CN116200422A - Transcription factor MaMYB4 and application thereof in synergistic regulation of scopolamine biosynthesis with MaTT8 gene - Google Patents

Abstract

The invention relates to the technical field of biology, in particular to trifolith gene MaMYB4 of white flower and application thereof, and provides transcription factor MaMYB4 for promoting plant organs to produce scopolamine and application thereof. The MaMYB4 is a MYB family transcription factor, the length of a complete coding frame is 870 bases, and the coding protein contains 290 amino acids. The excessive expression of the MaMYB4 causes the scopolamine glycoside in the hairy root of the sweet clover Bai Huacao to accumulate, and meanwhile, the MaTT8 can interact with the MaMYB4 to promote the generation of scopolamine glycoside, so that the result has important significance for the practical application of enriching the basic theory of accumulation of coumarin secondary metabolites, improving the quality defect of the existing sweet clover, improving the breeding efficiency of high-quality sweet clover and the like.

Description

Transcription factor MaMYB4 and application thereof in synergistic regulation of scopolamine biosynthesis with MaTT8 gene

[ field of technology ]

The invention relates to the technical field of biology, in particular to a transcription factor MaMYB4 and application thereof in synergetic regulation and control of scopolamine biosynthesis with a MaTT8 gene.

[ background Art ]

Scopoletin coumarin and its glycosylated scopoletin are natural products with aromatic smell, are important secondary metabolites from phenylpropane metabolic pathway, exist in plants in free state or glycoside form, and have important effects on plant growth and development and response to adverse stress. Not only is beneficial to plants to resist herbivorous insects and pathogens, regulate and control microbial community structures and promote plant health, but also is a scavenger of active oxygen, and plays an important role in abiotic stress. As the use of plant secondary metabolites has increased, it has become a global focus of attention, and studies on plant secondary metabolite biosynthesis have increased.

Sweet clover is rich in secondary metabolites such as scopoletin, however, the gene and molecular mechanism related to scopoletin biosynthesis have not been revealed yet. The gene related to the scopolamine synthesis of the white flower sweet clover is a biosynthesis candidate gene of a plant secondary metabolite, and has very important value.

MYB transcription factors are one of the largest and most important families of genes in plants, mainly regulating primary and secondary metabolism and plant response to stress. MYB transcription factors can bind to the promoter of a gene involved in phenylpropane synthesis to activate the activity of the phenylpropane synthesis gene. Salvia SmPAP1 can activate PAL gene of phenylpropane metabolic pathway and the expression of anthocyanin synthesis related gene C4H, CHS, and can regulate anthocyanin biosynthesis. The rice R2R3 MYB transcription factor OsC1 obviously up-regulates the expression of anthocyanin synthesis related genes (PAL, CHI, CHS, F3H, F3' H, DFR, ANS) and increases anthocyanin synthesis. Thus, MYB regulates phenylpropane metabolism by regulating the expression of key enzyme genes in the phenylpropane synthesis pathway. bHLH transcription factors often interact with MYB family proteins to form complexes that regulate the expression of downstream target genes. TT8 interacts with MYB protein present in WD40 repeats containing TTG1 to form a transcriptional regulatory complex that activates anthocyanin biosynthetic genes. GmMYBA2 interacts with GmTT8a to directly activate anthocyanin biosynthesis genes through MBW complexes. However, few reports have been made on the involvement of the transcription factor MYB in scopolamide regulation, and antagonism of the transcription factor MYB12 only promotes flavonol synthesis, MYB4 inhibits flavonol synthesis and thereby promotes scopolamide production. At present, no report exists on the function and molecular regulation mechanism of MYB4 for regulating scopolamine synthesis.

[ invention ]

In view of the above, it is necessary to provide a transcription factor MaMYB4 for promoting plant organs to produce scopolamine and application thereof, and the invention obtains the MaMYB4 gene with positive regulation and control function on the mamgt 79 promoter and the gene mapt 8 with interaction by researching the transcription factor MaMYB family, and the gene has good promotion function on accumulation of scopolamine in white flower sweet clover by researching, thus having important significance for improving the existing quality defect of white flower sweet clover, improving the breeding efficiency of high-quality white flower sweet clover and other practical applications.

In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:

the invention comprises application of a transcription factor MaMYB4 or a gene encoding the transcription factor MaMYB4 in regulating and controlling synthesis of scopolamine in hairy root of trifolith white, wherein the amino acid sequence of the transcription factor MaMYB4 is shown as SEQ ID NO. 1.

Further, the application is the application of the transcription factor MaMYB4 or the gene encoding the transcription factor MaMYB4 in activating the MaUGT79 gene and/or the MaUGT79 promoter.

Further, the nucleotide sequence of the coding transcription factor MaMYB4 is shown as SEQ ID NO.2.

Furthermore, the transcription factor MaMYB4 or the gene encoding the transcription factor MaMYB4 and the MaTT8 are co-transformed, so that the synthesis of scopolamine in the hairy root of trifolith of white flowers can be obviously improved; the amino acid sequence of MaTT8 is shown as SEQ ID NO. 3.

Furthermore, the transcription factor MaMYB4 or the gene encoding the transcription factor MaMYB4 and the MaTT8 are co-transformed, so that the effect of activating the MaUGT79 gene and/or the MaUGT79 promoter can be remarkably improved; the amino acid sequence of MaTT8 is shown as SEQ ID NO. 3.

Further, the nucleotide sequence of the coding transcription factor MaTT8 is shown as SEQ ID NO. 4.

The invention also comprises an expression vector, wherein the expression vector contains a coding sequence SEQ ID NO.2 of the transcription factor MaMYB4 gene.

The invention also comprises a method for promoting the synthesis of scopoletin in white flower and/or activating a MaUGT79 promoter by using the MaMYB4 expression vector, which is characterized in that the method comprises the following steps:

(1) Taking the recombinant plasmid T-MaMYB4 as a template, and carrying out PCR amplification by using a primer P5/a primer P6 through high-fidelity enzyme; recovering the MaMYB4 gene fragment by using a PCR product purification kit;

(2) The plant over-expression vector plasmid pBI121 is digested with XbaI and BamHI to obtain a linearization vector of pBI 121;

(3) Connecting the MaMYB4 gene fragment in the step (1) with a linearization vector of pBI121 to obtain a plant over-expression vector pBI121-MaMYB4;

(4) Transforming the plant overexpression vector pBI121-MaMYB4 in the step (3) into agrobacterium rhizogenes K599 by adopting an electric shock method, and infecting hairy roots of the sweet clover with the agrobacterium rhizogenes K599 to obtain the sweet clover;

the primer P5 sequence is shown as SEQ ID NO.9, and the primer P6 sequence is shown as SEQ ID NO. 10.

The invention also comprises a method for promoting the synthesis of scopoletin in white flower and/or activating a MaUGT79 promoter by using the cotransformation of the MaMYB4 gene and the MaTT8 gene, which is characterized in that the method comprises the following steps:

(1) Respectively constructing a MaMYB4 and a MaTT8 over-expression vector and naming the over-expression vectors as OE-MaMYB4 and OE-MaTT8; then adopting an electric shock method to transform agrobacterium rhizogenes K599 to obtain different agrobacterium;

(2) Cutting root tips of trifolitan seeds after germination of 7 seeds of trifolitan flowers by using a surgical knife, then slightly dipping in bacterial films of agrobacterium transformed with an OE-MaMYB4 vector and agrobacterium transformed with an OE-MaTT8 vector, and carrying out infection culture to obtain OE-MaTT8+OE-MaMYB4.

The invention has the following beneficial effects:

the invention provides a transcription factor MaMYB4 for promoting plant organs to produce scopolamine and application thereof. The MaMYB4 is a MYB family transcription factor, the length of a complete coding frame is 870 bases, and the coding protein contains 290 amino acids. The excessive expression of the MaMYB4 causes the scopolamine glycoside in the hairy root of the sweet clover Bai Huacao to accumulate, and meanwhile, the MaTT8 can interact with the MaMYB4 to promote the generation of scopolamine glycoside, so that the result has important significance for the practical application of enriching the basic theory of accumulation of coumarin secondary metabolites, improving the quality defect of the existing sweet clover, improving the breeding efficiency of high-quality sweet clover and the like.

[ description of the drawings ]

FIG. 1 is a diagram of the results of a yeast single hybridization experiment;

FIG. 2 is a graph of the results of a dual luciferase reporter assay;

FIG. 3 fluorescence expression of the MaMYB4 gene at different positions in Melilotus leucoanthus.

FIG. 4 is a diagram of PCR identification results of hairy roots of pBI121-MaMYB4 and RNAi-MaMYB4 genes; m in the figure is DL2000 DNAMaroker; (+) is a positive control, lanes 2-7 are pBI121-MaMYB4 transgenic hairy root PCR products; lanes 8-17 are RNAi-MaMYB4 transgenic hairy root PCR products;

FIG. 5 is a graph showing the relative expression levels of MaMYB4 obtained after infection of hairy roots of Melilotus officinalis with various Agrobacterium.

FIG. 6 is a graph showing the results of scopoletin content after infection of hairy roots of Melilotus officinalis with various Agrobacterium.

FIG. 7 is a graph showing the results of a yeast two-hybrid experiment;

FIG. 8 is a graph of the results of a bimolecular fluorescence complementation experiment;

FIG. 9 is a graph showing the result of expression level of gene MaUGT79 after co-transformation of MaMYB4 and MaTT8 with hairy root of Melilotus officinalis;

FIG. 10 is a graph showing the results of scopolamine content after the cotransformation of MaMYB4 with MaTT8 in the hairy root of Melilotus officinalis.

[ detailed description ] of the invention

In order that the above objects, features and advantages of the invention will be readily understood, a more particular description of the invention will be rendered by reference to the appended drawings. In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention. The invention may be embodied in many other forms than described herein and similarly modified by those skilled in the art without departing from the spirit or scope of the invention, which is therefore not limited to the specific embodiments disclosed below.

Example 1:

screening of transcription factors regulating scopolamine synthesis

In this example, the regulatory proteins of the MaUGT79 gene were screened using the yeast single hybrid system with the MaUGT79 promoter fragment, a key gene for the scopolamine luteolin synthesis pathway, a gene family member of the glycosyltransferase. In the results of the yeast single hybridization experiments, the transcription factor MaMYB4 which can regulate and control the biosynthesis of scopolamine was screened.

Subsequently, subsequent functional studies were performed with MaMYB4 as the target.

The results are shown in fig. 1 and 2; the yeast single hybridization of FIG. 1 shows that the MaMYB4 protein can bind to the MaUGT79 promoter and has certain interaction with the MaUGT79 gene promoter sequence.

As shown in the dual luciferase reporter experiments of FIG. 2, LUC/Rluc values in Agrobacterium-injected tobacco leaves. The interaction of the MaMYB4 protein and the MaUGT79 gene promoter sequence is reacted by taking the enzyme activity of Rlun as an internal reference and the ratio of the enzyme activities of LUC and Rluc. After injection of tobacco with Agrobacterium containing pGreenII 0800-MaUGT79 promoter+35S-MaMYB4 recombinant plasmid, the enzyme activity of LUC in leaves was 2.7 times that of Rluc, indicating that the MaMYB4 protein could activate expression of MaUGT 79.

In conclusion, it was demonstrated that MaMYB4 is a positive regulator that can bind directly to the scopolamine synthetic gene promoter.

Example 2:

cloning and expression analysis of scopolamine leucorrhea biosynthesis gene MaMYB 4:

based on the whole genome sequencing of the sweet clover, and carrying out a large amount of bioinformatics analysis and screening, the scopolamine glycoside biosynthesis gene MaMYB4 of the sweet clover is obtained. Then cloning and sequence analysis of scopolamine sodium biosynthesis gene MaMYB4 of white flower sweet clover.

Cloning vectors were purchased from Northenan using a 5min TA/Blunt-Zero Cloning Kit; coli competent cells DH 5. Alpha. Were purchased from Beijing full gold biotechnology Co. Reverse transcription kit TIANScript II RTKit (Tiangen), phanta Max Super-Fidelity DNA Polymerase (Norpran),

Plasmid Mini Kit Plasmid miniprep (next holy); sanPrep column type PCR product purification kit recovery kit (manufacturing engineering), sanPrep column type DNA gel recovery kit (manufacturing engineering), real-time fluorescence quantitative PCR qRT-PCR kit->

qPCR/>

Green Master Mix (No Rox) (next holy); primers were all synthesized by the company western anserinaceae; sequencing was performed by Oncology, inc.

1. Cloning of the gene MaMYB 4:

(1) The whole trifolith JiMa49 plant is used as a material, and the UNIQ-10 column type Trizol total RNA extraction kit (in the process of production) is used for extracting RNA. The extracted RNA was reverse transcribed into cDNA using the root reverse transcription kit TIANScript II RT Kit. Using this cDNA as a template, primers (primer P1 and primer P2) were designed for amplifying the MaMYB4 gene from the whole trifoliate plants. Wherein primer P1:5'-ATGGGAAGATCACCTTGTTGTGA-3' (SEQ ID NO. 5) and primer P2:5'-TCATTTCATTTCTAAGCCTTTGTAATC-3' (SEQ ID NO. 6) was amplified by PCR with high fidelity enzymes.

The reaction system for PCR amplification is as follows: 2×

Max Buffer 12.5. Mu. L, dNTP 0.5.0.5. Mu.L, forward and reverse primers (10. Mu.M) 1. Mu.L each, & lt/EN & gt>

Max Super-Fidelity DNAPolymerase (Noruzan high-fidelity DNA polymerase) 0.5 mu L, ddH ₂ O 8.5μL。

The PCR procedure was: 3min at 95 ℃;95℃30s,55℃30s,72℃1min30s,35 cycles; and at 72℃for 5min.

(2) After the reaction was completed, 5. Mu.l of the PCR product was subjected to 1.5% agarose gel electrophoresis, and a band of about 870bp was observed in the ultraviolet gel imaging system. And (5) recovering the MaMYB4 gene fragment by using a PCR product purification kit. The recovered and purified DNA fragment was ligated using 5min TA/Blunt-Zero Cloning Kit instructions. The obtained recombinant plasmid T-MaMYB4 is transformed into competent cells of the escherichia coli, ampicillin (100 mg/L) is used for marking and screening positive clones, and bacterial liquid PCR identification is carried out. Sequencing the bacterial liquid of the positive recombinant plasmid.

2. Expression analysis of the gene MaMYB4 in sweet clover:

collecting samples of different tissue parts of trifoliter JiMa49 roots, stems, leaves and flowers, extracting RNA, reversely transcribing into cDNA, and using the cDNA as a template to perform qRT-PCR to detect the expression quantity of the MaMYB4, wherein the upstream primer P3 of the qRT-PCR is as follows: 5'-AATTATCTCCGGCCAGACCT-3' (SEQ ID NO. 7), downstream primer P4:5'-GTAATCTTCCAGCTAT CAATGACC-3' (SEQ ID NO. 8).

As shown in FIG. 3, the qRT-PCR results show that the expression level of scopolamine sodium in white flower (MaMYB 4) is obviously higher than that of roots, stems and flowers in leaves as shown in FIG. 3.

Example 3:

application research of MaMYB4 gene in positive regulation of scopolamine biosynthesis

1. The specific method for constructing the over-expression vector is as follows:

(1) Using the plasmid of T-MaMYB4 as a template, and adopting an upstream primer P5 and a downstream primer P6 to carry out PCR amplification by high-fidelity enzyme, wherein the primer P5:5'-GAGAACACGGGGGACTCTAGAATGGGAAGATCACCTTGTTGTG A-3' (SEQ ID NO. 9), primer P6:5'-CCATGGTACCCCCGGGGATCCGTCATTTCATTTCT AAGCCTTTGTAATC-3' (SEQ ID NO. 10);

the reaction system for PCR amplification is as follows: 2×

Max Buffer 12.5. Mu. L, dNTP 0.5.0.5. Mu.L, forward and reverse primers (10. Mu.M) 1. Mu.L each, & lt/EN & gt>

Max Super-Fidelity DNAPolymerase (Noruzan high-fidelity DNA polymerase) 0.5 mu L, ddH ₂ O 8.5μL。

The PCR procedure was: 3min at 95 ℃;95℃30s,55℃30s,72℃1min30s,35 cycles; and at 72℃for 5min.

(2) The 5. Mu.LPCR product was subjected to 1.5% agarose gel electrophoresis, and the fragment consistent with the expected size was visualized using an ultraviolet gel imager, and the MaMYB4 gene fragment was recovered using a PCR product purification kit.

(3) Linearizing the carrier: the plant over-expression vector plasmid pBI121 was digested with XbaI and BamHI, and after digestion, the PCR product was purified using the PCR product purification kit. The enzyme digestion reaction system is as follows: vector plasmid 1. Mu.g, xbaI enzyme 1. Mu. L, bamHI enzyme 1. Mu.L, 10 Xbuffer 2. Mu. L, ddH ₂ O was made up to 20. Mu.L. The cleavage reaction was carried out at 37℃for 1h.

(4) Cloning kit of MaMYB4 gene fragment in step (2) and linearized pBI121 vector in step (3) by using one-step method

MultiS One Step Cloning Kit (Northenan), homologous recombination was performed as follows: linearized pBI121 vector 2.5. Mu. L, maMYB4 gene fragment 1. Mu.L, 5 XCE MultiS Buffer 1. Mu.L, exnase MultiS37℃for 30min; cooling to 4 ℃ or immediately cooling on ice. Coli DH 5. Alpha. Was transformed by heat shock. Selecting monoclonal to carry out bacterial liquid PCR detection, sequencing positive monoclonal, and carrying out sequencing on correct monoclonal to construct a successful vector, shaking bacteria, extracting plasmid by using a plasmid extraction kit to obtain a plant expression vector: pBI121-MaMYB4, stored at-20deg.C.

2. Construction of RNAi expression vectors

(1) Combining attB sites on the 5' ends of the gene-specific upstream and downstream primers according to Gateway instructions to obtain primer P7 and primer P8 using P7/P8 as primer pair and plasmid of T-MaMYB4 as template, using

And (3) carrying out PCR amplification by using Max Super-Fidelity DNAPolymerase high-fidelity enzyme, and recovering a PCR product by using a gel recovery kit. The entry vector was constructed by BP reaction using the entry vector pDONR-Zeo and the gel recovery product.

Wherein the P7 sequence is as follows: 5'-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGGGAAG ATCACCTTGTTGTGA-3' (SEQ ID NO. 11);

the P8 sequence is as follows: 5'-GGGGACCACTTTGTACAAGAAAGCTGGGTCTCATTTCATTTCTAA GCCTTTGTAATC-3' (SEQ ID NO. 12);

wherein, the BP reaction system is as follows: gateway BP II Clonase enzyme mixture 0.4. Mu.L, gel recovery product (50 ng/. Mu.L) 1. Mu. L, pDONR-Zeo (150 ng/. Mu.L) 0.6. Mu.L. The reaction was carried out overnight at 25 ℃.

(2) The ligation product was transformed into E.coli DH 5. Alpha. 100. Mu.L of the culture was plated on LB solid plates containing bleomycin Zeo antibiotic at a final concentration of 33 mg/L. The positive monoclonal detection is carried out by using universal primers M13F and M13R, the sequencing is carried out, the correct monoclonal shaking bacteria are sequenced, the plasmid is extracted by using a plasmid extraction kit, and the plasmid is preserved at the temperature of minus 20 ℃.

RNAi vector pK7 GWIGG 2 (II) RR and entry vector carry out LR reaction to construct RNAi recombinant vector, and the LR reaction system is as follows: gateway LRII Clonase enzyme mixture 0.4. Mu.L, entry Clone (150 ng/. Mu.L) 0.6. Mu.L, destination vector (150 ng/. Mu.L) 0.6. Mu. L, ddH ₂ O0.4. Mu.L. The reaction was carried out overnight at 25 ℃. The ligation product was transformed into E.coli DH 5. Alpha. After the completion of the transformation, 100. Mu.L of the culture was plated on LB solid plates containing spectinomycin Spe antibiotic at a final concentration of 50 mg/L. After the positive monoclonal bacteria liquid is detected, the positive monoclonal bacteria with a target band are shaken, and plasmids are extracted by a plasmid extraction kit, namely the constructed RNAi-MaMYB4 recombinant plasmids are successfully constructed and stored at the temperature of minus 20 ℃.

3. Transformation of hairy root of sweet clover with MaMYB4 gene

The pBI121-MaMYB4 plasmid, RNAi-MaMYB4 and empty vector were transformed into Agrobacterium rhizogenes K599 by electric shock. And (3) selecting a monoclonal to carry out bacterial liquid PCR detection, wherein the size of a product fragment is consistent with that of an expected target fragment, which shows that the agrobacterium with the MaMYB4 gene overexpression and RNAi vector is successfully obtained.

200 mu L of empty, maMYB4 over-expression vector and RNAi vector K599 bacterial liquid are coated on a flat plate, after the sweet clover seeds germinate 7, the sweet clover seeds are rapidly cut off at the position 5mm above the root tips of the sweet clover by a surgical knife, the seedlings with the cut roots are dipped in agrobacterium film lightly, the seedlings are placed on 1/2MS culture medium without antibiotics, and the dark culture is carried out outside the tinfoil paper. After co-cultivation for 3d, seedlings were taken out from the dark treatment condition, placed on 1/2MS solid medium, and placed in a filter paper-seedling-filter paper structure, and placed in a tissue culture chamber (16 h light/8 h dark) at 22 ℃ for 14d, and grown up to form hairy roots, which were then transferred to water for cultivation for 15-20 days to observe the growth for subsequent experiments.

4. Identification of molecular level of hairy root of trifolith in transgenic white flower

The identification of genome level and the identification of transcription level are carried out on the hairy root of the transgenic white flower sweet clover. Taking 1mm of root tip by using a super-light speed Mix, adding 20 mu L of partner, treating at 95 ℃ for 5min, centrifuging, absorbing 1 mu L of supernatant as a template, and detecting the target bands 35S-F by using a carrier upstream primer and a self primer for PCR detection by using an overexpression carrier: 5'-TGACGCACAATCC CACTATC-3' (SEQ ID NO. 13), P2:5'-TCATTTCATTTCTAAGCCTTTGTAATC-3' (SEQ ID NO. 6), RNAi vector primer for RNAi vector detection detects the target band: RNAi-F:5'-AAATTCGAT CGCACAAACTAG-3' (SEQ ID NO. 14), RNAi-R:5'-CTCTCTACCGTGATCAAGGT-3' (SEQ ID NO. 15), FIG. 4 shows that the target gene was successfully transformed into trifolith hairy roots. In addition, as shown in fig. 5, the expression of MaMYB4 in transgenic hairy roots was detected by qRT-PCR, which revealed that the expression level of MaMYB4 gene was higher in the overexpressed strain and lower in the RNAi strain, compared to the control.

5. Determination of scopolamine content in trifolith hairy root of transgenic white flower

100mg of fresh hairy root sample cultured for 60 days is weighed and ground by liquid nitrogen, 5mL of 80% ethanol is used for shaking and mixing uniformly, then ultrasonic extraction is carried out at room temperature for 30min, centrifugation is carried out at 12000rpm for 10min, filtrate is collected, 2 repeated extraction is carried out, the filtrate is combined after that, decompression concentration is carried out by a rotary evaporator, and the rotary evaporation temperature is 50 ℃. And (5) after the sample is distilled to a dry state, dissolving the sample by using methanol and fixing the volume to 10mL to obtain a sample extracting solution. HPLC was performed. Acetonitrile (eluent a) and 0.1% phosphoric acid water (eluent B) were used as mobile phases, and the detection wavelength was 346nm. The flow rate was 1mL/min, the column temperature was 30℃and the sample injection amount was 10. Mu.L. As shown in FIG. 6, the scopolamine content was higher in the overexpressing strain and lower in the RNAi strain than the control.

Example 4:

study of the regulatory mechanism of MaMYB4 protein:

1. yeast two-hybrid:

(1) The MaMYB4 is constructed into pGADT7 vector to obtain recombinant vector pGADT7-MaMYB4, the MaTT8 is constructed into pGBKT7 vector to obtain recombinant vector pGBKT7-MaTT8, yeast strain AH109 is transformed by using yeast transformation kit (Yeast Transformation Kit, clontechwww.clontech.com), solid defect culture medium SD-Trp/-Leu is coated, and the hybrid strain AD-MYB4+BD-TT8 is obtained by inversion culture at 30 ℃ for 3-5 days, and the transformation steps are according to the related specifications.

(2) After growing the monoclonal, 3-5 monoclonal cells were picked per plate, blown off into 20. Mu.L of sterilized double distilled water, and 2. Mu.L of spots were taken on SD-Trp/-Leu/-His/-Ade medium, and the interaction was verified, see FIG. 7, for specific results on SD/-Trp/-Leu/-His/-Ade defect medium, co-transformed yeast with MaTT8 could grow normally, indicating that MaMYB4 could interact with MaTT 8.

2. Bimolecular fluorescence complementation experiment:

MaMYB4 was constructed into pSAT1-cCFP-C (pE 3242) vector, maTT8 was constructed into pSAT1-nVenus-C (pE 3228) vector, agrobacterium GV3101 was transformed by electric shock method, agrobacterium was mixed in a ratio of 1:1, then tobacco was injected, and after 48 hours, observation was performed using a laser scanning confocal microscope and photographed. As shown in FIG. 8, the combination of nVenus-MaTT8+cCFP-MaMYB4 has a strong green fluorescent signal in the nucleus, indicating the interaction between MaMYB4 and MaTT 8.

3. Co-transformation of trifolith hairy roots with MaMYB4 and MaTT 8:

200 mu L of empty load (EV), a MaMYB4 over-expression vector (OE-MaMYB 4) and an RNAi vector (RNAi-MaMYB 4) as well as a MaTT8 over-expression vector (OE-MaTT 8) K599 bacterial liquid are coated on a flat plate, after germination of the sweet clover seeds 7, the sweet clover seeds are rapidly cut off at the position 5mm above the root tips of the sweet clover seeds by using a surgical knife, the seedlings after cutting root are slightly dipped in different agrobacterium film combinations, namely EV, RNAi-MaMYB4, OE-MaTT8+RNAi-MaMYB4, OE-MaMYB4 and OE-MaTT8+OE-MaMYB4, and the seedlings are placed on a 1/2MS medium without antibiotics, and are covered with tinfoil paper for dark culture for 3d. After co-cultivation for 3d, seedlings were taken out from the dark treatment condition, placed on 1/2MS solid medium, and placed in a filter paper-seedling-filter paper structure, and placed in a tissue culture chamber (16 h light/8 h dark) at 22 ℃ for 14d, and grown up to form hairy roots, which were then transferred to water for cultivation for 15-20 days to observe the growth for subsequent experiments.

After the MaMYB4 and the MaTT8 co-transform the hairy roots of the white flower sweet clover, the expression level of the MaUGT79 gene is obviously increased, particularly, the scopolamine content is obviously increased, particularly, the FIG. 9 can be seen, and particularly, the FIG. 10 can be seen, which shows that the interaction of the MaTT8 and the MaMYB4 can positively regulate the MaUGT79 gene, activate the MaUGT79 promoter and promote the biosynthesis of scopolamine of the white flower sweet clover.

In summary, the invention provides a novel transcription factor MaMYB4 for positively regulating scopolamine synthesis, which can promote accumulation of scopolamine in plant organs, such as accumulation of scopolamine in root of trifoliate sweet clover. The cloned MaMYB4 gene enriches regulatory genes in the existing scopolamine biosynthesis pathway.

The scopolamine content in hairy root of the transformed white flower sweet clover and the expression level of MaMYB4 are obviously and positively correlated. The MaMYB4 can be combined with a promoter of a scopolamine glycoside synthetic gene MaUGT79 to promote expression of the MaUGT79 gene, and can also be interacted with the MaTT8 to regulate and control biosynthesis of scopolamine glycoside. The invention is helpful for better understanding the molecular action mechanism of scopolamine biosynthesis.

The MaMYB4 gene belongs to MYB gene families, and the invention provides data support for further researching MYB gene families to regulate scopolamine glycoside accumulation.

The MaMYB4 gene provided by the invention is applied to biosynthesis of plant secondary metabolites, and provides a novel gene for molecular breeding of sweet clover.

The above examples merely represent a few embodiments of the present invention, which are described in more detail and are not to be construed as limiting the scope of the present invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of the invention should be assessed as that of the appended claims.

Claims (9)

1. The transcription factor MaMYB4 or the gene encoding the transcription factor MaMYB4 is applied to regulating and controlling the synthesis of scopolamine in hairy root of Melilotus leucovora, and the amino acid sequence of the transcription factor MaMYB4 is shown as SEQ ID NO. 1.

2. The use according to claim 1, characterized in that the use is of the transcription factor MaMYB4 or of a gene encoding the transcription factor MaMYB4 for activating the MaUGT79 gene and/or the MaUGT79 promoter.

3. The use according to claim 1 or 2, wherein the nucleotide sequence encoding the transcription factor MaMYB4 is shown in SEQ ID No.2.

4. The use according to claim 1, wherein the transcription factor MaMYB4 or the gene encoding the transcription factor MaMYB4 is co-transformed with mapt 8 to significantly enhance the synthesis of scopolamine in the hairy root of trifoliter white; the amino acid sequence of MaTT8 is shown as SEQ ID NO. 3.

5. The use according to claim 1, wherein the transcription factor MaMYB4 or the gene encoding the transcription factor MaMYB4 is co-transformed with mapt 8 to significantly enhance the effect of activating the MaUGT79 gene and/or the MaUGT79 promoter; the amino acid sequence of MaTT8 is shown as SEQ ID NO. 3.

6. The use according to claim 4 or 5, wherein the nucleotide sequence encoding said mapt 8 is set forth in SEQ ID No. 4.

7. An expression vector, wherein the expression vector comprises the coding sequence SEQ ID NO.2 of the transcription factor MaMYB4 gene as claimed in claim 3.

8. A method of promoting scopolamine synthesis and/or activating MaUGT79 promoter in white flower using the MaMYB4 expression vector of claim 5, wherein the method is:

(1) Taking the recombinant plasmid T-MaMYB4 as a template, and carrying out PCR amplification by using a primer P5/a primer P6 through high-fidelity enzyme; recovering the MaMYB4 gene fragment by using a PCR product purification kit;

(2) The plant over-expression vector plasmid pBI121 is digested with XbaI and BamHI to obtain a linearization vector of pBI 121;

(3) Connecting the MaMYB4 gene fragment in the step (1) with a linearization vector of pBI121 to obtain a plant over-expression vector pBI121-MaMYB4;

(4) Transforming the plant overexpression vector pBI121-MaMYB4 in the step (3) into agrobacterium rhizogenes K599 by adopting an electric shock method, and infecting hairy roots of the sweet clover with the agrobacterium rhizogenes K599 to obtain the sweet clover;

the primer P5 sequence is shown as SEQ ID NO.9, and the primer P6 sequence is shown as SEQ ID NO. 10.

9. A method for promoting scopolamine synthesis and/or activating MaUGT79 promoter in white flower by cotransformation of MaMYB4 gene and MaTT8 gene according to claim 4, characterized in that the method comprises:

(1) Respectively constructing a MaMYB4 and a MaTT8 over-expression vector and naming the over-expression vectors as OE-MaMYB4 and OE-MaTT8; then adopting an electric shock method to transform agrobacterium rhizogenes K599 to obtain different agrobacterium;

(2) Cutting root tips of trifolitan seeds after germination of 7 seeds of trifolitan flowers by using a surgical knife, then slightly dipping in bacterial films of agrobacterium transformed with an OE-MaMYB4 vector and agrobacterium transformed with an OE-MaTT8 vector, and carrying out infection culture to obtain OE-MaTT8+OE-MaMYB4.

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