Transcription factor MaMYB4 and its application in co-regulating scopoletin biosynthesis with MaTT8 gene

Abstract

The present invention relates to the field of biotechnology, and in particular to a gene MaMYB4 of Melilotus sutchuenensis and its application. The present invention provides a transcription factor MaMYB4 that promotes the production of scopoletin in plant organs and its application. The MaMYB4 of the invention is a MYB family transcription factor, with a complete coding frame length of 870 bases and an encoded protein containing 290 amino acids. Overexpression of MaMYB4 causes the accumulation of scopoletin in the hairy roots of Melilotus sutchuenensis, and at the same time, MaTT8 can interact with MaMYB4 to jointly promote the production of scopoletin. The results are of great significance for enriching the basic theory of coumarin secondary metabolite accumulation, improving the quality defects of existing Melilotus sutchuenensis, and improving the breeding efficiency of high-quality Melilotus sutchuenensis.

Description

[Technical field]

[0001] The invention relates to the field of biotechnology, and in particular to an application of a transcription factor MaMYB4 and a MaTT8 gene in synergistically regulating scopoletin biosynthesis. [Background technology]

[0002] The coumarin scopoletin and its glycosylated form scopoletin are natural products with aromatic odors. They are important secondary metabolites derived from the phenylpropanoid metabolic pathway. They exist in plants in free form or in the form of glycosides and play an important role in plant growth and development and response to adverse stress. They are not only beneficial for plants to resist herbivorous insects and pathogens, regulate the structure of microbial communities and promote plant health, but also act as scavengers of reactive oxygen species and play an important role in abiotic stress. As plant secondary metabolites are increasingly used, they have become the focus of global attention, and research related to the biosynthesis of plant secondary metabolites is increasing.

[0003] Melilotus alba is rich in secondary metabolites such as scopoletin, but the genes and molecular mechanisms related to the biosynthesis of scopoletin have not yet been revealed. It is of great value to explore the genes related to the biosynthesis of scopoletin in Melilotus alba as candidate genes for the biosynthesis of plant secondary metabolites.

[0004] As one of the largest and most important gene families in plants, MYB transcription factors mainly regulate primary and secondary metabolism and the response of plants to stress. MYB transcription factors can bind to the promoters of genes related to phenylpropanoid synthesis and activate the activity of phenylpropanoid synthesis genes. Salvia miltiorrhiza SmPAP1 can activate the expression of PAL genes in the phenylpropanoid metabolic pathway and anthocyanin synthesis-related genes C4H and CHS, positively regulating anthocyanin biosynthesis. Rice R2R3-class MYB transcription factor OsC1 significantly upregulates the expression of anthocyanin synthesis-related genes (PAL, CHI, CHS, F3H, F3'H, DFR, ANS) and increases anthocyanin synthesis. Therefore, MYB regulates phenylpropanoid metabolism by regulating the expression of key enzyme genes in the phenylpropanoid biosynthesis pathway. bHLH transcription factors often interact with MYB family proteins to form complexes, thereby regulating the expression of downstream target genes. TT8 interacts with MYB proteins containing WD40 repeat sequences of TTG1 to form a transcriptional regulatory complex that activates anthocyanin biosynthesis genes. GmMYBA2 interacts with GmTT8a and directly activates anthocyanin biosynthesis genes through the MBW complex. However, there are few reports on the involvement of transcription factor MYB in the regulation of scopolamine. Only antagonistic transcription factor MYB12 promotes flavonol synthesis, and MYB4 inhibits flavonol synthesis to promote scopolamine production. At present, there is no report on the function and molecular regulatory mechanism of MYB4 in regulating scopolamine synthesis. [Summary of the invention]

[0005] In view of the above, it is necessary to provide a transcription factor MaMYB4 that promotes the production of scopoletin in plant organs and its application. This invention obtains the MaMYB4 gene that has a positive regulatory effect on the MaUGT79 promoter and the gene MaTT8 that interacts with it by studying the MYB family transcription factors. Through research, it is concluded that the above genes have a good promoting effect on the accumulation of scopoletin in white sweet clover, which is of great significance for practical applications such as improving the quality defects of existing white sweet clover and improving the breeding efficiency of high-quality white sweet clover.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is:

[0007] The invention includes the application of transcription factor MaMYB4 or a gene encoding transcription factor MaMYB4 in regulating the synthesis of scopoletin in the hairy roots of Melilotus officinalis. The amino acid sequence of the transcription factor MaMYB4 is shown in SEQ ID NO.1.

[0008] Furthermore, the application is the application of the transcription factor MaMYB4 or the gene encoding the transcription factor MaMYB4 to activate the MaUGT79 gene and / or the MaUGT79 promoter.

[0009] Furthermore, the nucleotide sequence encoding the transcription factor MaMYB4 is shown as SEQ ID NO.2.

[0010] Furthermore, co-transformation of the transcription factor MaMYB4 or the gene encoding the transcription factor MaMYB4 with MaTT8 can significantly enhance the synthesis of scopoletin in the hairy roots of Melilotus officinalis. The amino acid sequence of MaTT8 is shown in SEQ ID NO.3.

[0011] Furthermore, co-transformation of the transcription factor MaMYB4 or the gene encoding the transcription factor MaMYB4 with MaTT8 can significantly enhance the effect of activating the MaUGT79 gene and / or the MaUGT79 promoter; the amino acid sequence of the MaTT8 is shown in SEQ ID NO.3.

[0012] Furthermore, the nucleotide sequence encoding the transcription factor MaTT8 is shown as SEQ ID NO.4.

[0013] The present invention also includes an expression vector, which contains the coding sequence SEQ ID NO.2 of the transcription factor MaMYB4 gene.

[0014] The present invention also includes a method for promoting the synthesis of scopoletin in Melilotus officinalis and / or activating the MaUGT79 promoter using the MaMYB4 expression vector, characterized in that the method is:

[0015] (1) Using the recombinant plasmid T-MaMYB4 plasmid as a template, PCR amplification was performed using primers P5 / primer P6 with a high-fidelity enzyme; the MaMYB4 gene fragment was recovered using a PCR product purification kit;

[0016] (2) Digesting the plant overexpression vector plasmid pBI121 with XbaI and BamHI to obtain a linearized vector of pBI121;

[0017] (3) connecting the MaMYB4 gene fragment of step (1) and the linearized vector of pBI121 to obtain the plant overexpression vector pBI121-MaMYB4;

[0018] (4) using an electroporation method to transform the plant overexpression vector pBI121-MaMYB4 of step (3) into Agrobacterium rhizogenes K599, and then infecting the hairy roots of the sweet clover with the Agrobacterium rhizogenes K599 to obtain;

[0019] The sequence of the primer P5 is shown in SEQ ID NO.9, and the sequence of the primer P6 is shown in SEQ ID NO.10.

[0020] The present invention also includes a method for promoting the synthesis of scopoletin in Melilotus officinalis and / or activating the MaUGT79 promoter by co-transforming the MaMYB4 gene and the MaTT8 gene, characterized in that the method comprises:

[0021] (1) MaMYB4 and MaTT8 overexpression vectors were constructed and named OE-MaMYB4 and OE-MaTT8, respectively; then, the rhizogenes Agrobacterium K599 was transformed by electroporation to obtain different Agrobacterium;

[0022] (2) After germination of 7 days, the root tip of the sweet clover seeds was cut off with a scalpel, and then lightly dipped into the bacterial film of Agrobacterium transformed with the OE-MaMYB4 vector and the Agrobacterium transformed with the OE-MaTT8 vector for infection culture to obtain OE-MaTT8+OE-MaMYB4.

[0023] The present invention has the following beneficial effects:

[0024] The present invention provides a transcription factor MaMYB4 that promotes the production of scopoletin in plant organs and its application. The MaMYB4 of the invention is a MYB family transcription factor, with a complete coding frame length of 870 bases and a coded protein containing 290 amino acids. Overexpression of MaMYB4 causes the accumulation of scopoletin in the hairy roots of Melilotus officinalis, and at the same time, MaTT8 can interact with MaMYB4 to jointly promote the production of scopoletin. The results are of great significance for enriching the basic theory of coumarin secondary metabolite accumulation, improving the quality defects of existing Melilotus officinalis, and improving the breeding efficiency of high-quality Melilotus officinalis.

Brief Description of the Drawings

[0025] Figure 1 Yeast one-hybrid assay results;

[0026] Figure 2 Figure 1. Results of dual luciferase reporter gene experiment.

[0027] Figure 3 Fluorescence expression of MaMYB4 gene in different parts of Melilotus officinalis.

[0028] Figure 4 The figure is the result of PCR identification of hairy roots of pBI121-MaMYB4 and RNAi-MaMYB4 genes; M in the figure is DL2000 DNA Marker; (+) is the positive control, lanes 2-7 are the PCR products of pBI121-MaMYB4 transgenic hairy roots; lanes 8-17 are the PCR products of RNAi-MaMYB4 transgenic hairy roots;

[0029] Figure 5 This is the relative expression result of MaMYB4 after different Agrobacterium infection of the hairy roots of Melilotus officinalis.

[0030] Figure 6 This is a graph showing the scopoletin content in the hairy roots of Clerodendrum tumefaciens infected with different Agrobacterium species.

[0031] Figure 7 This is a diagram of the yeast two-hybrid experiment results;

[0032] Figure 8 This is the result of the bimolecular fluorescence complementation experiment;

[0033] Fig. 9 This is the expression result of gene MaUGT79 after MaMYB4 and MaTT8 co-transformed into the hairy roots of Melilotus officinalis.

[0034] Fig.10 This is the result of co-transformation of MaMYB4 and MaTT8 into the hairy roots of Melilotus officinalis. [Specific implementation method]

[0035] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific embodiments of the present invention are described in detail below in conjunction with the accompanying drawings. In the following description, many specific details are set forth to facilitate a full understanding of the present invention. However, the present invention can be implemented in many other ways different from those described herein, and those skilled in the art can make similar improvements without violating the connotation of the present invention, so the present invention is not limited by the specific implementation disclosed below.

[0036] Embodiment 1:

[0037] Screening of transcription factors regulating scopolamine synthesis

[0038] In this example, the yeast one-hybrid system was used to screen the regulatory protein of the MaUGT79 gene using the promoter fragment of the glycosyltransferase gene family member of the key gene in the scopoletin biosynthesis pathway of Melilotus officinalis. In the results of the yeast one-hybrid experiment, the transcription factor MaMYB4 that may regulate the biosynthesis of scopoletin was screened.

[0039] Subsequently, MaMYB4 was used as the object for follow-up functional studies.

[0040] The results are as follows Figure 1 , Figure 2 shown by Figure 1 Yeast one-hybrid analysis showed that MaMYB4 protein could bind to the MaUGT79 promoter and interact with the MaUGT79 gene promoter sequence.

[0041] like Figure 2 The dual luciferase reporter gene experiment showed the LUC / Rluc value in tobacco leaves injected with Agrobacterium. The enzyme activity of Rlun was used as an internal reference, and the ratio of LUC and Rluc enzyme activities reflected the interaction between MaMYB4 protein and the promoter sequence of the MaUGT79 gene. After tobacco was injected with Agrobacterium containing the pGreenII 0800-MaUGT79 promoter+35S-MaMYB4 recombinant plasmid, the enzyme activity of LUC in the leaves was 2.7 times that of Rluc, indicating that MaMYB4 protein can activate the expression of MaUGT79.

[0042] In summary, it was shown that MaMYB4 is a positive regulatory factor that can directly bind to the promoter of the scopolamine biosynthesis gene.

[0043] Embodiment 2:

[0044] Cloning and expression analysis of the scopoletin biosynthesis gene MaMYB4 from Melilotus officinalis:

[0045] Based on the whole genome sequencing of Melilotus alba, a large number of bioinformatics analysis and screening were performed to obtain the scopoletin biosynthesis gene MaMYB4 of Melilotus alba. Then the scopoletin biosynthesis gene MaMYB4 of Melilotus alba was cloned and sequenced.

[0046] The cloning vector used was 5min TA / Blunt-Zero Cloning Kit, purchased from Novagen; Escherichia coli competent cells DH5α were purchased from Beijing Quanshijin Biotechnology Co., Ltd. Reverse transcription kit TIANScript II RTKit (Tiangen), Phanta Max Super-Fidelity DNA Polymerase (Novagen), Plasmid Mini Kit Plasmid Extraction Kit (Yisheng); SanPrep Column PCR Product Purification Kit Recovery Kit (Shengong), SanPrep Column DNA Gel Recovery Kit (Shengong), Real-time Fluorescence Quantitative PCR qRT-PCR Kit qPCR GreenMaster Mix (No Rox) (Yisheng); primers were synthesized by Xi'an Qingke Company; sequencing was completed by Aoke Biotechnology Co., Ltd.

[0047] 1. Cloning of the gene MaMYB4:

[0048] (1) RNA was extracted from the whole plant of Melilotus officinalis JiMa49 using the UNIQ-10 column Trizol total RNA extraction kit (Shenggong). The extracted RNA was reverse transcribed into cDNA using the Tiangen reverse transcription kit TIANScript II RT Kit. Using this cDNA as a template, primers (primer P1 and primer P2) were designed to amplify the MaMYB4 gene from the whole plant of Melilotus officinalis. Primer P1: 5'-ATGGGAAGATCACCTTGTTGTGA-3' (SEQ ID NO.5) and primer P2: 5'-TCATTTCATTTCTAAGCCTTTGTAATC-3' (SEQ ID NO.6) were amplified by PCR using a high-fidelity enzyme.

[0049] The reaction system of the above PCR amplification is as follows: 2× Max Buffer 12.5μL, dNTP 0.5μL, forward and reverse primers (10μM) 1μL each, Max Super-Fidelity DNA Polymerase (Novagen high-fidelity DNA polymerase) 0.5μL, ddH2O 8.5μL.

[0050] The PCR program was: 95°C for 3 min; 95°C for 30 s, 55°C for 30 s, 72°C for 1 min 30 s, 35 cycles; 72°C for 5 min.

[0051] (2) After the reaction, take 5 μl of PCR product for 1.5% agarose gel electrophoresis, and observe a band of about 870 bp in the UV gel imaging system. Recover the MaMYB4 gene fragment using a PCR product purification kit. Connect the recovered and purified DNA fragment using the 5min TA / Blunt-Zero Cloning Kit instructions. Transform the recombinant plasmid T-MaMYB4 into competent Escherichia coli cells, label with ampicillin (100 mg / L) to screen positive clones, and perform bacterial liquid PCR identification. Sequence the bacterial liquid of the positive recombinant plasmid.

[0052] 2. Expression analysis of gene MaMYB4 in Melilotus officinalis:

[0053] Samples from different tissue parts of the root, stem, leaf and flower of Melilotus officinalis JiMa49 were collected, RNA was extracted, and reverse transcribed into cDNA, which was used as a template for qRT-PCR to detect the expression level of MaMYB4, wherein the upstream primer P3 of qRT-PCR was 5'-AATTATCTCCGGCCAGACCT-3' (SEQ ID NO.7), and the downstream primer P4 was 5'-GTAATCTTCCAGCTAT CAATGACC-3' (SEQ ID NO.8).

[0054] The results of qRT-PCR are as follows Figure 3 As shown, from Figure 3 It can be seen that the expression level of the scopoletin biosynthesis gene MaMYB4 in Melilotus officinalis was significantly higher in leaves than in roots, stems and flowers.

[0055] Embodiment 3:

[0056] Study on the application of MaMYB4 gene in positive regulation of scopoletin biosynthesis

[0057] 1. Construct an overexpression vector. The specific method is as follows:

[0058] (1) Using the plasmid of T-MaMYB4 as a template, PCR amplification was performed using the upstream primer P5 and the downstream primer P6 with a high-fidelity enzyme, primer P5: 5'-GAGAACACGGGGGACTCTAGAATGGGAAGATCACCTTGTTGTG A-3' (SEQ ID NO. 9), primer P6: 5'-CCATGGTACCCCCGGGGATCCGTCATTTCATTTCT AAGCCTTTGTAATC-3' (SEQ ID NO. 10);

[0059] The reaction system for PCR amplification is as follows: 2× Max Buffer 12.5μL, dNTP 0.5μL, forward and reverse primers (10μM) 1μL each, Max Super-Fidelity DNA Polymerase (Novagen high-fidelity DNA polymerase) 0.5μL, ddH2O 8.5μL.

[0060] The PCR program was: 95°C for 3 min; 95°C for 30 s, 55°C for 30 s, 72°C for 1 min 30 s, 35 cycles; 72°C for 5 min.

[0061] (2) Take 5 μL of PCR product and perform 1.5% agarose gel electrophoresis. Use ultraviolet gel imaging to see the fragments consistent with the expected size, and use a PCR product purification kit to recover the MaMYB4 gene fragment.

[0062] (3) Linearized vector: Digest the plant overexpression vector plasmid pBI121 with XbaI and BamHI. After digestion, purify the product with a PCR product purification kit. The digestion reaction system is as follows: 1 μg of vector plasmid, 1 μL of XbaI enzyme, 1 μL of BamHI enzyme, 2 μL of 10× buffer, and ddH2O to 20 μL. Incubate the digestion reaction at 37°C for 1 h.

[0063] (4) The MaMYB4 gene fragment from step (2) and the pBI121 vector linearized in step (3) were cloned using a one-step cloning kit MultiS One Step Cloning Kit (Novozyme) was used for homologous recombination. The system was as follows: 2.5 μL of linearized pBI121 vector, 1 μL of MaMYB4 gene fragment, 1 μL of 5×CE MultiS Buffer, and Exnase MultiS were reacted at 37°C for 30 min; the mixture was cooled to 4°C or immediately placed on ice for cooling. Escherichia coli DH5α was transformed by heat shock method. Single clones were selected for bacterial liquid PCR detection, and positive single clones were sent for sequencing. The single clones with correct sequencing were the constructed vectors. The bacteria were shaken, and the plasmid was extracted with a plasmid extraction kit to obtain the plant expression vector: pBI121-MaMYB4, which was stored at -20°C.

[0064] 2. Construction of RNAi expression vector

[0065] (1) According to the Gateway instruction manual, the attB site was incorporated into the 5' end of the gene-specific upstream and downstream primers to obtain primers P7 and P8. P7 / P8 was used as a primer pair, and the plasmid of T-MaMYB4 was used as a template. Max Super-Fidelity DNA Polymerase was used for PCR amplification, and the PCR product was recovered using a gel recovery kit. The entry vector pDONR-Zeo and the gel recovery product were used for BP reaction to construct the entry vector.

[0066] Among them, the sequence of P7 is as follows: 5'-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGGGAAGATCACCTTGTTGTGA-3' (SEQ ID NO. 11);

[0067] The sequence of P8 is as follows: 5′-GGGGACCACTTTGTACAAGAAAGCTGGGTCTCATTTCATTTCTAAGCCTTTGTAATC-3′ (SEQ ID NO. 12);

[0068] The BP reaction system was as follows: 0.4 μL of Gateway BPⅡ Clonase enzyme mixture, 1 μL of gel recovery product (50 ng / μL), and 0.6 μL of pDONR-Zeo (150 ng / μL). The reaction was carried out at 25°C overnight.

[0069] (2) The ligation product was transformed into E. coli DH5α. 100 μL of the culture was spread on a LB solid plate containing bleomycin Zeo antibiotic at a final concentration of 33 mg / L. The positive single clone was detected using universal primers M13F and M13R and sent for sequencing. The single clone with the correct sequencing was shaken and the plasmid was extracted using a plasmid extraction kit and stored at -20°C.

[0070] RNAi vector pK7GWIWG2(II)RR and entry vector were used for LR reaction to construct RNAi recombinant vector. The LR reaction system was as follows: Gateway LRⅡClonase enzyme mixture 0.4μL, Entry Clone (150ng / μL) 0.6μL, target vector (150ng / μL) 0.6μL, ddH2O 0.4μL. React at 25℃ overnight. The ligation product was transformed into Escherichia coli DH5α. After the transformation, 100μL of culture was taken and spread on LB solid plate containing spectinomycin Spe antibiotic with a final concentration of 50mg / L. After the positive monoclonal bacterial solution was detected, the positive monoclonal with the target band was shaken and the plasmid was extracted with a plasmid extraction kit, that is, the successful RNAi-MaMYB4 recombinant plasmid was constructed and stored at -20℃.

[0071] 3. MaMYB4 gene transformation of sweet clover hairy roots

[0072] The pBI121-MaMYB4 plasmid, RNAi-MaMYB4 and empty vector were transformed into Agrobacterium rhizogenes K599 by electroporation. Single clones were picked for bacterial liquid PCR detection, and the product fragment was consistent with the expected target fragment size, indicating that Agrobacterium with MaMYB4 gene overexpression and RNAi vector was successfully obtained.

[0073] 200 μL of K599 bacterial solution containing empty vector, MaMYB4 overexpression vector and RNAi vector was applied on the plate. After 7 days of germination of Melilotus officinalis seeds, the root tip of Melilotus officinalis was quickly cut off with a scalpel 5 mm above it. The cut seedlings were lightly dipped in Agrobacterium bacterial film and placed on 1 / 2MS medium without antibiotics, wrapped with tin foil and cultured in the dark for 3 days. After 3 days of co-culture, the seedlings were taken out of the dark treatment condition, placed on 1 / 2MS solid medium, and placed in a filter paper-seedling-filter paper structure, and placed upright in a 22°C tissue culture room (16h light / 8h dark) for 14 days to start growing hairy roots, and then transferred to water for 15-20 days to observe the growth and conduct subsequent experiments.

[0074] 4. Molecular identification of transgenic sweet clover hairy roots

[0075] The transgenic white clover hairy roots were identified at the genomic and transcriptional levels. Using Superlight Mix, 1 mm of the root tip was taken, 20 μL of the chaperone was added, and the mixture was treated at 95°C for 5 min, then centrifuged. 1 μL of the supernatant was taken as a template. The overexpression vector was detected by PCR using the vector upstream primer and self primer to detect the target band 35S-F: 5'-TGACGCACAATCCCACTATC-3' (SEQ ID NO.13), P2: 5'-TCATTTCATTTCTAAGCCTTTGTAATC-3' (SEQ ID NO.6), and the RNAi vector was detected by RNAi vector primers to detect the target band: RNAi-F: 5'-AAATTCGAT CGCACAAACTAG-3' (SEQ ID NO.14), RNAi-R: 5'-CTCTCTACCGTGATCAAGGT-3' (SEQ ID NO.15), Figure 4 As shown in Figure 2, it indicates that the target gene has been successfully transformed into the hairy roots of Melilotus officinalis. Figure 5 As shown, the expression of MaMYB4 in transgenic hairy roots was detected by qRT-PCR, and it was found that compared with the control, the expression level of MaMYB4 gene was higher in the overexpression strain and lower in the RNAi strain.

[0076] 5. Determination of Scopolamine Content in Transgenic Clerodendrum truncatum Hairy Roots

[0077] Weigh 100 mg of fresh hairy root sample cultured for 60 days, grind it with liquid nitrogen, shake and mix with 5 mL of 80% ethanol, then perform ultrasonic extraction at room temperature for 30 min, centrifuge at 12000 rpm for 10 min, collect the filtrate, repeat the extraction twice, then combine the filtrates, and concentrate under reduced pressure using a rotary evaporator at a rotary evaporation temperature of 50°C. Wait for the sample to be rotary evaporated to a dry state, dissolve it in methanol and make up to 10 mL to obtain a sample extract. HPLC was used for detection. Acetonitrile (eluent A) and 0.1% phosphoric acid water (eluent B) were used as mobile phases, and the detection wavelength was 346 nm. The flow rate was 1 mL / min, the column temperature was 30°C, and the injection volume was 10 μL. The test results are as follows: Figure 6 As shown, the scopoletin content was higher in the overexpression line and lower in the RNAi line compared with the control.

[0078] Embodiment 4:

[0079] Research on the regulatory mechanism of MaMYB4 protein:

[0080] 1. Yeast two-hybrid:

[0081] (1) MaMYB4 was constructed into the pGADT7 vector to obtain the recombinant vector pGADT7-MaMYB4, and MaTT8 was constructed into the pGBKT7 vector to obtain the recombinant vector pGBKT7-MaTT8. The yeast strain AH109 was transformed using a yeast transformation kit (Yeast Transformation Kit, Clontech www.clontech.com), coated with solid defective medium SD-Trp / -Leu, and inverted cultured at 30°C for 3-5 days to obtain the hybrid strain AD-MYB4+BD-TT8. The transformation steps were carried out according to the relevant instructions.

[0082] (2) After monoclonal clones are grown, pick 3-5 monoclonal clones from each plate, blow them into 20 μL of sterilized double-distilled water, and spot 2 μL on SD-Trp / -Leu / -His / -Ade medium to verify the interaction. For specific results, see Figure 7 , on the defective medium of SD / -Trp / -Leu / -His / -Ade, yeast co-transfected with MaMYB4 and MaTT8 can grow normally, indicating that MaMYB4 can interact with MaTT8.

[0083] 2. Bimolecular fluorescence complementation experiment:

[0084] MaMYB4 was constructed into the pSAT1-cCFP-C (pE3242) vector, and MaTT8 was constructed into the pSAT1-nVenus-C (pE3228) vector. The Agrobacterium tumefaciens GV3101 was transformed by electroporation. The Agrobacterium tumefaciens were mixed at a ratio of 1:1 and then injected into tobacco. After 48 hours, the cells were observed and photographed using a laser scanning confocal microscope. Figure 8 As shown, the combination of nVenus-MaTT8+cCFP-MaMYB4 has a stronger green fluorescence signal in the cell nucleus, indicating that there is an interaction between MaMYB4 and MaTT8.

[0085] 3. MaMYB4 and MaTT8 co-transformation of Melilotus officinalis hairy roots:

[0086] 200 μL of K599 bacterial solution containing empty vector (EV), MaMYB4 overexpression vector (OE-MaMYB4), RNAi vector (RNAi-MaMYB4), and MaTT8 overexpression vector (OE-MaTT8) was applied on the plate. After 7 days of germination, the seeds of Melilotus officinalis were quickly cut off 5 mm above the root tip with a scalpel. The root-cut seedlings were lightly dipped in different Agrobacterium biofilm combinations, namely EV, RNAi-MaMYB4, OE-MaTT8+RNAi-MaMYB4, OE-MaMYB4, and OE-MaTT8+OE-MaMYB4, and placed on 1 / 2MS culture medium without antibiotics, wrapped in tin foil and cultured in the dark for 3 days. After 3 days of co-cultivation, the seedlings were taken out from the dark treatment conditions, placed on 1 / 2MS solid culture medium, and placed in a filter paper-seedling-filter paper structure. They were placed upright in a 22°C tissue culture room (16h light / 8h dark). After 14 days of cultivation, hairy roots began to grow. They were then transferred to water for 15-20 days to observe their growth and conduct subsequent experiments.

[0087] After MaMYB4 and MaTT8 were co-transformed into the hairy roots of Melilotus officinalis, the expression level of MaUGT79 gene was significantly increased. For details, please refer to Fig. 9 , the content of scopolamine increased significantly, for details, please refer to Fig.10 , indicating that the interaction between MaTT8 and MaMYB4 can positively regulate the MaUGT79 gene, activate the MaUGT79 promoter, and promote the biosynthesis of scopoletin in Melilotus officinalis.

[0088] In summary, the present invention provides a novel transcription factor MaMYB4 that positively regulates scopolamine synthesis, which can promote the accumulation of scopolamine in plant organs, such as promoting the accumulation of scopolamine in the roots of Melilotus officinalis. The cloned MaMYB4 gene of the present invention enriches the regulatory genes in the existing scopolamine biosynthesis pathway.

[0089] The scopolamine content in the hairy root transformed white sweet clover hairy root is significantly positively correlated with the expression of MaMYB4. MaMYB4 can bind to the promoter of the scopolamine synthesis gene MaUGT79 to promote the expression of the MaUGT79 gene, and can also interact with MaTT8 to jointly regulate the biosynthesis of scopolamine. The present invention helps to better understand the molecular mechanism of scopolamine biosynthesis.

[0090] The MaMYB4 gene of the present invention belongs to the MYB gene family, and the present invention provides data support for further studying the regulation of scopolamine accumulation by the MYB gene family.

[0091] The MaMYB4 gene of the invention is applied to the biosynthesis of plant secondary metabolites, and provides a new gene for molecular breeding of Melilotus officinalis.

[0092] The above-described embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the present invention. It should be pointed out that, for a person of ordinary skill in the art, several modifications and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the attached claims.

Claims

1. Application of transcription factor MaMYB4 or a gene encoding transcription factor MaMYB4 in regulating the synthesis of scopoletin in the hairy roots of Melilotus officinalis. The amino acid sequence of the transcription factor MaMYB4 is shown in SEQ ID NO.

1.

2. The use according to claim 1, characterized in that: The nucleotide sequence encoding the transcription factor MaMYB4 is shown in SEQ ID NO.

2.

3. The use according to claim 1, characterized in that: Co-transformation of the transcription factor MaMYB4 or the gene encoding the transcription factor MaMYB4 with MaTT8 can significantly increase the synthesis of scopoletin in the hairy roots of Melilotus officinalis. The amino acid sequence of MaTT8 is shown in SEQ ID NO.

3.

4. The use according to claim 3, characterized in that: The nucleotide sequence encoding the MaTT8 is shown in SEQ ID NO.

4.

5. A method for promoting the synthesis of scopoletin from Melilotus officinalis by using the transcription factor MaMYB4 as claimed in claim 1, characterized in that: The method is: (1) Using the recombinant plasmid T-MaMYB4 plasmid as a template, PCR amplification was performed using primers P5 / primer P6 with a high-fidelity enzyme; the MaMYB4 gene fragment was recovered using a PCR product purification kit; (2) Digesting the plant overexpression vector plasmid pBI121 with XbaI and BamHI to obtain a linearized vector of pBI121; (3) connecting the MaMYB4 gene fragment of step (1) and the linearized vector of pBI121 to obtain the plant overexpression vector pBI121-MaMYB4; (4) using an electroporation method to transform the plant overexpression vector pBI121-MaMYB4 of step (3) into Agrobacterium rhizogenes K599, and then infecting the hairy roots of the sweet clover with the Agrobacterium rhizogenes K599 to obtain; The sequence of the primer P5 is shown in SEQ ID NO.9, and the sequence of the primer P6 is shown in SEQ ID NO.

10.

6. A method for promoting the synthesis of scopoletin in Melilotus officinalis by co-transformation of the MaMYB4 gene and the MaTT8 gene as claimed in claim 3, characterized in that: The method is: (1) MaMYB4 and MaTT8 overexpression vectors were constructed and named OE-MaMYB4 and OE-MaTT8, respectively; then, the rhizogenes Agrobacterium K599 was transformed by electroporation to obtain different Agrobacterium; (2) After germination of 7 days, the root tip of the sweet clover seeds was cut off with a scalpel, and then lightly dipped into the bacterial film of Agrobacterium transformed with the OE-MaMYB4 vector and the Agrobacterium transformed with the OE-MaTT8 vector for infection culture to obtain OE-MaTT8+OE-MaMYB4.

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