KR102448762B1 - Molecular marker based on RVE5 gene from cabbage for discriminating anthocyanin deposition at low temperature and uses thereof - Google Patents

Abstract

The present invention relates to a cabbage-derived RVE5 gene-based molecular marker capable of discriminating anthocyanin accumulation at low temperature, and a use thereof, and the molecular marker of the present invention relates to anthocyanin accumulation in cabbage and anthocyanin, which have a low level of anthocyanin accumulation even in a low-temperature period, in which anthocyanin accumulation occurs mainly. Since high-level cabbage strains can be accurately identified, it is possible to reduce the time, cost, and effort required for breeding cabbage with low anthocyanin accumulation even by cultivating and harvesting cabbage at a low temperature, and it is also possible to reduce the time, cost, and effort required for breeding cabbage with low anthocyanin accumulation, and also for high-quality cabbage for growers and consumers. It is expected to enable production and supply.

Description

Molecular marker based on RVE5 gene from cabbage for discriminating anthocyanin deposition at low temperature and uses thereof

The present invention relates to a cabbage-derived RVE5 gene-based molecular marker capable of discriminating anthocyanin accumulation at a low temperature and a use thereof.

Plant pigments include three types (anthocyanin, carotenoid, beta line) except for chlorophyll, which is involved in photosynthesis, and these pigments are responsible for attracting animals for pollination and dispersing seeds. Prevents damage to plants by Anthocyanin is a water-soluble pigment of the flavonoid family, and determines the blue, purple and red colors of leaves, flowers and fruits of plants, and plays an important role in plant growth and development. Anthocyanins attract pollinators, such as insects, from petals, and help disperse seeds from fruits and seeds to provide ecological distribution. Flavonoid compounds including anthocyanins function to protect plants from damage to insects and external stress (that is, to give resistance to biological and abiotic stress), and in particular, function as an antioxidant to prevent damage caused by free radicals and the like. Anthocyanins also act as antioxidants in humans to reduce apoptosis and prevent cardiovascular diseases, cancer, diabetes, neurodegeneration, inflammation, viral infections and obesity. The qualitative and quantitative increase of anthocyanin content in fruits and vegetables through metabolic engineering is an important and industrially valuable research direction.

The accumulation of anthocyanins in plants is regulated by synthesis and degradation, and the process is regulated by genetic factors, developmental and environmental factors. In general, anthocyanin accumulation occurs by increased expression of anthocyanin biosynthesis genes and transcription factors regulating them, and anthocyanin accumulation is also caused by mutations in related genes. Purple cauliflower ( Brassica oleracea var. botrytis ) is produced by promoting expression by mutation of the BoMYB2 promoter in cabbage, and purple cabbage is produced due to lack of expression of the BoMYBL2-1 gene, a transcription repressor. Recently, it has been found that genes other than anthocyanin biosynthesis factors are related to anthocyanin accumulation in plants. LBD37/LBD39 (LATERAL ORGAN BOUNDARIES DOMAIN 37-39) gene is related to anthocyanin synthesis and nitrogen response, and RVE8/LCL5 (REVEILLE8/LHY/CCA1-LIKE5) gene, which is related to circadian rhythm, is related to anthocyanin accumulation. , LBD37L (LBD transcription factors 37-like) gene is also known to be associated with anthocyanin accumulation in cabbage.

The RVE/LCL gene encodes a novel MYB transcription factor that regulates the circadian clock. The first gene discovered was EPR1 (EARLY-PHYTOCHROME-RESPONSIVE 1)/ RVE7 , which was very similar to CCA1 (CIRCAIDAN CLOCK ASSOCIATE 1) and LHY (LATE ELONGATED HYPOCOTY), and CIR1 (CIRCADIAN 1)/ REV2 genes were also identified. The current model plant, Arabidopsis thaliana, has eight RVE/LCL genes, which are divided into two groups. One group includes RVE1 , RVE2/CIR1 , RVE7/EPR1 , and the other group includes RVE3/LCL3 , RVE5/LCL4 , RVE8/ LCL5 , RVE4 /LCL1 , RVE6 /LCL2 . Among them, the function of RVE8 is well known, and RVE8 is related to the regulation of histone 3 acetylation and the regulation of anthocyanin synthesis. RVE8 and similar RVE4 and RVE6 promote regulation of the circadian clock, but other RVEs have been reported to have low correlation with circadian clock. During anthocyanin biosynthesis, RVE8 regulates the anthocyanin synthesis inhibitors, phytochrome-interacting factors PIF4 and PIF. In addition, RVE8 promotes anthocyanin synthesis by binding to the promoters of anthocyanin biosynthesis-related genes CHS (chalcone synthase), ANS (anthocyanidin synthase) and UGT79B1 (UDP-sugar-dependent glycosyltransferase 79B1), whereas LNKs (NIGHT LIGHT- INDUCIBLE AND CLOCK-REULATED GENS) inhibits anthocyanin synthesis. In addition, it is recently known that RVE1 and RVE7 of Pyrus bretschneideri can promote anthocyanin synthesis by binding to PbDFR and PbANS promoters, but there is no study reporting that RVE5 is related to anthocyanin synthesis.

In spite of the positive effect of anthocyanins, the present inventors have developed a marker that can determine this, as in cabbage (especially green cabbage) breeding, varieties in which anthocyanins do not accumulate in leaves during harvest are preferred in major Asian countries including China. wanted to

On the other hand, Korean Patent No. 1680571 discloses 'SNP marker for identifying purple or green cabbage varieties at low temperature and their use', and Korean Patent No. 1724372 discloses 'SNP marker for selecting cabbage with low progoitrin content and Although the 'use thereof' is disclosed, there is no description about the cabbage-derived RVE5 gene-based molecular marker capable of discriminating anthocyanin accumulation at low temperature of the present invention and its use.

The present invention has been derived from the above needs, and the present inventors have developed green cabbage (low anthocyanin accumulators (LAA)), high anthocyanin accumulators (HAA, high anthocyanin accumulators) and purple cabbage at low temperatures. As a result of cloning and analyzing the BoRVE5 ( Brassica oleracea REVEILLE5 ) gene related to anthocyanin accumulation in the genome of It was confirmed that the InDel polymorphism was present. In addition, it was confirmed that LAA-type green cabbage has the BoRVE5a gene homozygous, HAA-type green cabbage has the BoRVE5b gene homozygous or heterozygous, and purple cabbage has the BoRVE5b gene homozygous. In addition, after developing a primer set capable of amplifying a partial region of a gene in which SNP and InDel sites affecting amino acid coding among various SNPs and InDel between the BoRVE5a and BoRVE5b alleles are located, using the developed primer set As a result of performing PCR on LAA and HAA type green cabbage and red cabbage, it was confirmed that the primer set of the present invention can discriminate between cabbage with a high level of anthocyanin accumulation and cabbage with a low level of anthocyanin accumulation at low temperatures. The invention was completed.

In order to solve the above problems, the present invention provides a cabbage-derived BoREV5a gene fragment consisting of the nucleotide sequence of SEQ ID NO: 1; And Cabbage-derived BoREV5b gene fragment consisting of the nucleotide sequence of SEQ ID NO: 2; provides a marker composition for determining anthocyanin accumulation in cabbage at low temperature, comprising one or more gene fragments selected from the group consisting of as an active ingredient.

In addition, the present invention is a primer set for determining anthocyanin accumulation in cabbage at low temperature for amplifying the cabbage-derived BoREV5a gene fragment consisting of the nucleotide sequence of SEQ ID NO: 1 or the cabbage-derived BoREV5b gene fragment consisting of the nucleotide sequence of SEQ ID NO: 2 provides

In addition, the present invention is the primer set; and a reagent for performing an amplification reaction. It provides a kit for determining anthocyanin accumulation in cabbage at low temperature.

In addition, the present invention comprises the steps of isolating genomic DNA from a cabbage sample; amplifying a target sequence by using the isolated genomic DNA as a template and performing an amplification reaction using the primer set; And it provides a method for determining the accumulation of anthocyanins in cabbage at a low temperature comprising a; and detecting the product of the amplification step.

Since the molecular marker of the present invention can accurately discriminate between a cabbage line with a low anthocyanin accumulation level and a cabbage line with a high anthocyanin accumulation level at a low temperature where anthocyanin accumulation mainly occurs, cabbage with a low anthocyanin accumulation even after cultivating and harvesting cabbage in a low temperature period It is expected that the time, cost and effort required for breeding can be reduced, and it is expected to enable the production and supply of high-quality cabbage to growers and consumers.

1 is a diagram showing the BoRVE5 3'-UTR sequence in which AT-SSR (simple sequence repeat) exists in the genome sequence of 10 cabbage lines (Table 1). Yellow indicates the AT-SSR region, and green and purple indicate SNPs found near the AT-SSR.
2 to 4 are shown by comparing the RVE5 genome sequence of 10 cabbage lines (Table 1), the exon region is shown in gray, the primer set for amplifying the BoRVE5a gene present in LAA-type green cabbage and HAA-type green The primer sets for amplifying the BoRVE5b gene present in cabbage and purple cabbage are indicated in orange.
FIG. 5 is a diagram illustrating marker positions capable of distinguishing LAA type or HAA type by comparing the BoRVE5a gene sequence present in LAA-type cabbage with the BoRVE5b gene sequence present in HAA-type cabbage. Orange: primer set positions for amplifying SNP or InDel sites between BoRVE5a and BoRVE5b gene sequences, purple: SNP and InDel (or GAC SSR) positions.
6 is a phylogenetic tree analysis result using the BoRVE5 base sequence (A) and the BoRVE5 amino acid sequence (B) of 10 cabbage lines (Table 1).
7A is a phylogenetic analysis result using the amino acid sequence of cabbage-derived BoRVE and Arabidopsis-derived AtRVE, and FIG. 7B is a diagram showing comparison of the BoRVE5a amino acid sequence of LAA-type cabbage with the BoRVE5b amino acid sequence of HAA-type cabbage. Red: Shows amino acid differences between BoRVE5a and BoRVE5b. *: Mark the major amino acid residues in the MYB domain, AtRVE1 (AT5G17300), AtRVE4 (AT5G02840), AtRVE5 (AT4G011280), AtRVE8 (AT3G09600), BoRVE1 (Bo3g012470), BoRVE2 (Bo6g057640), BoRVE3 (Bog002070), RVE3 (Bo5g002430) BoRVE5 (Bo9g003580), BoRVE6 (Bo9g108370), BoRVE7 (Bo5g025310), BoRVE8 (Bo1g144410).
8 is a LAA type (842, 2409, 2437, 09WH45, Golden Cross, Speed King) or HAA type ( 154, 337, CT154, T-523, T-530) and purple cabbage (7S4-51, 7S4-63) of the anthocyanin content measured and shown.
9 shows the association of anthocyanin synthesis with BoRVE5a and BoRVE5b genes in green cabbage and purple cabbage (7S4-51, 7S4-63) of cold-treated LAA type (2409, Golden Cross) or HAA type (T-523, T-530). It is the result of confirming the expression level of the gene ( BoCHS, BoF3H, BoDFR ). CHS : chalcone synthase, F3H : flavanone 3-hydroxylase, DFR : dihydroflavonol 4-reductase.
10 shows BoRVE5a and BoRVE5b genes and anthocyanin synthesis-related genes in LAA-type green cabbage (842) and HAA-type green cabbage (337) at morning (6 AM), daytime (2 PM) or night (10 PM) time zones ( BoCHS, BoF3H, BoDFR ) is the result of confirming the expression level.
11 and 12 are results of PCR performed on LAA-type or HAA-type green cabbage and purple cabbage using a primer set (Table 5) for amplifying the SNP or InDel region between the BoRVE5a and BoRVE5b gene sequences.

In order to achieve the object of the present invention, the present invention is a cabbage-derived BoREV5a gene fragment consisting of the nucleotide sequence of SEQ ID NO: 1; And Cabbage-derived BoREV5b gene fragment consisting of the nucleotide sequence of SEQ ID NO: 2; provides a marker composition for determining anthocyanin accumulation in cabbage at low temperature, comprising one or more gene fragments selected from the group consisting of as an active ingredient.

In the marker composition of the present invention, the cabbage-derived BoREV5a gene fragment consisting of the nucleotide sequence of SEQ ID NO: 1 or the cabbage-derived BoREV5b gene fragment consisting of the nucleotide sequence of SEQ ID NO: 2 is green cabbage (LAA type) having a low anthocyanin accumulation level at low temperature. , low anthocyanin accumulator) line, green cabbage (HAA type, high anthocyanin accumulator) line and purple cabbage line with high anthocyanin accumulation levels were compared with exons 4 and 5 in the RVE5 (REVEILLE5) gene between each line. Refers to a gene fragment containing an existing single nucleotide polymorphism (SNP) or insertion and deletion (InDel) polymorphism.

Specifically, the gene fragment consisting of the nucleotide sequence of SEQ ID NO: 1 is SNP (single nucleotide polymorphism) or InDel (insertion and deletion) present in exons 4 and 5 in the RVE5 gene of the cabbage line with a low level of anthocyanin accumulation at low temperature. As the BoREV5a gene fragment containing the polymorphism (see FIG. 5), using a marker composition including the BoREV5a gene fragment consisting of the nucleotide sequence of SEQ ID NO: 1, cabbage lines with low anthocyanin accumulation levels at low temperatures can be discriminated.

In addition, the gene fragment consisting of the nucleotide sequence of SEQ ID NO: 2 is a BoREV5b gene fragment containing SNP or InDel polymorphisms present in exons 4 and 5 in the RVE5 gene of cabbage lines and purple cabbage with high anthocyanin accumulation levels at low temperatures. As a result (refer to FIG. 5), using a marker composition comprising the BoREV5b gene fragment consisting of the nucleotide sequence of SEQ ID NO: 2, it is possible to discriminate a cabbage line with a high level of anthocyanin accumulation at a low temperature.

The gene fragment includes all of RNA, DNA and cDNA, and preferably refers to DNA.

In the marker composition of the present invention, the low temperature may be a minimum temperature of 2 to 10° C. for more than one month, but is not limited thereto.

In the marker composition of the present invention, the cabbage having a low level of anthocyanin accumulation at the low temperature may include anthocyanins of about 2 to 12 mg/100g FW content, and cabbage having a high level of anthocyanin accumulation at a low temperature is about 18 to 78 mg It may include anthocyanins of /100g FW content, but is not particularly limited thereto, and in view of an increase in anthocyanin content in plants under low temperature conditions (Photochemistry and Photobiology, 199, 70(1), 1-9), low temperature If the anthocyanin content in the cabbage after treatment is similar to or relatively less than before low-temperature treatment, it is desirable to set the cabbage as low anthocyanin content, and if the anthocyanin content in the cabbage after low-temperature treatment is relatively higher than before low-temperature treatment, it is preferable to set the cabbage as high anthocyanin content.

The present invention also provides a primer set for determining anthocyanin accumulation in cabbage at low temperature for amplifying the cabbage-derived BoREV5a gene fragment consisting of the nucleotide sequence of SEQ ID NO: 1 or the cabbage-derived BoREV5b gene fragment consisting of the nucleotide sequence of SEQ ID NO: 2 to provide.

In the primer set of the present invention, the primer set for amplifying the cabbage-derived BoREV5a gene fragment consisting of the nucleotide sequence of SEQ ID NO: 1 is an oligonucleotide primer set consisting of the nucleotide sequences of SEQ ID NO: 20 and 21, and the nucleotide sequence of SEQ ID NO: 2 The primer set for amplifying the cabbage-derived BoREV5b gene fragment consisting of the nucleotide sequence may be an oligonucleotide primer set consisting of the nucleotide sequences of SEQ ID NOs: 22 and 23, but is not limited thereto.

The primers include oligonucleotides consisting of fragments of 20 or more, 21 or more, 22 or more, 23 or more, 24 or more consecutive nucleotides in the sequence of SEQ ID NOs: 20 to 23 according to the sequence length of each primer can do. For example, the primer of SEQ ID NO: 20 (27 oligonucleotides) may include an oligonucleotide consisting of a fragment of 24 or more, 25 or more, 26 or more consecutive nucleotides in the sequence of SEQ ID NO: 20. In addition, the primer may also include an addition, deletion or substitution of the nucleotide sequence of SEQ ID NO: 20 to SEQ ID NO: 23. The even-numbered oligonucleotide primers in the SEQ ID NO: are forward primers, and the odd-numbered oligonucleotide primers are reverse primers.

The two primer sets consisting of the oligonucleotides of SEQ ID NOs: 20 to 23 according to the present invention are exons in the RVE5 gene of a cabbage line with a low anthocyanin content (LAA type) and a cabbage line with a high anthocyanin content (HAA type) among low-temperature-treated cabbage As a primer set that can confirm the SNP or InDel polymorphism present in Nos. 4 and 5, when the two primer sets are used simultaneously, the results of each primer set are combined to form an LAA-type green cabbage line and an HAA-type green cabbage and It can efficiently identify purple cabbage.

In the present invention, "primer" refers to a single-stranded oligonucleotide sequence complementary to a nucleic acid strand to be copied, and can serve as a starting point for synthesis of a primer extension product. The length and sequence of the primers should allow synthesis of the extension product to begin. The specific length and sequence of the primer will depend on the conditions of use of the primer, such as temperature and ionic strength, as well as the complexity of the desired DNA or RNA target.

As used herein, the oligonucleotide used as a primer may also contain a nucleotide analogue, for example, a phosphorothioate, an alkylphosphorothioate or a peptide nucleic acid. An intercalating agent may be included.

The present invention also includes the primer set; and a reagent for performing an amplification reaction. It provides a kit for determining anthocyanin accumulation in cabbage at low temperature.

In the kit of the present invention, the primer set and cabbage are as described above.

In one embodiment of the present invention, the reagent for performing the amplification reaction may include, but is not limited to, DNA polymerase, dNTPs, and a buffer.

In one embodiment of the present invention, the kit may further include a user guide describing optimal conditions for performing the reaction. The handbook is a printout explaining how to use the kit, eg, how to prepare reverse transcription buffer and PCR buffer, and suggested reaction conditions. Instructions include 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 published or provided through an electronic medium such as the Internet.

The present invention also

isolating genomic DNA from the cabbage sample;

amplifying a target sequence by using the isolated genomic DNA as a template and performing an amplification reaction using the primer set; and

It provides a method for determining the accumulation of anthocyanins in cabbage at a low temperature, including; detecting the product of the amplification step.

In the method according to an embodiment of the present invention, the primer set and the cabbage line are as described above.

The method of the present invention comprises isolating genomic DNA from a cabbage sample. The cabbage sample is not limited thereto, but may be leaves, stems, roots, or products processed in a dried form. As a method for isolating the genomic DNA, a method known in the art may be used, for example, the CTAB method may be used, or a Wizard prep kit (Promega) may be used. The target sequence may be amplified by performing an amplification reaction using the isolated genomic DNA as a template and using the oligonucleotide primer set according to an embodiment of the present invention as a primer. Methods for amplifying a target nucleic acid include polymerase chain reaction (PCR), ligase chain reaction, nucleic acid sequence-based amplification, and transcription-based amplification systems. 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 them, PCR is a method of amplifying a target nucleic acid from a primer pair that specifically binds 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 method for determining anthocyanin accumulation in cabbage at low temperature comprises detecting a product of the amplification step, and the detection of the product of the amplification step is gel electrophoresis, high resolution melting (HRM) ) analysis, fluorescence measurement, capillary electrophoresis, DNA chip, radiometric measurement, or phosphorescence measurement, but is not limited thereto.

Gel electrophoresis may use agarose gel electrophoresis or acrylamide gel electrophoresis depending on the size of the amplification product. In addition, HRM analysis techniques can be performed, which is a relatively new post-PCR analysis method used to identify variations in nucleic acid sequences. This method is based on detecting small differences in PCR dissociation curves. It is connected to a PCR device and the difference in the degree of dissociation according to the temperature of dsDNA having the used dye is analyzed and processed using software specifically designed for HRM analysis. In addition, as one of the methods for detecting the amplification product, capillary electrophoresis may be performed. Capillary electrophoresis may use, for example, an ABi Sequencer.

In addition, in the fluorescence measurement method, when PCR is performed by labeling FAM, HEX, VIC, JOE, ROX, TAMRA, Cy3 or Cy5 at the 5'-end of the primer, the target sequence is labeled with a detectable fluorescent labeling material, and thus labeled Fluorescence can be measured using a fluorescence meter. In addition, the radioactive measurement method is a radioactive isotope such as ³² P or ³⁵ S is added to the PCR reaction solution during PCR to label the amplification product, and then a radioactive measuring instrument, for example, a Geiger counter (Geiger counter) or liquid scintillation The radioactivity can be measured using a liquid scintillation counter.

In the method according to an embodiment of the present invention, in the presence of cabbage genomic DNA, PCR is performed with the primer sets of SEQ ID NOs: 20 and 21 or the primer sets of SEQ ID NOs: 22 and 23 of the present invention, and then bands through gel electrophoresis By detecting the form and confirming the presence of these bands, the presence or absence of the BoREV5a or BoREV5b gene, which can determine whether the cabbage sample has high and low anthocyanin accumulation levels under low-temperature cultivation conditions, can be confirmed.

More specifically, the method of the present invention is a single band of 214 bp for the BoREV5a gene (homozygous) when PCR is performed using the primer sets of SEQ ID NOs: 20 and 21 on low-temperature-treated green or purple cabbage. It can be identified as an anthocyanin low content cabbage line (LAA type green cabbage), and PCR is performed using the primer sets of SEQ ID NOs: 22 and 23 to show a single band of 235 bp (homozygous) for the BoREV5b gene. It can be identified as a high-content cabbage line (HAA type green cabbage or purple cabbage), and PCR is performed using both the primer sets of SEQ ID NOs: 20 and 21 and the primer sets of SEQ ID NOs: 22 and 23 to 214 bp and 235 If two bands of bp ( BoREV5a / BoREV5b , heterozygote) are shown, it can be discriminated as an anthocyanin-rich cabbage line (HAA type green cabbage).

Hereinafter, the present invention will be described in detail by way of Examples. However, the following examples are only illustrative of the present invention, and the content of the present invention is not limited to the following examples.

Materials and Methods

1. Plant materials and growth conditions

Green cabbage and purple cabbage ( Brassica oleracea var. capitata f. alba and - f. rubra ) seeds used for gene sequencing were obtained from Asia Seedry Co., Ltd. Biotechnology Breeding Research Institute (Icheon-si, Gyeonggi-do) and Korea Seedling Co., Ltd. (Pyeongtaek-si, Gyeonggi-do) was provided and used. The characteristics of the four inner hybrids of green cabbage (154, 2409, 2437 and 09WH45) and the four inner hybrids of purple (red) cabbage (7S5-51, 7S5-63, B90 and B98) are summarized in Table 1 below. Cabbage materials used for marker verification are summarized in Table 2 below, and were provided by domestic seed companies and Suncheon University GSP Horticultural Seed Business Group. Some seeds were germinated and cultured in the plant growth phase, and the conditions of growth temperature of 22°C, 16 hours (light)/8 hours (dark), and 140 μmol m ^-2 ·s ^-1 were maintained. Inner hybrid 337 and 842 seeds were germinated in Petri dishes and maintained for 3 days.

2. BoRVE5 gene sequencing

Genomic DNA of 10 cabbage lines in Table 1 was extracted using DNeasy Plant Mini Kit (QIAGEN, Hilden, Germany). The AT-repeat (AT SSR, simple sequence repeats) portion present in the 3'-UTR portion was amplified with the primer sets (SEQ ID NOs: 3 and 4) of Table 3 below, and the length of the fragment was 101-121 bp (Fig. 1). ). Using this sequence, it was confirmed through NCBI Blast analysis that the main body of this gene would be BoRVE5 . /Brassica_oleracea/Info/Index) was searched to determine the primer sets (SEQ ID NOs: 5 and 6 or SEQ ID NOs: 5 and 7) of Table 3 below.

After denaturing at 94°C for 5 minutes, PCR was repeated 30 cycles of 94°C for 30 seconds, 58°C for 30 seconds, and 72°C for 2 minutes, and finally synthesized at 72°C for 7 minutes. The PCR product was electrophoresed on a 1% agarose gel, and DNA was purified using LaboPass Gel Extraction Kit (COSMOGENETECH, Korea). The purified DNA was inserted into a T&A cloning vector (RBC, Taiwan) and transformed into E. coli DH5α. Plasmid DNA from E. coli was isolated using LaBoPass Plasmid Mini Kit (COSMOGENETECH), and nucleotide sequence analysis was commissioned by Macrogen (Korea). At least 10 clones were analyzed in order to avoid errors that may occur in the PCR reaction and sequencing process and to find out the existence of a plurality of genes. The comparison of the sequences obtained from the above was performed using the multiple sequence alignment program CLUSTAL Omega (EMBL-EBI, UK).

3. BoRVE5 Expression analysis of genes and anthocyanin biosynthesis-related genes

Total RNA was isolated using leaf samples (Tables 1 and 2) and TRIzol Reagent (Thermo Scientific, USA), and DNA was removed by treatment with RQ1 RNase-free DNase (Promaga, USA) at 65°C for 10 minutes. The first strand of cDNA was synthesized with the Revertra Ace Kit (TOYOBO, Japan) using the oligo dT primer, and expression analysis was performed using it, and the primer information used at this time is shown in Table 4 below.

qRT-PCR was analyzed with a MiniOpticon system (Bio-Rad, USA) using SYBR Green Realtime Master Mix (TOYOBO). PCR was denatured at 95°C for 10 minutes, followed by 94°C 30 seconds, 58°C 30 seconds, 72°C. The 30 second cycle was repeated 40 times, and the fluorescence value was measured at the last step of each cycle. All analyzes were performed in triplicate, and the transcript values of the target genes were quantified relative to the BoAactin2 gene, and analyzed by the 2 ^-ΔΔC T method (Methods., 2001, 25(4), 402-408).

4. Anthocyanin Extraction and Quantification

To measure the anthocyanin content, 1 ml of 1% HCl-methanol (v/v) was added to 0.3 g of leaf powder, reacted in the dark for 24 hours while mixing well, centrifuged at 13,000 x g conditions, and the supernatant was taken 530, Absorbance was measured at 620 and 650 nm. The relative anthocyanin content was obtained by the following formula. A ₅₃₀ , A ₆₂₀ , and A ₆₅₀ mean absorbance values.

Relative anthocyanin content=(A ₅₃₀ -A ₆₂₀ )-0.1(A ₆₅₀ -A ₆₂₀ )

Total anthocyanin content (mg anthocyanin/100 g fresh weight) was calculated by the following formula based on cyanidin-3-glucoside, a control sample. MW is the molecular weight of cyanidin-3-glucoside (449.2 g/mol), DF is the dilution factor, and ∋ is the diameter of the cuvette (1 cm).

Total anthocyanin content = Relative anthocyanin content × MW × DF × 1000 × ∋

5. Validation of Markers

To distinguish between BoRVE5a (LAAs, genotype with low anthocyanin accumulation at low temperature) and BoRVE5b (HAAs, genotype with high anthocyanin accumulation at low temperature), each gene-specific primer based on the genetic polymorphism present in exon 4 and exon 5 was prepared (Table 5). For PCR, 10 ng of the genome, 10 pmol of each forward and reverse primer, and 20 μl of 5X PCR Master Mix (Elpisbiotech, Korea) were adjusted, denatured at 94°C for 5 minutes, 94°C for 30 seconds, 60°C for 30 seconds, 72 ℃ 30 sec was repeated 28 times and reacted at 72 ℃ for 5 minutes. The presence of genomic DNA was determined by amplification of BoActin 2 , and the PCR product was confirmed by electrophoresis on a 1.2% agarose gel.

6. Statistical analysis

Anthocyanin content and gene expression level were statistically analyzed using one-way analysis of variance (ANOVA) and Tukey's pair-wise comparisons in Minitab v.17 (Minitab Inc., USA), and all data were obtained from 3 replicates of the sample. and expressed as the mean and standard deviation.

Example 1. Anthocyanin accumulation and BoRVE5 Gene related studies

A gene inversely correlated with anthocyanin accumulation and AT-SSR length was discovered through RNA_seq analysis using cabbage from 8 strains derived from Asian seedlings. It was found that the 3'-UTR (untranslated region) of the gene (Table 1 and FIG. 1). AT-repeats varied from 9 to 19 (AT9-19), and cabbage with a high level of anthocyanin accumulation had a short number of repetitions. This gene was named BoRVE5 , and it was decided to be cloned and analyzed from several cabbage lines.

Example 2. BoRVE5 Gene cloning and sequencing

To clone the BoRVE5 gene, PCR was performed with the primer combinations of Table 3 (SEQ ID NOs: 5 and 6 or SEQ ID NOs: 5 and 7) to obtain the entire gene sequence. Genes were found in 4 cabbage lines with low anthocyanin accumulation at low temperature (LAAs; 842, 2409, 2437, 09WH45), 2 cabbage lines with high anthocyanin accumulation (HAAs; 154, 337) and 4 purple cabbage lines (7S4-51, 7S4-63). , B90, B90), the average length from the ATG start codon to 30 nucleotides after AT-SSR was 1913 bp (1899 to 1947 bp), and consisted of 8 exons and 8 introns, and a large number of InDel and SNPs were present ( FIGS. 2 to 4 ). There were several SNP or InDel polymorphisms capable of distinguishing cabbage with low anthocyanin accumulation from cabbage with high anthocyanin accumulation (Fig. 5), and the genes contrasting according to the level of anthocyanin accumulation were named as follows: Genes present in LAA cabbage The gene present in BoRVE5a , HAA cabbage and purple cabbage is BoRVE5b .

In addition, when phylogenetic analysis was performed using the BoRVE5 base sequence of 10 cabbage lines (Table 1), the B90 line, a purple cabbage, was grouped with the LAA type cabbage, but when the lineage analysis was performed using the BoRVE5 amino acid sequence, purple It was confirmed that the cabbage line B90 was grouped with purple cabbage (FIG. 6). Through this, many polymorphisms between LAA-type and HAA-type cabbage exist in introns, which were not expected to be significantly associated with anthocyanin accumulation traits.

Example 3. BoRVE5 amino acid composition analysis

To infer the diversity and function of BoRVE5, a phylogenetic tree was created based on the amino acid sequences of 8 cabbage-derived BoRVE and 4 Arabidopsis-derived AtRVE. As a result, as shown in Figure 7A, it appeared in three clades, one of which consists of RVE4 and RVE8, which are related to the biological clock and anthocyanin accumulation, and the other is REV3, whose function is not well known, It consists of RVE5 and RVE6, and the other consists of RVE1, RVE2 and RVE7. Through this, the REV function revealed in Arabidopsis was expected to be similar to that of cabbage.

In addition, as a result of listing the amino acid sequence using the gene sequence cloned from cabbage, there was a portion showing an amino acid difference between the amino acid sequences of BoRVE5a and BoRVE5b, but there was no difference in the MYB domain and the LCL domain, which are the main domains of the MYB protein (FIG. 7B). Through this, it was expected that there would be no significant difference in the functions of BoRVE5a and BoRVE5b proteins.

Example 4. Analysis of anthocyanin accumulation pattern in cabbage by low-temperature treatment

In order to determine the anthocyanin accumulation trait at low temperature, green cabbage and purple of LAA type (842, 2409, 2437, 09WH45, Golden Cross, Speed King) or HAA type (154, 337, CT154, T-523, T-530) Anthocyanin accumulation in cabbage (7S4-51, 7S4-63) grown at normal growth temperature (CK: 20-30°C) and cabbage grown for one month at low temperature ( see Agronomy , 2020, 10, 602, Song et al.) was measured and compared.

As a result, the anthocyanin content of cabbage grown under the low temperature (LT) condition was higher than that of the normal growth temperature (CK) condition. In particular, the level of anthocyanin accumulation was higher in HAA type green cabbage and purple cabbage than in LAA type green cabbage. It was confirmed that it significantly increased at a lower temperature (FIG. 8).

Example 5. Anthocyanin synthesis and BoRVE5 Allele relationship analysis

As a result of checking the expression levels of BoRVE5a and BoRVE5b in green and purple cabbage (7S4-51, 7S4-63) of cold-treated LAA type (2409, Golden Cross) or HAA type (T-523, T-530), BoRVE5a The gene was expressed only in LAA type green cabbage and at very low levels in HAA type green cabbage and purple cabbage. On the other hand, it was confirmed that the BoRVE5b gene was hardly expressed in LAA type green cabbage, and the expression level was significantly increased in HAA type green cabbage and purple cabbage. This expression pattern was similar to that of BoDFR , a key gene for anthocyanin synthesis (FIG. 9).

In addition, in order to investigate the association between BoRVE5 expression and circadian rhythm, in the LAA-type green cabbage (842) and HAA-type green cabbage (337), the BoRVE5a and BoRVE5b genes and the anthocyanin synthesis-related genes ( BoCHS, BoF3H, BoDFR ) were As a result of analyzing the expression at morning (6AM), daytime (2PM) or nighttime (10PM) time zones, the BoRVE5a and BoRVE5b genes showed high expression levels of BoRVE5a in LAA-type cabbage, and BoRVE5b in HAA-type cabbage, regardless of the specific time period. It was confirmed that the expression level of genes and anthocyanin synthesis-related genes were high (FIG. 10). Through this, it was found that the BoRVE5a and BoRVE5b genes associated with the anthocyanin accumulation level of the present invention are not related to the circadian rhythm.

Example 6. Molecular Marker Development and Verification - Distinguishing Cabbage with Low Anthocyanin Accumulation at Low Temperature (LAA) and High Cabbage (HAA)

Genetic polymorphisms existed in exons 4 and 5 of the BoRVE5a and BoRVE5b genes, and primers based on SNP and InDel that change the amino acid encoding were prepared (Table 5) and then PCR was performed to verify whether they are universal.

As a result, the BoRVE5a gene was detected only in LAA-type green cabbage (2437, 09WH45, 2409, 842), and the BoRVE5b gene was detected in HAA-type green cabbage (154, 337) and purple cabbage (7S4-63, 7S4-51). was detected only in ( FIG. 11A ), and among HAA types, it was confirmed that CT-415 was detected as heterozygous ( FIG. 11B ). Through this, it was found that when the BoRVE5a gene is homozygous, it is LAA type cabbage, and when the BoRVE5b gene is homozygous or heterozygous, it can be identified as HAA type cabbage.

In addition, as a result of expanding the coverage of 47 green cabbage lines and 57 purple cabbage lines (Table 2), it was concluded that the LAA type green cabbage was the BoRVE5a isoform, and the HAA type green cabbage was the BoRVE5b isoform or heterozygous. Alternatively, it was found that red cabbage has an isoform of BoRVE5b (FIG. 12).

Through this, it was considered that the degree of anthocyanin accumulation could be accurately determined in low-temperature-treated green cabbage by using a primer set capable of amplifying InDel or SNP polymorphisms between the BoRVE5a and BoRVE5b genes of the present invention.

<110> The Industry & Academic Cooperation in Chungnam National University (IAC) <120> Molecular marker based on RVE5 gene from cabbage for discriminating anthocyanin deposition at low temperature and uses it <130> PN20364 <160> 23 <170> KoPatentIn 3.0 <210> 1 <211> 248 <212> DNA <213> Brassica oleracea <400> 1 aatcatgaat cgacaaatct gccaatacct gtgattcaag gtatatggtt tttgatttta 60 gtttgttgta tctatgaatg gtgtggtgtt taaccatata tggtgtaatg aatattagag 120 gaaccaggag tctcggccac agctctccca aagaatcgct gttacagcac cagtaataag 180 gaagttaagc caaatttacc agctgtgaca cacccgaaca atgaagaaag ttgtgaaaag 240 acacctag 248 <210> 2 <211> 260 <212> DNA <213> Brassica oleracea <400> 2 aatcatgaat cgacaaatct gccaatacct gcgattcaag gtatagtgac atcatggttt 60 ttgattttag tttgttgtat ctatgaatgg tgtggtgttt aaccatatat ggtgtaatga 120 atattagagg aaccgggagt ctcggccaca gctctcccaa agaatcgctg ttacagcacc 180 agtaataagg aagttaagcc aaatttacca gctgtgacga cagacccgaa caatgaagaa 240 agttgtgaaa agacacgtag 260 <210> 3 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 tggtttgtcc tattttgtct aata 24 <210> 4 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 acattgtcct tatcaacaca tatc 24 <210> 5 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 atggtgtcgc taaacgctag acc 23 <210> 6 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 acaaaatagg acaaaccact gcgctt 26 <210> 7 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 7 cagcgattct ttgggagagc tgtg 24 <210> 8 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 8 cctgtgattc aagaggaacc agga 24 <210> 9 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 9 cacaactttc ttcattgttc gggtgtg 27 <210> 10 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 10 aatacctgcg attcaagagg aaccg 25 <210> 11 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 11 aactttcttc attgttcggg tctgtcg 27 <210> 12 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 12 gttcacaagg agtgcaagtt tcgg 24 <210> 13 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 13 acaaacttcc tggtctcaac ctgc 24 <210> 14 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 14 agtatacgtg gcttcttgac acgg 24 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 15 acgaarggga caaagccacg 20 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 16 tcccacgaat gagaacatga 20 <210> 17 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 17 caccgtcaac cctaggaaaa 20 <210> 18 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 18 tgccaatcta cgagggtttc 20 <210> 19 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 19 agcttctcct tgatgtctct cac 23 <210> 20 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 20 gaatcgacaa atctgccaat acctgtg 27 <210> 21 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 21 acaactttct tcattgttcg ggtgtg 26 <210> 22 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 22 cgacaaatct gccaatacct gcg 23 <210> 23 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 23 caactttctt cattgttcgg gtctgtc 27

Claims (7)

Cabbage-derived BoREV5a gene fragment consisting of the nucleotide sequence of SEQ ID NO: 1; And Cabbage-derived BoREV5b gene fragment consisting of the nucleotide sequence of SEQ ID NO: 2; A marker composition for determining anthocyanin accumulation in cabbage at a low temperature of 2 to 10 ℃, comprising one or more gene fragments selected from the group consisting of as an active ingredient. For amplifying the cabbage-derived BoREV5a gene fragment consisting of the nucleotide sequence of SEQ ID NO: 1 or the cabbage-derived BoREV5b gene fragment consisting of the nucleotide sequence of SEQ ID NO: 2, a primer set for determining anthocyanin accumulation in cabbage at a low temperature of 2 to 10 ° C. According to claim 2, wherein the primer set is an oligonucleotide primer set consisting of the nucleotide sequences of SEQ ID NOs: 20 and 21; or an oligonucleotide primer set consisting of the nucleotide sequences of SEQ ID NOs: 22 and 23; The primer set of claim 2 or 3; and a kit for determining anthocyanin accumulation in cabbage at a low temperature of 2 to 10° C., comprising a reagent for performing an amplification reaction. The kit according to claim 4, wherein the reagents for performing the amplification reaction include DNA polymerase, dNTPs and a buffer. isolating genomic DNA from the cabbage sample;
using the isolated genomic DNA as a template and amplifying a target sequence by performing an amplification reaction using the primer set of claim 2 or 3; and
Detecting the product of the amplification step; comprising, a method of determining anthocyanin accumulation in cabbage at a low temperature of 2 to 10 ℃. The method according to claim 6, wherein the detection of the product of the amplification step is performed through a DNA chip, gel electrophoresis, radiometric measurement, fluorescence measurement, or phosphorescence measurement.

Images