Transcription factor DcMYB56 related to high anthocyanin as well as coding gene and application of transcription factor DcMYB56

Abstract

The invention discloses a transcription factor DcMYB56 related to high anthocyanin as well as a coding gene and application of the transcription factor DcMYB56. The amino acid sequence of the transcription factor DcMYB56 related to high anthocyanin is as shown in SEQ ID No. 1. The nucleotide sequence of the coding gene of the transcription factor DcMYB56 related to high anthocyanin is as shown in SEQ ID No. 2. The invention also provides a recombinant expression vector, an expression cassette or a recombinant thallus of the coding gene of the high anthocyanin related transcription factor DcMYB56. The invention also provides application of the transcription factor DcMYB56 or the coding gene of the transcription factor DcMYB56 in cultivation of high-anthocyanin transgenic plants. The invention also provides an application of a recombinant expression vector, an expression cassette or a recombinant thallus containing the coding gene of the transcription factor DcMYB56 related to the high anthocyanin in cultivation of a high-anthocyanin transgenic plant. According to the invention, the dendrobium officinale DcMYB56 gene is over-expressed, so that a plant with high anthocyanin content is obtained.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a high-anthocyanin-related transcription factor DcMYB56 and a coding gene and application thereof. Background Art

[0002] Dendrobium candidum belongs to the orchid family and mainly grows in tropical and subtropical regions of Asia, Oceania and Europe. According to the Chinese Pharmacopoeia, Dendrobium candidum is a traditional precious Chinese medicinal material. The stems of Dendrobium candidum are rich in flavonoids, polysaccharides, phenanthrene, bibenzyl and polyphenol compounds. Dendrobium candidum is divided into red stem Dendrobium candidum and green stem Dendrobium candidum according to the color of the stems. High-quality Dendrobium candidum stems are rich in anthocyanins and contain a variety of anthocyanin components. Anthocyanins are flavonoid water-soluble pigments. In plants, they can attract insects for pollination and help plants resist a variety of biological and abiotic stresses. They are easily absorbed by the human body and have a positive effect on human health.

[0003] Red stem Dendrobium candidum is rich in anthocyanins, which has become one of the main characteristics of high-quality Dendrobium candidum. High-anthocyanin plant breeding has become an important direction of current breeding. Therefore, the prior art urgently needs a high-anthocyanin-related transcription factor DcMYB56, its encoding gene, and its application in cultivating high-anthocyanin transgenic plants. Summary of the invention

[0004] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a high-anthocyanin-related transcription factor DcMYB56 and its encoding gene and application.

[0005] The objective of the present invention is achieved through the following technical solutions: The present invention first excavates a transcription factor related to high anthocyanin from Dendrobium officinale, marked as DcMYB56, whose amino acid sequence is shown in SEQ ID No.1, with a total of 213 amino acids. The coding gene of the transcription factor related to high anthocyanin DcMYB56, whose nucleotide sequence is shown in SEQ ID No.2, has a full length of 642bp in the coding region.

[0006] The present invention also provides a recombinant expression vector, an expression cassette or a recombinant bacterial cell containing a coding gene of a high-anthocyanin-related transcription factor DcMYB56.

[0007] The present invention also provides the use of transcription factor DcMYB56 or a gene encoding transcription factor DcMYB56 in cultivating high anthocyanin transgenic plants. Further, the plant is Arabidopsis thaliana. The results show that the anthocyanin content in transgenic Arabidopsis thaliana is significantly increased, and the expression level of related genes involved in anthocyanin synthesis is significantly increased.

[0008] The present invention also provides the use of a recombinant expression vector, an expression cassette or a recombinant bacterial cell containing a gene encoding a high-anthocyanin-related transcription factor DcMYB56 in cultivating a high-anthocyanin transgenic plant, wherein the plant is Arabidopsis thaliana.

[0009] The beneficial effect of the present invention is that the present invention regulates the expression of anthocyanin synthesis-related genes by overexpressing the Dendrobium officinale DcMYB56 gene, and further regulates anthocyanin synthesis, thereby obtaining plants with high anthocyanin content, which has great production and application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 The results of RNA agarose gel electrophoresis; Figure 2 Agarose gel test results of PCR products amplified from DcMYB56 gene; Figure 3 This is the detection image of PCR colony agarose gel electrophoresis; Figure 4 The phenotypes and anthocyanin contents of the two species of Dendrobium officinale are shown; Figure 5 is the expression level of DcMYB56 gene in two species of Dendrobium officinale; Figure 6 Overexpression of the DcMYB56 gene in Dendrobium officinale increases the anthocyanin content in Arabidopsis thaliana. DETAILED DESCRIPTION

[0011] The present invention is described in detail below with reference to the accompanying drawings.

[0012] Observe the phenotypes of red stem Dendrobium officinale and green stem Dendrobium officinale, such as Figure 4 In A, there are two plants on the left and right. The left plant is a red-stem Dendrobium officinale, and the right plant is a green-stem Dendrobium officinale. There are obvious phenotypic differences between the green-stem Dendrobium officinale and the red-stem Dendrobium officinale. The stems and leaves of the red-stem Dendrobium officinale are red, while the stems and leaves of the green-stem Dendrobium officinale are green.

[0013] UPLC MS / MS was used to detect the content and types of anthocyanins in green and red stems of Dendrobium officinale. Figure 4B shows a bar graph obtained by UPLC-MS / MS determination of the types and contents of anthocyanins in two types of Dendrobium officinale, where Del represents delphinidin, Del-3-G represents delphinidin-3-O-β-D-glucoside, Del-3,5-D represents delphinidin-3,5-diglucoside, Del-3-R represents delphinidin-3-O-rutinoside, Mal represents malvidin, Mal-3,5-D represents malvidin glycoside, Mal-3-G represents malvidin-3-galactoside, Pet represents petunia pigment, and Pet-3- G represents petunia cyanidin-3-O-β-D-glucoside, Cya represents cyanidin, Cya-3,5-D represents cyanidin-3,5-diglucoside, Cya-3-G represents cyanidin glucoside, Pel-3-gal represents pelargonidin, Pel represents pelargonidin glycoside, Pel-3-G represents asteridin, Peo represents peonydin, Peo-3,5-D represents peonydin-3,5-diglucoside, Peo-3-G represents peonydin-3-O-glucoside; biological determination was repeated three times (***p < 0.001, *p < 0.05); the results showed that the types and contents of anthocyanins in red-stem Dendrobium officinale were significantly higher than those in green-stem Dendrobium officinale.

[0014] By designing corresponding fluorescence quantitative PCR primers, the gene expression levels of DcMYB56 gene related to high anthocyanin in green stem Dendrobium officinale and red stem Dendrobium officinale were detected. Figure 5 As shown, the results showed that the expression level of this gene in red-stem Dendrobium officinale was 410 times that in green-stem Dendrobium officinale, and this gene is involved in the regulation of anthocyanin synthesis.

[0015] Example 1 Obtaining the full-length CDS sequence of the DcMYB56 gene of Dendrobium officinale The reference sequence was downloaded directly from the NCBI database (XP_020698982). The gene sequence shown in SEQ ID NO: 1 was used to design primers using the IDTDNA online tool (https: / / sg.idtdna.com / pages / tools / primerquest), and the upstream and downstream primers were as follows: Upstream primer DcMYB56-F1: 5'-ATG GAA ACT AGA GAG CTT CAT AAT GTG -3' (SEQ ID No. 3); Downstream primer DcMYB56-R2: 5'- CTA AGT TGC TCC AAC ACC TAA GAA ATC AAT G-3' (SEQ ID No. 4).

[0016] The primers used in the present invention were synthesized by Beijing Qingke Biotechnology Co., Ltd.

[0017] Cloning of DcMYB56 gene 1. RNA extraction and detection 1.1 RNA extraction Use the EASYspin Universal Plant RNA Rapid Extraction Kit / DNase I from Beijing Adele Company, and the operation steps are as follows: 1) Take the leaf tissue of Dendrobium officinale, grind it into fine powder in liquid nitrogen, take 100 mg of the fine powder and transfer it into a centrifuge tube containing 500 μL lysis solution RPA, and immediately shake it vigorously by hand for 20 seconds to fully lyse it; 2) Electric homogenization for 30 seconds to help lyse and shear DNA, reduce viscosity and increase yield; 3) Centrifuge the lysate at 13,000 rpm for 8 minutes to precipitate the insoluble fragments; 4) Transfer the supernatant of the lysate to a new centrifuge tube and add half the volume of anhydrous ethanol. When a precipitate appears, mix it immediately. 5) Add the mixture (less than 720 μL each time) to an adsorption column RA (the adsorption column is placed in a collection tube), centrifuge at 13,000 rpm for 2 minutes, and discard the waste liquid; 6) Add 350 μL of deproteinized solution RW1, place at room temperature for 1 minute, centrifuge at 13,000 rpm for 30 seconds, and discard the waste liquid; 7) Add 50 μl of DNase I working solution to the center of the adsorption column RA and leave at room temperature (20-30°C) for 15 minutes; 8) Add 350 μl of deproteinized solution RW1 to the adsorption column RA, centrifuge at 12,000 rpm for 30 seconds, discard the waste liquid, and return the adsorption column to the collection tube; 9) Add 500 μL of Rinse Buffer RW, centrifuge at 13,000 rpm for 30 seconds, discard the waste liquid, add 500 μL of Rinse Buffer RW, and repeat; 10) Place the adsorption column RA back into the empty collection tube and centrifuge at 13,000 rpm for 2 minutes to remove as much rinse solution as possible to prevent residual ethanol in the rinse solution from inhibiting downstream reactions; 11) Take out the adsorption column RA and put it into an RNase free centrifuge tube. Add 30-50 μL RNase free water to the middle of the adsorption membrane according to the expected RNA yield. Incubate at room temperature for 1 minute and centrifuge at 12,000 rpm for 1 minute.

[0018] 2.2 RNA detection 1) Detection using an ultra-trace nucleic acid detector (DeNovix-DS-11+) Use a micropipette to draw 1 μL RNA, click into the measurement hole, click the measurement button, and the result shows that the RNA concentration is: 995.838 ng / μL, A260 / 230=2.326, A260 / 280=2.202. The RNA concentration and purity fully meet the requirements of the subsequent reverse transcription experiment. (2) Agarose gel electrophoresis detection The extracted RNA was detected by 1% agarose gel electrophoresis. Figure 1 As shown, the RNA bands are clear, the gel wells are clean, and there is no tailing degradation phenomenon, which further indicates that the extracted RNA can be used for the next reverse transcription experiment.

[0019] 2.3 PCR amplification Reverse transcription method: 1) Thaw the RNA template and the components in the kit on ice for later use; 2) Prepare the reaction system on ice in a nuclease-free microcentrifuge tube as shown in Table 1:

[0020] 3) Use a pipette to mix the system and centrifuge briefly, then incubate at 65°C for 5 min; 4) The reaction solution was quickly cooled on ice, centrifuged briefly, and then the components shown in Table 1 were added:

[0021] 5) Use a pipette to mix the system and centrifuge briefly, then incubate at 50°C for 30-50 min; 6) Incubate at 85℃ for 5 min and refrigerate the reverse transcription product for later use.

[0022] DcMYB56 gene coding region (CDS) cloning Amplification was performed using 2×Q5 hot start ultra-fidelity polymerase premix, the PCR system is shown in Table 3, and the reaction procedure is shown in Table 4;

[0023] The PCR amplification products of DcMYB56 gene were detected by 1% agarose gel. The PCR amplification products of DcMYB56 gene were detected by 1% agarose gel. The detection results are shown in Figure 2 Lane 1 has a bright and clear band near 600 bp, which is similar in size to the target band. It is inferred that this band is the target PCR product. The PCR product is further recovered by gel excision.

[0024] PCR product gel excision recovery steps: 1) Cut the target DNA band of DcMYB56 gene from the agarose gel, put it into a clean centrifuge tube, weigh and calculate the gel weight, the gel weight is about 200 mg; 2) Add 200 μl Buffer PG to the gel block; 3) Incubate in a 50°C water bath until the gel is completely dissolved; 4) Column equilibration: Add 200 μl Buffer PS to the adsorption column (Spin Columns DM) in the collection tube, centrifuge at 13,000 rpm for 1 minute, discard the waste liquid in the collection tube, and put the adsorption column back into the collection tube; 5) Add the solution obtained in step 3 to the adsorption column in the collection tube, place at room temperature for 2 minutes, centrifuge at 13,000 rpm for 1 minute, pour out the waste liquid in the collection tube, and put the adsorption column back into the collection tube; 6) Add 450 μl Buffer PW to the adsorption column, centrifuge at 13,000 rpm for 1 minute, discard the waste liquid in the collection tube, and put the adsorption column back into the collection tube; 7) Repeat step 6; 8) Centrifuge at 13,000 rpm for 1 minute and discard the waste liquid in the collection tube; 9) Place the adsorption column in a new 1.5 ml centrifuge tube, add 50 μl of Buffer EB to the middle of the adsorption membrane, and leave it at room temperature for 2 minutes; centrifuge at 13,000 rpm for 1 minute to collect the DNA solution; and store at -20°C.

[0025] 2. Construction of Sequencing Vector DcMYB56 gene coding region gene fragment connection vector: The cloning reaction system catalyzed by Topoisomerase is shown in Table 5:

[0026] After adding the reagents in Table 5, mix them by gently blowing with a pipette, centrifuge at low speed to collect all the liquid at the bottom of the centrifuge tube, and carry out the ligation reaction at room temperature (20℃-30℃) for 5 minutes.

[0027] Transformation of ligation products: 1) Take 100 μL of competent cells melted on ice, add the ligation product, mix gently, and let stand on ice for 30 min; 2) Heat shock in a 42℃ water bath for 60s, quickly transfer to an ice bath, and let stand for 2 min. Do not shake the sample during the standing time on ice. 3) Add 700 μL of sterile liquid LB medium without antibiotics to the centrifuge tube, mix well and resuscitate at 37°C, 200 rpm for 60 min; 4) Take 700 μL of the recovery solution and spread it evenly on the LB medium containing Amp antibiotics, then place the plate upside down in a 37°C incubator for overnight culture.

[0028] 3.DcMYB56 gene sequencing verification Positive clone identification: The LB medium plate cultured overnight was taken out, and it was found that the plate was evenly covered with single clones. Two clones were randomly selected for colony PCR identification. The reaction system was shown in Table 6, and the reaction procedure was shown in Table 7.

[0029]

[0030] The colony PCR results were tested by 1% agarose gel electrophoresis. Figure 3 As shown, the detection found that the sizes of the amplified fragments of the two randomly selected clones were consistent with expectations, and it can be judged that the ligation reaction was correct.

[0031] To further confirm that the fragment of the DcMYB56 gene was correct, a correct clone was selected and sent to Qingke Biotechnology Co., Ltd. for first-generation sequencing. After analysis of the sequencing results, it was shown that the sequence was completely consistent with SEQ ID NO: 1. At the same time, the sequencing plasmid was saved for use in subsequent steps.

[0032] Example 2 Subcellular localization of DcMYB56 gene 1. Using the correctly sequenced positive clone plasmid obtained in Example 1 as a template, the ORF region of DcMYB56 was amplified using the following primers: DcMYB56-GFP-F1: 5'-CCT ACT AGT ATG GAA ACT AGA GAG CTT CAT AAT GTG-3' (SEQ ID No. 5); DcMYB56-GFP-R1: 5'-CCT ACG CGT AGT TGC TCC AAC ACC TAA GAA ATC AAT G-3' (SEQ ID No. 6).

[0033] The PCR amplification reaction system is shown in Table 8, and the reaction procedure is shown in Table 9.

[0034]

[0035] The PCR amplification product was electrophoresed on a 1% agarose gel at 5 v / cm for 20 minutes. The electrophoresis fragment of the DcMYB56 gene PCR amplification was cut under ultraviolet light and recovered using a gel recovery kit. The specific steps for PCR product gel cutting and recovery are as follows: 1) Cut the target DNA band of DcMYB56 gene from the agarose gel, put it into a clean centrifuge tube, weigh and calculate the gel weight, the gel weight is about 200 mg; 2) Add 200 μl Buffer PG to the gel block; 3) Incubate in a 50°C water bath until the gel is completely dissolved; 4) Column equilibration: Add 200 μl Buffer PS to the adsorption column (Spin Columns DM) in the collection tube, centrifuge at 13,000 rpm for 1 minute, discard the waste liquid in the collection tube, and put the adsorption column back into the collection tube; 5) Add the solution obtained in step 3 to the adsorption column in the collection tube, place at room temperature for 2 minutes, centrifuge at 13,000 rpm for 1 minute, pour out the waste liquid in the collection tube, and put the adsorption column back into the collection tube; 6) Add 450 μl Buffer PW to the adsorption column, centrifuge at 13,000 rpm for 1 minute, discard the waste liquid in the collection tube, and put the adsorption column back into the collection tube; 7) Repeat step 6; 8) Centrifuge at 13,000 rpm for 1 minute and discard the waste liquid in the collection tube; 9) Place the adsorption column in a new 1.5 ml centrifuge tube, add 50 μl of Buffer EB to the middle of the adsorption membrane, and leave it at room temperature for 2 minutes; centrifuge at 13,000 rpm for 1 minute to collect the DNA solution; and store at -20°C.

[0036] 2. Connect the recovered product of the DcMYB56 gene shown in SEQ ID No. 1 to the 3302Y3 vector containing GFP (Greenfluorescence protein), and digest it with SpeI / MluI. Purify the vector digestion product and the recovered fragment digestion product together with a PCR purification kit. Digest the PCR reaction system shown in Table 10 at 37°C for 1h. Purify the vector digestion product with a PCR purification kit (recover the plasmid after double digestion), reconstitute it at 16°C for 2h according to the recombinant PCR reaction system shown in Table 11, and transform the ligation product into competent cells.

[0037]

[0038] 3. Transform competent cells with 15 μL of the ligation product, transform to a resistant plate containing kanamycin, and culture at 37°C for 12 hours. Pick 10 plaques for colony PCR identification. The identification primers are DcMYB56-GFP-F1 and DcMYB56-GFP-R1 in Example 1. Sequence the bacterial solution corresponding to the three positive bands and extract the target plasmid. The PCR identification reaction system is shown in Table 12, and the reaction procedure is shown in Table 13.

[0039]

[0040] 4. The obtained recombinant expression vector with the correct sequence was named 3302Y3-DcMYB56-GFP. The constructed GFP fusion protein was transiently expressed in tobacco leaves. At the same time, tobacco leaves transformed with the 3302Y3-GFP empty vector were used as controls. GFP fluorescence was detected using a laser confocal microscope. The specific steps are as follows: The constructed fusion vector 3302Y3-DcMYB56-GFP was transformed into Agrobacterium GV3101, and then injected into tobacco (Nicotiana Benthamiana) leaves to transiently express it in epidermal cells to determine the subcellular localization of DcMYB56. 4-Diamino-2-phenylindole (DAPI) staining was used to observe the fluorescence signal in tobacco leaves using a laser confocal microscope (SP8, Leica Microsystems). The results showed that the green fluorescent protein signal of DcMYB56 fused to GFP overlapped with the signal of DAPI staining, indicating that DcMYB56 was localized in the cell nucleus. In summary, when the recombinant expression vector 3302Y3-DcMYB56-GFP vector was transferred into tobacco leaves, the expressed DcMYB56 protein was specifically localized in the cell nucleus. This result indicates that DcMYB56 is a nuclear-localized protein.

[0041] Example 3 Obtaining DcMYB56 Gene-Transgenic Arabidopsis and Detecting Its Anthocyanin Content The gene sequence shown in SEQ ID NO: 1 was used to design primers using the IDTDNA online tool (https: / / sg.idtdna.com / pages / tools / primerquest). The upstream and downstream primers are as follows: Upstream primer DcMYB56-F2: 5'- CCT ACT AGT ATG GAA ACT AGA GAG CTT CAT AAT GTG -3' (SEQ IDNo.7); Downstream primer DcMYB56-R2: 5'- CCT ACG CGT CTA AGT TGC TCC AAC ACC TAA GAA ATC AAT G-3' (SEQ ID No. 8).

[0042] The above primers were synthesized by Beijing Qingke Biotechnology Co., Ltd.

[0043] Primers DcMYB56-F2 and DcMYB56-R2 were used, cDNA was used as a template, and high-fidelity DNA polymerase (Q5 High-Fidelity DNA Polymerase, NEB) was used for PCR amplification. After amplification, 1% agarose gel electrophoresis was performed to recover the target fragment. The specific steps are as follows: 1) Cut the target DNA band of DcMYB56 gene from the agarose gel, put it into a clean centrifuge tube, weigh and calculate the gel weight, the gel weight is about 200 mg; 2) Add 200 μl Buffer PG to the gel block; 3) Incubate in a 50°C water bath until the gel is completely dissolved; 4) Column equilibration: Add 200 μl Buffer PS to the adsorption column (Spin Columns DM) in the collection tube, centrifuge at 13,000 rpm (-16,200 × g) for 1 minute, discard the waste liquid in the collection tube, and put the adsorption column back into the collection tube; 5) Add the solution obtained in step 3 to the adsorption column in the collection tube, place at room temperature for 2 minutes, centrifuge at 13,000 rpm for 1 minute, pour out the waste liquid in the collection tube, and put the adsorption column back into the collection tube; 6) Add 450 μl Buffer PW to the adsorption column, centrifuge at 13,000 rpm for 1 minute, discard the waste liquid in the collection tube, and put the adsorption column back into the collection tube; 7) Repeat step 6; 8) Centrifuge at 13,000 rpm for 1 minute and discard the waste liquid in the collection tube; 9) Place the adsorption column in a new 1.5 ml centrifuge tube, add 50 μl of Buffer EB to the middle of the adsorption membrane, and leave it at room temperature for 2 minutes; centrifuge at 13,000 rpm for 1 minute to collect the DNA solution; and store at -20°C.

[0044] The enzyme digestion and ligation reaction steps of the overexpression vector are as follows: The 3302Y3 vector plasmid stored in the laboratory was used to transform Arabidopsis thaliana. The 3302Y3 vector plasmid and the full-length recovered fragment of DcMYB56 were double-digested with the high-fidelity restriction endonucleases SpeⅠ and MluⅠ from New England Biolabs (NEB), respectively, at 37°C for 3-4h, and then the product was recovered using a gel recovery kit. The steps were the same as above. The enzyme digestion reaction system is shown in Table 14;

[0045] The DNA fragment and the vector were ligated using T4 DNA ligase (NEB) at 16 °C for 2 h. The reaction system is shown in Table 15;

[0046] The ligation product was transformed into Escherichia coli competent cells DH5α, and finally spread on LB solid culture medium containing kanamycin (50 mg / L) and cultured at 37°C for 12 hours.

[0047] When colonies grow on the plate and are of appropriate size, single clones are picked and placed in LB liquid culture medium containing kanamycin (50 mg / L), placed in an incubator for shaking culture, and the incubator setting parameters are 37°C 200rpm. After the bacterial solution becomes turbid, take it out for PCR reaction (see Table 16 for the reaction system and Table 17 for the reaction program). The PCR reaction products are identified by 1% agarose gel electrophoresis, and finally the screened positive clones are sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing.

[0048]

[0049] Plasmid extraction: Collect the cells of the correctly sequenced bacterial solution and use the Kangwei Century Plasmid Extraction Kit to extract the plasmid. The specific steps are as follows: 1) Take 4 ml of overnight culture solution and add it to a centrifuge tube. Centrifuge at 12000 rpm for 1 minute to collect the bacterial precipitate and discard the supernatant. 2) Add 250 µl of Buffer P1 (with RNase A added) to the centrifuge tube containing the bacterial pellet and mix thoroughly with a pipette to suspend the bacterial pellet; 3) Add 250 µl of Buffer P2 to the centrifuge tube and gently mix by inverting 4-6 times to thoroughly lyse the bacteria. 4) Add 350µl Buffer N3 to the centrifuge tube and immediately mix by gently inverting up and down 8-10 times. Mix thoroughly until a white flocculent precipitate appears. Centrifuge at 12000rpm for 10 minutes. 5) Transfer the supernatant obtained in step 4 to the adsorption column (Spin Columns DM) in the collection tube, centrifuge at 12000rpm for 1 minute, pour out the waste liquid in the collection tube, and put the adsorption column back into the collection tube; 6) Add 150 µl Buffer PB to the adsorption column and centrifuge at 12,000 rpm for 1 minute; 7) Add 400 µl Buffer PW to the adsorption column, centrifuge at 12,000 rpm for 1 minute, and discard the waste liquid in the collection tube; 8) Place the adsorption column in a new centrifuge tube, add 50µl Buffer EB to the middle of the adsorption membrane, centrifuge at 12000rpm for 1 minute, collect the plasmid solution into the centrifuge tube, and store the plasmid at -20℃.

[0050] Transformation of Agrobacterium competent cells (GV3101) The transformation method of Agrobacterium tumefaciens GV3101 is as follows: Place the competent cells on ice and slowly thaw. Add 3 µL of plasmid DNA to 100 µL of Agrobacterium competent cells and mix thoroughly. Place the centrifuge tube in liquid nitrogen and freeze for 5 min. After taking it out, place it in a 37 ℃ water bath for heat shock for 5 min. Finally, add 900 µL of LB liquid culture medium and shake and culture it in a shaker at 30℃ and 220rpm for 2h. Take 300 µL of the transformation product and spread it on LB solid culture medium containing 50 µg / mL Kan and 25 µg / mL Amp, and culture it at 30℃ for 2d.

[0051] Obtaining Arabidopsis thaliana transformed with DcMYB56 gene: The Arabidopsis thaliana transformation method adopts the inflorescence immersion method (Clough and Bent, 1998). The fresh Agrobacterium cultured overnight was centrifuged at 4000 g for 10 min at room temperature, the supernatant was discarded, and the Arabidopsis inflorescence was resuspended and diluted to an OD600 value of 0.8 with transformation solution (5% sucrose, 200 µL / L Swilet-77). Then the Arabidopsis inflorescence was immersed in the bacterial solution for transformation. The seeds obtained from the transformed Arabidopsis thaliana were the T1 generation. The T1 generation seeds were screened in the culture soil containing glufosinate-ammonium (60 mg / L), and the transgenic plants contained the herbicide resistance gene Bar. When the plants grew for 4-5 weeks, the phenotype was observed and the anthocyanin content was determined.

[0052] Overexpression of DcMYB56 increases the content of anthocyanins in Arabidopsis. In this experiment, the model plant Arabidopsis was used to verify the role of DcMYB56 in the regulation of anthocyanin biosynthesis. In wild-type Arabidopsis, the CaMV35S promoter drove DcMYB56 overexpression. Then, its gene expression and anthocyanin content were detected in transgenic plants. In this experiment, a total of two DcMYB56 overexpression (DcMYB56-OE) lines were obtained, and OE1 and OE2 were analyzed.

[0053] See also Figure 6 , Figure 6 A is the RT-PCR quantitative analysis of DcMYB56 overexpression in wild-type Arabidopsis thaliana, AtActin is a housekeeping gene; Figure 6 B shows 20-day-old wild-type Arabidopsis seedlings (left), DcMYB56-OE #1 seedlings (middle, DcMYB56 overexpression line 1), and DcMYB56-OE #2 seedlings (right, DcMYB56 overexpression line 2); Figure 6 C is the anthocyanin content of wild-type Arabidopsis, DcMYB56-OE#1 and DcMYB56-OE#2 plants; Figure 6 D is the expression of anthocyanin biosynthesis-related genes in wild-type Arabidopsis plants, DcMYB56-OE#1 and DcMYB56-OE#2 plants. The gene expression level of wild-type Arabidopsis is 1, AtActin is a housekeeping gene, and the data are the average of three biological replicates of the above plants (**p < 0.01).

[0054] The results showed that the expression level of DcMYB56 in the overexpression lines was significantly higher than that in the wild-type plants, and the leaf color of the overexpression lines was darker than that of the wild-type plants (see Figure 6 A. Figure 6 B). In addition, the anthocyanin content of the overexpression line was significantly higher than that of the wild-type plant (see Figure 6 C). qRT-PCR was used to detect the transcription levels of anthocyanin biosynthesis-related genes in wild-type and DcMYB56-OE plants. AtCHS, AtF3H, AtF3'H, AtDFR, AtANS, and AtUF3GT were upregulated in DcMYB56-OE plants (see Figure 6 D). Therefore, DcMYB56 positively regulates anthocyanin biosynthesis.

[0055] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions of the technical solution of the present invention by ordinary technicians in this field do not deviate from the essence and scope of the technical solution of the present invention.

Claims

1. A high anthocyanin-related transcription factor DcMYB56, characterized in that: Its amino acid sequence is shown in SEQ ID No.

1.

2. The coding gene of the high anthocyanin-related transcription factor DcMYB56 according to claim 1, characterized in that: Its nucleotide sequence is shown in SEQ ID No.

2.

3. A recombinant expression vector, expression cassette or recombinant bacterial cell containing the coding gene of the high-anthocyanin-related transcription factor DcMYB56 according to claim 2.

4. Use of the transcription factor DcMYB56 according to claim 1 or the encoding gene according to claim 2 in cultivating high anthocyanin transgenic plants.

5. The use according to claim 4, characterized in that: The plant is Arabidopsis thaliana.

6. Use of the recombinant expression vector, expression cassette or recombinant bacteria containing the coding gene of the high-anthocyanin-related transcription factor DcMYB56 as claimed in claim 3 in cultivating high-anthocyanin transgenic plants, wherein the plant is Arabidopsis thaliana.

Images