Mint McTRY gene and its expressed protein and application

Abstract

The present invention discloses a mint McTRY gene, its expression protein and application, and belongs to the technical field of plant genetic engineering. The nucleotide sequence of the mint McTRY gene disclosed in the present invention is shown in SEQ ID NO.1; the amino acid sequence of its expression protein is shown in SEQ ID NO.2. The present invention uses mint leaves as materials, obtains the mint McTRY gene by cloning, constructs its overexpression vector 35S::McTRY on this basis, and transfers it into Arabidopsis to obtain transgenic plants. No epidermal hairs are formed on the leaves and stems of the transgenic strains, and the anthocyanin content is extremely low. The McTRY gene can down-regulate the expression of positive regulatory genes for epidermal hair formation, down-regulate the expression of anthocyanin synthesis genes, down-regulate the expression of related transcription factors for anthocyanin synthesis, and up-regulate the expression of negative regulatory genes for epidermal hair formation at the molecular level.

Description

Technical Field

[0001] The invention belongs to the technical field of plant genetic engineering, and more specifically, relates to a mint McTRY gene and its expression protein and application. Background Art

[0002] Mint (Mentha haplocalyx Briq.) belongs to the Lamiaceae family and the genus Mentha (Mentha L.). It is a plant that can be used for both medicine and food. The mint essential oil it produces has antibacterial, antibacterial and fresh-keeping effects. It is used in the development of a variety of pharmaceuticals. It is also widely used in the fields of food and daily chemicals as a spice and flavor, and plays an important economic role. my country is a major exporter of mint essential oil, and mint occupies an important position in the national economy.

[0003] Epidermal hairs are a kind of hair-like epidermal structure differentiated during the morphogenesis of plant epidermal cells, which can be composed of single or multiple cells. Epidermal hairs are divided into non-glandular hairs and glandular hairs according to whether they have the function of synthesis and secretion. Non-glandular hairs are natural physical barriers for plants to block herbivores, resist pathogen attacks, reduce transpiration, and prevent ultraviolet rays. Glandular hairs can also synthesize, accumulate and secrete some secondary metabolites to provide chemical protection for plants. Peppermint epidermal hairs include three types: non-glandular hairs, capitate glandular hairs and peltate glandular hairs, among which peltate glandular hairs are the most important essential oil synthesis and storage organs.

[0004] Anthocyanins are glycosylated polyphenolic compounds belonging to the class of flavonoid compounds. They are widely found in plant leaves, flowers, fruits and other organs and have a variety of biological functions. Anthocyanins make plants colorful and give them ornamental value. They are also conducive to pollination and seed dispersal. Anthocyanins can improve plant resistance and help plants resist a variety of biological and abiotic stresses. In addition, anthocyanins have antioxidant properties and have important medical value in repairing memory damage, alleviating myopia, preventing cancer, anti-aging, preventing cardiovascular disease and lowering blood lipids. They provide humans with rich nutritional value and medical and health care functions, and are of great benefit to human health.

[0005] Transcription factors are important regulatory factors of gene expression. They can specifically bind to the cis-acting elements of target genes, inhibit or enhance their transcription to regulate transcription efficiency, and play an important role in plant growth and development. MYB is one of the largest transcription factor families in plants. It plays an important regulatory role in regulating plant growth and development, synthesis of primary and secondary metabolites, and signal transduction. It has been reported to be closely related to plant epidermal hair development and anthocyanin biosynthesis.

[0006] At present, MYB transcription factors related to epidermal hair development or anthocyanin synthesis have been identified in other plants, but there are few reports on related MYB transcription factors in mint, especially transcriptional inhibitory MYB transcription factors that simultaneously inhibit epidermal hair development and anthocyanin synthesis. Summary of the invention

[0007] In view of the above-mentioned problems existing in the prior art, the technical problem to be solved by the present invention is to provide a mint McTRY gene for constructing a mint transgenic strain; another technical problem to be solved by the present invention is to provide an expression protein of the mint McTRY gene; and another technical problem to be solved by the present invention is to provide an application of the mint McTRY gene for obtaining a new mint germplasm without epidermal hairs and with a low anthocyanin content.

[0008] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows:

[0009] The mint McTRY gene, whose nucleotide sequence is shown in SEQ ID NO.1.

[0010] The amino acid sequence of the expressed protein of the mint McTRY gene is shown in SEQ ID NO.2.

[0011] The use of the mint McTRY gene in inhibiting the formation of Arabidopsis epidermal hairs comprises the following steps:

[0012] (1) constructing a plant expression vector of the mint McTRY gene;

[0013] (2) transforming the constructed plant expression vector of the mint McTRY gene into Arabidopsis thaliana;

[0014] (3) Cultivate and screen transgenic Arabidopsis thaliana without epidermal hair formation.

[0015] The plant expression vector is 35S::McTRY.

[0016] The invention discloses an application of the mint McTRY gene in inhibiting the expression of positive regulatory genes for epidermal hair formation, wherein the positive regulatory genes for epidermal hair formation are AtMYB23, AtMYB5, AtGL2 and AtTTG2.

[0017] The invention discloses an application of the mint McTRY gene in promoting the expression of negative regulatory genes for the formation of epidermal hairs. The negative regulatory genes for the formation of epidermal hairs are AtTRY, AtTCL1, AtTCL2, AtETC2 and AtETC3.

[0018] Application of mint McTRY gene in reducing anthocyanin content in Arabidopsis thaliana.

[0019] The invention discloses an application of the mint McTRY gene in inhibiting the expression of anthocyanin synthesis genes, wherein the anthocyanin synthesis genes are AtPAL1, AtPAL2, AtC4H, At4CL, AtCHS, AtCHI, AtF3H, AtF3'H, AtDFR, AtLDOX and AtUF3GT.

[0020] The invention discloses an application of the mint McTRY gene in inhibiting the expression of anthocyanin synthesis-related transcription factors, wherein the anthocyanin synthesis-related transcription factors are AtPAP1, AtPAP2, AtMYB113, AtMYB114, AtMYB4, AtTT2, AtMYB5, AtGL2 and AtTT8.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] 1) The present invention uses mint leaves as materials, obtains the mint McTRY gene by cloning, constructs its overexpression vector 35S::McTRY on this basis, and transfers it into Arabidopsis to obtain transgenic plants.

[0023] 2) Compared with the wild type, the McTRY gene expression level in the transgenic lines OE1 and OE3 was high, and no epidermal hairs were formed on the leaves and stems; the McTRY gene expression level in the transgenic line OE4 was low, and epidermal hairs were still formed on the leaves and stems, and the inhibitory effect on epidermal hair formation was low.

[0024] 3) McTRY gene can inhibit the formation of epidermal hairs in transgenic Arabidopsis thaliana at the molecular level by downregulating the expression of AtMYB23, AtMYB5, AtGL2, AtTTG2 genes, and upregulating the expression of AtTRY, AtTCL1, AtTCL2, AtETC2 and AtETC3 genes.

[0025] 4) Compared with the wild-type WT Arabidopsis line, the anthocyanin content in the McTRY transgenic Arabidopsis lines OE1 and OE3 was very low.

[0026] 5) Compared with the wild type, the expression levels of anthocyanin biosynthesis genes AtPAL1, AtC4H, AtCHS, AtCHI, AtF3H, AtF3'H, AtDFR, AtLDOX and AtUF3GT were significantly downregulated in the McTRY transgenic Arabidopsis lines.

[0027] 6) The expression levels of anthocyanin synthesis-related transcription factors AtPAP1, AtPAP2, AtMYB113, AtMYB114, AtMYB4, AtTT2, AtMYB5, AtGL2 and AtTT8 genes in the McTRY transgenic lines were significantly lower than those in the wild type WT, and overall, the expression levels of these genes in lines OE1 and OE3 decreased more than that in OE4. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is the electrophoresis detection diagram of McTRY gene CDS clone;

[0029] Figure 2 This is the identification diagram of the McTRY gene overexpression vector (M: DNA Marker; 1: recombinant plasmid pHellsgate8-McTRY-GFP; 2: XhoI-digested recombinant plasmid pHellsgate8-McTRY-GFP);

[0030] Figure 3 This is a GFP fluorescence observation image of pHellsgate8-McTRY-GFP vector in the root of transgenic Arabidopsis seedlings;

[0031] Figure 4 This is the RNA level identification diagram of McTRY transgenic Arabidopsis;

[0032] Figure 5 The diagrams are for the development of leaf epidermal hairs of Arabidopsis wild-type WT and McTRY transgenic lines OE1, OE3 and OE4 (A is a surface image of leaves of each line under a stereo microscope; B is a surface image of leaves of each line taken by a scanning electron microscope; C is a surface image of stems of each line under a stereo microscope; D is a statistical diagram of the number of leaf epidermal hairs of each line);

[0033] Figure 6 The expression level analysis diagram of epidermal hair development-related genes in Arabidopsis wild-type WT and McTRY transgenic lines OE1, OE3 and OE4;

[0034] Figure 7 The anthocyanin synthesis analysis diagram of Arabidopsis wild-type WT and McTRY transgenic lines OE1, OE3 and OE4 (A is a growth diagram of plants of each line after six weeks of growth, B is a color diagram of plants of each line after six weeks of growth and anthocyanin extracted therefrom, and C is an analysis diagram of the relative content of anthocyanin in plants of each line after six weeks of growth);

[0035] Figure 8 The expression level analysis diagram of anthocyanin synthesis genes in Arabidopsis wild type WT and McTRY transgenic lines OE1, OE3 and OE4;

[0036] Fig. 9 This is an analysis of the gene expression levels of transcription factors that regulate anthocyanin synthesis in Arabidopsis wild-type WT and McTRY transgenic lines OE1, OE3 and OE4. DETAILED DESCRIPTION

[0037] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described below in conjunction with specific embodiments. In the following embodiments, unless otherwise specified, the technical means used are conventional means well known to those skilled in the art.

[0038] Embodiment 1:

[0039] 1. Amplification of the CDS sequence of the mint McTRY gene

[0040] Take an appropriate amount of leaf tissue samples of the test material mint variety '68-7', and extract RNA from the samples according to the plant tissue total RNA extraction kit provided by Nanjing Novozymes Biotech Co., Ltd. The extracted RNA was synthesized into cDNA according to the third-generation high-efficiency cDNA single-strand synthesis kit (genome-free) of Novozymes Biotech Co., Ltd. According to the transcriptome sequence of the mint McTRY gene, the following primers were designed: McTRY-F: 5'-ATGGATAAGTCATCTAGAAAGAAGC-3'; McTRY-R: 5'-TTATCCTTTTGGTTTGCGACTAGTTC-3'. The CDS sequence of the McTRY gene was obtained by PCR, and the PCR amplification results were detected by agarose gel electrophoresis system ( Figure 1 ). The CDS sequence of the McTRY gene is shown in the sequence listing as SEQ ID NO 1, which is 240 bp in total and encodes 79 amino acids in total, as shown in the sequence listing as SEQ ID NO 2.

[0041] 2. Construction of fusion expression vector

[0042] According to the CDS sequence of the McTRY gene and the multiple cloning site of the overexpression vector pHellsgate8-GFP (XhoI was selected as the restriction site), the amplification primers were designed: McTRY-GFP-F: 5'-CATTTGGAGAGGACACGCTCGAGATGGATAAGTCATCTAGA-3'; McTRY-GFP-R: 5'-CTTGCTCACCATGAATTCCTCGAGTCCTTTTGGTTTGCGACT-3', and the target fragment of the McTRY gene was amplified by PCR. The pHellsgate8-GFP vector was digested with XhoI at 37℃ for 3h. The PCR product and the digestion product were recovered by gel recovery using the Shanghai Biotech Gel Recovery Kit to obtain the target fragment and the linearized vector fragment. The target fragment was connected to the pHellsgate8-GFP linear vector using the ClonExpress II One Step Cloning Kit homologous recombination kit to construct the 35S::McTRY overexpression vector. The connection system was as follows: 5×CEII buffer 2μL, linearized vector about 100-200ng, insert fragment about 50-100ng, ExnaseII 1μL, ddH2O supplemented to 10μL, placed at 37℃ for 30min and immediately transferred to ice. After the E. coli DH5α competent cells were thawed on ice, the above recombinant product was added to the competent cells and mixed, incubated on ice for 30min, then heat-shocked at 42℃ for 45sec, cooled on ice for 5min, added to 500μL LB medium, placed in a 37℃ shaker for 1h, and the bacterial solution was spread on a plate containing 100mg / L spectinomycin, and incubated in an incubator at 37℃ for 12-16h. Single colonies were verified by colony PCR, and positive colonies were picked and inoculated into LB liquid medium containing 100 mg / L spectinomycin, and cultured at 200 rpm and 37°C for 24 h. Plasmid enzyme digestion verification ( Figure 2 ), and then sequenced after verification. The correct sequence is the 35S::McTRY overexpression vector.

[0043] 3. Agrobacterium infection of Arabidopsis

[0044] The 35S::McTRY overexpression vector plasmid was electroporated to transform Agrobacterium GV3101 competent cells, and the positive strains were verified by colony PCR, and Arabidopsis was transformed using the floral dip method. First, the transformation medium was prepared. The components included 1 / 2MS culture medium, 0.01μg / mlBAP, 5% sucrose, 0.02% silwetL-77, and the pH was adjusted to 5.7 with KOH. The 35S::McTRY Agrobacterium positive strain was picked and placed in YEP liquid culture medium containing 100mg / L spectinomycin and 50mg / L rifampicin, and the culture was shaken at 28℃, 200rpm, for 16h. Take 0.5ml of the above bacterial solution and inoculate it into 50ml YEP liquid culture medium containing the above antibiotics, and incubate at 28℃, 200rpm to make the OD 600 The value reached 1.8-2.0. Collect the bacterial solution in a centrifuge tube, centrifuge at 5000rpm for 5min, discard the supernatant, and resuspend the precipitate with transformation medium until the resuspended liquid OD 600 = 0.8. The Arabidopsis plants to be transformed were placed flat, and the flower buds were immersed in the transformation medium for 10 seconds, followed by dark culture for 16 to 24 hours, and then normal culture was resumed.

[0045] 4. Positive plant screening

[0046] The infected seeds were sterilized with chlorine and cultured on MS medium containing 50 mg / L kanamycin. The resistant positive seedlings were selected and transplanted into the soil. After the seeds were harvested, they were further screened with MS medium containing 50 mg / L kanamycin. The transformed seedlings with a 3:1 separation ratio were selected and planted into the soil. After the seeds were harvested, they were further screened with MS medium containing 50 mg / L kanamycin. The transformed seedlings with 100% kanamycin resistance were selected and further observed for GFP fluorescence ( Figure 3 ), and the positive seedlings with GFP fluorescence were screened out as transgenic positive homozygous lines. The McTRY transgenic homozygous lines were named OE1, OE3, and OE4. RNA was extracted from the leaves of homozygous transgenic positive lines, and its cDNA was obtained by reverse transcription. Quantitative primers were designed, and qRT-PCR verification was performed ( Figure 4). The quantitative primers are as follows: 35S::McTRY-F: 5'-CCTGCCATGGAGAGGTAAGC-3'; 35S::McTRY-R: 5'-GCTGCACTTCTTCCTGGGAT-3'. The 20μL PCR reaction system is: 1μL of upstream and downstream primers, 10μL of 2×GS AntiQ qPCR SYBR Fast Mix, 1μL of cDNA, and 7μL of ddH2O; the PCR reaction is performed using a fluorescent quantitative PCR instrument BIO-RAD CFX-Opus 96. The operating program is based on the instruction manual: 95℃ pre-denaturation for 30s, 95℃ denaturation for 10s, 60℃ annealing and extension for a total of 30s, denaturation, annealing and extension for a total of 40 cycles, and fluorescence signals are collected at 72℃.

[0047] 5. Effect of McTRY gene on epidermal hair formation in transgenic Arabidopsis

[0048] Arabidopsis seeds (WT, OE1, OE3, and OE4) were disinfected with 75% ethanol and 2% sodium hypochlorite solution, placed on 1 / 2MS solid culture medium, and cultured in a light incubator for 7 days. After the seedlings were transferred to soil and grown for 3-4 weeks, the epidermal hairs of the McTRY transgenic Arabidopsis lines and wild-type Arabidopsis were observed using a stereomicroscope.

[0049] The results are as follows Figure 5 As shown, compared with the wild type, the McTRY gene expression level in the transgenic lines OE1 and OE3 was high, and no epidermal hairs were formed on the leaves and stems; the McTRY gene expression level in the transgenic line OE4 was low, and epidermal hairs were still formed on the leaves and stems, and the inhibitory effect on epidermal hair formation was low.

[0050] 6. Regulatory mechanism of McTRY gene on epidermal hair formation in transgenic Arabidopsis leaves

[0051] AtMYB23, AtMYB5, AtGL2, and AtTTG2 are positive regulatory genes for epidermal hair formation. Upregulation of these genes promotes the formation of epidermal hairs, while downregulation reduces the formation of epidermal hairs. AtTRY, AtTCL1, AtTCL2, AtETC2, and AtETC3 are negative regulatory genes for epidermal hair formation. Upregulation of these genes inhibits the formation of epidermal hairs, while downregulation increases the formation of epidermal hairs. The expression levels of genes related to epidermal hair formation (AtMYB23, AtMYB5, AtGL2, AtTTG2, AtTRY, AtTCL1, AtTCL2, AtETC2, and AtETC3) in Arabidopsis wild-type and McTRY transgenic Arabidopsis lines were detected by qRT-PCR.

[0052] The results are as follows Figure 6As shown in the figure, compared with the wild type, AtMYB23, AtMYB5, AtGL2, and AtTTG2 genes were significantly down-regulated in McTRY transgenic Arabidopsis lines, and the degree of expression decrease in transgenic lines OE1 and OE3 was lower than that in transgenic line OE4. There was no significant difference between the OE4 line and the wild type in AtETC3 gene. AtTRY, AtTCL1, AtTCL2, and AtETC2 genes were significantly up-regulated in McTRY transgenic Arabidopsis lines, and the degree of expression increase of these genes in lines OE1 and OE3 was higher than that in OE4. This shows that McTRY can inhibit the formation of epidermal hairs in transgenic Arabidopsis at the molecular level by down-regulating the expression of AtMYB23, AtMYB5, AtGL2, and AtTTG2, and up-regulating the expression of AtTRY, AtTCL1, AtTCL2, AtETC2, and AtETC3.

[0053] 7. Effect of McTRY gene on anthocyanin synthesis in transgenic Arabidopsis

[0054] After disinfection, Arabidopsis seeds (WT, OE1, OE3 and OE4) were planted on 1 / 2MS solid culture medium, placed at 4°C for two days, and then placed in a light incubator for 7 days. After the seedlings were moved to soil and grown for 6 weeks, rosette leaves were collected to extract anthocyanins. The components of the anthocyanin extract include n-propanol, concentrated hydrochloric acid and water (n-propanol: concentrated hydrochloric acid: water / 18:1:81, v:v:v). About 0.2g of plant leaf tissue was weighed, quickly frozen and ground with liquid nitrogen, and then 2ml of anthocyanin extract was added. It was placed at room temperature away from light, extracted overnight, centrifuged at 13000g for 20min, and the supernatant was transferred to a clean centrifuge tube and diluted twice for determination. The absorbance values ​​at 535nm and 650nm were measured using a spectrophotometer, and the relative anthocyanin content per gram of fresh weight of plant tissue was calculated according to the formula 2X(A535-A650).

[0055] The results are as follows Figure 7 As shown, the leaves of the wild type WT are more purple-red than those of strains OE1, OE3, and OE4. OE1 and OE3 have almost no purple-red leaves, while OE4 has a little purple-red leaves ( Figure 7 A). From the color of anthocyanins extracted from OE1, OE3, OE4 and WT, it can be seen that the anthocyanin extract of wild-type WT has the darkest color, while the anthocyanin extract of OE4 is lighter in color. The extracts from OE1 and OE3 have almost no color, indicating that the wild-type WT has the highest anthocyanin content, followed by a small amount of anthocyanins in OE4, and OE1 and OE3 contain almost no anthocyanins ( Figure 7 B) Compared with the wild-type WT Arabidopsis line, the anthocyanin content in McTRY transgenic Arabidopsis lines OE1 and OE3 was very low, while the anthocyanin content in the transgenic line OE4 was slightly higher than that in OE1 and OE3.

[0056] 8. Regulatory mechanism of McTRY gene on anthocyanin synthesis in transgenic Arabidopsis

[0057] The proteins encoded by AtPAL1, AtPAL2, AtC4H, At4CL, AtCHS, AtCHI, AtF3H, AtF3'H, AtDFR, AtLDOX and AtUF3GT genes are important synthases in the anthocyanin synthesis pathway. The upregulation of these gene expressions will promote the synthesis of anthocyanins, and the downregulation of their expressions will lead to a reduction in anthocyanin synthesis. The present invention uses qRT-PCR technology to detect the expression levels of anthocyanin synthesis genes (AtPAL1, AtPAL2, AtC4H, At4CL, AtCHS, AtCHI, AtF3H, AtF3'H, AtDFR, AtLDOX and AtUF3GT) in Arabidopsis wild type and McTRY transgenic Arabidopsis strains.

[0058] The results are as follows Figure 8 As shown in the figure, compared with the wild type, the expression levels of AtPAL1, AtC4H, AtCHS, AtCHI, AtF3H, AtF3'H, AtDFR, AtLDOX and AtUF3GT were significantly down-regulated in McTRY transgenic Arabidopsis lines, and except for AtC4H, the expression levels of other genes in lines OE1 and OE3 were lower than that in OE4. There was no significant difference between AtPAL1 and At4CL in line OE4 and WT, but their expression levels in lines OE1 and OE3 were significantly down-regulated compared with WT.

[0059] The qRT-PCR technology was used to detect the gene expression levels of related transcription factors (AtPAP1, AtPAP2, AtMYB113, AtMYB114, AtMYB4, AtTT2, AtMYB5, AtGL2 and AtTT8) that regulate anthocyanin synthesis. These genes positively regulate anthocyanin synthesis.

[0060] The results are as follows Fig. 9 As shown, the expression levels of AtPAP1, AtPAP2, AtMYB113, AtMYB114, AtMYB4, AtTT2, AtMYB5, AtGL2 and AtTT8 genes in the McTRY transgenic lines were significantly lower than those in the wild type WT, and overall, the expression levels of these genes in lines OE1 and OE3 decreased more than that in OE4.

[0061] Therefore, it was concluded that the McTRY gene down-regulates the expression levels of transcription factors related to anthocyanin synthesis regulation at the molecular level and further reduces the expression of anthocyanin synthesis genes, thereby leading to reduced anthocyanin synthesis in McTRY transgenic Arabidopsis lines.

Claims

1. The mint McTRY gene, whose nucleotide sequence is shown in SEQ ID NO.

1.

2. The expressed protein of the mint McTRY gene according to claim 1, whose amino acid sequence is shown in SEQ ID NO.

2.

3. Use of the mint McTRY gene according to claim 1 in inhibiting the formation of Arabidopsis epidermal hairs.

4. The use of the mint McTRY gene in inhibiting the formation of Arabidopsis epidermal hairs according to claim 3, characterized in that: The following steps are involved: (1) constructing a plant expression vector of the mint McTRY gene; (2) transforming the constructed plant expression vector of the mint McTRY gene into Arabidopsis thaliana; (3) Cultivate and screen transgenic Arabidopsis thaliana without epidermal hair formation.

5. The use of the mint McTRY gene in inhibiting the formation of Arabidopsis epidermal hairs according to claim 4, characterized in that: The plant expression vector is 35S::McTRY.

6. The use of the mint McTRY gene according to claim 1 in inhibiting the expression of positive regulatory genes for epidermal hair formation, characterized in that: The positive regulatory genes for epidermal hair formation are AtMYB23, AtMYB5, AtGL2 and AtTTG2.

7. The use of the mint McTRY gene according to claim 1 in negatively regulating gene expression to promote epidermal hair formation, characterized in that: The negative regulatory genes for epidermal hair formation are AtTRY, AtTCL1, AtTCL2, AtETC2 and AtETC3.

8. Use of the mint McTRY gene according to claim 1 in reducing the anthocyanin content in Arabidopsis thaliana.

9. The use of the mint McTRY gene according to claim 1 in inhibiting the expression of anthocyanin synthesis genes, characterized in that: The anthocyanin synthesis genes are AtPAL1, AtPAL2, AtC4H, At4CL, AtCHS, AtCHI, AtF3H, AtF3'H, AtDFR, AtLDOX and AtUF3GT.

10. The use of the mint McTRY gene according to claim 1 in inhibiting the expression of transcription factors related to anthocyanin synthesis, characterized in that: The anthocyanin synthesis-related transcription factors are AtPAP1, AtPAP2, AtMYB113, AtMYB114, AtMYB4, AtTT2, AtMYB5, AtGL2 and AtTT8.

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