required to supply susceptible plants in laboratories throughout the year to develop the detection technique for white rust. Pathogens sampled from susceptible plants were identified as chrysanthemum white rust through investigation of shape and size of basidiospore using microscope and analysis of rDNA sequence of the pathogen. In addition, the specific gene for chrysanthemum white rust was selected from genome database of Puccinia horiana and 10 to 20 pg of its genomic DNA could be obtained by PCR using primers designed to target the specific gene of chrysanthemum white rust. Six pg of its genomic DNA could be also amplified by RT-qPCR. In the near future, we will confirm if chrysanthemum white rust can be detected through the PCR method mentioned above before symptom caused by infection of white rust is become visible.
We observe the mycelia to penetrate through messophy cell 7 days after inoculation in susceptible cultivar by confocal scanning microscope. and can select 37 genes to be increased gene expression after pathogen inoculation by transcriptome analysis. One of 37 genes is confirmed the differences of gene expression between resistant and susceptible cultivars. We will find more candidate genes possibly involved in the interaction of chrysanthemum and chrysanthemum white rust.
<제2세부과제>
Chrysanthemum is an economically important horticultural plant in many countries. The white rust is one of the most devastating diseases caused by an obligate fungal pathogen Puccinia horiana, which is a species of the genus Puccinia, the family Pucciniaceae within the order Uredinales. Puccinia horiana is an autoecious microcyclic fungi (Bonde et al., 2015), a highly specialized obligately biotrophic pathogen (De Backer et al., 2013), hence this fungus cannot be cultured to obtain sufficient biomass for DNA extraction (Wang et al., 2009).
The fungus was isolated from chrysanthemum leaf in Seocheon, South Korea. P. horiana species was identified by specific primers (Pedley, 2009): forward Ph F2 (5’-CCC CTT TTT TAT TAT ATA ACA CAA G-3’) and reverse Ph R1 (5’-CAA AAA TTA TTT TGT GAG AGG G-3’) and in a Taq polymerase driven PCR on crude genomic DNA prepared by using PCR premix (Bioneer, Korea). This Product is 340 bp. Genomic DNA was extracted using DNeasy Plant Mini Kit (Qiagen, Valencia, CA, USA). The DNA was constructed into insert libraries and sequenced by the Illumina HiSeq2000 or the Pacific Bioscience (PacBio) single Molecule, Real-Time (SMRT) sequencing technology. Sequencings were run on corresponding flat forms and resulted in 281,206,002 reads (HiSeq) and 3,535,482 reads (PacBio). The read bases were assembled by the AllPathLG or FALCON. The resulting draft genome consisted of 66,403,302 nucleotides and 87 contigs. Open reading frames of the assembled genome were predicted using MAKER version 2.31.8 (Cantarel et al., 2008) and carried out blasted with UniProt Swiss-Prot (2010506) (Bairoch and Apweiler, 2000). Predicted Number of transcripts is 7,589 and its total length is 17,284,841 bp.
The chromosome contains 66,403,302 nucleotides with 43.2% GC content. The genome contains 7,589 predicted transcripts, 903 RNA genes. Among the 7,589 predicted protein coding genes. Most predicted genes were validated by RNA-Seq data with half supported over the entire gene length. The genes were categorized into 32 fuctions.
The disease is being controlled mostly by application of chemicals. In Korea, 26 items are registered and 10 items contain 6 triazole compounds. To identify and to obtain the information of the drug target for triazoles, possible sterol 14-demethylase orthologues were extracted. From the draft genome information, the nucleotide sequence of the sterol 14-demethylase gene was identified. The amino acid sequence was deduced and the tertiary structure of the enzyme was predicted. This protein showed no less than 84% amino acid sequence identities to those of genus Puccinia and no more than 68% to those of other genus.
※ AI-Helper는 부적절한 답변을 할 수 있습니다.