CN109486831B - Carmine radish anthocyanin biosynthesis regulatory gene RsAN1 and application thereof - Google Patents

Abstract

The invention discloses a carmine radish anthocyanin biosynthesis regulatory geneRsAN1And the application thereof,RsAN1the nucleotide sequence of (A) is shown in SEQ ID NO.1, and the coded amino acid sequence is shown in SEQ ID NO. 2.RsAN1The expression quantity of the nopaline is in positive correlation with the content of the nopaline, and the nopaline is obtained by performing the expression on the nopalineRsAN1Cloning, sequence analysis, expression analysis and plant transfection of gene to obtain transient over-expressionRsAN1The nopaline radish has the advantages that the cotyledon and the leaf of the nopaline radish can synthesize and accumulate a large amount of anthocyanin, and the wild type nopaline radish leaf and the cotyledon cannot synthesize the anthocyanin. Functional analysis of the genes shows that the accumulation amount of anthocyanin can be obviously improved after the RsAN1 gene is singly over-expressed in the nopal radish. Demonstration of the nopal GeneRsAN1Is a positive regulation factor of anthocyanin, and provides excellent gene resources for further improvement of carmine radish and other plants. Provides a new idea for molecular breeding in the future and provides theoretical basis and materials for researching variety breeding of carmine radish and the like.

Description

Carmine radish anthocyanin biosynthesis regulatory gene RsAN1 and application thereof

Technical Field

The invention belongs to the technical field of plant genetic engineering, and particularly relates to a carmine radish anthocyanin biosynthesis regulatory gene RsAN1 and application thereof.

Background

Carmine radish also called red heart radish is a local variety with the characteristics of Fuling area in Chongqing city, and is famous for rich anthocyanin because fleshy root of carmine radish is red from the epidermis to the interior. The radish pickle has crisp meat, is suitable for processing various radish products, is not rotten after being soaked for a long time, and is bright red, tender and crisp. Fuling, Wulong, Nanchuan and other counties in Chongqing are the main producing areas of carmine radish, and the cultivation history has been over 100 years. The carmine radish has high anthocyanin content compared with other red heart radish varieties, and in Chongqing areas, the anthocyanin is usually extracted from the carmine radish to be used as a natural colorant. However, the effect is not good due to soil and climate reasons when introduced in other provinces and cities. The mechanism influencing the anthocyanin accumulation is not clear, so that the production and popularization of the carmine radish are greatly limited. Early researches show that the biosynthesis of plant anthocyanin is mainly regulated at the transcription level, and MYB and bHLH transcription factors play a very important regulating and controlling role in anthocyanin biosynthesis and influence the accumulation level of anthocyanin. Whether the large accumulation of the nopaline anthocyanin is also regulated by the two transcription factors is not clear at present.

Domestic and foreign researches show that anthocyanin synthesis is mainly realized by regulating and controlling the expression of structural genes in the anabolism process of anthocyanin, so that the synthesis of enzyme in the anabolism process is influenced. Three classes of transcription factors that regulate anthocyanin synthesis have been recognized, R2R3-MYB, bHLH (basic Hlix-Loop-Hlix) and WD40, which interact to regulate anthocyanin biosynthesis.

Plant MYB transcription factors are a very large family that regulate plant secondary metabolic processes. MYB transcription factors are known for their structural existence of a conserved DNA binding domain. Most conserved as DBD (DNA Binding domain), generally comprises 1-3 incomplete repeats R (repeat), each repeat R consisting of 51-52 conserved amino acid residues and a spacer sequence. The R3 region contains a typical structural domain [ D/E]Lx₂[R/K]x₃Lx₆Lx₃R, called bHLH Motif, is a region capable of interacting with a class of bHLH proteins; the C end has a structure domain of motif 6 [ R/K]Px[P/A/R]xx[F/Y]. At least 154 MYBs in Arabidopsis constitute a large family of transcription factors, which consists of 25 subfamilies. These transcription factors have ten aspects in regulating secondary metabolism, controlling cell morphology, mediating pathogen resistance, responding to hormone signals and the likeThe PAP1 and PAP2 which are involved in transcriptional regulation of arabidopsis anthocyanin biosynthesis belong to the 6 th subfamily. Recent studies have shown that MYB involved in transcriptional regulation of anthocyanin synthesis belongs mainly to the R2R3-MYB class.

Although the former people also report the regulation gene MYB related to anthocyanin biosynthesis in carrot, the functions of the genes are generally expressed and analyzed in model plants such as tobacco, arabidopsis and the like, and the anthocyanin accumulation amount is not large when the MYB gene is expressed alone. No related report of anthocyanin biosynthesis regulatory genes in carmine radish exists so far.

Disclosure of Invention

Aiming at the defects in the prior art, the invention aims to provide a nopaline Anthocyanin biosynthesis regulatory gene RsAN1 (Anthochynin 1) and application thereof, provides a key gene for regulating Anthocyanin content in nopaline, and provides a new gene resource for genetic modification of a large amount of Anthocyanin accumulated in nopaline.

In order to achieve the purpose, the invention adopts the following technical scheme: a carmine anthocyanin biosynthesis regulatory gene RsAN1, the nucleotide sequence of which is shown in SEQ ID NO. 1; the coded amino acid sequence is shown in SEQ ID NO. 2.

A recombinant expression vector comprising the nucleotide sequence of RsAN 1.

A plant cell line or plant transformant comprising the recombinant expression vector.

The gene RsAN1 is applied to regulation and control of synthesis of herbal anthocyanin.

A cultivation method of a high-content anthocyanin carmine radish comprises the following steps:

1) taking the fleshy root of the carmine radish to extract total RNA, and synthesizing first strand cDNA by reverse transcription by taking the total RNA as a template;

2) taking the cDNA obtained in the step 1) as a template, taking nucleotide sequences shown by SEQ ID NO 3 and SEQ ID NO 4 as primers for amplification, obtaining a nucleotide sequence of a PCR amplification product shown in SEQ ID NO.1, and connecting the PCR amplification product into an expression vector to construct a recombinant expression vector;

3) and (3) transforming the constructed recombinant expression vector to agrobacterium GV3101, infecting and transforming the nopal radish with the agrobacterium GV3101, and culturing for 4-5 days to obtain the high-content anthocyanin nopal radish.

Further, the expression vector is pEAQ-HT.

Compared with the prior art, the invention has the following beneficial effects:

1. the invention firstly identifies the key MYB transcription factor RsAN1 of anthocyanin biosynthesis in the carmine radish, and the expression quantity of RsAN1 is in positive correlation with the content of the carmine radish anthocyanin. The carmine radish gene RsAN1 is proved to be a positive regulatory factor of anthocyanin, and provides excellent gene resources for further improvement of carmine radish and other plants.

2. According to the invention, the nopaline radish with the transient overexpression RsAN1 is obtained by cloning, sequence analysis and plant transfection of the nopaline radish RsAN1 gene, a large amount of anthocyanin can be synthesized and accumulated in leaves and cotyledons of the nopaline radish 4-5 days later, but wild type nopaline radish leaves and cotyledons cannot synthesize anthocyanin. The result shows that the accumulation amount of anthocyanin can be obviously improved after the RsAN1 gene is singly overexpressed in the nopal radish. Provides theoretical basis and material for researching variety cultivation of carmine radish and the like. In addition, the RsAN1 gene is generally suitable for herbaceous plants, provides an effective technical means for regulating and controlling anthocyanin of the herbaceous plants, and has wide application value.

Drawings

FIG. 1 is an electrophoretogram of RsAN1 fragment obtained by PCR amplification;

m is a DNA Marker; a is an open reading frame fragment of RsAN1 gene;

FIG. 2 is a diagram showing the structural analysis of the deduced amino acid sequence of radish RsAN 1;

FIG. 3 is a graph of the amino acid sequence of radish RsAN1 with the evolutionary analysis of MYB proteins from other plants;

FIG. 4 shows the expression levels of RsAN1 in different parts of wild type nopaline radish plants;

FIG. 5 shows the anthocyanin content in the nopal radish leaves and cotyledons;

a and C are cotyledons and leaves of transient transformation RsAN1, and B and D are cotyledons and leaves of a negative control group.

Detailed Description

The present invention will be described in further detail with reference to the following specific embodiments and the accompanying drawings. The experimental procedures described in the examples are not specifically described, i.e., they are carried out according to conventional molecular biological experimental procedures.

The carmine radish 'Red Heart No. 1' used in the following examples was provided by agricultural science and technology development company of Fuling Green, Chongqing; coli DH5 α and Agrobacterium GV3101 were purchased from Shanghai Diego Biotechnology Ltd, and the expression vector pEAQ-HT was stored in the laboratory.

1 XTBE buffer, 6 loading buffer, Goldview^TMNucleic acid dye, RNase inhibitor, dNTP Mix, AMV reverse transcriptase, MgCl₂DNA polymerase and agarose were purchased from Shanghai bioengineering, Inc. The other raw materials are ordinary and commercially available unless otherwise specified.

Example 1 cloning of nopal radish Gene RsAN1 and construction of expression vector

(1) Extraction and detection of total RNA of carmine radish

The total RNA of the carmine radish is extracted by adopting a plant RNA extraction kit (the specific method refers to the specification) of Beijing Ederly Biotech company Limited. The residual DNA in the total RNA was treated with DNase I (TaKaRa) according to the protocol. RNA concentration determination and purity were determined on a nucleic acid protein analyzer.

2. mu.L of RNA was pipetted and detected on 1.5% agarose gel electrophoresis, OD of extracted RNA₂₆₀/OD₂₈₀The ratio of (A) to (B) is 1.80-2.00, the integrity is good, and the reverse transcription can be used.

(2) Synthesis of first Strand cDNA

The first strand cDNA was synthesized using MLV-Reverse Transcriptase kit (Invitrogen) using total RNA extracted from nopal radish as a template, and the detailed procedures were performed according to the kit instructions.

Preparation of the mixture in the centrifuge tube (6. mu.L) in the following template RNA/primer sequence: template RNA 2. mu.L, oligo (dT) Primer 3. mu.L, RNase-free ddH₂O1. mu.L. Keeping the temperature at 70 ℃ for 10min on a PCR instrument, and rapidly cooling the PCR instrument on ice for more than 2 min. Centrifugation was carried out for several seconds to obtain a template RNA/primer denaturing solution.

Reverse transcription reaction solution (10 μ L) was prepared: template RNA/primer denaturing solution 6. mu.L, 5 XM-MLV Buffer 2. mu.L, dNTP mix (10mM) 0.5. mu.L, RNase Inhibitor 0.25. mu.L, RTase M-MLV 0.5. mu.L, Nase-free dH₂O0.75. mu.L. On the PCR instrument, the temperature is kept at 42 ℃ for 1 h. And (3) keeping the temperature of the PCR instrument at 70 ℃ for 15min, and then placing the PCR instrument on ice for cooling to obtain a cDNA solution.

(3) Cloning of the Gene RsAN1

Blast homology alignment was performed using MYB gene sequences from other species. Corresponding primers RsAN 1-F and RsAN1-R are designed, and the specific steps are as follows:

RsAN1-F：5’-ATGGAGGGTTCGTCCAAAGG-3’

RsAN1-R：5’-CTAATCAAGTTCAACAGTCTCTCC-3’

using the obtained cDNA as a template, and using PrimeScript^TMThe RT-PCR Kit (TaKaRa Co.) was used for PCR amplification.

The PCR amplification system (50. mu.L) was: mu.L of 10 XPCR buffer, 2. mu.L of dNTP mix 0.5. mu.L of RsAN 1-F (forward primer), 0.5. mu.L of RsAN1-R (reverse primer), 3. mu.L of cDNA template, 39. mu.L of nucleic-free Water.

PCR procedure: 5min at 98 ℃; 30s at 98 ℃, 30s at 57 ℃, 1min at 72 ℃ and 40s, and 35 cycles; 7min at 72 ℃.

After the PCR reaction, a small amount of the PCR product (about 2. mu.L) was collected and subjected to agarose gel electrophoresis, and the results are shown in FIG. 1.

As can be seen from FIG. 1, the size of the PCR-amplified fragment was about 800 bp. The target fragment was recovered with a DNA Gel recovery Kit (Agare Gel DNA Purification Kit, TaKaRa Co.), and the recovery method was carried out basically by referring to the procedures described in the Kit.

According to the size and effective concentration of the recovered product fragment, connecting a proper amount of the recovered and purified product with a cloning vector, wherein the vector is a pMD19-T vector (TaKaRa), and the molar ratio of the target DNA to the cloning vector is controlled to be 3: about 1.

A connection system: mu.L of pMD19-T vector, 4. mu.L of purified DNA, 5. mu.L of Ligation solution I. After mixing, the phases were incubated overnight at 16 ℃.

Taking out the competent cells DH5 alpha from-80 ℃, placing the cell in ice for melting, taking 2 mu L of the ligation product, adding the ligation product into a centrifuge tube containing 50 mu L of the competent cells, carrying out ice bath for 30min, carrying out water bath heat shock for 90s at 42 ℃, immediately carrying out ice bath for more than 3min, adding 800 mu L of LB liquid culture medium without antibiotics at 37 ℃, and carrying out shake culture at 200rpm at 37 ℃ for 1 h. Centrifuging at 4000rpm for 2min, discarding 900 μ L supernatant, suspending and precipitating the rest liquid with a gun head, mixing, spreading appropriate amount of conversion solution on LB solid plate culture medium (containing 100 μ g mL of X-gal/IPTG)^-1Amp), performing inverted culture in a 37 ℃ incubator overnight after closing a plate, screening white colonies through an X-gal/IPTG blue white spot, and selecting correct clones to Shenzhen Hua Dagen technology company for sequencing verification after PCR detection of bacterial liquid.

(4) Sequence analysis

Amino acid analysis was performed on the protein encoded by the LcMYB1 gene using the ExPASy on-line analysis tool PI/Mw (http:// www.expasy.ch/tools/dnahtml). The results are shown in fig. 2, where RsAN1 has a relatively conserved R2R3 domain compared to MYB transcription factors from other plants, and contains a site on the R3 domain that interacts with bHLH transcription factors. Overall, the N-terminal part of the protein has high homology with other proteins, but the C-terminal part has large variation.

As a result of alignment of amino acid sequences of homologous genes between different species and construction of phylogenetic trees using MEGA 5.0 software, RsAN1 has a close relationship with Arabidopsis AtPAP1 and AtPAP2 as shown in FIGS. 2 and 3.

(5) Construction of recombinant expression vectors

And (3) treating the expression vector pEAQ-HT by using Age I and Xho I double enzyme digestion, and recovering the large fragment of the expression vector pEAQ-HT according to a Takara agarose gel recovery kit after enzyme digestion is finished. And performing double enzyme digestion on RsAN1 with a correct sequence by Age I and Xho I, connecting by using a Gibson assembly technology, finally transforming a connecting product into escherichia coli DH5a, selecting positive clones from a screening LB culture plate containing kanamycin (100mg/L), and performing PCR detection and sequencing verification to obtain a recombinant expression vector pEAQ-RsAN 1.

Wherein, the single enzyme cutting system is as follows: 5 μ L of plasmid pEAQ-HT or 19T-RsAN 1; age I1 muL; xho I1. mu.L; buffer 2. mu.L; sterile ddH₂Make up to 20. mu.L of O. React at 37 ℃ for 3 h.

Gene RsAN1 amplification primers:

pEAQRsAN1-F:5'-TATTCTGCCCAAATTCGCGAATGGAGGGTTCGTCCAAAG-3'

pEAQRsAN1-R:5'-TGAAACCAGAGTTAAAGGCCTCAAGTTCCAGTCTCTCCATC-3

connecting a reaction system: 3 μ L of RsAN1 fragment; 1-2 μ L of linear vector (large fragment recovered from pEAQ-HT); solution I5 μ L; sterile ddH₂Make up to 10. mu.L of O. Ligation was carried out at 16 ℃ for 2 h.

(6) RsAN1 expression level detection

The expression level of RsAN1 in wild type red heart No.1 nopaline radish was measured by Real-Time PCR reaction (ABI 7500Real-Time PCR System). The reaction volume was 20. mu.L according to Real-Time PCR Master Mix (TOYOBO, Japan) instructions; the reaction conditions were as follows: 50 ℃ for 60 s; 95 ℃, 60s, 95 ℃, 15s, 56 ℃, 20s, 72 ℃, 35s, 40 cycles; then, the analysis of melting curve at 55-95 ℃ is carried out. Each sample was set to 3 replicates. After the reaction is finished, the specificity of the product is identified through a melting curve. Analysis and application of Gene relative expression amount 2^-△△CTMethods data were processed (Livak and Schmittgen 2001) and expression levels of RsAN1 were calculated. All the experiments are repeated for 3 times, and ddH is used for the experiments₂O is negative control; the results are shown in FIG. 4.

Primers for fluorescent quantitative PCR:

QRsAN1-F：5'-CGTTGTTCCTCTATGCCTTC-3'

QRsAN1-R：5'-TAGCAAACTCTCCCACCACA-3'

as can be seen from fig. 4, RsAN1 is expressed at the location where anthocyanin is accumulated (skin, meat, petiole), and the more the accumulation amount of anthocyanin is, the higher the gene expression amount is, and it can be seen that the expression amount of RsAN1 is in positive correlation with the accumulation amount of anthocyanin; on the other hand, no RsAN1 is expressed in the wild type nopaline radish leaves, and no anthocyanin is synthesized and accumulated.

(7) Genetic transformation of recombinant expression vectors

The constructed recombinant expression vector pEAQ-RsAN1 is transferred into agrobacterium EHA105 by a conventional freeze thawing method, and pEAQ-RsAN1 is transferred into wild type carmine radish by an agrobacterium-mediated method, and the method comprises the following specific steps:

agrobacterium containing the pEAQ-RsAN1 expression vector was inoculated into a liquid LB medium containing kanamycin (100mg/L) and cultured at 28 ℃ and 300rpm to OD₆₀₀About 2, centrifuging the cultured broth at 4000rpm for 5min, and collecting the thallus with MAA (10mM MES (2- [ N-morpholino)]ethanesulfonic acid)pH 5.6，10mM MgCl ₂100. mu.M Acetostyringone) suspension of microbial cells OD₆₀₀To about 1.0; agrobacterium containing the pEAQ-RsAN1 expression vector was injected into the backs of nopaline radish cotyledons and leaves using a syringe without a needle, while Agrobacterium containing the empty vector pEAQ-HT was injected into the nopaline radish cotyledons and leaves as a negative control. The results of photographing after 4-5 days of injection are shown in FIG. 5.

As can be seen from FIG. 5, transient overexpression of RsAN1 in the cotyledons and leaves of nopaline radish can lead to accumulation of a large amount of anthocyanin in the leaves and cotyledons, while no anthocyanin is synthesized in the leaves and cotyledons of the negative control plants. The result shows that the transient overexpression of RsAN1 in nopal radish can promote the synthesis and accumulation of anthocyanin.

Collecting the sample to analyze the anthocyanin content, wherein the anthocyanin content measuring method comprises the following steps: the anthocyanin content is measured by a pH differential method according to the test method of Wrolstad et al (1982), and the method comprises the following specific steps: a sample of radish tissue (0.1 g) was extracted in 3mL of an extract (methanol; water: concentrated hydrochloric acid 85: 12: 3) sufficiently in the dark at room temperature (about 5 to 6 hours). Preparing Buffer1 and Buffer 2, and preparing Buffer 1: 0.2mol/L KCl-0.2mol/L HCl (25: 67), pH 1; buffer 2: 1mol/L NaAc-0.4mol/L HCl (100: 150), pH 5. 0.5mL of the extract was added to a test tube with 2mL of Buffer1 and Buffer 2, respectively, and their absorbance at 530nm was measured with an ultraviolet spectrophotometer.

The results were calculated from empirical formulas as follows: anthocyanin contentAmount (mg/g) ═ Δ OD₅₃₀X 5X 3X 445.2/29600X 0.1, wherein 5 is dilution factor, 3 is volume of the extract, and 445.2 is relative molecular mass of cyanidin-3-glucoside; 29600 is the absorption coefficient (mol) of cyanidin-3-glucoside^-1.ml^-1) And 0.1 represents the sample weight.

The anthocyanin content in the transiently transformed RsAN1 radish leaves and cotyledons is calculated to be 0.45mg/g and 0.28mg/g respectively, while no anthocyanin can be detected in the negative control radish leaves and cotyledons.

In conclusion, when the RsAN1 gene is expressed in the carmine radish alone, the synthesis and accumulation of anthocyanin in the carmine radish can be obviously promoted, so that the content of anthocyanin in the carmine radish is increased, and the quality of the carmine radish is improved.

Finally, the above embodiments are only for illustrating the technical solutions of the present invention and not for limiting, although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all of them should be covered in the claims of the present invention.

SEQUENCE LISTING

<110> Changjiang university academy;

<120> nopaline anthocyanin biosynthesis regulatory gene RsAN1 and application thereof

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<170> SIPOSequenceListing 1.0

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<213> carmine radish (Raphanus sativus L.)

<400> 1

atggagggtt cgtccaaagg gttgagaaaa ggtgcatgga ctgctgaaga agatagtctc 60

ttgaggcaat gcattgataa gtatggagaa gggaaatggc accaagttcc tttaagagct 120

gagctcaatc ggtgcaggaa gagttgtaga ctaagatggt tgaactattt gaagccaagt 180

atcaagagag ggaaacttaa ctctgatgaa gttgatcttc ttcttcgcct tcataaactt 240

ttgggaaaca ggtggtcttt aattgctggt agattacccg gtcggactgc caatgatgta 300

aaaaattact ggaacaccca tttgagtaag aaacatgaac caggttgtaa gacccagatg 360

aaaaaagaga agagaaacat tccttgctct tctactacac tagcccaaaa aatcgacgtt 420

ttcaaacctc gacctcgatc cttcaccgtt aacaacggct gcagccatat cattggcatg 480

ccaaaacctg acgttgttcc tctatgcctt cgattcaaca acaccaaaaa tgtttgtgaa 540

aatattgcta catgtaacaa agatgacgat aaatctgagc ttgtttgtaa tttaatggat 600

ggtcagaata tgtggtggga gagtttgcta gatgagagcc aagatccagc tgctctctat 660

ccagaagcta cagcaacaaa aaaggccgta acctccgagt ttgacgttga tcacctttgg 720

agcctgttgg atggagagac tgttgaactt gattag 756

<210> 2

<211> 251

<212> PRT

<213> carmine radish (Raphanus sativus L.)

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MEGSSKGLRK GAWTAEEDSL LRQCIDKYGE GKWHQVPLRA ELNRCRKSCR LRWLNYLKPS 60

IKRGKLNSDE VDLLLRLHKL LGNRWSLIAG RLPGRTANDV KNYWNTHLSK KHEPGCKTQM 120

KKEKRNIPCS STTLAQKIDV FKPRPRSFTV NNGCSHIIGM PKPDVVPLCL RFNNTKNVCE 180

NIATCNKDDD KSELVCNLMD GQNMWWESLL DESQDPAALY PEATATKKAV TSEFDVDHLW 240

SLLDGETVEL D 251

<210> 3

<211> 39

<212> DNA

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tattctgccc aaattcgcga atggagggtt cgtccaaag 39

<210> 4

<211> 41

<212> DNA

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tgaaaccaga gttaaaggcc tcaagttcca gtctctccat c 41

<210> 5

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<212> DNA

<213> Artificial sequence

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cgttgttcct ctatgccttc 20

<210> 6

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<212> DNA

<213> Artificial sequence

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tagcaaactc tcccaccaca 20

Claims (3)

1. Nopaline anthocyanin biosynthesis regulatory geneRsAN1The application of the gene in regulating and controlling the synthesis of anthocyanin in carmine radish is shown in SEQ ID No. 1.

2. The cultivation method of the high-content anthocyanin carmine radish is characterized by comprising the following steps:

1) the biosynthesis regulatory gene of nopaline anthocyanin according to claim 1RsAN1Connecting into an expression vector to construct a recombinant expression vector;

2) and (3) transforming the constructed recombinant expression vector to agrobacterium GV3101, infecting and transforming the nopal radish with the agrobacterium GV3101, and culturing for 4-5 days to obtain the high-content anthocyanin nopal radish.

3. The method for cultivating the carmine radish with high anthocyanin content according to claim 2, wherein the expression vector is pEAQ-HT.

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