SmWRKY13 gene and its application in increasing the content of salvianolic acid in Salvia miltiorrhiza.

Abstract

This invention discloses the SmWRKY13 gene and its application in increasing the salvianolic acid content in Salvia miltiorrhiza, belonging to the field of genetic engineering technology. By constructing a recombinant expression vector containing the SmWRKY13 gene and transforming it into hairy roots of Salvia miltiorrhiza, transgenic hairy root lines overexpressing SmWRKY13 were obtained. The salvianolic acid content of these transgenic hairy root lines was detected. The results showed that overexpression of SmWRKY13 significantly increased the content of salvianolic acid components such as rosmarinic acid and salvianolic acid B in the hairy roots of Salvia miltiorrhiza, with the total salvianolic acid content reaching up to 5.72 times that of the control group. This invention provides an effective strategy for increasing the salvianolic acid content in Salvia miltiorrhiza using genetic engineering methods, which is of great significance for meeting market demand and promoting the large-scale preparation of salvianolic acid.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology and relates to the SmWRKY13 gene and its application in increasing the content of tanshinone in danshen. Background Technology

[0002] Danshen is a plant of the Lamiaceae family (Salvia miltiorrhiza). Salvia miltiorrhiza The dried roots and rhizomes of Danshen (Salvia miltiorrhiza). Danshen has the effects of promoting blood circulation and removing blood stasis, regulating menstruation and relieving pain, clearing the heart and relieving irritability, cooling the blood and reducing swelling. Clinically, Danshen and its preparations are widely used in the treatment of cardiovascular diseases, cerebrovascular diseases, liver diseases, etc., and the market demand for Danshen exceeds supply.

[0003] Tanshinone is the second largest group of major chemical components in Salvia miltiorrhiza, mainly including rosmarinic acid, tanshinone A, and tanshinone B. Phenolic acids possess various biological activities, including antioxidant, anticoagulant, antithrombotic, antitumor, and anticoagulant effects. Clinically, they are mainly used for antibacterial and anti-inflammatory purposes, liver and kidney protection, and the treatment of cardiovascular and cerebrovascular diseases, possessing high economic and medicinal value. Therefore, increasing the content of tanshinone in Salvia miltiorrhiza is of great significance.

[0004] Current research on the regulation of the salvianolic acid biosynthesis pathway mainly focuses on key enzymes and transcription factors. Existing studies have shown that some WRKY family transcription factors can regulate the synthesis of secondary metabolites in tanshinone. This invention utilizes genetic engineering technology to overexpress... SmWRKY13 The gene can significantly increase the content of tanshinone in danshen. Currently, no other method utilizing this gene has been found that is relevant to the subject matter of this invention. SmWRKY13 There are reports on gene-based enhancement of salvianolic acid in Salvia miltiorrhiza. Therefore, this invention has positive significance in increasing the content of active ingredients in Salvia miltiorrhiza, improving its quality, and practically solving the problem of salvianolic acid shortage. Summary of the Invention

[0005] To address the above problems, the present invention provides a... SmWRKY13 Genes and their application in increasing the content of tanshinone in salvia miltiorrhiza hairs.

[0006] The objective of this invention can be achieved through the following technical solutions: In a first aspect, the present invention provides a tanshinone transcription factor SmWRKY13, the nucleotide sequence of which is shown in SEQ ID NO. 1.

[0007] Secondly, the present invention provides a gene expression cassette comprising the aforementioned nucleotide sequence.

[0008] Thirdly, the present invention provides a recombinant expression vector containing the nucleotide sequence of the SmWRKY13.

[0009] Fourthly, the present invention provides a transgenic engineered bacterium containing the aforementioned recombinant expression vector.

[0010] Fifthly, the present invention also provides an application of the tanshinone transcription factor SmWRKY13, the gene expression cassette, the recombinant expression vector, or the transgenic engineered bacteria in increasing the content of tanshinone in tanshinone.

[0011] Furthermore, the SmWRKY13 gene was overexpressed in Salvia miltiorrhiza.

[0012] Furthermore, hairy root culture was employed.

[0013] Sixthly, the present invention provides an application of the tanshinone transcription factor SmWRKY13 in plant breeding, wherein the plant breeding involves selecting tanshinone varieties with increased tanshinone content.

[0014] Furthermore, the SmWRKY13 gene was overexpressed in Salvia miltiorrhiza.

[0015] The beneficial effects of this invention are: (1) In this invention, it was found that after CMV infection of Salvia miltiorrhiza, the content of salvianolic acid in Salvia miltiorrhiza increased, and SmWRKY13 The expression was downregulated, but subsequent independent research was conducted. SmWRKY13 The effect of salvianolic acid content in tanshinone was found to be... SmWRKY13 It can directly and positively regulate the synthesis of salvianolic acid.

[0016] (2) This invention proposes a method through overexpression SmWRKY13 A method for increasing the content of salvianolic acid in Salvia miltiorrhiza using genetic engineering resulted in transgenic hairy root systems with significantly increased contents of rosmarinic acid and salvianolic acid B, and a total salvianolic acid content 5.72 times higher than the control group. This invention provides a promising technical strategy for the efficient production of salvianolic acid, and is of great significance for meeting market demand and promoting the large-scale preparation of salvianolic acid.

[0017] (3) This invention has successfully cultivated a new variety of Salvia miltiorrhiza with significantly increased content of salvianolic acid, which effectively reduces the cost of obtaining salvianolic acid and the production process is environmentally friendly. Attached Figure Description

[0018] Figure 1 for SmWRKY13 Gene bioinformatics analysis diagram; Figure 2 for SmWRKY13 Subcellular localization analysis results; Figure 3 A flowchart illustrating the rooting process of the hairy roots of the relevant transgenic Salvia miltiorrhiza. Figure 4 Image showing the positive identification results of overexpression of Salvia miltiorrhiza hairy roots; Figure 5 for SmWRKY13 Figure showing the results of quantitative analysis of gene expression in hairy roots; Figure 6 The figure shows the results of quantitative analysis of key enzyme genes in the phenolic acid pathway in the roots of positive hairy roots. Figure 7 for SmWRKY13 The activation effect of proteins on key enzyme target genes in the phenolic acid pathway is verified by the Dual-LUC experimental analysis results.

[0019] Figure 8 for SmWRKY13 The results of the detection of salvianolic acid content in the hairy roots of genetically modified Salvia miltiorrhiza are shown in the figure. Detailed Implementation

[0020] The invention will be further illustrated below with specific implementation examples.

[0021] Example 1: Salvia miltiorrhiza SmWRKY13 Acquisition of genes Based on the transcriptome sequencing annotation information and transcription factor prediction information, the FPKM values ​​of SmWRKY family transcription factor members were extracted from the CMV-infected Salvia miltiorrhiza transcriptome. The sequence of the gene SmWRKY13, which was downregulated after CMV infection, had a large differential value, and was highly expressed in the root, was selected.

[0022] Retrieved from laboratory transcriptome databases SmWRKY13 The CDS sequence was obtained, yielding a full-length 666 bp sequence. SmWRKY13 Genes, sequences as shown in SEQ ID NO.1. Phylogenetic analysis of the amino acid sequences of Arabidopsis WRKY transcription factors and related genes was performed using MEGA 6.0 software. Figure 1 A). Using DNAMAN, a bioinformatics software, to... SmWRKY13 Sequence alignment analysis was performed with five WRKY transcription factors in Arabidopsis thaliana (see...). Figure 1 B).

[0023] Example 2: Salvia miltiorrhiza SmWRKY13 Construction and transformation of gene expression vectors in plants Based on the already cloned Salvia miltiorrhiza SmWRKY13 Gene sequence, primers for constructing plant overexpression vectors (sequences see SEQ ID NO.3 and SEQ ID NO.4), construct 2306- SmWRKY13 -flag carrier, and 2306- SmWRKY13 The -flag plasmid was transformed into Agrobacterium rhizogenes C58C1. Single colonies were picked for PCR verification. The results showed that it contained... SmWRKY13 Plant overexpression vector 2306- SmWRKY13 -flag has been successfully transformed into Agrobacterium rhizogenes C58C1 and can be used for subsequent genetic transformation and hair root infection experiments.

[0024] The specific primer design is as follows:

[0025] Example 3: Salvia miltiorrhiza SmWRKY13 Subcellular localization analysis of genes 3.1 Salvia miltiorrhiza expression vector pHB- SmWRKY13 -YFP construction Based on the already cloned Salvia miltiorrhiza SmWRKY13 Gene sequence, primers for designing plant overexpression vectors (sequences shown in SEQ ID NO. 5 and SEQ ID NO. 6), and construction of pHB- SmWRKY13 -YFP vector.

[0026] The specific primer design is as follows:

[0027] 3.2 pHB- SmWRKY13 -YFP vector transformed Agrobacterium tumefaciens GV3101 Take the pHB- obtained in Example 3.1 SmWRKY13 Agrobacterium tumefaciens GV3101 was transformed with the YFP and empty vector pHB-YFP plasmids, and single colonies were picked for PCR verification. The results showed that it contained... SmWRKY13 plant overexpression vector pHB- SmWRKY13 -YFP has been successfully transformed into Agrobacterium tumefaciens GV3101.

[0028] 3.3 Transient expression in tobacco Using a sterile syringe, aspirate the pHB- carrier-containing sample constructed in step 2.1. SmWRKY13 Agrobacterium tumefaciens GV3101 containing the vectors -YFP and empty vector pHB-YFP was used to transiently transform well-grown tobacco leaves. The leaves were first cultured in the dark for 24 hours, followed by 24 hours of light exposure. Subcellular localization was observed using a laser confocal microscope. Tobacco leaves were placed face down on a slide with distilled water, covered with a coverslip, and observed under a laser confocal microscope. The results are as follows. Figure 2 As shown in the figure, YFP represents yellow fluorescent protein; DAPI represents blue fluorescent protein; BF represents bright field; Merged is a combined image of yellow fluorescent protein and bright field; pHB- SmWRKY13 -YFP is SmWRKY13 The transient fluorescence expression of the empty vector pHB-YFP was observed; pHB-YFP was the fluorescence expression of the empty vector. The figure shows that the fluorescence of the empty vector pHB-YFP was distributed in the cytoplasm and nucleus; while... SmWRKY13The fluorescence of the -YFP fusion protein was limited to the cell nucleus. The results indicate that... SmWRKY13 They are expressed in the cell nucleus, consistent with their function as transcription factors.

[0029] Example 4

[0030] 4.1 Obtaining monoclonal hairy roots based on genetic transformation Pre-culture of sterile Salvia miltiorrhiza seedlings: The explants of sterile Salvia miltiorrhiza seedlings were spread on 1 / 2 MS solid medium and placed for 48 hours; Co-culture: The pre-cultured explants were mixed with Agrobacterium rhizogenes for infection. The mixing time was 10 min. After that, the surface bacterial solution was blotted dry with sterile paper and placed on 1 / 2 MS solid medium for 48 h. Antibiotic reduction: Co-cultured explants were placed sequentially on 1 / 2 MS solid medium containing 300 mg / L Cb, 500 mg / L Cef, and 200 mg / L Cef, and antibiotics were reduced every week until no antibiotics were present. Separate individual clones: Cut off individual hairy roots on antibiotic-free 1 / 2 MS solid medium and place them individually on 1 / 2 MS solid medium; Subculturing: A portion of the grown single clone is cut off for subculturing, eventually obtaining a stable root system (see...). Figure 3 ).

[0031] 4.2 Extraction of DNA from the hairy roots of Salvia miltiorrhiza Genomic DNA from the transgenic hairy roots obtained in section 4.1 was extracted using the CTAB method. For specific operating procedures, please refer to the CTAB operating instructions.

[0032] 4.3 PCR positive identification of transgenic hairy roots overexpressing the gene Upstream primers were selected from clones. SmWRKY13 Gene primer 2306- SmWRKY13 -F, a reverse primer pair 2306-R was designed. This primer pair was used for PCR reaction to achieve PCR positive identification of transgenic hairy roots overexpressing the gene. Specific primer design: 2306- SmWRKY13 -F is the upstream primer and 2306-R is the downstream primer, with sequences as shown in SEQ ID NO. 9 and SEQ ID NO. 10.

[0033] All positive transgenic hairy roots and control hairy roots need to be identified using the rolB gene, with primers rolB-F and rolB-R, sequences shown in SEQ ID NO. 7 and SEQ ID NO. 8, to confirm that the Ri plasmid has indeed integrated into the *Salvia miltiorrhiza* genome. Figure 4 ).

[0034] In this embodiment, the overexpressing root system OE- that was positive was selected. SmWRKY13 -1、OE- SmWRKY13 -7 and OE- SmWRKY13 Three clones of -8 and the empty transgenic hairy root line C58C1-EV of 2306 were used as controls. The empty transgenic root line C58C1-EV and the overexpressing root line OE- were compared. SmWRKY13 -1、OE- SmWRKY13 -7 and OE- SmWRKY13 Gene expression analysis of SmWRKY13 was performed at -8, and the results showed that the expression level of SmWRKY13 in the three overexpression root systems was higher than that in the empty transgenic hairy root system. Figure 5 ).

[0035] The specific primer design is as follows:

[0036] Example 5: Quantitative PCR detection of related gene expression in the hairy roots of transgenic Salvia miltiorrhiza. 5.1 Shake-flask culture of hairy roots In Example 4, the expression level of the SmMYCL1 gene in positive hairy roots was quantitatively analyzed to screen for its expression. Three overexpression-positive hairy roots were cultured in the dark for approximately 50 days before harvesting. A suitable amount of fresh hairy roots were blotted dry with absorbent paper, wrapped in aluminum foil, and frozen in liquid nitrogen at -80°C for RNA extraction. The remaining hairy roots were dried and used for extracting salvianolic acid.

[0037] 5.2 RNA extraction and cDNA synthesis Total RNA was extracted and its purity and concentration were determined. It was then reverse transcribed into cDNA for quantitative PCR analysis. The reaction system used the Tiangen Polysaccharide and Polyphenol Plant Total RNA Extraction Kit, with SmActin as the internal control gene.

[0038] 5.3 Primer Design and Synthesis

[0039] According to Danshen gene SmWRKY13 Primers were designed using Primer 5.0 software to detect the expression of related genes in the hairy roots of Salvia miltiorrhiza, and the coding sequences of the genes related to the biosynthesis of salvianolic acid were used. The Actin gene was used as an internal control.

[0040] The specific primer design is as follows:

[0041] 5.4 Quantitative PCR detection of hairy roots of transgenic Salvia miltiorrhiza Using the same amount of the first strand of cDNA as a template, quantitative PCR amplification was performed using the primers designed above (SEQ ID NO. 13-28). The reaction system is as follows:

[0042] The results showed that, in terms of the salvianolic acid biosynthesis pathway, in the three overexpression lines OE- SmWRKY13 -1、OE- SmWRKY13 -7 and OE- SmWRKY13 In OE-8, the expression levels of the vast majority of key enzyme genes were promoted to varying degrees. SmWRKY13 Compared with the control group, the -1 strain SmC4H Expression levels increased significantly by 3.57-fold, OE- SmWRKY13 Compared with the control group, the -7 strain SmTAT1 The expression level increased significantly by 2.27 times. This indicates... SmWRKY13 Overexpression of [a substance] has a significant promoting effect on the biosynthetic pathway of tanshinone (see [reference]). Figure 6 ).

[0043] Example 6: Dual-luciferase assay verification SmWRKY13 Targets of transcription factors The promoter of the key enzyme gene for the biosynthesis of salvianolic acid was constructed into the pGreen II 0800 vector. The constructed vector was then transformed into Agrobacterium GV3101 (pSoup). pHB- SmWRKY13 -YFP vector, and transformed into GV3101. Bacterial cultures with different promoters were then mixed with pH B- SmWRKY13 YFP bacterial suspensions were mixed at a 1:1 ratio, suspended in permeate, and injected into tobacco. Dual-LUC detection was performed. SmWRKY13 The activation effect of transcription factors on promoters. Results showed that: SmWRKY13 right SmC4H1 The promoter inhibition effect is significant (see Figure 7 ).

[0044] Example 7 Overexpression SmWRKY13 Detection of salvianolic acid content in transgenic hairy roots In this embodiment, the overexpressing root system OE- that was positive was selected. SmWRKY13 -1、OE- SmWRKY13 -7 and OE- SmWRKY13 -8 and 2306 empty transgenic hairy root C58C1-EV were used as controls.

[0045] In this embodiment, the content of salvianolic acid in the hairy roots of transgenic Salvia miltiorrhiza was determined by HPLC, as follows: 7.1 Extraction of Tanshinone Wrap the hairy root sample in absorbent paper and place it in an oven until completely dry. Grind the sample into powder using a mortar and pestle. Place 50 mg of the powder into a 50 mL centrifuge tube. Add 10 mL of a 4:1 mixture of ethanol and water to the centrifuge tube and sonicate at room temperature for 0.5 h. Centrifuge at 8000 rpm for 20 min at room temperature. Transfer the supernatant to a distillation flask and rotary evaporate at 65 °C until the supernatant is dry. Dissolve the residue in the distillation flask with 2 mL of double-distilled water and transfer the dissolved solution to a new 2 mL EP tube. Centrifuge at 12000 rpm for 10 min. Use a 1 mL syringe to aspirate the sample and filter it through a 0.22 μm organic filter membrane. Store the sample at 4 °C for subsequent analysis.

[0046] 7.2 Preparation of Danshensu acid standard Caffeic acid, rosmarinic acid, salvianolic acid B, and salvianolic acid A powders were accurately determined and dissolved separately in chromatographically pure methanol to prepare standard stock solutions with a concentration of 1 mg / mL. Subsequently, 100 μL of each of these stock solutions was taken and mixed to prepare a mixed standard solution with a concentration of 0.25 mg / mL. The mixed standard was serially diluted with chromatographically pure methanol to obtain six standard solutions of different concentrations for plotting a standard curve.

[0047] 7.3 Liquid Chromatographic Detection Conditions for Tanshinone Acids A C-18 reversed-phase silica gel column was used. The chromatographic parameters were set as follows: flow rate 1.0 mL / min, sample injection volume 10 μL, column temperature maintained at 35℃, and detection wavelength 281 nm. The eluents were acetonitrile (B) and an aqueous solution containing 0.03% trifluoroacetic acid (D). The specific elution procedure is as follows:

[0048] The results are as follows Figure 8 In the figure, EV represents the empty vector. In this invention, the contents of rosmarinic acid, salvianolic acid B, and total salvianolic acid were significantly increased in the overexpressed hairy root system. Compared with the control root system, the overexpressed root system OE- SmWRKY13 -1 and OE- SmWRKY13 The contents of rosmarinic acid and salvianolic acid B were significantly increased at -7, and OE- SmWRKY13 The total salvianolic acid content in the -8 root system was 5.72 times higher than that in the control group. This invention makes it possible to commercialize salvianolic acid in large quantities and provides an important source to meet market demand for salvianolic acid.

[0049] This invention employs overexpression SmWRKY13Metabolic engineering strategies have yielded transgenic hairy root systems of Salvia miltiorrhiza that produce high levels of salvianolic acid, making it possible to commercialize salvianolic acid in large quantities and providing an important source to meet market demand for salvianolic acid.

[0050] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. Those skilled in the art can readily make various modifications to these embodiments and apply the general principles described herein to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the invention should be within the protection scope of the present invention.

[0051] Sequence List: SEQ ID NO. 1 SmWRKY 13 ATGTCTTCCCAAGCCATGCTGAATCAGAGCTTGTTTGAAGAGCAAGAAAT GATAATCTCATCACAGCTTGGGATTTTCCCTTTCCACACAAACTTGTCAT CATCTCTCCCACTATTAGGGTTCAACCAGCCCCTCAAAACCCTCTCTGCA GATTCACCTCTCATAACCCTTTCTGATCACTCCACACTTACCAAGCAAAA GGAAGACTTCACACCTCACTTTTCTGGACTGCCCCACCTTCTTTCTTTGC AAAGATCAGCTGCTTCAAATTACTGGGCATGGGGTGAGTTGAGCGACTGC ATGAGCATGAAGAGAAGCGGAGGGGAGGATCACCTGGGCGTGTCGGCGAT GAAGATGAAGAAGATCAAGGCAAGAAGAAAGGTGAGAGAGCCCAGGTTTT GCTTCAAGACCATGAGCGATGTAGATGTTCTTGATGATGGTTACAAATGG AGAAAATACGGTCAGAAAGTCGTCAAGAACACACAACATCCTAGGAGCTA CTACAGATGCACACAAGATAATTGTCGTGTGAAGAAACGCGTAGAGCGAC TAGCAGACGATCCTAGAATGGTGATAACGACATACGAGGGAAGGCACATT CACTCTCCATCACACGATGAAGACGATTCTCAAGCTTCTGCCCAACTCAC TAACTTCTTCTGGTAG SEQ ID NO. 2 MSSQAMLNQSLFEEQEMIISSQLGIFPFHTNLSSSLPLLGFNQPLKTLSADSPLITLSDHSTLTKQKEDFTPHFSGLPHLLSLQRSAASNYWAWGELSDCMSMKRSGGEDHLGVSAMKMKKIKARRKVREPRFCFKTMSDVDVLDDGYKWRKYGQKVVKNTQHPRSYYRCTQDNCRVKKRVERLADDPRMVITTYEGRHIHSPSHDEDDSQASAQLTNFFW

Claims

1. A Salvia miltiorrhiza transcription factor SmWRKY13, characterized in that, The nucleotide sequence of SmWRKY13 is shown in SEQ ID NO.

1.

2. A gene expression cassette, characterized in that, It comprises the nucleotide sequence of claim 1.

3. A recombinant expression vector, characterized in that, The nucleotide sequence comprising SmWRKY13 as described in claim 1.

4. A genetically engineered bacterium, characterized in that, It includes the recombinant expression vector as described in claim 3.

5. The application of the tanshinone transcription factor SmWRKY13 of claim 1, the gene expression cassette of claim 2, the recombinant expression vector of claim 3, or the transgenic engineered bacteria of claim 4 in increasing the content of tanshinone in tanshinone.

6. The application according to claim 5, characterized in that, The SmWRKY13 gene was overexpressed in Danshen.

7. The application according to claim 5, characterized in that, Hairy root culture was used.

8. The application of the Tanshinone transcription factor SmWRKY13 as described in claim 1 in plant breeding, characterized in that, The plant breeding involves selecting and breeding *Salvia miltiorrhiza* varieties with increased *tanshinone* content.

9. The application according to claim 8, characterized in that, The SmWRKY13 gene was overexpressed in Danshen.

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