KR20230057921A - NAC013 gene controlling pithiness in radish taproots and uses thereof - Google Patents
NAC013 gene controlling pithiness in radish taproots and uses thereof Download PDFInfo
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Abstract
Description
본 발명은 무 저장뿌리의 바람들이(pithiness)를 유도하는 NAC013 (NAC domain-containing protein 013) 유전자 및 이의 용도에 관한 것이다.The present invention relates to a NAC013 (NAC domain-containing protein 013) gene that induces pithiness in radish storage roots and uses thereof.
무(Raphanus sativus L.)는 배추에 이어 국내 채소 종자 시장(약 2,000억)에서 두 번째로 중요한 채소 작물이며, 중국, 인도를 비롯한 아시아 국가에서 널리 소비되고 있다. 그러나 소비량이 많은 채소 작물임에도 불구하고 무는 유전체 정보에 기반한 분자 육종 기술 수준은 타 대표작물에 비하여 낮은 편이다.Radish ( Raphanus sativus L.) is the second most important vegetable crop in the domestic vegetable seed market (approximately 200 billion won) after Chinese cabbage, and is widely consumed in Asian countries including China and India. However, despite being a vegetable crop with high consumption, radish has a lower level of molecular breeding technology based on genetic information than other representative crops.
수확된 무를 잘랐을 때 속이 푸석푸석하고 빈공간이 생긴 경우를 종종 발견할 수 있는데, 이러한 현상을 바람들이(pithiness 또는 sponginess)라고 한다. 바람들이가 일어난 무는 상품적 가치가 없어 재배농가 소득에 큰 피해를 일으킬 수 있기 때문에, 무 육종가들은 바람들이가 일어나지 않는 무 품종을 개발하려는 노력을 기울여 왔다. 그럼에도 불구하고, 바람들이는 재배 환경과 유전적 요인이 복합적으로 작용하여 일어나는 특성이고 수확하여 뿌리를 절단해야 발생 여부를 확인이 가능하기 때문에 육종 과정에서 선별하여 배제시키기에 매우 어려운 특성으로 알려져 있다.When harvested radish is cut, it is often found that the inside is crumbly and empty spaces are formed. This phenomenon is called pithiness or sponginess. Radish breeders have made efforts to develop radish varieties that do not wind up because wind-infested radish has no commercial value and can cause great damage to growers' income. Nevertheless, it is known as a very difficult characteristic to select and exclude in the breeding process because it is a characteristic that is caused by a combination of the cultivation environment and genetic factors, and it is possible to check whether it occurs only by harvesting and cutting the roots.
본 발명에서는 무 바람들이를 촉진하는 QTL (Quantitative Trait Loci) 분석과 바람들이 발생 시 발현되는 유전자를 유전체 수준에서 분석하여 전사인자인 NAC013 (NAC domain-containing protein 013) 유전자의 유전형에 따라 바람들이가 유도됨을 발견하였다.In the present invention, QTL (Quantitative Trait Loci) analysis that promotes radish winds and genes expressed when winds occur are analyzed at the genome level, so that winds can be winded according to the genotype of the transcription factor NAC013 (NAC domain-containing protein 013) gene. found to be induced.
한편, 한국공개특허 제2015-0019300호에는 'NAC016 유전자를 이용한 비생물적 스트레스 내성이 증가된 기능성 녹색 지속 변이 식물체의 제조 방법 및 그에 따른 식물체'가 개시되어 있고, 한국공개특허 제2011-0073869호에는 지베렐린과 6-벤질아데닌 합제를 사용한 '배의 바람들이 및/또는 무름 현상을 감소시키는 방법 및 그의 조성'이 개시되어 있으나, 본 발명의 '무 저장뿌리의 바람들이를 유도하는 NAC013 유전자 및 이의 용도'에 대해서는 기재된 바가 없다.On the other hand, Korean Patent Publication No. 2015-0019300 discloses 'a method for producing a functional green persistent mutant plant with increased resistance to abiotic stress using the NAC016 gene and the resulting plant', and Korean Patent Publication No. 2011-0073869 discloses 'a method for reducing windage and/or brittleness of pears and a composition thereof' using a mixture of gibberellin and 6-benzyladenine, but ' NAC013 gene inducing windage of radish storage roots and its composition' of the present invention. There is nothing described about 'use'.
본 발명은 상기와 같은 요구에 의해 도출된 것으로서, 본 발명자들은 무 바람들이를 촉진하는 QTL (Quantitative Trait Loci) 분석과 바람들이 발생 시 발현되는 유전자를 유전체 수준에서 분석하여 바람들이 연관 유전자를 동정하고자 하였다.The present invention has been derived from the above needs, and the present inventors are trying to identify genes related to wind by analyzing QTL (Quantitative Trait Loci) that promote wind blowing and genes expressed when winds occur at the genome level. did
이를 위해, 본 발명에서는 무 바람들이 발생 시 발현이 증가하는 전사체 프로파일링 데이터를 생산 및 분석하고 이를 QTL 분석 결과와 종합하여 RsNAC013 (Raphanus sativus NAC domain-containing protein 013) 유전자가 바람들이 유도에 관여하는 것임을 최초로 보고하였다. 또한, 본 발명에서는 RsNAC013의 유전형에 따라 바람들이 발생 빈도가 달라짐을 분리집단과 그 외 원교(WonKyo) 계통들에 대한 유전형 분석을 통하여 보여주었으며, RsNAC013 유전형의 기능 분석을 통하여 RsNAC013의 전사인자 활성도가 높을수록 바람들이 가능성이 높아짐을 제시하였다.To this end, the present invention produces and analyzes transcriptome profiling data, in which expression increases when windless winds occur, and combines them with QTL analysis results to determine that the RsNAC013 ( Raphanus sativus NAC domain-containing protein 013) gene is involved in winds induction. It was first reported that In addition, the present invention showed that the frequency of occurrence of wind changes according to the genotype of RsNAC013 through genotyping analysis of segregated groups and other WonKyo strains, and through functional analysis of RsNAC013 genotype, the transcription factor activity of RsNAC013 It was suggested that the higher the wind, the higher the possibility.
또한, 본 발명에서는 RsNAC013 유전자의 InDel 다형성에 기반하여 분자마커를 개발하였고, 개발한 분자마커를 이용한 유전형 판별 결과와 바람들이 표현형을 분석한 결과, 상기 분자마커가 무의 바람들이 경향성을 예측할 수 있음을 확인함으로써, 본 발명을 완성하였다.In addition, in the present invention, a molecular marker was developed based on the InDel polymorphism of the RsNAC013 gene, and as a result of genotype discrimination using the developed molecular marker and wind phenotype analysis, the molecular marker can predict the wind trend of radish. By confirming, the present invention was completed.
상기 과제를 해결하기 위해, 본 발명은 서열번호 1 또는 서열번호 2의 염기서열로 이루어진 RsNAC013 (Raphanus sativus NAC domain-containing protein 013) 유전자를 유효성분으로 포함하는 무의 바람들이를 조절하기 위한 조성물을 제공한다.In order to solve the above problems, the present invention is a composition for controlling wind blowing of radish containing RsNAC013 ( Raphanus sativus NAC domain-containing protein 013) gene consisting of the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2 as an active ingredient to provide.
또한, 본 발명은 서열번호 3 및 서열번호 4의 올리고뉴클레오티드 프라이머를 포함하는, 무의 바람들이 예측용 프라이머 세트를 제공한다.In addition, the present invention provides a primer set for prediction of winds of nothing, including the oligonucleotide primers of SEQ ID NO: 3 and SEQ ID NO: 4.
또한, 본 발명은 상기 프라이머 세트 및 증폭 반응을 수행하기 위한 시약을 포함하는 무의 바람들이 예측용 키트를 제공한다.In addition, the present invention provides a kit for predicting winds of radish containing the primer set and reagents for performing the amplification reaction.
또한, 본 발명은 무 시료로부터 게놈 DNA를 분리하는 단계; 상기 분리된 게놈 DNA를 주형으로 하고, 본 발명의 프라이머 세트를 이용하여 증폭 반응을 수행하여 표적 서열을 증폭하는 단계; 및 상기 증폭 단계의 산물을 검출하는 단계;를 포함하는 무의 바람들이 예측 방법을 제공한다.In addition, the present invention comprises the steps of isolating genomic DNA from a radish sample; Amplifying a target sequence by performing an amplification reaction using the isolated genomic DNA as a template and using the primer set of the present invention; And detecting the product of the amplification step; winds of nothing including provide a prediction method.
또한, 본 발명은 무 시료로부터 분리한 핵산에서 RsNAC013 유전자의 하위 유전자 발현수준을 측정하는 단계;를 포함하는 무의 바람들이 예측 방법을 제공한다.In addition, the present invention provides a method for predicting the winds of radish, including measuring the expression level of the lower gene of the RsNAC013 gene in nucleic acids isolated from radish samples.
본 발명의 RsNAC013 유전자는 무의 분자 육종에 유용하게 활용될 수 있어 육종 산업에서 실용화가 용이할 것으로 사료되며, 본 발명에서는 바람들이를 유도하는 유전자의 유전형을 판별할 수 있는 분자 마커를 제시하고 있으므로 정확한 분자 정보에 기반한 육종에 바로 적용될 수 있다. The RsNAC013 gene of the present invention can be usefully used for molecular breeding of radish, so it is considered that it will be easy to put into practical use in the breeding industry. It can be directly applied to breeding based on precise molecular information.
도 1은 전사체 분석을 이용한 무 바람들이 과정의 이해에 관한 것으로, (a)는 무 원교10040호(WK40)의 바람들이 여부에 따라 차등발현되는 3,966개 DEG (Differentially Expressed Genes) 발현 패턴을 K-means clustering으로 분류한 결과이고, (b)는 (a)의 두 cluster를 구성하는 유전자들에 대한 GO (Gene Ontology) term enrichment 분석 결과이며, (c)는 무 저장뿌리 내부에서 선별된 유전자의 발현 패턴 분석을 위해 총 RNA를 분리한 부위의 모식도이고, (d)는 세포사멸 관련 9개 유전자를 바람들이 정도가 다른 원교10040호 무 개체에서 분리한 RNA를 이용하여 qRT-PCR로 발현 측정한 결과이다. 바람들이 index 값이 클수록 바람들이가 심함을 의미한다.
도 2는 무 바람들이 형질의 세포사멸 관여 유전자 RsNAC013과의 연관성에 관한 것으로, (a)는 무 바람들이 관련 분리집단 제작에 사용한 원교10040호(WK40, 바람들이가 잘 일어남)와 원교10029호(WK29, 바람들이가 잘 일어나지 않음)의 모습과 F2 분리집단의 바람들이 형질 측정에 대한 index 값과 이를 기준으로 형질 분석한 바람들이 분포 그래프이고, (b)는 (a)의 F2에 대하여 GBS (Genotyping by Sequencing)를 진행, 1:2:1로 분리되는 biparental SNPs를 선별하고 이들과 바람들이 형질 간의 상관 관계를 분석한 결과로, qPITH-1을 발견한 것을 보여주는 것이며, (c)는 qPITH-1 주변에 세포사멸 관련 DEG를 탐색한 결과 RsNAC013를 발견하였음을 보여주는 것으로 RsNAC013은 WK40에서 3' UTR이 일부 소실되어 있고 2개의 아미노산 서열이 WK29와 차이를 보임을 확인하였다. (d)는 RsNAC013과 그 하위 유전자들의 발현 분석을 위해 선발한 WK40과 WK29 개체의 바람들이 분석 결과이고, (e)는 (d)의 무 개체에서 분리한 총 RNA를 이용하여 RsNAC013과 하위 유전자들의 발현 정도를 qRT-PCR로 분석한 결과이다.
도 3은 RsNAC013의 유전형과 바람들이의 연관성 분석 결과로, (a)는 원교10040호(WK40)와 원교10029호(WK29)의 RsNAC013 염기서열을 alignment한 결과로 보라색 박스 부위가 (b)의 PCR 마커로 활용한 구간이다. (b)는 46bp의 길이 차이를 이용하여 RsNAC013의 유전형을 PCR로 분석한 결과이고, (c)는 46bp 길이 차이를 보이는 구간과 더불어 아미노산 배열의 차이를 일으키는 SNP가 두 개 존재함을 보여주는 Sanger sequencing chromatogram이고, (d)는 원교10040호와 원교10029호를 양친으로 제작한 F2의 바람들이와 RsNAC013의 유전형을 종합 분석한 결과이며, (e)는 24개 원교들의 바람들이와 RsNAC013의 유전형을 분석한 결과이다.
도 4는 RsNAC013 유전형의 분자적 기능을 분석한 것으로, (a) 및 (b)는 RsNAC013의 3' UTR의 46bp과 기타 Indel의 차이가 전사 후 프로세스(posttranscriptional process)에 영향을 미치는지를 분석하기 위해 원교10029호(WK29)와 10040호(WK40)의 3' UTR 구간(WK29는 258bp, WK40는 204bp)을 각각 분리하여 핵으로 타겟되는 nlsGFP 아래 부착, GL2 프로모터 하에 발현시키도록 제작한 벡터(pGL2::nlsGFP:3'UTR)로 형질전환시킨 애기장대 뿌리에서의 nlsGFP의 발현 정도를 비교한 결과이다. (c) 내지 (f)는 RsNAC013 코딩 부위에 존재하는 두 아미노산 차이가 세포 내의 단백질 위치에 영향을 주는지를 확인하고자 RsNAC013 cDNA를 WK40과 WK29에서 분리하여 N 말단에 GFP를 부착, 35S 프로모터 하에 발현시킨 애기장대 형질전환체의 뿌리에서의 GFP-RsNAC013 발현을 공초점 현미경으로 분석한 결과로, (c) 및 (e)는 Murashige & Skoog Agar 배지에서 발아 후 발현을 분석한 결과이고, (d) 및 (f)는 각각 (c)와 (e)의 개체를 20mM의 과산화수소로 1시간 처리한 후 이미징한 결과이다. (g)는 노지에서 키운 원교10029호와 10040호의 뿌리 RNA에서 RsAOX1a와 RsNAC013의 발현 비율을 qRT-PCR로 분석한 결과이고, (h)는 온실에서 원교10029호와 10040호를 8주간 키운 후 300mM의 과산화수소를 하루 처리하여 RsAOX1a와 RsNAC013의 발현 비율을 qRT-PCR로 분석한 결과이며, (i)는 (c-f)의 애기장대 형질전환체를 활용하여 RsNAC013와 ANAC013이 애기장대 AOX1a (AtAOX1a)의 발현을 촉진하는 정도를 digital droplet RT-PCR로 측정하여 계산한 결과이다.1 relates to the understanding of the windless process using transcriptome analysis, (a) is the result of K-means clustering classification of 3,966 DEG (Differentially Expressed Genes) expression patterns that are differentially expressed depending on whether or not the winds of Muwonkyo 10040 (WK40) This is the result of GO (Gene Ontology) term enrichment analysis for the genes constituting the cluster, (c) is a schematic diagram of the region where total RNA was isolated to analyze the expression pattern of genes selected inside the radish storage root, (d) is the result of measuring the expression of nine genes related to apoptosis by qRT-PCR using RNA isolated from Wongyo 10040 radish individuals with different degrees of windage. The higher the winds index value, the stronger the winds.
Figure 2 relates to the relationship between the gene RsNAC013 involved in apoptosis of the windless trait, (a) shows the appearance of Wongyo 10040 (WK40, wind blowing well) and Wonkyo 10029 (WK29, wind blowing does not occur well) and the wind characteristics of the F 2 segregation group used in the production of the related segregation group. The index value for the measurement and the winds analyzed based on it are a distribution graph, and (b) is a GBS (Genotyping by Sequencing) for F 2 of (a), biparental SNPs separated by 1:2:1 As a result of selecting and analyzing the correlation between these traits and winds, it shows that qPITH-1 was found, and (c) shows that RsNAC013 was found as a result of searching for apoptosis-related DEGs around qPITH-1 . It was confirmed that RsNAC013 has a partial loss of 3' UTR in WK40 and differs from WK29 in two amino acid sequences. (d) is the result of the wind analysis of WK40 and WK29 individuals selected for the expression analysis of RsNAC013 and its subgenes, and (e) is the analysis result of RsNAC013 and subgenes using total RNA isolated from the radish individuals in (d). This is the result of analyzing the expression level by qRT-PCR.
Figure 3 is the result of the analysis of the correlation between the genotype of RsNAC013 and the affair, (a) is the result of alignment of the RsNAC013 base sequences of Wonkyo 10040 (WK40) and Wonkyo 10029 (WK29), and the purple box is the section used as a PCR marker in (b). (b) is the result of PCR analysis of the genotype of RsNAC013 using a 46bp length difference, and (c) is Sanger sequencing showing that there are two SNPs that cause a difference in amino acid sequence along with a section showing a 46bp length difference. (d) is the result of a comprehensive analysis of the winds and RsNAC013 genotypes of F 2 produced by Wonkyo 10040 and Wonkyo 10029 as parents, and (e) is the wind and RsNAC013 genotypes of 24 Wonkyos. is the result of the analysis.
Figure 4 analyzes the molecular function of the RsNAC013 genotype, (a) and (b) are 3 of Wonkyo No. 10029 (WK29) and No. 10040 (WK40) to analyze whether the difference between the 46 bp of the 3' UTR of RsNAC013 and other indels affects the posttranscriptional process. Arabidopsis thaliana roots transformed with a vector ( pGL2::nlsGFP:3'UTR ) designed to separate the 'UTR section (258 bp for WK29, 204 bp for WK40), attach under nlsGFP targeted to the nucleus, and express under the GL2 promoter. This is the result of comparing the expression level of nlsGFP in . (c) to (f) show whether the two amino acid differences in the coding region of RsNAC013 affect the location of the protein in the cell. RsNAC013 cDNA was isolated from WK40 and WK29, GFP was attached to the N-terminus, and expressed under the 35S promoter. As a result of analyzing the expression of GFP-RsNAC013 in the roots of Arabidopsis thaliana transformants by confocal microscopy, (c) and (e) are the results of analyzing the expression after germination in Murashige & Skoog Agar medium, (d) and (f) is the result of imaging after treating the objects of (c) and (e) with 20 mM hydrogen peroxide for 1 hour, respectively. (g) is the result of qRT-PCR analysis of the expression ratio of RsAOX1a and RsNAC013 in the root RNA of Wongyo 10029 and 10040 grown in the field, and (h) is the result of 300 mM This is the result of analyzing the expression ratio of RsAOX1a and RsNAC013 by qRT-PCR by treating with hydrogen peroxide for one day . This is the result calculated by measuring the degree of promotion by digital droplet RT-PCR.
본 발명의 목적을 달성하기 위하여, 본 발명은 서열번호 1 또는 서열번호 2의 염기서열로 이루어진 RsNAC013 (Raphanus sativus NAC domain-containing protein 013) 유전자를 유효성분으로 포함하는 무의 바람들이를 조절하기 위한 조성물을 제공한다.In order to achieve the object of the present invention, the present invention is to control the wind of radish containing the RsNAC013 ( Raphanus sativus NAC domain-containing protein 013) gene consisting of the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2 as an active ingredient composition is provided.
본 발명에 따른 서열번호 1의 염기서열은 바람들이가 잘 일어나지 않는 무의 RsNAC013 유전자이고, 서열번호 2의 염기서열은 바람들이가 잘 일어나는 무의 RsNAC013 유전자로, 서열번호 2의 염기서열은 서열번호 1의 염기서열에서 1,985번째부터 2,030번째 까지의 염기(46bp)가 결손된 것이 주요 특징이다. 상기 서열번호 2의 염기서열로 이루어진 RsNAC013 유전자는 서열번호 1의 염기서열로 이루어진 RsNAC013 유전자보다 전사 활성이 보다 우수하며, 스트레스 환경에 반응한 세포사멸을 더 잘 유도하여 무 바람들이 현상을 촉진하게 된다.According to the present invention, the nucleotide sequence of SEQ ID NO: 1 is the RsNAC013 gene of radish that does not wind easily, and the nucleotide sequence of SEQ ID NO: 2 is the RsNAC013 gene of radish that does not wind easily, and the base sequence of SEQ ID NO: 2 is SEQ ID NO: The main feature is that the 1,985th to 2,030th bases (46bp) in the nucleotide sequence of No. 1 are missing. The RsNAC013 gene composed of the nucleotide sequence of SEQ ID NO: 2 has a higher transcriptional activity than the RsNAC013 gene composed of the nucleotide sequence of SEQ ID NO: 1, and better induces apoptosis in response to a stress environment, thereby promoting the windless phenomenon. .
본 발명의 일 구현 예에 있어서, 상기 서열번호 1의 염기서열로 이루어진 RsNAC013 유전자형을 포함하고 있는 무 자원은 바람들이가 잘 일어나지 않는 경향을 보였으며, 서열번호 2의 염기서열로 이루어진 RsNAC013 유전자형을 포함하고 있는 무 자원은 바람들이가 잘 일어나는 경향을 보여주었다.In one embodiment of the present invention, the radish resource containing the RsNAC013 genotype consisting of the nucleotide sequence of SEQ ID NO: 1 showed a tendency to be less prone to wind, including the RsNAC013 genotype consisting of the nucleotide sequence of SEQ ID NO: 2 The radish resource that is doing showed a tendency to wind well.
본 발명은 또한, 서열번호 3 및 서열번호 4의 올리고뉴클레오티드 프라이머를 포함하는, 무의 바람들이 예측용 프라이머 세트를 제공한다.The present invention also provides a primer set for prediction of winds of nothing, including the oligonucleotide primers of SEQ ID NO: 3 and SEQ ID NO: 4.
상기 프라이머는 각 프라이머의 서열 길이에 따라, 서열번호 3 및 4의 서열 내의 16개 이상, 17개이상, 18개 이상, 19개 이상, 20개 이상의 연속 뉴클레오티드의 절편으로 이루어진 올리고뉴클레오티드를 포함할 수 있다. 예를 들면, 서열번호 3의 프라이머(21개 올리고뉴클레오티드)는 서열번호 4의 서열 내의 16개 이상, 17개 이상, 18개 이상, 19개 이상, 20개 이상의 연속 뉴클레오티드의 절편으로 이루어진 올리고뉴클레오티드를 포함할 수 있다. 또한, 상기 프라이머는 서열번호 3 및 4의 염기서열의 부가, 결실 또는 치환된 서열도 포함할 수 있다.The primers may include oligonucleotides consisting of segments of at least 16, at least 17, at least 18, at least 19, at least 20 consecutive nucleotides in the sequences of SEQ ID NOs: 3 and 4, depending on the sequence length of each primer. there is. For example, the primer of SEQ ID NO: 3 (21 oligonucleotides) is an oligonucleotide consisting of segments of at least 16, at least 17, at least 18, at least 19, at least 20 contiguous nucleotides in the sequence of SEQ ID NO: 4 can include In addition, the primers may also include added, deleted or substituted sequences of the nucleotide sequences of SEQ ID NOs: 3 and 4.
본 발명에 있어서, "프라이머"는 카피하려는 핵산 가닥에 상보적인 단일 가닥 올리고뉴클레오티드 서열을 말하며, 프라이머 연장 산물의 합성을 위한 개시점으로서 작용할 수 있다. 상기 프라이머의 길이 및 서열은 연장 산물의 합성을 시작하도록 허용해야 한다. 프라이머의 구체적인 길이 및 서열은 요구되는 DNA 또는 RNA 표적의 복합도(complexity)뿐만 아니라 온도 및 이온 강도와 같은 프라이머 이용 조건에 의존할 것이다.In the present invention, "primer" refers to a single-stranded oligonucleotide sequence complementary to a nucleic acid strand to be copied, and may serve as a starting point for synthesis of a primer extension product. The length and sequence of the primers should allow for the synthesis of extension products to begin. The specific length and sequence of the primer will depend on the complexity of the DNA or RNA target required, as well as the conditions of use of the primer, such as temperature and ionic strength.
본 명세서에 있어서, 프라이머로서 이용된 올리고뉴클레오티드는 또한 뉴클레오티드 유사체(analogue), 예를 들면, 포스포로티오에이트(phosphorothioate), 알킬포스포로티오에이트 또는 펩티드 핵산(peptide nucleic acid)를 포함할 수 있거나 또는 삽입 물질(intercalating agent)를 포함할 수 있다.In this specification, oligonucleotides used as primers may also include nucleotide analogs, such as phosphorothioates, alkylphosphorothioates, or peptide nucleic acids, or Intercalating agents may be included.
본 발명은 또한, 상기 프라이머 세트 및 증폭 반응을 수행하기 위한 시약을 포함하는 무의 바람들이 예측용 키트를 제공한다.The present invention also provides a kit for predicting winds of radish containing the primer set and reagents for performing the amplification reaction.
본 발명의 일 구현 예에 있어서, 상기 증폭 반응을 수행하기 위한 시약은 DNA 폴리머라제, dNTPs 및 버퍼를 포함할 수 있으나, 이에 제한되지 않는다.In one embodiment of the present invention, reagents for performing the amplification reaction may include DNA polymerase, dNTPs, and buffers, but are not limited thereto.
본 발명의 일 구현 예에 있어서, 상기 키트는 또한 최적의 반응 수행 조건을 기재한 사용자 안내서를 추가로 포함할 수 있다. 안내서는 키트 사용법, 예를 들면, 역전사 완충액 및 PCR 완충액 제조 방법, 제시되는 반응 조건 등을 설명하는 인쇄물이다. 안내서는 팜플렛 또는 전단지 형태의 안내 책자, 키트에 부착된 라벨, 및 키트를 포함하는 패키지의 표면상에 설명을 포함한다. 또한, 안내서는 인터넷과 같이 전기 매체를 통해 공개되거나 제공되는 정보를 포함한다.In one embodiment of the present invention, the kit may further include a user guide describing optimal reaction performance conditions. The guide is a printout that explains how to use the kit, for example, how to prepare reverse transcription buffer and PCR buffer, and the reaction conditions to be presented. The guide includes a brochure in the form of a pamphlet or leaflet, a label affixed to the kit, and instructions on the surface of the package containing the kit. In addition, the guide includes information disclosed or provided through electronic media such as the Internet.
본 발명은 또한, 무 시료로부터 게놈 DNA를 분리하는 단계; 상기 분리된 게놈 DNA를 주형으로 하고, 본 발명의 프라이머 세트를 이용하여 증폭 반응을 수행하여 표적 서열을 증폭하는 단계; 및 상기 증폭 단계의 산물을 검출하는 단계;를 포함하는 무의 바람들이 예측 방법을 제공한다.The present invention also includes the steps of isolating genomic DNA from radish samples; Amplifying a target sequence by performing an amplification reaction using the isolated genomic DNA as a template and using the primer set of the present invention; And detecting the product of the amplification step; winds of nothing including provide a prediction method.
본 발명의 방법은 무 시료에서 게놈 DNA를 분리하는 단계를 포함한다. 상기 게놈 DNA를 분리하는 방법은 당업계에 공지된 방법을 이용할 수 있으며, 예를 들면, CTAB 방법을 이용할수도 있고, Wizard prep 키트(Promega 사)를 이용할 수도 있다. 상기 분리된 게놈 DNA를 주형으로 하고, 본 발명의 일 실시예에 따른 올리고뉴클레오티드 프라이머 세트를 프라이머로 이용하여 증폭 반응을 수행하여 표적 서열을 증폭할 수 있다. 표적 핵산을 증폭하는 방법은 중합효소연쇄반응(polymerase chain reaction; PCR), 리가아제 연쇄반응(ligase chain reaction), 핵산 서열 기재 증폭(nucleic acid sequence-based amplification), 전사 기재 증폭시스템(transcription-based amplification system), 가닥 치환 증폭(strand displacement amplification) 또는 Qβ 복제효소(replicase)를 통한 증폭 또는 당업계에 알려진 핵산 분자를 증폭하기 위한 임의의 기타 적당한 방법이 있다. 이 중에서, PCR이란 중합효소를 이용하여 표적 핵산에 특이적으로 결합하는 프라이머 쌍으로부터 표적 핵산을 증폭하는 방법이다. 이러한 PCR 방법은 당업계에 잘 알려져 있으며, 상업적으로 이용가능한 키트를 이용할 수도 있다.The method of the present invention includes the step of isolating genomic DNA from a radish sample. As a method for isolating the genomic DNA, a method known in the art may be used, and for example, a CTAB method may be used or a Wizard prep kit (Promega) may be used. A target sequence may be amplified by performing an amplification reaction using the isolated genomic DNA as a template and an oligonucleotide primer set according to an embodiment of the present invention as primers. Methods for amplifying a target nucleic acid include polymerase chain reaction (PCR), ligase chain reaction, nucleic acid sequence-based amplification, transcription-based amplification system), strand displacement amplification or amplification via Qβ replicase or any other suitable method for amplifying nucleic acid molecules known in the art. Among these, PCR is a method of amplifying a target nucleic acid from a pair of primers that specifically bind to the target nucleic acid using a polymerase. Such PCR methods are well known in the art, and commercially available kits may be used.
본 발명의 일 구현 예에 있어서, 상기 증폭된 표적 서열은 검출가능한 표지 물질로 표지될 수 있다. 상기 표지물질은 형광, 인광 또는 방사성을 발하는 물질일 수 있으나, 이에 제한되지 않는다. 바람직하게는, 상기 표지물질은 Cy-5 또는 Cy-3이다. 표적 서열의 증폭시 프라이머의 5'-말단에 Cy-5 또는 Cy-3를 표지하여 PCR을 수행하면 표적 서열이 검출가능한 형광 표지 물질로 표지될 수 있다. 또한, 방사성 물질을 이용한 표지는 PCR 수행 시 32P 또는 35S 등과 같은 방사성 동위원소를 PCR 반응액에 첨가하면 증폭 산물이 합성되면서 방사성이 증폭 산물에 혼입되어 증폭 산물이 방사성으로 표지될 수 있다. 표적 서열을 증폭하기 위해 이용된 올리고뉴클레오티드프라이머 세트는 전술한 것과 같다.In one embodiment of the present invention, the amplified target sequence may be labeled with a detectable labeling material. The labeling material may be a material that emits fluorescence, phosphorescence or radioactivity, but is not limited thereto. Preferably, the label is Cy-5 or Cy-3. When the target sequence is amplified, PCR is performed by labeling the 5'-end of the primer with Cy-5 or Cy-3, and the target sequence can be labeled with a detectable fluorescent labeling material. In addition, when a radioactive isotope such as 32 P or 35 S is added to the PCR reaction solution during PCR, the radioactive material is incorporated into the amplification product as the amplification product is synthesized, and the amplification product can be radioactively labeled. The oligonucleotide primer set used to amplify the target sequence is the same as described above.
본 발명의 일 구현 예에 있어서, 상기 무의 바람들이 예측 방법은 상기 증폭 단계의 산물을 검출하는 단계를 포함하며, 상기 증폭 단계 산물의 검출은 겔 전기영동, DNA 칩, 모세관 전기영동, 방사성 측정, 형광 측정 또는 인광 측정을 통해 수행될 수 있으나, 이에 제한되지 않는다. 증폭 산물을 검출하는 방법 중의 하나로서, 모세관 전기영동을 수행할 수 있다. 모세관 전기영동은 예를 들면, ABi Sequencer를 이용할 수 있다. 또한, 겔 전기영동을 수행할 수 있으며, 겔 전기영동은 증폭 산물의 크기에 따라 아가로스 겔 전기영동 또는 아크릴아미드 겔 전기영동을 이용할 수 있다. 또한, 형광 측정 방법은 프라이머의 5'-말단에 Cy-5 또는 Cy-3를 표지하여 PCR을 수행하면 표적 서열이 검출가능한 형광 표지 물질로 표지되며, 이렇게 표지된 형광은 형광 측정기를 이용하여 측정할 수 있다. 또한, 방사성 측정 방법은 PCR 수행 시 32P 또는 35S 등과 같은 방사성 동위원소를 PCR 반응액에 첨가하여 증폭 산물을 표지한 후, 방사성 측정기구, 예를 들면, 가이거 계수기(Geiger counter) 또는 액체섬광계수기(liquid scintillation counter)를 이용하여 방사성을 측정할 수 있다.In one embodiment of the present invention, the method of predicting winds of radish includes detecting the product of the amplification step, and the detection of the product of the amplification step is gel electrophoresis, DNA chip, capillary electrophoresis, radioactivity measurement , but may be performed through fluorescence measurement or phosphorescence measurement, but is not limited thereto. As one of the methods for detecting amplification products, capillary electrophoresis can be performed. Capillary electrophoresis can use, for example, an ABi Sequencer. In addition, gel electrophoresis can be performed, and for gel electrophoresis, agarose gel electrophoresis or acrylamide gel electrophoresis can be used depending on the size of the amplification product. In addition, in the fluorescence measurement method, when PCR is performed by labeling Cy-5 or Cy-3 at the 5'-end of the primer, the target sequence is labeled with a detectable fluorescent labeling material, and the labeled fluorescence is measured using a fluorescence meter. can do. In addition, the radioactive measurement method is to label the amplification product by adding a radioactive isotope such as 32 P or 35 S to the PCR reaction solution during PCR, and then use a radioactive measuring instrument, for example, a Geiger counter or liquid scintillation Radioactivity can be measured using a liquid scintillation counter.
본 발명은 또한, 무 시료로부터 분리한 핵산에서 RsNAC013 (Raphanus sativus NAC domain-containing protein 013) 유전자의 하위 유전자 발현수준을 측정하는 단계;를 포함하는 무의 바람들이 예측 방법을 제공한다.The present invention also provides a method for predicting radish winds, including measuring the expression level of a subgene of RsNAC013 ( Raphanus sativus NAC domain-containing protein 013) gene in nucleic acid isolated from a radish sample.
본 발명의 일 구현 예에 따른 상기 방법에 있어서, RsNAC013 유전자의 하위 유전자는 이에 한정되는 것은 아니나, AOX1a (ALTERNATIVE OXIDASE1a), MnSOD (MANGANESE SUPEROXIDE DISMUTASE 1), GPX2 (GLUTATHIONE PEROXIDASE 2) 또는 GST1 (GLUTATHIONE STRANSFERASE 1)일 수 있다.In the method according to an embodiment of the present invention, subgenes of the RsNAC013 gene are, but are not limited to, AOX1a (ALTERNATIVE OXIDASE1a), MnSOD (MANGANESE SUPEROXIDE DISMUTASE 1), GPX2 (GLUTATHIONE PEROXIDASE 2) or GST1 (GLUTATHIONE STRANSFERASE) 1) can be.
본 발명의 예에서, 상기 RsNAC013 유전자의 하위 유전자는 서열번호 1의 염기서열로 이루어진 RsNAC013 유전자형(바람들이가 잘 일어나지 않는 무의 RsNAC013 유전자)일 경우보다 서열번호 2의 염기서열로 이루어진 RsNAC013 유전자형(바람들이가 잘 일어나는 무의 RsNAC013 유전자)일 때 전사체의 발현 수준이 현저하게 높은 것이 특징이며, 유전자의 발현수준 측정은 역전사 중합효소반응(RT-PCR), 경쟁적 역전사 중합 효소반응(Competitive RT-PCR), 실시간 역전사 중합효소반응(Real-time RT-PCR), RNase 보호 분석법(RNase protection assay), DNA 칩 등이 있으나 이로 제한되는 것은 아니다.In the example of the present invention, the lower gene of the RsNAC013 gene is the RsNAC013 genotype consisting of the nucleotide sequence of SEQ ID NO: 2 than the case of the RsNAC013 genotype (non-breathing RsNAC013 gene) consisting of the nucleotide sequence of SEQ ID NO: 1 It is characteristic that the expression level of the transcript is remarkably high when it is the RsNAC013 gene of radish, which occurs well, and the expression level of the gene is measured by reverse transcription polymerase reaction (RT-PCR) and competitive reverse transcription polymerase reaction (Competitive RT-PCR). ), Real-time RT-PCR, RNase protection assay, DNA chip, etc., but are not limited thereto.
이하, 본 발명을 실시예에 의해 상세히 설명한다. 단, 하기 실시예는 본 발명을 예시하는 것일 뿐, 본 발명의 내용이 하기 실시예에 한정되는 것은 아니다.Hereinafter, the present invention will be described in detail by examples. However, the following examples are only to illustrate the present invention, and the content of the present invention is not limited to the following examples.
실시예 1. 바람들이(pithiness)는 뿌리 물관 유세포가 세포사멸(PCD: programmed cell death)된 결과이다.Example 1. Pithiness is a result of programmed cell death (PCD) of root xylem flow cells.
1.1 바람들이에 약한 무 원교10040호(WK40)의 전사체 분석1.1 Transcriptome analysis of Wongyo 10040 (WK40), which is weak to wind
원교10040호를 국립원예특작과학원의 노지(field grown)와 서울대학교 생명과학부 온실(greenhouse grown)에서 키우면서 파종 후 6주부터 10주 사이에 무 뿌리에 바람들이가 일어났는지의 여부를 microCT scanning을 이용하여 분석하고, 해당 뿌리로부터 총 RNA를 분리하였다. 분리한 총 RNA를 마크로젠에 의뢰하여 Illumina paired-end RNA sequencing한 결과, 총 17개의 무 개체로부터 4억 7천 7백 만개의 paired-end read들을 확보하였고, 이를 50,032개의 무 유전자 코딩 시퀀스에 mapping하였다. Differentially Expressed Gene (DEG; false discovery rate, FDR,≤0.05; |Fold Change|≥2)에 해당하는 3,966개의 유전자를 발견하였고 이들의 발현 양상과 기능을 K-means clusterning과 gene ontology (GO) term enrichment 분석(FDR ≤0.05)을 통해 유추하였다.While Wongyo No. 10040 was grown in the field grown of the National Institute of Horticultural and Herbal Science and in the greenhouse grown of the Department of Life Sciences, Seoul National University, microCT scanning was used to determine whether wind blowing occurred to the radish roots between 6 and 10 weeks after sowing. and analyzed, and total RNA was isolated from the root. As a result of Illumina paired-end RNA sequencing by commissioning the isolated total RNA to Macrogen, 477 million paired-end reads were obtained from a total of 17 radish individuals, which were mapped to 50,032 radish gene coding sequences. . 3,966 genes corresponding to Differentially Expressed Genes (DEG; false discovery rate, FDR,≤0.05; |Fold Change|≥2) were discovered, and their expression patterns and functions were analyzed by K-means clusterning and gene ontology (GO) term enrichment Inferred through analysis (FDR ≤0.05).
도 1a는 3,966개의 DEG를 발현 양상에 따라 K-means clustering 한 결과이며, 도 1b는 이렇게 분류한 두 cluster 구성 유전자에 대하여 GO term enrichment 분석한 결과이다. Cluster 1은 노지에서 자란 무에서 발현이 증가하며 이 가운데 특히 바람들이를 보여주는 개체에서의 발현이 더 증가하는 양상을 보여준다. Cluster 1을 구성하는 2,107개의 유전자들 가운데에는 세포사멸(cell death), 다양한 스트레스 반응성(response to stress/biotic stimulus), 항산화반응(antioxidant activity) 조절자들이 다수 분포하는 것을 확인할 수 있었다.Figure 1a is the result of K-means clustering according to the expression pattern of 3,966 DEGs, and Figure 1b is the result of GO term enrichment analysis for the genes constituting the two clusters classified as above.
1.2 세포 사멸 관련 유전자들의 발현 양상 분석1.2 Analysis of expression patterns of apoptosis-related genes
상기 1.1의 결과에 근거하여 세포 사멸에 관여하는 유전자들의 발현 변화가 바람들이 정도와 어떠한 상관관계를 갖는지를 측정하였다. 9개의 대표 유전자를 선발하여 바람들이가 관찰된 원교10040호 뿌리 횡절단면의 바깥쪽과 안쪽을 따로 분리하여 총 RNA를 정제하고 quantitative(q) realtime (RT)-polymerase chain reaction (PCR)을 실시하였다. qRT-PCR에 사용된 프라이머의 정보 및 반응 조건은 Hoang 등(Plant J. 2022 Jan;109(1):144-163)에 자세히 기술되어 있다.Based on the results of 1.1 above, it was measured how the expression changes of genes involved in cell death correlate with the degree of wind. After selecting 9 representative genes, the outside and inside of the transverse section of the root of Wongyo No. 10040 where wind was observed were separately separated, total RNA was purified, and quantitative (q) realtime (RT)-polymerase chain reaction (PCR) was performed. . The information and reaction conditions of the primers used for qRT-PCR are described in detail in Hoang et al. (Plant J. 2022 Jan; 109(1):144-163).
도 1c는 RNA를 분리한 바깥쪽과 안쪽 부위를 보여주고 있으며, 도 1d는 바람들이 정도가 약한(index 3) 또는 심한(index 9) 무에서의 9개 유전자 발현 양상을 바깥쪽과 안쪽 무 조직에서 측정한 결과를 보여주고 있다. 도 1d에서 알 수 있듯이 바람들이 정도가 심할수록 세포사멸에 관여하는 유전자의 발현량이 안쪽 부위에서 급격히 증가하는 것을 확인할 수 있었다. 무 안쪽 부위는 물관조직으로 구성되어 있으며, 이는 대부분 유세포(parenchyma cell)로 이루어져 있다. 이상의 결과를 통해 무 바람들이는 물관조직 내의 유세포들이 세포사멸을 겪어 일어난 결과로 결론지을 수 있었다.Figure 1c shows the outer and inner regions from which RNA was isolated, and Figure 1d shows the expression patterns of 9 genes in radishes with weak wind (index 3) or severe wind (index 9) outside and inner radish tissue. The measured results are shown. As can be seen in Figure 1d, it was confirmed that the expression level of genes involved in apoptosis increased rapidly in the inner region as the degree of wind increased. The inner part of the radish is composed of xylem tissue, which is mostly composed of parenchyma cells. Through the above results, it can be concluded that the flow cells in the xylem tissue undergo apoptosis.
실시예 2. 무 바람들이 형질은 세포사멸에 관여하는 NAC 도메인 전사인자인 RsNAC013과 연관되어 있다.Example 2. The radish wind trait is associated with RsNAC013, a NAC domain transcription factor involved in apoptosis.
무 바람들이의 유전적 요인을 탐색하기 위하여 바람들이가 잘 일어나는 원교10040호(WK40)와 바람들이가 잘 일어나지 않는 원교10029호(WK29)를 양친으로 사용하여 F2 분리집단을 제작하였다. F2를 노지에서 키운 후 수확하여 절단면의 바람들이 정도를 도 2(a)의 index에 근거하여 측정하고 바람들이의 분포를 분석하였다. 동일한 분리집단 내 92개 개체에 대하여 게노믹 DNA를 분리하고, 이를 양친 간의 단일염기다형성(single nucleotide polymorphism, SNP)을 이용하여 바람들이 형질과 연관된 유전좌위를 찾기 위한 genotyping by sequencing (GBS) 라이브러리 제작에 사용하였다. GBS 라이브러리를 Illumina 시스템으로 시퀀싱하여 무 유전체 v2.20 (GenBank: GCA_002197605.1)에 대하여 mapping하고, 찾아낸 biparental SNP 가운데 F2에서 1:2:1의 비율로 분리하는 240개의 SNPs를 선발하였다. 240개의 SNPs를 바람들이 형질과 같이 rQTL을 사용하여 분석하여 logarithm of odds (LOD)가 5 이상인 구간을 찾아내었다(도 2b). 구간 내에서 전술한 실시예 1에서 밝힌 세포사멸 관련 DEG을 탐색한 결과, RsNAC013을 발견하였고, RsNAC013이 두 양친 간에 3번 엑손 말단에 다형성을 보여주고 있음을 밝혀내었다(도 2c). RsNAC013의 발현 양상이 바람들이와 연관성을 갖고 있는지를 알아보기 위하여 바람들이 정도가 다른 원교10040호와 바람들이가 없는 원교10029호 무(도 2d)에서의 RsNAC013과 이의 하위 유전자들의 발현을 quantitative RT-PCR로 분석하였다(도 2e). 분석 결과, RsNAC013과 하위 유전자들이 바람들이가 일어나는 원교10040호에서 훨씬 많이 발현되는 것을 확인할 수 있었다. 이는 QTL 분석 결과와 더불어 RsNAC013이 무 바람들이 형질을 유도하는 유전적 요인으로 작용하고 있음을 제시한다.In order to explore the genetic factors of incest, an F 2 segregation group was created using Wongyo 10040 (WK40), which is prone to incest, and Wongyo 10029 (WK29), which is not infrequent, as parents. F 2 was harvested after growing in the open field, and the degree of wind blowing on the cut surface was measured based on the index of FIG. 2 (a), and the distribution of wind blowing was analyzed. Isolate genomic DNA from 92 individuals in the same segregation group, and use single nucleotide polymorphism (SNP) between parents to create a genotyping by sequencing (GBS) library to find genetic loci associated with traits used in The GBS library was sequenced with the Illumina system and mapped against genome-free v2.20 (GenBank: GCA_002197605.1), and among the found biparental SNPs, 240 SNPs separated at a ratio of 1:2:1 in F 2 were selected. 240 SNPs were analyzed using rQTL as in the wind trait, and a logarithm of odds (LOD) of 5 or more was found (Fig. 2b). As a result of searching for apoptosis-related DEGs identified in Example 1 within the section, RsNAC013 was found, and it was found that RsNAC013 showed a polymorphism at the end of
실시예 3. Example 3. RsNAC013RsNAC013 유전형의 차이를 구별하는 분자 마커는 바람들이 발생 가능성을 예측한다. Molecular markers that distinguish genotype differences predict the likelihood of winds.
도 3a는 원교10040호와 원교10029호의 RsNAC013의 염기서열을 시퀀싱하여 정렬한 결과이다. 여러 SNPs와 더불어 원교10040호에서 총 46개의 염기서열이 3' untranslated region (3' UTR)에서 소실된 것을 확인할 수 있었다. 이러한 염기서열 소실 구간을 포함한 PCR 프라이머 세트[Forward: 5'-TTCAGGAAGGTCTTTGCTGTG-3' (서열번호 3), Reverse: 5'-ATGTACAACTTGAAGAGTTGAACTTATTGA-3' (서열번호 4)]를 제작, 원교10029호와 10040호의 게노믹 DNA를 증폭하여 이를 전기영동으로 분리하였을 때, 도 3b에서 제시하듯이 RsNAC013의 유전형을 쉽게 구별할 수 있었다. 더불어 단백질 코딩 구간 내의 2개의 SNP는 아미노산 서열을 변화시킴을 알 수 있었다(도 3c). 따라서, 두 원교 간의 바람들이 발생의 차이가 아미노산 치환에 의한 기능의 변화에 기인할 가능성을 배제할 수 없었다. 원교10040호와 10029호의 F2 개체들의 바람들이와 RsNAC013의 유전형 차이가 상관관계를 갖는지를 분석하고자 바람들이가 분석된 90개의 F2 개체들의 유전형 분석을 PCR 프라이머 세트를 이용하여 실시하였다(도 3d). 그 결과, 원교10040호의 유전형을 지닌 개체의 바람들이 정도가 심해지는 경향성을 확인할 수 있었다. 또한, 국립원예특작과학원이 보유한 다른 원교(inbred line)들의 RsNAC013 유전형과 바람들이 여부를 비교 분석하였을 때에도 원교10040호의 유전형을 지닌 라인들이 바람들이를 보이는 경향성을 발견할 수 있었다(도 3e).Figure 3a shows the result of sequencing and aligning the nucleotide sequences of RsNAC013 of Wonkyo 10040 and Wonkyo 10029. In addition to several SNPs, it was confirmed that a total of 46 base sequences in Wonkyo 10040 were deleted from the 3' untranslated region (3' UTR). A PCR primer set [Forward: 5'-TTCAGGAAGGTCTTTGCTGTG-3' (SEQ ID NO: 3), Reverse: 5'-ATGTACAACTTGAAGAGTTGAACTTATTGA-3' (SEQ ID NO: 4)] including the missing nucleotide sequence was prepared, and Wonkyo No. 10029 and No. 10040 When genomic DNA was amplified and separated by electrophoresis, the genotype of RsNAC013 could be easily distinguished as shown in FIG. 3B. In addition, it was found that two SNPs in the protein coding region change the amino acid sequence (FIG. 3c). Therefore, it could not be ruled out that the difference in the generation of winds between the two bridges was due to changes in function due to amino acid substitution. In order to analyze whether there is a correlation between the windage of F2 individuals of Wongyo 10040 and 10029 and the genotype difference of RsNAC013 , genotyping of 90 F2 individuals whose windage was analyzed was performed using a PCR primer set (FIG. 3d ). As a result, it was confirmed that the degree of cheating of individuals with the genotype of Wongyo 10040 increased. In addition, when the RsNAC013 genotype and inbred of other inbred lines owned by the National Institute of Horticultural and Herbal Science were compared and analyzed, the lines with the genotype of inbred No. 10040 tended to show inbred (Fig. 3e).
실시예 4. Example 4. RsNAC013RsNAC013 의 다형성은 세포 사멸 관련 하위 유전자의 발현에 영향을 미친다.Polymorphisms in affect the expression of apoptosis-related subgenes.
상기 실시예 3의 결과에서 제시하듯이 RsNAC013은 원교10040호과 원교10029호의 유전형 사이에 3' 뿐만 아니라, 아미노산 서열의 차이를 보여주고 있다(도 3a-c). RsNAC013 단백질은 카르복실기 말단 쪽에 endoplasmic reticulum (ER) 멤브레인에 삽입될 것으로 예측되는 소수성 아미노산 서열을 지니고 있다. 애기장대에서 NAC013은 membrane anchored region이 외부 자극시 절단되어야 핵으로 이동하여 하위유전자의 전사를 조절하는 것으로 알려져 있다.As shown in the results of Example 3, RsNAC013 shows differences in amino acid sequence as well as 3' between the genotypes of Wonkyo 10040 and Wonkyo 10029 (Fig. 3a-c). The RsNAC013 protein has a hydrophobic amino acid sequence at its carboxyl terminus that is predicted to be inserted into the endoplasmic reticulum (ER) membrane. In Arabidopsis, NAC013 is known to regulate subgene transcription by moving to the nucleus only when the membrane anchored region is cleaved upon external stimulation.
본 실시예에서는 RsNAC013의 유전형 차이가 바람들이와 관련된 세포사멸 하위 유전자의 조절을 어떻게 다르게 유도하는지를 분석하고자 하였다. 첫째, 3'의 길이 차이가 전사 후 조절(posttranscriptional regulation)을 다르게 유도하여 RsNAC013 발현량을 다르게 하는지를 살펴보기 위해, 두 원교의 RsNAC013의 3'을 따로 분리하여 nlsGFP 하단에 부착시킨 후 이를 애기장대 뿌리의 비뿌리털세포(atrichoblast)에서 발현하는 유전자인 GLABRA2 (GL2)의 프로모터 조절을 받도록 발현시켰다(도 4a 및 4b). 그 결과, nlsGFP의 발현정도에 있어서 유의미한 차이를 발견할 수 없었다. 둘째, RsNAC013의 아미노산 서열의 차이가 단백질의 세포 내 위치 이동이나 전사인자 활성도에 영향을 주는 지를 살펴보았다. 먼저, 세포 내 위치 이동의 차이 여부를 35S 프로모터 하에 두 유전형의 단백질 코딩 부위를 GFP와 부착시켜 분석하였다(도 4c-f). RsNAC013의 핵으로의 이동은 활성산소 자극에 의해 유도됨에 근거하여, 본 발명에서는 과산화수소를 처리하여 자극시켜 보았다. 그 결과, 자극이 없는 상태(Mock)에서는 GFP-RsNAC013이 ER에 주로 자리하는 것을 보여준 반면, 20mM의 과산화수소를 뿌려 한 시간 처리한 개체의 뿌리에서는 핵에 위치하는 것을 확인할 수 있었다. 서로 다른 유전형의 RsNAC013 행동이 차이를 보이지 않는 것으로 보아, 활성 산소에 의한 핵으로의 이동성에는 차이가 없는 것으로 보였다.In this example, we tried to analyze how the difference in genotype of RsNAC013 induces the regulation of apoptosis-related subgenes differently. First, in order to examine whether the 3' length difference induces posttranscriptional regulation differently and causes the RsNAC013 expression level to differ, the 3' of RsNAC013 of the two bridges was separated and attached to the bottom of nlsGFP, and then Arabidopsis thaliana roots It was expressed under the promoter control of GLABRA2 ( GL2 ), a gene expressed in atrichoblast of ( FIGS. 4a and 4b ). As a result, no significant difference was found in the expression level of nlsGFP. Second, we examined whether differences in the amino acid sequence of RsNAC013 affect the movement of the protein in cells or the activity of transcription factors. First, the difference in intracellular positional movement was analyzed by attaching the protein coding regions of the two genotypes to GFP under the 35S promoter (FIG. 4c-f). Based on the fact that the movement of RsNAC013 to the nucleus is induced by active oxygen stimulation, in the present invention, it was stimulated by treatment with hydrogen peroxide. As a result, it was shown that GFP-RsNAC013 was mainly located in the ER in the absence of stimulation (Mock), whereas it was confirmed that GFP-RsNAC013 was located in the nucleus in the roots of individuals treated with 20 mM hydrogen peroxide for 1 hour. As there was no difference in RsNAC013 behavior of different genotypes, there seemed to be no difference in the mobility to the nucleus by reactive oxygen species.
마지막으로 SNP에 의한 아미노산 서열의 차이가 전사인자 활성도의 차이를 초래하는지를 확인하였다(도 4g-i). 이를 위해, NAC013의 직접적인 표적인 AOX1a (ALTERNATIVE OXIDASE1a)의 NAC013에 대한 상대적 발현량(AOX1a 발현량/NAC013 발현량)을 전사활성도를 측정하는 기준으로 삼았다. 세 가지 다른 접근법으로 분석을 진행하였다. 도 4g는 앞서 도 2d에서 분리한 RNA로부터 측정한 RsAOX1a와 RsNAC013의 비율을 보여주고 있다. 원교10040호의 RsNAC013의 활성이 훨씬 높은 것을 확인할 수 있다. 도 4h는 온실에서 원교10040호와 원교10029호를 키우면서 과산화수소를 처리하여 활성산소 자극을 주었을 경우와 주지 않았을 경우의 비율을 측정한 것이다. 활성산소 스트레스 자극 시 두 원교 모두에서 RsNAC013의 활성이 증가하였으나, 이 경우에도 원교10040호의 RsNAC013 활성이 높았다. 도 4i에서는 두 RsNAC013 유전형을 각각 애기장대에 형질전환하여 발현시켰을 때 AOX1a의 발현에 미치는 영향을 측정한 것이다. 이 경우에도 역시 원교10040호의 RsNAC013 활성이 더 높은 것을 확인할 수 있었다. 이상의 결과들을 종합해 보면, RsNAC013 가운데 원교10040호 유전형은 원교10029호 유전형에 비하여 전사인자 활성도가 높고, 전사활성도가 높은 RsNAC013 원교10040호 유전형은 스트레스 환경에 반응한 세포사멸을 더 잘 유도함으로써 바람들이가 일어나는 유전적 환경을 제공한다고 결론지을 수 있었다.Finally, it was confirmed whether differences in amino acid sequences by SNPs lead to differences in transcription factor activity (FIG. 4g-i). To this end, the relative expression level ( AOX1a expression level/ NAC013 expression level) of AOX1a (ALTERNATIVE OXIDASE1a), a direct target of NAC013 , to NAC013 was used as a criterion for measuring transcriptional activity. The analysis proceeded with three different approaches. Figure 4g shows the ratio of RsAOX1a and RsNAC013 measured from the RNA isolated in Figure 2d above. It can be confirmed that the activity of RsNAC013 of Wonkyo 10040 is much higher. Figure 4h is a measurement of the ratio of the case where active oxygen stimulation was given and the case where it was not given by treating hydrogen peroxide while growing Wongyo 10040 and Wongyo 10029 in a greenhouse. The activity of RsNAC013 increased in both Wonkyo when stimulated by reactive oxygen species stress, but even in this case, the RsNAC013 activity of Wonkyo 10040 was high. In Figure 4i, the effect on the expression of AOX1a was measured when the two RsNAC013 genotypes were respectively transformed and expressed in Arabidopsis thaliana. In this case, it was also confirmed that the RsNAC013 activity of Wongyo 10040 was higher. Summarizing the above results, among RsNAC013 , the Wongyo 10040 genotype has higher transcription factor activity than the Wongyo 10029 genotype, and the high transcriptional activity of the RsNAC013 Wongyo 10040 genotype induces apoptosis in response to the stress environment better, thereby reducing the wind. It can be concluded that it provides the genetic environment in which
<110> Seoul National University R&DB Foundation <120> NAC013 gene controlling pithiness in radish taproots and uses thereof <130> PN22043 <160> 4 <170> KoPatentIn 3.0 <210> 1 <211> 2501 <212> DNA <213> Raphanus sativus <400> 1 atggagactt caaggggttc gcgtccggct ggagacagag ttctagctcc aggttttagg 60 tttcatccaa ccgacgagga actggttgtc tattatctca agagaaagat tcgccgcaag 120 aaacttagag ttgctgcaat cggcgagacg gatgtttaca agtttgatcc cgaggagttg 180 cccggtacac ttttatcaga tcttctcctt cctaccattt tcattttatt ttgcaaacag 240 tttcctgaaa gttctgatct tttcctctgt tttcagtttc ctgaaagttg tgatcttctg 300 ctctgttttc agtttcctga aagttgagat cttttgctct gttttgctct gttttactct 360 gttttagata aaatttgtat agttgaagta tcgattttgg ttactctatt ataacttgag 420 atgttatgtg atcgttgtgt gtttaaaagg ttagcttttg atggactgat gtttttttgt 480 aggcaaagcg ttgtataata ctggagatcg tcagtggttc tttttcagct cgagggacag 540 gaaacaagga ggcaggtcaa gcagagcaac tggccatgga tactggaaag caacaggggt 600 ggacagagtc atcaagtgca attctcgtcc cgttggagag aagaagacgc ttgttttcca 660 cagaggcagg gctcccaagg gagagaggac taactgggtc atgcacgagt acaccttgca 720 caaagaggaa ctcaagatgt gtggagatgt tacgcataat tatgttcttt acaagatttt 780 taagaagagc gggtctggtc ctaagaatgg tgaacagtat ggagctcctt ttgttgaaga 840 agagtgggct gaggatgatg atgaggtcga tgaggctgat gcagtccatg ttcctacaaa 900 tcagctcatg gtttcagctg gtatgggaaa taataataac atatgggcgg atggtggtgg 960 gcttaaccag tctgagttga atgaaaacga cattcaagaa ctgatgagac aggttaccga 1020 agagactgga gtgagctctc atgtggcaaa tgataatccg gtaaacttgg cagaagatga 1080 gtatttggaa attgacgatc ttttgctccc tggacctgaa ccaagttatg tggatcaaga 1140 agagtctgct gtgctgaatg gtaatgattt ctttgatgtt gattcatata ttggtgattt 1200 tgatgccaca aatcctcaga cagagcctgt cggtgttgtt ggtttgaaca atggggttgt 1260 acatagtctt ccagtgactg accaggctaa taacaaccag tttcaacagc aaacatggaa 1320 gaaccaagat agcaactggc cactccgtaa cagctatacc agaaagataa gtagtgaatc 1380 gtggacgcat gagctgataa acaatgatga agttgctgtt tgcggatatg gtgagacccc 1440 tggtacaggt aaaaccaaca acacattttc tctgtaaact ctccaataga gctcttctga 1500 tgatccttta tgtttagaca taggacatgt tcctaatcat atttacttgg caggtgacgc 1560 atctgaattc acaaaccctc tgacttgtgc tgtaaatata gcaaagggag aagaagccac 1620 aaaagacgag tcaagtcagt ttacttctag tgtatggtct ttcctggagt cgattcctgc 1680 aagtccagca tttgcttcag agagtccaat cgttaacctg aacatagtca ggatatcaag 1740 ccttggtggc cgctaccggt ttaattctaa aagcacaagt agcaatgttg ttgtaaggaa 1800 gaagtctgga gaaaacaaca aggagaagaa ccacaagggt ttcctctgct tatctatcat 1860 tggggctctg tgtgctttat cttgggtgat gttgggaaca atgggagttt caggaaggtc 1920 tttgctgtgg tgagaaccct aatctcaaag acaatgtgga catcaacaga aagaatggct 1980 tagagttggg tttagcatga gccttaacca agttaaatct tttgtaattg ttttactagc 2040 tttagaatcg taaactgttt agtatcggtt tgttgttttg gtttgaacct ttatgcagaa 2100 aacaaagctt tataaccatt gcttactaaa cacagtatca ataagttcaa ctctcaagtt 2160 gtacatagag tggtgatatc ctcaagccgc ttcttcacac tcatatcaat aagcttagaa 2220 cctgaaccac cgtacctgat agtgaaccca gcgatcaacc tctcgtcgat caccgtccta 2280 actctcacgt tcctagctcc cgtgagtctc tgcacatgct tcgccacctg agccagatgc 2340 tgagaatcaa gcctcacaac cgacttcaca accacgacct ccgtcttcgt catcttgttg 2400 tacaccgctt cgaactcctt catgatctcc ttcactagat ctatccgatc cgaatcgatc 2460 agcacgttca agaagttgga cgtgtgtgga agcagggata a 2501 <210> 2 <211> 2446 <212> DNA <213> Raphanus sativus <400> 2 atggagactt caaggggttc gcgtccggct ggagacagag ttctagctcc aggttttagg 60 tttcatccaa ccgacgagga actggttgtc tattatctca agagaaagat tcgccgcaag 120 aaacttagag ttgctgcaat cggcgagacg gatgtttaca agtttgatcc cgaggagttg 180 cccggtacac ttttatcaga tcttctcctt cctaccattt tcattttatt ttgcaaacag 240 tttcctgaaa gttctgatct tttcctctgt tttcagtttc ctgaaagttg tgatcttctg 300 ctctgttttc agtttcctga aagttgagat cttttgctct gttttgctct gttttactct 360 gttttagata aaatttgtat agttgaagta tcgattttgg ttactctatt ataacttgag 420 atgttatgtg atcgttgtgt gtttaaaagg ttagcttttg atggactgat gtttttttgt 480 aggcaaagcg ttgtataata ctggagatcg tcagtggttc tttttcagct cgagggacag 540 gaaacaagga ggcaggtcaa gcagagcaac tggccatgga tactggaaag caacaggggt 600 ggacagagtc atcaagtgca attctcgtcc cgttggagag aagaagacgc ttgttttcca 660 cagaggcagg gctcccaagg gagagaggac taactgggtc atgcacgagt acaccttgca 720 caaagaggaa ctcaagatgt gtggagatgt tacgcataat tatgttcttt acaagatttt 780 taagaagagc gggtctggtc ctaagaatgg tgaacagtat ggagctcctt ttgttgaaga 840 agagtgggct gaggatgatg atgaggtcga tgaggctgat gcagtccatg ttcctacaaa 900 tcagctcatg gtttcagctg gtatgggaaa taataataac atatgggcgg atggtggtgg 960 gcttaaccag tctgagttga atgaaaacga cattcaagaa ctgatgagac aggttaccga 1020 agagactgga gtgagctctc atgtggcaaa tgataatccg gtaaacttgg cagaagatga 1080 gtatttggaa attgacgatc ttttgctccc tggacctgaa ccaagttatg tggatcaaga 1140 agagtctgct gtgctgaatg gtaatgattt ctttgatgtt gattcatata ttggtgattt 1200 tgatgccaca aatcctcaga cagagcctgt cggtgttgtt ggtttgaaca atggggttgt 1260 acatagtctt ccagtgactg accaggctaa taacaaccag tttcaacagc aaacatggaa 1320 gaaccaagat agcaactggc cactccgtaa cagctatacc agaaagataa gtagtgaatc 1380 gtggacgcat gagctgataa acaatgatga agttgctgtt tgcggatatg gtgagacccc 1440 tggtacaggt aaaaccaaca acacattttc tctgtaaact ctccaataga gctcttctga 1500 tgatccttta tgtttagaca taggacatgt tcctaatcat atttacttgg caggtgacgc 1560 atctgaattc acaaaccctc tgacttgtgc tgtaaatata gcaaagggag aagaagccac 1620 aaaagacgag tcaagtcagt ttacttctag cgtatggtct ttcctggagt cgatgcctgc 1680 aagtccagca tttgcttcag agagtccaat cgttaacctg aacatagtca ggatatcaag 1740 ccttggtggc cgctaccggt ttaattctaa aagcacaagt agcaatgttg ttgtaagaaa 1800 gaagtctgga gaaaacaaca agaagaagaa ccacaagggt ttcctctgct tatctatcat 1860 tggggctctg tgtgctttat cttgggtgat gttgggaaca atgggagttt caggaaggtc 1920 tttgctgtgg tgagaacccc aaaactcaaa gacaatgtgg acatcaacag aaagaatggc 1980 ttagatttta ttagctttag aatcataaac tgtttagtat cggtttgttg ttttggtttg 2040 aacctttatg cagaacaaag ctttaaacca ttgtttatga tgatcaataa gttcaactct 2100 caagttgtac atgagtggtg atatcctcaa gccgcttctt cacactcata tcaataagct 2160 tagaacctga accaccgtac ctgatagtga acccagcgat caagctctcg tcgatcaccg 2220 tcctaactct cacattccta gctcccgtga gtctctgcac gtgcttcgcc acctgagcca 2280 gatgctgaga atcaagcctc acaaccgact tcacaaccac cagctccgtc ttggtcatct 2340 tgttgtacac agcttcgaac tccttcatga tctccttcac cagatctatc cgatccgaat 2400 cgatcagcac gttcaagaag ttggacgtgt gtggaagcag ggataa 2446 <210> 3 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 ttcaggaagg tctttgctgt g 21 <210> 4 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 atgtacaact tgaagagttg aacttattga 30 <110> Seoul National University R&DB Foundation <120> NAC013 gene controlling pithiness in radish taproots and uses its <130> PN22043 <160> 4 <170> KoPatentIn 3.0 <210> 1 <211> 2501 <212> DNA <213> Raphanus sativus <400> 1 atggagactt caaggggttc gcgtccggct ggagacagag ttctagctcc aggttttagg 60 tttcatccaa ccgacgagga actggttgtc tattatctca agagaaagat tcgccgcaag 120 aaacttagag ttgctgcaat cggcgagacg gatgtttaca agtttgatcc cgaggagttg 180 cccggtacac ttttatcaga tcttctcctt cctaccattt tcattttatttgcaaacag 240 tttcctgaaa gttctgatct tttcctctgt tttcagtttc ctgaaagttg tgatcttctg 300 ctctgttttc agtttcctga aagttgagat cttttgctct gttttgctct gttttactct 360 gttttagata aaatttgtat agttgaagta tcgattttgg ttactctatt ataacttgag 420 atgttatgtg atcgttgtgt gtttaaaagg ttagcttttg atggactgat gtttttttgt 480 aggcaaagcg ttgtataata ctggagatcg tcagtggttc tttttcagct cgagggacag 540 gaaacaagga ggcaggtcaa gcagagcaac tggccatgga tactggaaag caacaggggt 600 ggacagagtc atcaagtgca attctcgtcc cgttggagag aagaagacgc ttgttttcca 660 cagaggcagg gctcccaagg gagagaggac taactgggtc atgcacgagt acaccttgca 720 caaagaggaa ctcaagatgt gtggagatgt tacgcataat tatgttcttt acaagatttt 780 taagaagagc gggtctggtc ctaagaatgg tgaacagtat ggagctcctt ttgttgaaga 840 agagtgggct gaggatgatg atgaggtcga tgaggctgat gcagtccatg ttcctacaaa 900 tcagctcatg gtttcagctg gtatgggaaa taataataac atatgggcgg atggtggtgg 960 gcttaaccag tctgagttga atgaaaacga cattcaagaa ctgatgagac aggttaccga 1020 agagactgga gtgagctctc atgtggcaaa tgataatccg gtaaacttgg cagaagatga 1080 gtatttggaa attgacgatc ttttgctccc tggacctgaa ccaagttatg tggatcaaga 1140 agagtctgct gtgctgaatg gtaatgattt ctttgatgtt gattcatata ttggtgattt 1200 tgatgccaca aatcctcaga cagagcctgt cggtgttgtt ggtttgaaca atggggttgt 1260 acatagtctt ccagtgactg accaggctaa taacaaccag tttcaacagc aaacatggaa 1320 gaaccaagat agcaactggc cactccgtaa cagctatacc agaaagataa gtagtgaatc 1380 gtggacgcat gagctgataa acaatgatga agttgctgtt tgcggatatg gtgagacccc 1440 tggtacaggt aaaaccaaca acacattttc tctgtaaact ctccaataga gctcttctga 1500 tgatccttta tgtttagaca taggacatgt tcctaatcat atttacttgg caggtgacgc 1560 atctgaattc acaaaccctc tgacttgtgc tgtaaatata gcaaagggag aagaagccac 1620 aaaagacgag tcaagtcagt ttacttctag tgtatggtct ttcctggagt cgattcctgc 1680 aagtccagca tttgcttcag agagtccaat cgttaacctg aacatagtca ggatatcaag 1740 ccttggtggc cgctaccggt ttaattctaa aagcacaagt agcaatgttg ttgtaaggaa 1800 gaagtctgga gaaaacaaca aggagaagaa ccacaagggt ttcctctgct tatctatcat 1860 tggggctctg tgtgctttat cttgggtgat gttgggaaca atgggagttt caggaaggtc 1920 tttgctgtgg tgagaaccct aatctcaaag acaatgtgga catcaacaga aagaatggct 1980 tagagttggg tttagcatga gccttaacca agttaaatct tttgtaattg ttttactagc 2040 tttagaatcg taaactgttt agtatcggtt tgttgttttg gtttgaacct ttatgcagaa 2100 aacaaagctt tataaccatt gcttactaaa cacagtatca ataagttcaa ctctcaagtt 2160 gtacatagag tggtgatatc ctcaagccgc ttcttcacac tcatatcaat aagcttagaa 2220 cctgaaccac cgtacctgat agtgaaccca gcgatcaacc tctcgtcgat caccgtccta 2280 actctcacgt tcctagctcc cgtgagtctc tgcacatgct tcgccacctg agccagatgc 2340 tgagaatcaa gcctcacaac cgacttcaca accacgacct ccgtcttcgt catcttgttg 2400 tacaccgctt cgaactcctt catgatctcc ttcactagat ctatccgatc cgaatcgatc 2460 agcacgttca agaagttgga cgtgtgtgga agcagggata a 2501 <210> 2 <211> 2446 <212> DNA <213> Raphanus sativus <400> 2 atggagactt caaggggttc gcgtccggct ggagacagag ttctagctcc aggttttagg 60 tttcatccaa ccgacgagga actggttgtc tattatctca agagaaagat tcgccgcaag 120 aaacttagag ttgctgcaat cggcgagacg gatgtttaca agtttgatcc cgaggagttg 180 cccggtacac ttttatcaga tcttctcctt cctaccattt tcattttatttgcaaacag 240 tttcctgaaa gttctgatct tttcctctgt tttcagtttc ctgaaagttg tgatcttctg 300 ctctgttttc agtttcctga aagttgagat cttttgctct gttttgctct gttttactct 360 gttttagata aaatttgtat agttgaagta tcgattttgg ttactctatt ataacttgag 420 atgttatgtg atcgttgtgt gtttaaaagg ttagcttttg atggactgat gtttttttgt 480 aggcaaagcg ttgtataata ctggagatcg tcagtggttc tttttcagct cgagggacag 540 gaaacaagga ggcaggtcaa gcagagcaac tggccatgga tactggaaag caacaggggt 600 ggacagagtc atcaagtgca attctcgtcc cgttggagag aagaagacgc ttgttttcca 660 cagaggcagg gctcccaagg gagagaggac taactgggtc atgcacgagt acaccttgca 720 caaagaggaa ctcaagatgt gtggagatgt tacgcataat tatgttcttt acaagatttt 780 taagaagagc gggtctggtc ctaagaatgg tgaacagtat ggagctcctt ttgttgaaga 840 agagtgggct gaggatgatg atgaggtcga tgaggctgat gcagtccatg ttcctacaaa 900 tcagctcatg gtttcagctg gtatgggaaa taataataac atatgggcgg atggtggtgg 960 gcttaaccag tctgagttga atgaaaacga cattcaagaa ctgatgagac aggttaccga 1020 agagactgga gtgagctctc atgtggcaaa tgataatccg gtaaacttgg cagaagatga 1080 gtatttggaa attgacgatc ttttgctccc tggacctgaa ccaagttatg tggatcaaga 1140 agagtctgct gtgctgaatg gtaatgattt ctttgatgtt gattcatata ttggtgattt 1200 tgatgccaca aatcctcaga cagagcctgt cggtgttgtt ggtttgaaca atggggttgt 1260 acatagtctt ccagtgactg accaggctaa taacaaccag tttcaacagc aaacatggaa 1320 gaaccaagat agcaactggc cactccgtaa cagctatacc agaaagataa gtagtgaatc 1380 gtggacgcat gagctgataa acaatgatga agttgctgtt tgcggatatg gtgagacccc 1440 tggtacaggt aaaaccaaca acacattttc tctgtaaact ctccaataga gctcttctga 1500 tgatccttta tgtttagaca taggacatgt tcctaatcat atttacttgg caggtgacgc 1560 atctgaattc acaaaccctc tgacttgtgc tgtaaatata gcaaagggag aagaagccac 1620 aaaagacgag tcaagtcagt ttacttctag cgtatggtct ttcctggagt cgatgcctgc 1680 aagtccagca tttgcttcag agagtccaat cgttaacctg aacatagtca ggatatcaag 1740 ccttggtggc cgctaccggt ttaattctaa aagcacaagt agcaatgttg ttgtaagaaa 1800 gaagtctgga gaaaacaaca agaagaagaa ccacaagggt ttcctctgct tatctatcat 1860 tggggctctg tgtgctttat cttgggtgat gttgggaaca atgggagttt caggaaggtc 1920 tttgctgtgg tgagaacccc aaaactcaaa gacaatgtgg acatcaacag aaagaatggc 1980 ttagatttta ttagctttag aatcataaac tgtttagtat cggtttgttg ttttggtttg 2040 aacctttatg cagaacaaag ctttaaacca ttgtttatga tgatcaataa gttcaactct 2100 caagttgtac atgagtggtg atatcctcaa gccgcttctt cacactcata tcaataagct 2160 tagaacctga accaccgtac ctgatagtga acccagcgat caagctctcg tcgatcaccg 2220 tcctaactct cacattccta gctcccgtga gtctctgcac gtgcttcgcc acctgagcca 2280 gatgctgaga atcaagcctc acaaccgact tcacaaccac cagctccgtc ttggtcatct 2340 tgttgtacac agcttcgaac tccttcatga tctccttcac cagatctatc cgatccgaat 2400 cgatcagcac gttcaagaag ttggacgtgt gtggaagcag ggataa 2446 <210> 3 <211> 21 <212> DNA <213> artificial sequence <220> <223> primer <400> 3 ttcaggaagg tctttgctgt g 21 <210> 4 <211> 30 <212> DNA <213> artificial sequence <220> <223> primer <400> 4 atgtacaact tgaagagttg aacttattga 30
Claims (7)
상기 분리된 게놈 DNA를 주형으로 하고, 제2항의 프라이머 세트를 이용하여 증폭 반응을 수행하여 표적 서열을 증폭하는 단계; 및
상기 증폭 단계의 산물을 검출하는 단계;를 포함하는 무의 바람들이 예측 방법.Isolating genomic DNA from the radish sample;
Amplifying a target sequence by performing an amplification reaction using the isolated genomic DNA as a template and using the primer set of claim 2; and
Detecting the product of the amplification step; wind prediction method comprising a.
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