WO2023068618A1 - Gène nac013 induisant la présence de moelle dans les racines de stockage du radis et son utilisation - Google Patents

Gène nac013 induisant la présence de moelle dans les racines de stockage du radis et son utilisation Download PDF

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WO2023068618A1
WO2023068618A1 PCT/KR2022/015215 KR2022015215W WO2023068618A1 WO 2023068618 A1 WO2023068618 A1 WO 2023068618A1 KR 2022015215 W KR2022015215 W KR 2022015215W WO 2023068618 A1 WO2023068618 A1 WO 2023068618A1
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rsnac013
radish
seq
gene
winds
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Korean (ko)
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이지영
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서울대학교산학협력단
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae

Definitions

  • the present invention relates to a NAC013 (NAC domain-containing protein 013) gene that induces pithiness in radish storage roots and uses thereof.
  • NAC013 NAC domain-containing protein 013
  • 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.
  • radish has a lower level of molecular breeding technology based on genetic information than other representative crops.
  • 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.
  • 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'
  • 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
  • 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.
  • 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.
  • 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.
  • RsNAC013 Raphanus sativus NAC domain-containing protein 013
  • 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.
  • the present invention provides a kit for predicting winds of radish containing the primer set and reagents for performing the amplification reaction.
  • 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.
  • 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.
  • (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
  • (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.
  • 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.
  • (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.
  • 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.
  • RsNAC013 Raphanus sativus NAC domain-containing protein 013
  • the nucleotide sequence of SEQ ID NO: 1 is the RsNAC013 gene of radish that does not wind easily
  • the nucleotide sequence of SEQ ID NO: 2 is the RsNAC013 gene of radish that does not wind easily
  • 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. .
  • 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.
  • 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
  • the primers may also include added, deleted or substituted sequences of the nucleotide sequences of SEQ ID NOs: 3 and 4.
  • 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.
  • 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.
  • reagents for performing the amplification reaction may include DNA polymerase, dNTPs, and buffers, but are not limited thereto.
  • 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.
  • 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.
  • 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 Q ⁇ replicase or any other suitable method for amplifying nucleic acid molecules known in the art.
  • 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.
  • PCR methods are well known in the art, and commercially available kits may be used.
  • 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.
  • the label is Cy-5 or Cy-3.
  • 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.
  • 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.
  • 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.
  • capillary electrophoresis can be performed.
  • Capillary electrophoresis can use, for example, an ABi Sequencer.
  • 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.
  • 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.
  • 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.
  • RsNAC013 Raphanus sativus NAC domain-containing protein 013
  • 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.
  • AOX1a ALTERNATIVE OXIDASE1a
  • MnSOD MANGANESE SUPEROXIDE DISMUTASE 1
  • GPX2 GLUTATHIONE PEROXIDASE 2
  • GST1 GLUTATHIONE STRANSFERASE
  • 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
  • RT-PCR reverse transcription polymerase reaction
  • Competitive RT-PCR competitive reverse transcription polymerase reaction
  • Example 1 Pithiness is a result of programmed cell death (PCD) of root xylem flow cells.
  • PCD programmed cell death
  • Figure 1a is the result of K-means clustering according to the expression pattern of 3,966 DEGs
  • 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.
  • 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.
  • Figure 1c shows the outer and inner regions from which RNA was isolated
  • 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.
  • 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).
  • Figure 3a shows the result of sequencing and aligning the nucleotide sequences of RsNAC013 of Wonkyo 10040 and Wonkyo 10029.
  • SNPs SNPs
  • 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.
  • 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.
  • ER endoplasmic reticulum
  • NAC013 is known to regulate subgene transcription by moving to the nucleus only when the membrane anchored region is cleaved upon external stimulation.
  • 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.
  • 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.
  • 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.
  • 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

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Abstract

La présente invention concerne un gène NAC013 induisant la formation de moelle dans les racines de stockage du radis, ainsi que son utilisation et, plus particulièrement, une composition destinée à lutter contre la formation de moelle dans les radis, comprenant comme principe actif RsNAC013( protéine 013 Raphanus sativus contenant un domaine NAC) codée par une séquence génétique de SEQ ID NO : 1 ou SEQ ID NO : 2, et un ensemble d'amorces, pour prédire si les radis ont de la moelle, comprenant une amorce oligonucléotidique de SEQ ID NO : 3 et SEQ ID NO : 4.
PCT/KR2022/015215 2021-10-22 2022-10-07 Gène nac013 induisant la présence de moelle dans les racines de stockage du radis et son utilisation WO2023068618A1 (fr)

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KR20210141532 2021-10-22
KR10-2021-0141532 2021-10-22
KR10-2022-0031259 2022-03-14
KR1020220031259A KR20230057921A (ko) 2021-10-22 2022-03-14 무 저장뿌리의 바람들이를 유도하는 nac013 유전자 및 이의 용도

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR101091494B1 (ko) * 2009-12-24 2011-12-07 장인국 배의 바람들이 및/또는 무름 현상을 감소시키는 방법 및 그의 조성
KR20150019300A (ko) * 2013-08-13 2015-02-25 서울대학교산학협력단 Nac016 유전자를 이용한 비생물적 스트레스 내성이 증가된 기능성 녹색 지속 변이 식물체의 제조 방법 및 그에 따른 식물체

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR101091494B1 (ko) * 2009-12-24 2011-12-07 장인국 배의 바람들이 및/또는 무름 현상을 감소시키는 방법 및 그의 조성
KR20150019300A (ko) * 2013-08-13 2015-02-25 서울대학교산학협력단 Nac016 유전자를 이용한 비생물적 스트레스 내성이 증가된 기능성 녹색 지속 변이 식물체의 제조 방법 및 그에 따른 식물체

Non-Patent Citations (3)

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Title
DATABASE Nucleotide 4 October 2016 (2016-10-04), ANONYMOUS : "PREDICTED: Raphanus sativus NAC domain-containing protein 13 (LOC108813214), mRNA", XP093058180, retrieved from ncbi Database accession no. XM_018585704.1 *
HOANG NAM V., PARK SUHYOUNG, PARK CHULMIN, SUH HANNAH, KIM SANG‐TAE, CHAE EUNYOUNG, KANG BYOUNG‐CHEORL, LEE JI‐YOUNG: "Oxidative stress response and programmed cell death guided by NAC013 modulate pithiness in radish taproots", THE PLANT JOURNAL, BLACKWELL SCIENTIFIC PUBLICATIONS, OXFORD., GB, vol. 109, no. 1, 1 January 2022 (2022-01-01), GB , pages 144 - 163, XP093058327, ISSN: 0960-7412, DOI: 10.1111/tpj.15561 *
MARCELIS L, HEUVELINK E, DIJK D VAN: "Pithiness and Growth of Radish Tubers as Affected by Irradiance and Plant Density", ANNALS OF BOTANY, ACADEMIC PRESS, LONDON., GB, vol. 79, no. 4, 1 April 1997 (1997-04-01), GB , pages 397 - 402, XP093058187, ISSN: 0305-7364, DOI: 10.1006/anbo.1996.0357 *

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