A transcription factor RsRAP2-12 regulating radish anthocyanin synthesis and its application

Abstract

The present invention belongs to the field of genetic engineering technology and discloses a transcription factor RsRAP2-12 for regulating radish anthocyanin synthesis and its application. The nucleotide sequence of the RsRAP2-12 gene is such as SEQ ID No. 1, which can be used to regulate the synthesis of radish anthocyanins. The present invention also obtains an overexpression vector and a VIGS silencing vector for the RsRAP2-12 gene; and proves that transient overexpression of RsRAP2-12 on radish leaves can increase the anthocyanin content on the leaves, and injecting the constructed VIGS silencing vector into the roots of red-skinned, red-fleshed radishes can reduce the accumulation of anthocyanins. Therefore, the RsRAP2-12 gene plays an important role in regulating the synthesis of radish anthocyanins. The present invention is of great significance for breeding new radish varieties with high anthocyanin content and provides strong technical support for improving radish quality.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, relates to a transcription factor RsRAP2-12 for regulating radish anthocyanin synthesis, and particularly relates to the application of the RsRAP2-12 gene in regulating radish anthocyanin synthesis. Background Art

[0002] Radish (Raphanus sativus L.) is an important root vegetable crop in the genus Raphanus, part of the Brassicaceae family. Anthocyanins, which impart rich color and antioxidant activity to radish, are beneficial to human health. Radish varieties rich in anthocyanins are popular with consumers for their vibrant color and high nutritional value. Anthocyanins are water-soluble flavonoid compounds found throughout plant parts, contributing to their rich color (Chaves-Silva et al., 2018; Alappat and Alappat, 2020). They also possess numerous benefits, including antioxidant, anti-cardiovascular, anti-cancer, metabolic, vision-protecting, blood sugar control, anti-obesity and anti-inflammatory properties, and anti-aging properties. They are considered natural health ingredients beneficial to the human body (Zhang et al., 2023; Nomi et al., 2019). Over 650 anthocyanins have been discovered in nature, each exhibiting slightly different colors. Three anthocyanidins—cyanidin, pelargonidin, and delphinidin—are among the most important pigments in plants (Naing et al., 2018). Pelargonidin and its derivatives appear red or orange, while cyanidin and its derivatives primarily appear brick red or magenta. Delphinidin and its derivatives also appear blue or purple (Liu et al., 2018b; Liu et al., 2018c). Anthocyanidins are also involved in numerous biological processes, such as plant responses to environmental stress and responses to pathogens and insects.

[0003] Anthocyanin biosynthesis in plants is a key branch of the flavonoid biosynthesis pathway, a process that is relatively conserved in higher plants and involves multiple complex enzymatic reactions. Anthocyanin biosynthesis in plants is regulated by various transcription factors, including members of the MYB, WD40, WRKY, ERF, bHLH, BBX, and bZIP families (González et al., 2008; Jaakola et al., 2010). The expression of structural genes is controlled by both transcription factors and environmental cues. The three most important and widely studied transcription factor families are MYB, bHLH, and WD40. These transcription factors can regulate structural genes individually or in combination with each other to form the MBW complex, thereby regulating anthocyanin biosynthesis (Hichri et al., 2011; Song et al., 2021; Petroni et al., 2011; Xu et al., 2015).

[0004] In Arabidopsis, RAP2-12 is an ethylene-responsive transcription factor (RAP2.12), encoding a member of the ERF (ethylene response factor) subfamily B-2 of the ERF / AP2 transcription factor family. This protein contains an AP2 domain. Transcription factors influence anthocyanin biosynthesis by regulating the expression of structural genes (D'Amelia et al., 2014; Liu et al., 2016). Recent studies have demonstrated a regulatory role for RAP2 family genes in anthocyanin biosynthesis. Transient overexpression of RAP2-4 in crabapple leaves and apple fruit increased anthocyanin content in leaves and fruit (Li et al., 2020). RAP2-7 and RAP2-7 are ethylene-responsive transcription factors that are regulated by miR172b, thereby regulating anthocyanin biosynthesis (Hinz et al., 2010). RAP2.10 (related to AP2.10; TR18328|c0_g1) and BBX28 (TR1858|c0_g1) were highly correlated with the expression of ANS, CHI, F3H, and F3′H, suggesting that RAP2.10 may be a candidate transcription factor involved in the biosynthesis of anthocyanins in sweet cherry (Guo et al., 2020). Summary of the Invention

[0005] The present invention aims to provide an application of the RsRAP2-12 gene in regulating the synthesis of anthocyanins in radish, so as to accelerate the breeding process of radish and quickly cultivate radish varieties with high anthocyanin content, thereby providing a favorable technology for breeding new radish varieties with high anthocyanin content and improving radish quality.

[0006] The present invention provides a transcription factor RsRAP2-12 gene for regulating radish anthocyanin synthesis, wherein the nucleotide sequence of the RsRAP2-12 gene is as SEQ ID No.1.

[0007] The present invention also provides the encoded protein of the RsRAP2-12 gene, and the amino acid sequence of the encoded protein is shown as SEQ ID No. 2.

[0008] The present invention also provides a primer pair for amplifying the RsRAP2-12 gene, comprising a forward primer having a nucleotide sequence as shown in SEQ ID No. 3 and a reverse primer having a nucleotide sequence as shown in SEQ ID No. 4.

[0009] The present invention also provides the use of the RsRAP2-12 gene or the protein encoded by the RsRAP2-12 gene in regulating the synthesis of radish anthocyanins.

[0010] The present invention also provides a method for increasing the anthocyanin content in radish leaves. The method comprises constructing an overexpression vector to transiently overexpress the RsRAP2-12 gene in the radish leaves.

[0011] Furthermore, the method comprises the following steps:

[0012] S11. Amplify RsRAP2-12 gene by PCR

[0013] The amplification primer pair is the amplification primer pair according to claim 3;

[0014] S12. Construction of recombinant overexpression vector

[0015] The RsRAP2-12 gene amplified in step S11 was connected to the plant overexpression vector 1008 by homologous recombination, and the recombinant vector was transformed into Agrobacterium tumefaciens GV3101, and a positive strain was obtained after resistance screening;

[0016] S13. Transformation and screening of radish leaves with high anthocyanin content

[0017] The positive strain obtained in step 12 is prepared into an impregnation solution, which is thoroughly shaken and the OD600 value is adjusted to 0.6-0.8 using a spectrophotometer. The solution is injected into the true leaves of radish. After culturing in the dark at 28° C. for 2 days, the solution is transferred to normal conditions for culturing. The phenotype of the injection site is observed after about a week for screening new radish varieties.

[0018] Furthermore, in step S11, the PCR amplification procedure is as follows: total volume 20 μL, forward primer 0.5 μL, reverse primer 0.5 μL, template DNA 2 μL, high-fidelity enzyme Mix 10 μL, ddH2O 7 μL;

[0019] The PCR reaction procedure was as follows: pre-denaturation at 98°C for 30 seconds; denaturation at 98°C for 10 seconds, annealing at 60°C for 5 seconds, extension at 72°C for 10 seconds, 35 cycles; extension at 72°C for 2 minutes; and storage of the PCR amplified product at 4°C.

[0020] Furthermore, in step S13, the infection solution comprises 5 ml MgCl2, 5 ml MES and 50 uL AS, and the volume is fixed to 100 ml with sterile water.

[0021] The present invention also provides a method for reducing the anthocyanin content in radish flesh, which comprises constructing a VIGS silencing vector to cause the RsRAP2-12 gene to lose its function.

[0022] Furthermore, the method comprises the following steps:

[0023] S21. Construction of VIGS vector for radish

[0024] S22, transforming the VIGS vector constructed in step S21 into competent E. coli DH5α for propagation, shaking the cells after blue-white spot screening, and extracting the plasmid using a plasmid extraction kit. The extracted plasmid was stored at 20°C;

[0025] S23. When the roots of red-skinned and red-fleshed radish swell to a diameter of about 3 cm, start the first injection. Place the extracted plasmid at room temperature and thaw it for later use. Use a 1m sterile syringe to draw up an appropriate amount of plasmid and inject it into the middle of the fleshy root of the radish. Inject multiple sites each time, infect once every other week, and cut the radish roots one week after three infections to observe the phenotype.

[0026] The following are the sequences involved in the present invention:

[0027] Coding sequence of RsRAP2-12 gene (SEQ ID NO.1):

[0028] ATGTGTGGAGGAGCTATAATCTCCGATTTCATCCCGCCGCCGAGGTCTCGCC

[0029] GCGTCACCAGCGAGTTTCTCTGGCCGGATCTGAAGAAGAAGAAGAGCTCGAGGAAACGCTCCTCGAGTTTCTTCGATCTTGACGATGAGTTCGAGGCTGACTTCCAGGGCTTCAACGACGATTCCTTCATCGACTGCGATGATGCGAAACCGTTCGTTTTCGCCGGGGCTCGTAAACCCCCCGCCGCCACTGCCGCCGATTCAGCTTTTGGCAAGAAAGTTGCTGACGGAGAAGGTGAGAGATCTGCAAAGAGGAAGAGGAAGAGCCAGTACCGAGGTATAAGACAACGTCCTTGGGGAAAATGGGCTGCTGAGATTCGTGATCCAAGGGAAGGTTCAAGAGTGTGGCTTGGAACTTTCAAAACTGCCGAGGAAGCTGCAAGAGCTTACGATGCTGCAGCTCGTAGAATCCGTGGTTCCAAAGCTAAGGTGAATTTCCCAGAGGAGAACCCACTTGCCAAGAAGGTGGCTAAACCGAACCCAAACCCAACTCTGGTTCAGAACGTGGACAACTCCTTTGACAATATATGTTTCATGGAGGAGAAACAAGAAGTTAACAGCAGCAACAACAACAACAATCAATTTGGTAATGGGTATCATCAGTTATTCAGCTCAGACCAGGGTAGTAACTCATTTGGTTGTTCTGAGTTTGGTTGGAACGATCAAGCTCCTATAACTCCTGAGATCTCTTCGGCGTTTATCAACAACAACAACTCTGCTCTATTCGCCGAGGAAGCTGATCCAGCTAAGAAGCTCAAGTCCATGGATTTCGAGACACCTTACAACAACACTGAATGGGATTCTTCACTTGATTTCTTCAACGAAGACGCCGTGGCGACTCAGGACAATGGTGCAAACCCTATGGAACTATGGAGCATTGATGAGATCGATTCCATGATTGGAGGAGTCTTCTGA

[0030] Protein sequence of RsRAP2-12 gene (SEQ ID NO.2):

[0031] MCGGAIISDFIPPPRSRRVTSEFLWPDLKKKKSSRKRSSSFFDLDDEFEADFQGFNDDSFIDCDDAKPFVFAGARKPPAATAADSAFGKKVADGEGERSAKRKRKSQYRGIRQRPWGKWAAEIRDPREGSRVWLGTFKTAEEAARAYDAAARRIRGSKAK VNFPEENPLAKKVAKPNPNPTLVQNVDNSFDNICFMEEKQEVNSSNNNNNQFGNGYHQLFSSDQGSNSFGCSEFGWNDQAPITPEISSAFINNNNSALFAEEADPAKKLKSMDFETPYNNTEWDSSLDFFNEDAVATQDNGANPMELWSIDEIDSMIGGVF

[0032] SEQ ID NO.3:

[0033] ATGTGTGGAGGAGCTATAATCTCCG

[0034] SEQ ID NO.4:

[0035] GAAGACTCCTCCAATCATGGAA

[0036] In summary, the present invention has the following beneficial effects:

[0037] The RsRAP2-12 gene provided by the present invention is a transcription factor. By cloning the coding sequence of RsRAP2-12 in radish and constructing an overexpression vector, the expression in radish leaves can promote the synthesis of anthocyanins in the leaves. The VIGS silencing vector is constructed and injected into the roots of red-skinned and red-fleshed radish to reduce the accumulation of anthocyanins. Therefore, the radish RsRAP2-12 gene plays an important role in the regulation of radish anthocyanin synthesis. The isolation and identification of the RsRAP2-12 gene from radish has important breeding significance for cultivating new radish varieties with high anthocyanin content, and provides a reference basis for cultivating new radish varieties with high anthocyanin content. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 A and B are phenotypic analyses of transiently overexpressed RsRAP2-12 in the examples of the present invention, C is anthocyanin accumulation determination, and D is gene expression analysis;

[0039] Figure 2 This is an analysis diagram of the expression pattern of anthocyanin synthesis-related genes in leaves of the transiently overexpressed RsRAP2-12 gene in an embodiment of the present invention;

[0040] Figure 3 A and B are phenotypic analyses of VIGS-silenced RsRAP2-12 in the present invention, C is anthocyanin accumulation assay, and D is gene expression analysis.

[0041] Figure 4 This is a diagram analyzing the expression pattern of anthocyanin synthesis-related genes in root flesh after VIGS silencing the RsRAP2-12 gene in an embodiment of the present invention. DETAILED DESCRIPTION

[0042] To illustrate the technical content, achieved objectives and effects of the present invention in detail, the following description is given in conjunction with the embodiments and accompanying drawings.

[0043] Implementation Cases:

[0044] First, the present invention cloned the RsRAP2-12 gene from radish XHT. Its coding sequence is 966 base pairs long and encodes 321 amino acids. Transient overexpression of this gene in radish leaves effectively increases anthocyanin synthesis and content, while silencing the RsRAP2-12 gene to disable its function reduces anthocyanin accumulation.

[0045] Coding sequence of RsRAP2-12 gene (SEQ ID NO.1):

[0046] ATGTGTGGAGGAGCTATAATCTCCGATTTCATCCCGCCGCCGAGGTCTCGCCGCGTCACCAGCGAGTTTCTCTGGCCGGATCTGAAGAAGAAGAAGAGCTCGAGGAAACGCTCCTCGAGTTTCTTCGATCTTGACGATGAGTTCGAGGCTGACTTCCAGGGCTTCAACGACGATTCCTTCATCGACTGCGATGATGCGAAACCGTTCGTTTTCGCCGGGGCTCGTAAACCCCCCGCCGCCACTGCCGCCGATTCAGCTTTTGGCAAGAAAGTTGCTGACGGAGAAGGTGAGAGATCTGCAAAGAGGAAGAGGAAGAGCCAGTACCGAGGTATAAGACAACGTCCTTGGGGAAAATGGGCTGCTGAGATTCGTGATCCAAGGGAAGGTTCAAGAGTGTGGCTTGGAACTTTCAAAACTGCCGAGGAAGCTGCAAGAGCTTACGATGCTGCAGCTCGTAGAATCCGTGGTTCCAAAGCTAAGGTGAATTTCCCAGAGGAGAACCCACTTGCCAAGAAGGTGGCTAAACCGAACCCAAACCCAACTCTGGTTCAGAACGTGGACAACTCCTTTGACAATATATGTTTCATGGAGGAGAAACAAGAAGTTAACAGCAGCAACAACAACAACAATCAATTTGGTAATGGGTATCATCAGTTATTCAGCTCAGACCAGGGTAGTAACTCATTTGGTTGTTCTGAGTTTGGTTGGAACGATCAAGCTCCTATAACTCCTGAGATCTCTTCGGCGTTTATCAACAACAACAACTCTGCTCTATTCGCCGAGGAAGCTGATCCAGCTAAGAAGCTCAAGTCCATGGATTTCGAGACACCTTACAACAACACTGAATGGGATTCTTCACTTGATTTCTTCAACGAAGACGCCGTGGCGACTCAGGACAATGGTGCAAACCCTATGGAACTATGGAGCATTGATGAGATCGATTCCATGATTGGAGGAGTCTTCTGA

[0047] Protein sequence of RsRAP2-12 gene (SEQ ID NO.2):

[0048] MCGGAIISDFIPPPRSRRVTSEFLWPDLKKKKSSRKRSSSFFDLDDEFEADFQGFNDDSFIDCDDAKPFVFAGARKPPAATAADSAFGKKVADGEGERSAKRKRKSQYRGIRQRPWGKWAAEIRDPREGSRVWLGTFKTAEEAARAYDAAARRIRGSKAK VNFPEENPLAKKVAKPNPNPTLVQNVDNSFDNICFMEEKQEVNSSNNNNNQFGNGYHQLFSSDQGSNSFGCSEFGWNDQAPITPEISSAFINNNNSALFAEEADPAKKLKSMDFETPYNNTEWDSSLDFFNEDAVATQDNGANPMELWSIDEIDSMIGGVF

[0049] 2. Cloning of Radish RsRAP2-12 Gene and Construction of Expression Vector

[0050] Based on the RsRAP2-12 sequence and the characteristics of the 1008 vector, specific primers of RsRAP2-12 with homology arms were designed.

[0051] PCR amplification was performed using total RNA extracted from the root bark of 'XHT' as a template. The reaction system and procedure were set up according to the instruction manual of 2X Hieff Canace AdvanceFast PCR Master MIX (YESEN, China).

[0052] The PCR amplification program was as follows: pre-denaturation at 98°C for 30 s; denaturation at 98°C for 10 s, annealing at 60°C for 5 s, extension at 72°C for 10 s, 35 cycles; extension at 72°C for 2 min; the total PCR amplification volume was 20 μL, 0.5 μL of forward primer, 0.5 μL of reverse primer, 2 μL of template DNA, 10 μL of high-fidelity enzyme mix, and 7 μL of ddH2O; the PCR amplification product was stored at 4°C.

[0053] Amplification primers are as follows:

[0054] Forward primer (SEQ ID NO. 3): ATGTGTGGAGGAGCTATAATCTCCG

[0055] Reverse primer (SEQ ID NO. 4): GAAGACTCCTCCAATCATGGAA

[0056] The target gene product was recovered, and the 1008 vector plasmid was double-digested with enzymes. The prepared reaction system was incubated on ice for 30 minutes for recombination, and then transformed into competent E. coli DH5α cells. After selection with CRM antibiotics, single colonies were isolated and positive clones were sent to the company for sequencing, resulting in the plant recombinant plasmid 1008-RsRAP2-12. The 1008-RsRAP2-12 vector plasmid was extracted and transformed into Agrobacterium tumefaciens GV3101. After selection with CRM and Rif antibiotics, positive clones were isolated.

[0057] 3. Transient overexpression of radish RsRAP2-12 in radish leaves.

[0058] Preparation of resuspension: Shake the transformed Agrobacterium until turbid and collect it in a sterilized 50 mL centrifuge tube. Centrifuge for 5 min (12000 rpm) and discard the supernatant.

[0059] First add 10 mL of resuspension solution, vortex mix (protect from light), and adjust the OD600 value to about 0.7-0.8 using an ultra-micro spectrophotometer. Place it in a shaker at 28°C and 90 rpm in the dark and incubate for 4 hours before use.

[0060] Water the materials to be transformed sufficiently the day before transformation, and select radish leaves with two leaves and two hearts that are in good growth; ensure that the leaf surface is clean and free of water before injection, aspirate 500 μL of infection solution and inject the radish leaves with a 1 mL disposable sterile syringe; the injected radish seedlings are placed in a light incubator for dark culture for 2 days, cover the injected radish plants with plastic wrap, and then transfer them to a normal artificial climate chamber for culture. After 10 days, closely observe the color changes at the injection site.

[0061] 4. Construction of VIGS vector for radish RsRAP2-12 gene

[0062] Nanjing GenScript Biotech Co., Ltd. was commissioned to construct a VIGS vector for radish. The vector was transformed into competent Escherichia coli DH5α cells for propagation. After blue-white spot screening, the cells were shaken and the plasmid was extracted using a plasmid extraction kit and stored at 20°C. The RsRAP2-12 gene was silenced in 5-day-old red radish. The extracted plasmid was then injected into the root flesh of the radish. The injected radishes were maintained in a climatic chamber at 25°C / 22°C with a 16-hour / 8-hour light / dark cycle. Injections were performed once a week for a total of three times, and phenotypes were assessed after the fourth week.

[0063] 5. Determination of anthocyanin content

[0064] Refer to the pH differential method described by Niu Shanshan (2011). Radish material was thoroughly ground into a powder in liquid nitrogen. 1 g of the thoroughly ground powder sample was weighed and transferred to a 10 mL centrifuge tube. 8 mL of 0.05% 4°C pre-cooled hydrochloric acid-methanol solution was added, vortexed to mix thoroughly, and placed in a 4°C refrigerator protected from light. After 12 hours of centrifugation, the supernatant was transferred to a 25 mL volumetric flask. 8 mL of the same hydrochloric acid-methanol solution was added to the centrifuge tube, vortexed to mix thoroughly, and placed in a 4°C refrigerator for 6 hours of centrifugation. The supernatant was transferred to a 25 mL volumetric flask. This step was repeated once. The volume was adjusted to 0.05% 4°C pre-cooled hydrochloric acid-methanol solution. Two test tubes (each containing 1 mL of the extract) were set up, and 4 mL of 0.4 mol / L citric acid / disodium hydrogen phosphate buffer (pH 5.0) and 0.4 mol / L KCl-HCl buffer (pH 1.0) were added, respectively. The mixture was mixed thoroughly and allowed to stand at room temperature for 20 minutes. The absorbance at 530 nm and 700 nm was measured using a dual-wavelength UV spectrophotometer, with 0.05% hydrochloric acid-methanol solution as a control. Each sample was repeated three times.

[0065] VI. Analysis of expression patterns of RsRAP2-12 genes and anthocyanin synthesis-related genes

[0066] Total RNA was extracted and real-time quantitative PCR (RT-qPCR) analysis was performed using SYBR Green Master Mix using a real-time fluorescence quantitative PCR instrument. RsActin was used as an internal control. The relative expression levels of candidate genes were calculated using Formula 2 -△△Ct (Livak and Schmittgen, 2001). For semi-quantitative RT-PCR, PCR products were separated by electrophoresis on a 1.2% agarose gel and visualized under UV light. All reactions were performed in triplicate. The expression levels of 10 anthocyanin biosynthesis-related genes, including RsRsRAP2-12, including RsMYB1, RsZIP60, RsDFR, RsUFGT, RsANS, RsC4H, RsTT8, Rs3GT, and RsINC13249, were measured.

[0067] 7. Overexpression of the RsRAP2-12 gene can promote the synthesis of anthocyanins

[0068] Depend on Figure 1 It can be seen that RsRAP2-12 was transiently overexpressed in radish leaves. The leaf colors of the experimental group and the control group were compared, and it was found that the injection site of the transgenic line leaves turned red significantly ( Figure 1 A, B), the anthocyanin content of the leaves was measured, and the results showed that the anthocyanin content in the leaves overexpressing the RsRAP2-12 gene was significantly higher than that in the control group ( Figure 1C), the expression level of RsRAP2-12 gene in the transgenic lines was analyzed, and the results showed that the expression level of RsRAP2-12 gene in the leaves of the transgenic lines was significantly different compared with the control group ( Figure 1 D) These results indicate that RsRAP2-12 can increase the content of anthocyanins in leaves and promote the accumulation of anthocyanins in leaves.

[0069] 8. Analysis of anthocyanin synthesis-related gene expression in transient overexpression leaves

[0070] Total RNA was extracted from the leaves of the transgenic lines and reverse transcribed into cDNA. The results of analysis of the expression of anthocyanin synthesis-related genes in the transgenic lines showed that compared with the control group, the expression levels of anthocyanin synthesis-related genes RsMYB1, RsZIP60, RsDFR, RsUFGT, RsANS, RsC4H, RsTT8, Rs3GT, and RsINC13249 in the transgenic lines were significantly increased ( Figure 2 ), these results indicate that RsRAP2-12 can regulate the synthesis of anthocyanins by activating the activity of structural genes and key transcription factors for anthocyanin synthesis.

[0071] 9. Silencing RsRAP2-12 reduces anthocyanin accumulation in radish fleshy roots

[0072] A VIGS silencing vector was constructed and injected into red flesh and red skin radish. The root flesh color of the experimental group was compared with that of the control group. It was found that the injection site of the transgenic line was obviously whitened ( Figure 3 A), the content of proanthocyanidins in the root flesh was measured, and the results showed that the anthocyanidin content in the experimental group was significantly lower than that in the control group ( Figure 1 C), the expression level of RsRAP2-12 gene in the VIGS silenced strain was analyzed, and the results showed that the expression level of RsRAP2-12 gene in the VIGS silenced strain was significantly different from that in the control group ( Figure 3 D). These results indicate that silencing RsRAP2-12 can reduce the content of anthocyanins and inhibit anthocyanin accumulation.

[0073] 10. Analysis of expression of genes related to anthocyanin synthesis in radish after silencing the RsRAP2-12 gene

[0074] Total RNA was extracted from radish root pulp and reverse transcribed into cDNA. The results of analysis of the expression of anthocyanin synthesis-related genes showed that compared with the control group, the expression of anthocyanin synthesis-related genes RsMYB1, RsZIP60, RsDFR, RsUFGT, RsANS, RsC4H, RsTT8, Rs3GT, and RsINC13249 in the transgenic lines was significantly reduced ( Figure 4), these results indicate that the RsRAP2-12 gene positively regulates anthocyanin biosynthesis in radish.

[0075] In summary, the present invention clones the coding sequence of RsRAP2-12 in radish, constructs an overexpression vector, and expresses it in radish leaves to promote the synthesis of anthocyanins in the leaves. Constructing a VIGS silencing vector and injecting it into the roots of red-skinned and red-fleshed radish can reduce the accumulation of anthocyanins. It can be seen that the RsRAP2-12 gene provided by the present invention plays an important role in the regulation of radish anthocyanin synthesis. The isolation and identification of the RsRAP2-12 gene from radish has important breeding significance for cultivating new radish varieties with high anthocyanin content, and can provide a reference basis for cultivating new radish varieties with high anthocyanin content.

[0076] The above is only an embodiment of the present invention, and common knowledge such as the specific technical solutions or characteristics in the solution is not described in detail here. It should be pointed out that for those skilled in the art, without departing from the technical solution of the present invention, several variations and improvements can be made, which should also be regarded as the scope of protection of the present invention, and these will not affect the effect of the implementation of the present invention and the practicality of the patent. The scope of protection required by this application shall be based on the content of its claims, and the specific implementation methods and other records in the description can be used to interpret the content of the claims.

Claims

1. Application of the RsRAP2-12 gene or the protein encoded by the RsRAP2-12 gene in regulating radish anthocyanin synthesis, characterized in that: The regulation of radish anthocyanin synthesis is to promote the biosynthesis of anthocyanins by overexpressing the RsRAP2-12 gene or to inhibit the accumulation of anthocyanins by silencing the RsRAP2-12 gene; the nucleotide sequence of the RsRAP2-12 gene is such as SEQ ID No.1, and the amino acid sequence of the encoded protein is such as SEQ ID No.

2.

2. The use according to claim 1, characterized in that The primer pair for amplifying the RsRAP2-12 gene includes a forward primer having a nucleotide sequence as shown in SEQ ID No. 3 and a reverse primer having a nucleotide sequence as shown in SEQ ID No.

4.

3. A method for increasing the anthocyanin content in radish leaves, characterized in that: The method comprises constructing an overexpression vector to transiently overexpress the RsRAP2-12 gene according to claim 1 on radish leaves.

4. The method for increasing anthocyanin content in radish leaves according to claim 3, wherein: The method comprises the following steps: S11. Amplify RsRAP2-12 gene by PCR The amplification primer pair is the amplification primer pair according to claim 2; S12. Construction of recombinant overexpression vector The RsRAP2-12 gene amplified in step S11 was connected to the plant overexpression vector 1008 by homologous recombination, and the recombinant vector was transformed into Agrobacterium tumefaciens GV3101, and a positive strain was obtained after resistance screening; S13. Transformation and screening of radish leaves with high anthocyanin content Prepare the infection solution with the positive strain obtained in step 12, shake it thoroughly, adjust the OD600 value to 0.6-0.8 using a spectrophotometer, inject it into the true leaves of radish, culture it in the dark at 28°C for 2 days, move it to normal conditions for culture, and observe the phenotype of the injection site after about a week.

5. The method for increasing anthocyanin content in radish leaves according to claim 4, wherein: In step S11, the PCR amplification system: total volume 20 μL, forward primer 0.5 μL, reverse primer 0.5 μL, template DNA 2 μL, high-fidelity enzyme Mix 10 μL, ddH2O 7 μL; The PCR reaction procedure was as follows: pre-denaturation at 98°C for 30s; denaturation at 98°C for 10s, annealing at 60°C for 5s, extension at 72°C for 10s, 35 cycles; extension at 72°C for 2min; and storage of the PCR amplified product at 4°C.

6. The method for increasing anthocyanin content in radish leaves according to claim 4, characterized in that: In step S13, the infection solution contains 5 ml MgCl2, 5 ml MES and 50 uL AS, and the volume is adjusted to 100 ml with sterile water.

7. A method for reducing anthocyanin content in radish flesh, characterized in that: The method comprises constructing a VIGS silencing vector to cause the RsRAP2-12 gene function as claimed in claim 1 to be lost.

8. The method for reducing anthocyanin content in radish flesh according to claim 7, characterized in that: The method comprises the following steps: S21. Construction of VIGS vector for radish S22, transforming the VIGS vector constructed in step S21 into competent E. coli DH5α for propagation, shaking the cells after blue-white spot screening, and extracting the plasmid using a plasmid extraction kit. The extracted plasmid was stored at 20°C; S23. When the roots of red-skinned and red-fleshed radish swell to a diameter of about 3 cm, start the first injection. Place the extracted plasmid at room temperature and thaw it for later use. Use a 1m sterile syringe to draw up an appropriate amount of plasmid and inject it into the middle of the fleshy root of the radish. Inject multiple sites each time, infect once every other week, and cut the radish roots one week after three infections to observe the phenotype.

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