Radish rsmyb308 gene and application thereof

Abstract

The application discloses a radish RsMYB308 gene and application thereof, and relates to the technical field of plant genes.The radish RsMYB308 gene is obtained by a homologous cloning method, and overexpression vectors and silencing vectors of the RsMYB308 gene are respectively transformed into radish leaves and red-fleshed red-skin radishes by an agrobacterium-mediated method.The results show that the expression of genes related to anthocyanin synthesis in the radish leaves with overexpression of the RsMYB308 gene is significantly increased, and the anthocyanin content is obviously higher than that of a control group, the RsMYB308 gene can increase the anthocyanin content in the radish leaves and promote anthocyanin accumulation, the expression of genes related to anthocyanin synthesis in the radish root flesh with silencing expression of the RsMYB308 gene is down-regulated, and the anthocyanin content is obviously lower than that of the control group.The RsMYB308 gene provided by the application can accelerate the breeding process of the radish, rapidly cultivate radish varieties with high anthocyanin content, and provide a theoretical basis for analyzing anthocyanin synthesis of the radish.

Description

Technical Field

[0001] This invention relates to the field of plant gene technology, specifically to the radish RsMYB308 gene and its applications. Background Technology

[0002] Radish (Raphanus sativus L., 2n=2x=18), also known as turnip or daikon, is an important vegetable crop belonging to the genus Raphanus in the Brassicaceae family. It possesses high nutritional and medicinal value. Radish varieties rich in anthocyanins are popular among consumers due to their vibrant color and high nutritional value. Anthocyanins, also known as anthocyanin glycosides, are natural water-soluble pigments in plants, primarily stored in the vacuoles of plant cells, playing a crucial role in pigment deposition in plant organs. For example, the NsMYB1 gene promotes anthocyanin accumulation in the black fruits of Siberian white thorn. Transient overexpression of DbMYB2 significantly promotes anthocyanin accumulation in tobacco leaves. These findings consistently demonstrate a close correlation between enhanced color and increased anthocyanin levels.

[0003] Anthocyanin biosynthesis in plants is an important branch of the flavonoid biosynthesis pathway and is relatively conserved in higher plants, involving multiple complex enzymatic reactions. Anthocyanin biosynthesis in plants is regulated by various transcription factors. The MYB-bHLH-WD40 ternary protein complex (MBW), composed of MYB (v-myb avian myeloblastosis viral oncogene homolog), bHLH (basic Helix-Loop-Helix), and WD40 (WD repeat) transcription factors, binds to corresponding cis-regulatory elements in the promoters of structural genes, promoting or inhibiting the expression of downstream structural genes. This complex is the most common transcriptional activation mode in the anthocyanin biosynthesis pathway.

[0004] However, existing technologies lag behind in radish color improvement breeding. The gene RsMYB308 cloned in this invention belongs to the MYB transcription factor family. Studies have shown that overexpression of the RsMYB308 gene can increase anthocyanin content in plants, while loss of function of the RsMYB308 gene can decrease anthocyanin content. Therefore, this invention provides a radish gene RsMYB308 that can be applied to the breeding of new radish varieties, accelerating the radish breeding process, rapidly cultivating radish varieties with high anthocyanin content, and providing a theoretical basis for understanding radish anthocyanin synthesis. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides the radish RsMYB308 gene and its applications.

[0006] To achieve the above objectives, the technical solution of the present invention is as follows:

[0007] This invention provides the radish RsMYB308 gene, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0008] The present invention also provides the protein encoded by the above-mentioned RsMYB308 gene, the amino acid sequence of which is shown in SEQ ID NO.2.

[0009] The present invention also provides a recombinant expression vector containing the above-mentioned RsMYB308 gene, and a host cell or engineered bacteria containing the recombinant expression vector.

[0010] This invention also provides the application of the above-mentioned RsMYB308 gene in promoting anthocyanin synthesis / reducing anthocyanin accumulation in radishes.

[0011] Preferably, the method for promoting anthocyanin synthesis in radish is to increase the expression level of the RsMYB308 gene in radish, specifically by linking the RsMYB308 gene to an expression vector and transforming it into radish via Agrobacterium-mediated transformation, thereby overexpressing the RsMYB308 gene in transgenic radish.

[0012] Preferably, the method for reducing anthocyanin accumulation in radish is to reduce the expression level of the RsMYB308 gene in radish, specifically by linking the RsMYB308 gene to a silencing vector and transforming it into radish via Agrobacterium-mediated transformation, thereby silencing the expression of the RsMYB308 gene in the transgenic radish.

[0013] Preferably, the Agrobacterium-mediated transformation into radish specifically involves introducing an overexpression vector or silencing vector of the RsMYB308 gene into plant cells, tissues, or organs.

[0014] The present invention also provides a method for preparing the RsMYB308 gene of claim 1, comprising the following steps:

[0015] S1. Design primers for PCR amplification, wherein the primers include a forward primer with a nucleotide sequence as shown in SEQ ID NO.3 and a reverse primer with a nucleotide sequence as shown in SEQ ID NO.4;

[0016] S2. Using red-skinned, red-fleshed radish cDNA as a template, PCR amplification was performed to obtain the RsMYB308 gene.

[0017] Preferably, the PCR amplification program is as follows: 98℃ pre-denaturation for 30s; 98℃ denaturation for 10s, 65℃ annealing for 5s, 72℃ extension for 10s, cycle number 35; 72℃ extension for 2min; the PCR amplification system includes 1μL of forward primer, 1μL of reverse primer, 2μL of template cDNA, 10μL of high-fidelity enzyme Mix and 6μL of ddH2O.

[0018] The present invention also provides a method for promoting anthocyanin synthesis / reducing anthocyanin accumulation in radishes. The method involves ligating the above-mentioned RsMYB308 gene into an overexpression vector / silencing vector and introducing it into radish cells, tissues or organs to obtain transgenic plants.

[0019] Compared with existing technologies, the beneficial effects of this solution are:

[0020] This invention cloned the RsMYB308 gene from red-skinned, red-fleshed radish. Its full-length coding sequence is 600 bp, encoding 199 amino acids. Transient overexpression of this gene in radish leaves increased the accumulation of anthocyanins and the expression levels of genes related to anthocyanin biosynthesis. Silencing the expression of RsMYB308 in the root tissue reduced anthocyanin accumulation and the expression levels of anthocyanin biosynthesis genes in the root. This can accelerate the radish breeding process, rapidly cultivate radish varieties with high anthocyanin content, and provide a theoretical basis for understanding anthocyanin synthesis in radishes. Attached Figure Description

[0021] Figure 1 The effect of RsMYB308 gene overexpression on anthocyanin synthesis in this invention is shown in Figure 1. (A represents the phenotypic analysis of radish leaves in the control group and transiently overexpressed RsMYB308; B represents the expression level of anthocyanin synthesis-related genes in leaves with transiently overexpressed RsMYB308; C represents the expression level of RsMYB308 gene in leaves with transiently overexpressed RsMYB308; D represents the anthocyanin content in the control group and OE-RsMYB308 tissues; pFGC1008 is the empty vector control, and OE-MYB308 is the overexpression line.)

[0022] Figure 2 The phenotypes of RsMYB308 gene overexpression and silencing, as well as the effects on anthocyanins, are shown in this invention (A is the phenotypic analysis of the root of the control group and RsMYB308-pTY inoculated radish; B is the expression level of anthocyanin synthesis-related genes in RsMYB308-pTY; C is the expression level of RsMYB308 gene in RsMYB308-pTY; D is the anthocyanin content in the tissues of the control group and RsMYB308-pTY; pTY is the empty vector control, and pTY-MYB308 is the interference line). Detailed Implementation

[0023] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0024] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0025] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0026] Example 1: Cloning and expression vector construction of the radish RsMYB308 gene

[0027] Based on the RsMYB308 sequence and the characteristics of the 004 vector, specific primers with homologous arms for RsMYB308 were designed.

[0028] 1. Cloning of the RsMYB308 gene

[0029] 1) PCR amplification was performed using red-skinned, red-fleshed radish cDNA as a template. The reaction system and program were set according to the 2X Hieff Canace Advance Fast PCR Master MIX manual (YESEN, China). The PCR amplification program was: 98℃ pre-denaturation for 30 s; 98℃ denaturation for 10 s, 65℃ annealing for 5 s, 72℃ extension for 10 s, cycle number 35; 72℃ extension for 2 min. The total PCR amplification volume was 20 μL, 1 μL of forward primer, 1 μL of reverse primer, 2 μL of template cDNA, 10 μL of high-fidelity enzyme mix, and 6 μL of ddH2O. The PCR amplification products were stored at 4℃. The amplification primers are as follows:

[0030] Forward primer (SEQ ID NO.3):

[0031] TTACAATTACCATGGGGCGCGCCATGAACAAAATTCGCCACCG;

[0032] Reverse primer (SEQ ID NO.4):

[0033] AACATCGTATGGGTAGGTACCCTGGAAAAGAAGAAGAGTTTC.

[0034] 2) After purification by agarose gel electrophoresis, the PCR product was sent to Sangon Biotech (Shanghai) Co., Ltd. for Sanger sequencing. If the sequencing was correct, the RsMYB308 gene was obtained.

[0035] The coding sequence of the RsMYB308 gene (SEQ ID NO.1):

[0036] ATGAACAAAATTCGCCACCGCGGTCTTTCTCGGCCTTCCGGAATGATACACCGTGCCAAGAGGTATAGAATAAGAGGGAGAAAGTACGCAAAGCCAGAACTTAAACAAAGCAACTTTTCAAAAGATGAGGACGATCTCATCCTCAAGCTTCATGCACTTCTTGGCAATAGATGGTCATTGATAGCGGGAAGATTGCCTGGACTAACCGACAACGAAGTAAGAATCCATTGGGAAACTTACTTAGAGAAGAAACTAATGAAAATGGGAATCGATCCAACCAATCATCGTCTCTACCATCACACCAACTACACTTCTAGACGATACATCAATGCCTCGCATAAGGAACATGAAACCGATATTATTAGTGATCAATCTTCTTCGGTATCCGAATCATGTGATGATATGACACTATTACCCGTTTCAAGAACCATTTGCTCTGAGGATAATGCTAGTGCCGGACATAACTGGTTGCCTGACCTCAACATCGGTCTCGTCCCGATGAAGACAGCGACTTCTTTGCCAGTTGGCTCCCTTCAAGAACCTAGCGAATCCTCTAACCATGGTTCAACGAGTCAAGAAACTCTTCTTCTTTTCCAGTAA。

[0037] Protein sequence of RsMYB308 gene (SEQ ID NO.2):

[0038] MNKIRHRGLSRPSGMIHRAKRYRIRGRKYAKPELKQSNFSKDEDDLILKLHALLGNRWSLIAGRLPGLTDNEVRIHWETYLEKKLMKMGIDPTNHRLYHHTNYTSRRYINASHKEHETDIISDQSSSVSESCDDMTLLPVSRTICSEDNASAGHNWLPDLNIGLVPMKTATSLPVGSLQEPSESSNHGSTSQETLLLFQ。

[0039] 3) The RsMYB308 gene obtained in step 2) was ligated to the plant overexpression vector 004 via homologous recombination. The prepared reaction system was incubated on ice for 30 minutes for recombination, and then transformed into *E. coli* DH5α competent cells. After screening for the target gene, single clones were selected and cultured. Positive clones were sent to the company for sequencing to form the plant recombinant plasmid 004-RsMYB308. The 004-RsMYB308 vector plasmid was extracted and transformed into *Agrobacterium tumefaciens* GV3101. After screening with CRM and Rif antibiotics, positive *Agrobacterium* clones were obtained.

[0040] Example 2: Phenotypic effects of transient overexpression and silencing of radish RsMYB308 on anthocyanins

[0041] 1 Experimental Methods

[0042] 1.1 Preparation of Radish Leaves with Transient Overexpression of RsMYB308

[0043] 1) Preparation of resuspension: The transformed Agrobacterium was shaken until turbid and collected in a sterile 50 mL centrifuge tube. Centrifuged for 10 min (12000 rpm) and discarded the supernatant. 10 mL of the resuspension was added and vortexed (protected from light). The OD value was measured using a micro spectrophotometer. 600 Adjust the value to around 0.7-0.8, place it in a 28℃, 90rpm shaker and incubate in the dark for 4 hours before use.

[0044] 2) Water the material to be transformed thoroughly the day before transformation, and select radish leaves with two leaves and two buds that are growing well. Before injection, ensure that the leaf surface is clean and dry. Draw 500μL of infection solution and inject the radish leaf with a 1mL disposable sterile syringe. After injection, place the radish seedlings in a light incubator at 28℃ in the dark for 2 days. Cover the injected radish plants with plastic wrap and then transfer them to a normal artificial climate chamber for cultivation. After 10 days, closely observe the color change at the injection site.

[0045] 1.2 Construction of a virus-induced gene silencing (VIGS) vector for the radish RsMYB308 gene

[0046] A VIGS vector for radish was constructed by Nanjing GenScript Biotech Co., Ltd. The VIGS vector was transformed into competent E. coli DH5α cells for propagation. After blue-white screening, the cells were cultured and plasmids were extracted using a plasmid extraction kit. The extracted plasmids were stored at 20℃. The RsMYB308 gene was silenced in 5-day-old red-fleshed radishes, and the extracted plasmids were injected into the root flesh of purple radishes. The injected radishes were kept in an artificial climate chamber at 25℃ / 22℃ with a 16-hour / 8-hour light / dark cycle. Injections were performed weekly for a total of three weeks, and phenotypes were evaluated at week four.

[0047] 1.3 Determination of anthocyanin content (pH differential method)

[0048] Radish material was thoroughly ground into powder in liquid nitrogen. 1g of the thoroughly ground powder sample was weighed and transferred to a 10mL centrifuge tube. 8mL of 0.05% pre-chilled hydrochloric acid-methanol solution (4℃) was added, vortexed, and placed in a 4℃ refrigerator protected from light. After 12 hours, the sample was centrifuged, and the supernatant was transferred to a 25mL volumetric flask. 8mL of the same hydrochloric acid-methanol solution was added to the centrifuge tube, vortexed, and the sample was centrifuged again at 4℃ for 6 hours. The supernatant was transferred to a 25mL volumetric flask, and this step was repeated once. The sample was then brought to volume with 0.05% pre-chilled hydrochloric acid-methanol solution (4℃). Two test tubes were prepared (1mL of extract in each). 4mL of 0.4mol / L citrate / disodium hydrogen phosphate buffer (pH 5.0) and 0.4mol / L KCl-HCl buffer (pH 1.0) were added to each tube, respectively. The mixtures were mixed and allowed to stand at room temperature for 20 minutes. Using a 0.05% hydrochloric acid-methanol solution as a control, the absorbance values ​​at 530 nm and 700 nm were measured using a dual-wavelength UV spectrophotometer. Each sample was tested three times.

[0049] 1.4 Analysis of the expression patterns of the RsMYB308 gene and anthocyanin synthesis-related genes

[0050] Total RNA was extracted and analyzed using real-time quantitative PCR (RT-qPCR) on SYBR Green Master Mix (GeneStar, China) using a real-time quantitative PCR instrument (Hangzhou, China). RsActin was used as an internal control. The relative expression levels of candidate genes were calculated using Equation 2. -△△Ct (Livak and Schmittgen, 2001). All reactions were performed in triplicate. The expression levels of nine anthocyanin synthesis-related genes, including RsDFR, Rs3GT, RsTT8, RsC4H, RsANS, RsUFGT, RsCHS, and RsF3H, were detected.

[0051] 2 Experimental Results

[0052] 2.1 Overexpression of the RsMYB308 gene can promote anthocyanin synthesis.

[0053] Depend on Figure 1 It can be seen that transient overexpression of RsMYB308 in radish leaves, when compared with the control group, revealed that the injection site of the transgenic line leaves turned significantly red. Figure 1A) The relative expression levels of anthocyanin biosynthesis-related genes were studied using RT-qPCR. The results showed that when RsMYB308 was overexpressed, the expression of RsDFR, Rs3GT, RsTT8, RsC4H, RsANS, RsUFGT, RsCHS, RsF3H, and RsMYB308 were all significantly increased. Figure 1 B, C). The anthocyanin content of the leaves was measured, and the results showed that the anthocyanin content in leaves overexpressing the RsMYB308 gene was significantly higher than that in the control group. Figure 1 D). These results indicate that RsMYB308 can increase the anthocyanin content in radish leaves and promote anthocyanin accumulation.

[0054] 2.2 Silencing RsMYB308 reduces the accumulation of anthocyanins in the fleshy roots of radishes.

[0055] A VIGS silencing vector was constructed and injected into red-fleshed, red-skinned radishes. The root flesh color of the experimental group and the control group was compared, and it was found that the injection site of the transgenic lines showed a significant whitening. Figure 2 A) The relative expression levels of genes related to anthocyanin biosynthesis were studied using RT-qPCR. The results showed that when the expression of RsMYB308 was silenced in root mesophyll, the expression of RsDFR, Rs3GT, RsTT8, RsC4H, RsANS, RsUFGT, RsCHS, RsF3H, and RsMYB308 were all downregulated. Figure 2 B, C). The anthocyanin content of the root mesophyll was measured, and the results showed that the anthocyanin content in the experimental group was significantly lower than that in the control group. Figure 2 D).

[0056] The above specific embodiments are merely explanations of the present invention and are not intended to limit the present invention. After reading this specification, those skilled in the art can make modifications to these embodiments without contributing any inventive step, but as long as they are within the scope of the claims of the present invention, they are protected by patent law.

Claims

1. The radish RsMYB308 gene, characterized by, The nucleotide sequence of the RsMYB308 gene is shown in SEQ ID NO.

1.

2. The protein encoded by the RsMYB308 gene according to claim 1, characterized in that, The amino acid sequence of the protein is shown in SEQ ID NO.

2.

3. A recombinant expression vector containing the RsMYB308 gene as described in claim 1.

4. A host cell containing the recombinant expression vector of claim 3.

5. An engineered bacterium containing the recombinant expression vector of claim 3.

6. The application of the RsMYB308 gene as described in claim 1 in promoting anthocyanin synthesis or reducing anthocyanin accumulation in radish.

7. The application according to claim 6, characterized in that, The method to promote anthocyanin synthesis in radish is to increase the expression level of the RsMYB308 gene in radish. Specifically, the RsMYB308 gene is linked to an expression vector, and the overexpression vector of the RsMYB308 gene is transformed into radish cells, tissues or organs through Agrobacterium-mediated transformation, so that the RsMYB308 gene is overexpressed in transgenic radish.

8. The application according to claim 6, characterized in that, The method to reduce anthocyanin accumulation in radishes is to reduce the expression level of the RsMYB308 gene in radishes. Specifically, the RsMYB308 gene is linked to a silencing vector, and the silencing vector of the RsMYB308 gene is transformed into radish cells, tissues or organs through Agrobacterium-mediated transformation, so that the RsMYB308 gene is silenced in transgenic radishes.

9. A method for preparing the RsMYB308 gene according to claim 1, characterized in that, Includes the following steps: S1. Design primers for PCR amplification, wherein the primers include a forward primer with a nucleotide sequence as shown in SEQ ID NO.3 and a reverse primer with a nucleotide sequence as shown in SEQ ID NO.4; S2. Using red-skinned, red-fleshed radish cDNA as a template, PCR amplification was performed to obtain the RsMYB308 gene. The PCR amplification program was as follows: 98℃ pre-denaturation for 30s; 98℃ denaturation for 10s, 65℃ annealing for 5s, 72℃ extension for 10s, cycle number 35; 72℃ extension for 2min. The PCR amplification system includes 1 μL of forward primer, 1 μL of reverse primer, 2 μL of template cDNA, 10 μL of high-fidelity enzyme mix, and 6 μL of ddH2O.

10. A method for promoting anthocyanin synthesis or reducing anthocyanin accumulation in radishes, characterized in that, The method involves linking the RsMYB308 gene described in claim 1 to an overexpression vector or a silencing vector, and introducing it into the cells, tissues, or organs of radish to obtain transgenic plants.

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