WO2023068618A1 - Nac013 gene inducing pithiness in radish storage roots, and use thereof - Google Patents

Abstract

The present invention relates to an NAC013 gene inducing pithiness in radish storage roots, and the use thereof and, more specifically, to a composition, for controlling pithiness in radishes, comprising as an active ingredient RsNAC013 (Raphanus sativus NAC domain-containing protein 013) encoded by a gene sequence of SEQ ID NO: 1 or SEQ ID NO: 2, and a primer set, for predicting pithiness in radishes, comprising an oligonucleotide primer of SEQ ID NO: 3 and SEQ ID NO: 4.

Description

NAC013 gene for inducing airflow in radish storage roots and its use

The present invention relates to a NAC013 (NAC domain-containing protein 013) gene that induces pithiness in radish storage roots and uses thereof.

This result was researched with the support of the GoldenSeed Project of the Ministry of Agriculture, Food and Rural Affairs and the Personal Basic Research Project of the Ministry of Science and ICT (Task Nos. 213006055SBK30 and 2021R1A2C3006061).

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.

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.

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.

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'.

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

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.

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.

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.

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.

In addition, the present invention provides a kit for predicting winds of radish containing the primer set and reagents for performing the amplification reaction.

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.

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.

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 accurate molecular information.

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.

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.

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. .

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.

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.

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.

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.

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.

The present invention also provides a kit for predicting winds of radish containing the primer set and reagents for performing the amplification reaction.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Example 1. Pithiness is a result of programmed cell death (PCD) of root xylem flow cells.

1.1 Transcriptome analysis of Wongyo 10040 (WK40), which is weak to wind

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).

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. Cluster 1 shows an increase in expression in radish grown in the open field, especially in individuals showing wind. Among the 2,107 genes constituting Cluster 1, it was confirmed that a large number of regulators of cell death, various responses to stress/biotic stimulus, and antioxidant activity were distributed.

1.2 Analysis of expression patterns of apoptosis-related genes

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).

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.

Example 2. The radish wind trait is associated with RsNAC013, a NAC domain transcription factor involved in apoptosis.

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 exon 3 between the two parents (FIG. 2c). In order to examine whether the expression pattern of RsNAC013 has a correlation with wind blowing, quantitative RT-quantitative RT- It was analyzed by PCR (Fig. 2e). As a result of the analysis, it was confirmed that RsNAC013 and its subgenes were much more expressed in Wongyo 10040, where wind blowing occurred. This, along with the QTL analysis results, suggests that RsNAC013 acts as a genetic factor inducing windless traits.

Example 3. RsNAC013 Molecular markers that distinguish genotype differences predict the likelihood of winds.

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).

Example 4. RsNAC013 Polymorphisms in affect the expression of apoptosis-related subgenes.

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.

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.

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

Claims (7)

1. A composition for regulating wind blowing in radish, comprising a gene of RsNAC013 ( Raphanus sativus NAC domain-containing protein 013) consisting of the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2 as an active ingredient.
2. A primer set for predicting winds of nothing, including the oligonucleotide primers of SEQ ID NO: 3 and SEQ ID NO: 4.
3. A kit for predicting winds of radish containing reagents for carrying out the primer set and the amplification reaction of claim 2.
4. The kit according to claim 3, wherein the reagents for performing the amplification reaction include DNA polymerase, dNTPs, and a buffer.
5. 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.
6. The method of claim 5, wherein the detection of the product of the amplification step is performed through gel electrophoresis, DNA chip, radioactivity measurement, fluorescence measurement or phosphorescence measurement.
7. 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;

Images