A method for improving andrographolide content based on methyl jasmonate and apmyc2-aphsfb2b module

Abstract

The application provides a method for improving androstane content based on methyl jasmonate and ApMYC2-ApHSFB2b module, and belongs to the field of plant biotechnology. The application constructs an overexpression vector of ApMYC2 gene and an overexpression vector of ApHSFB2b gene respectively, and transforms the two overexpression vectors into andrographis plants; after transformation, the andrographis plants are treated with methyl jasmonate again. Through the transformation of the overexpression vectors and the treatment of methyl jasmonate, the two work together to directly activate the expression of ApCPS1, a gene in the andrographolide biosynthesis pathway, and then promote the expression of other genes downstream of the synthesis pathway, so as to improve the androstane content, and provide an effective technical means for the planting and development of andrographis.

Description

Technical Field

[0001] This invention belongs to the field of plant biotechnology, specifically relating to a method for increasing the content of andrographolide based on methyl jasmonate and the ApMYC2-ApHSFB2b module. Background Technology

[0002] Andrographis paniculata, an important medicinal plant, contains andrographolide (AD), its main active ingredient, which exhibits significant anti-inflammatory, antibacterial, and antiviral pharmacological activities, showing broad application prospects in the pharmaceutical field. However, the natural content of andrographolide in Andrographis paniculata is low, which greatly limits its application in drug development and production. Currently, the chemical synthesis of andrographolide faces many challenges, such as complex synthesis processes, high costs, and numerous side reactions, making large-scale, efficient production difficult. Therefore, exploring a method to effectively increase the andrographolide content in Andrographis paniculata is of significant practical importance. Summary of the Invention

[0003] In order to solve the problems existing in the prior art, the purpose of this invention is to provide a method for increasing the content of andrographolide based on methyl jasmonate and ApMYC2-ApHSFB2b module, which can increase the content of andrographolide by 101.5%.

[0004] The present invention also aims to provide the application of the above method in the cultivation of Andrographis paniculata or the production of andrographolide.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0006] This invention provides a method for increasing the content of andrographolide based on methyl jasmonate and the ApMYC2-ApHSFB2b module, comprising the following steps: constructing an overexpression vector of the ApMYC2 gene and an overexpression vector of the ApHSFB2b gene respectively, transforming the two overexpression vectors into andrographolide plants; and treating the andrographolide plants again with methyl jasmonate after transformation.

[0007] Preferably, the two overexpression vectors are transformed into the Andrographis paniculata plants 6 days before harvesting; and the Andrographis paniculata plants are treated with methyl jasmonate 1 day before harvesting.

[0008] Preferably, the ApMYC2 gene is inserted between the AgeI and XhoI restriction sites of the pEAQ-HT vector to construct an ApMYC2 gene overexpression vector; the nucleotide sequence of the ApMYC2 gene is shown in SEQ ID No. 1.

[0009] Preferably, the ApHSFB2b gene is inserted between the AgeI and XhoI restriction sites of the pEAQ-HT vector to construct an ApHSFB2b gene overexpression vector; the nucleotide sequence of the ApHSFB2b gene is shown in SEQ ID No. 2.

[0010] Preferably, the overexpression vector is introduced into Agrobacterium and transformed into Andrographis paniculata plants, wherein the Agrobacterium is GV3101 and the transformation method is injection.

[0011] Preferably, Agrobacterium containing the ApMYC2 gene overexpression vector and Agrobacterium containing the ApHSFB2b gene overexpression vector are mixed in equal proportions and left to stand in the dark for 3 hours before injection.

[0012] Preferably, the injection is a leaf injection, and the injection area is not less than 80% of the leaf area.

[0013] Preferably, the treatment method for methyl jasmonate is spraying; the concentration of methyl jasmonate is 200 μM.

[0014] Preferably, the Andrographis paniculata plant is in the 6-8 leaf stage.

[0015] This invention also provides the application of the above method in the cultivation of Andrographis paniculata or the production of andrographolide.

[0016] Compared with the prior art, the beneficial effects of the technical solution of the present invention are as follows:

[0017] This invention reveals that ApMYC2 and ApHSFB2b interact and, mediated by methyl jasmonate, play a crucial role in activating the andrographolide biosynthesis pathway gene ApCPS1, thereby influencing andrographolide accumulation. The method provided by this invention significantly increases the andrographolide content in Andrographis paniculata by 101.5% through the synergistic effect of the methyl jasmonate (MeJA) signaling pathway and the ApMYC2-ApHSFB2b transcriptional module, providing a core technology for the high-value utilization of Andrographis paniculata. Attached Figure Description

[0018] Figure 1 Effects of overexpression of ApMYC2, ApHSFB2b and ApMYC2-ApHSFB2b and methyl jasmonate spray on andrographolide;

[0019] Figure 2 Yeast two-hybrid experiments confirmed the in vitro interaction between ApMYC2 and ApHSFB2b;

[0020] Figure 3Firefly luciferase complementation experiments confirmed the interaction between ApMYC2 and ApHSFB2b in vivo;

[0021] Figure 4 Yeast one-hybrid experiments confirmed the interaction between the ApCPS1 promoter and ApMYC2;

[0022] Figure 5 Dual-luciferase assays were used to investigate the activation of the ApCPS1 promoter by the ApMYC2 and ApMYC2-ApHSFB2b complex in tobacco leaves. Detailed Implementation

[0023] This invention provides a method for increasing the content of andrographolide based on methyl jasmonate and the ApMYC2-ApHSFB2b module, comprising the following steps: constructing an overexpression vector of the ApMYC2 gene and an overexpression vector of the ApHSFB2b gene respectively, transforming the two overexpression vectors into andrographolide plants; and treating the andrographolide plants again with methyl jasmonate after transformation.

[0024] In this invention, the treatment is preferably carried out 6 days before harvesting the Andrographis paniculata plants to obtain andrographolide, that is, 6 days before harvesting the Andrographis paniculata plants, two overexpression vectors are transformed into the Andrographis paniculata plants; and 1 day before harvesting the Andrographis paniculata plants, the Andrographis paniculata plants are treated with methyl jasmonate.

[0025] In this invention, the ApMYC2 gene is inserted between the AgeI and XhoI restriction sites of the pEAQ-HT vector to construct an ApMYC2 gene overexpression vector; the nucleotide sequence of the ApMYC2 gene is shown in SEQ ID No.1, which is the CDS sequence of ApMYC2; the construction is preferably performed using homologous recombination.

[0026] In this invention, the ApHSFB2b gene is inserted between the AgeI and XhoI restriction sites of the pEAQ-HT vector to construct an ApHSFB2b gene overexpression vector; the nucleotide sequence of the ApHSFB2b gene is shown in SEQ ID No. 2, which is the CDS sequence of ApHSFB2b; the construction is preferably performed using homologous recombination.

[0027] The present invention preferably involves introducing the overexpression vector into Agrobacterium, transforming it into Andrographis paniculata plants, and establishing a transient overexpression system; the Agrobacterium is GV3101, and the transformation method is injection. The present invention preferably involves mixing Agrobacterium containing the ApMYC2 gene overexpression vector and Agrobacterium containing the ApHSFB2b gene overexpression vector in an equal ratio (1:1), incubating in the dark for 3 hours, and then injecting. The OD of the Agrobacterium bacterial suspension used for injection is... 600The preferred concentration is 0.7–0.8. The injection is a foliar injection, preferably involving 1–2 pairs of leaves, with the injection area being no less than 80% (i.e., ≥80%) of the total leaf area. Alternatively, the injection is performed using a 1 mL syringe, where a small hole is gently punctured (without breaking the leaf) on the underside of the *Andrographis paniculata* leaf before injecting the bacterial solution. The injected *Andrographis paniculata* is then cultured in a room at 22°C with a photoperiod of 12 / 12.

[0028] The treatment method of methyl jasmonate described in this invention is by spraying, preferably foliar spraying, which can be selected as spraying on the front of the leaf, spraying on the back of the leaf, or spraying on both sides of the leaf; the concentration of methyl jasmonate is 200 μM, preferably sprayed until the leaves are moistened (in this invention, "moistened" means that the sprayed liquid is evenly distributed on the surface of the plant leaves, so that the leaf surface is covered by the liquid and forms a thin liquid film, but the liquid film will not gather into water droplets and drip). As an optional embodiment, the methyl jasmonate sprayed in this invention is an aqueous solution of methyl jasmonate. This invention uses methyl jasmonate solution to spray the leaves of Andrographis paniculata plants to precisely activate the plant hormone jasmonic acid (JA) signaling pathway. Through methyl jasmonate induction, it promotes the activation of the promoter region of the Andrographolide Synthesis Pathway Enzyme Gene ApCPS1 by the ApMYC2-ApHSFB2b transcriptional complex. The ApMYC2-ApHSFB2b transcriptional complex activates ApCPS1 in a cascade manner under the signal of methyl jasmonate, forming a highly efficient regulatory chain of "signal sensing-module assembly-gene activation". Through the synergistic effect of gene overexpression and induction by exogenous signaling molecules, it breaks through the bottleneck of traditional single regulation methods and achieves a multiple-level increase in andrographolide synthesis.

[0029] The preferred Andrographis paniculata plant used in this invention is at the 6-8 leaf stage.

[0030] This invention also provides the application of the above method in the cultivation of Andrographis paniculata or the production of andrographolide.

[0031] The technical solutions of this invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0032] The primers used in specific embodiments of this invention are shown in the table below:

[0033] Table 1 Primers used to construct the vector in this invention.

[0034]

[0035]

[0036] In the table, lowercase letters represent vector homologous arms in homologous recombination primers, and uppercase letters represent gene cloning primers.

[0037] Unless otherwise specified, the following embodiments are all conventional methods.

[0038] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0039] Example 1

[0040] 1. Increased andrographolide levels by spraying methyl jasmonate and overexpressing the ApMYC2-ApHSFB2b module:

[0041] (1) Experimental materials

[0042] Andrographis paniculata seeds from Guigang, Guangxi were washed with pure water and soaked for 24 hours. They were then sown in moist soil containing vermiculite and nutrient soil in a 1:4 ratio for germination. The soil was placed at 28℃ with 12 / 12 hours of light / dark conditions, and treated with 100 μmol·m⁻²·g·mol⁻¹. -2 ·s -1 They were cultured in an artificial climate chamber with light intensity and 60% relative humidity until they grew 6-8 leaves, and then used for experimental treatment.

[0043] (2) Construct overexpression vectors for the ApMYC2 gene and ApHSFB2b gene.

[0044] The CDS sequences of the ApMYC2 gene (SEQ ID No. 1) and the ApHSFB2b gene (SEQ ID No. 2) were obtained using a Takara rapid PCR polymerase assay kit. PCR amplification was performed using Max DNA Polymerase (R045A) (primers: SEQ ID No. 33, SEQ ID No. 34, SEQ ID No. 35, SEQ ID No. 36). The overexpression vector pEAQ-HT was double-digested with AgeI / XhoI. The digestion system consisted of 3 μg of vector plasmid, 2 μL of each enzyme, and 10 μL of 10×Cutsmart. The digestion conditions were 37℃ for 1 h. The digestion products were verified by gel electrophoresis and then recovered from the gel. The PCR amplified and recovered products of ApMYC2 and ApHSFB2b were ligated with the digested products of the overexpression vector pEAQ-HT, respectively. The ligation reaction was performed according to the instructions of the Vazyme ClonExpress II One Step Cloning Kit to obtain the overexpression vectors of the ApMYC2 and ApHSFB2b genes.

[0045] (3) Overexpression vector was transformed into Agrobacterium GV3101

[0046] Select the positive plasmids from step (2) (overexpression vectors of the ApMYC2 gene or overexpression vectors of the ApHSFB2b gene), and transform them into Agrobacterium GV3101 according to the instructions of Weidi Bio's GV3101 Chemically Competent Cell product to obtain successfully transformed Agrobacterium.

[0047] (4) Select single clones of Agrobacterium that have been successfully transformed in step (3) and add them to 3m LYEB culture medium (containing 25 μg / mLRif and 50 μg / mL Kan) and culture with shaking (28℃, 200 rpm) for 24 h. Then, take 1 mL of the above culture solution and add it to 30m LYEB culture medium (containing 25 μg / mLRif and 50 μg / mL Kan) for expansion culture for 6 h until OD is reached. 600 The concentration should be between 0.6 and 0.8. Collect the bacteria by centrifugation at 4000 rpm for 6 minutes, resuspend the cells in buffer (preparation method shown in Table 2), centrifuge again for 3 minutes (to wash), and finally resuspend the cells in buffer to OD. 600 At 0.7–0.8, Agrobacterium-mediated transformation was obtained containing an overexpression vector of the ApMYC2 gene (pEAQ-ApMYC2) and an overexpression vector of the ApHSFB2b gene (pEAQ-ApHSFB2b).

[0048] Table 2 Buffer Formulation

[0049] reagents Mother liquor concentration per 100mL <![CDATA[MgCl2]]> 500mmol / L 2mL MES 500mmol / L 2mL AS 200mmol / L 100μL <![CDATA[ddH2O]]> Up to 100mL

[0050] (5) Two bacterial solutions, pEAQ-ApMYC2 and pEAQ-ApHSFB2b, were mixed at a 1:1 ratio and left to stand in the dark for 3 hours before injection. A control group was injected with GV3101 strain transformed by pEAQ-HT (empty vector) using the same transformation method. A 1 mL syringe was used to lightly puncture the underside of the Andrographis paniculata leaves (without puncturing them) and inject the bacterial solution to cover 80% of the leaf area. After injection, the Andrographis paniculata was cultured in a room at 22℃ with a photoperiod of 12 / 12. Five days after injection, a 200 μM methyl jasmonate aqueous solution was sprayed until the leaves were moist. Samples were taken six days after injection.

[0051] 2. Analysis of the effects of co-expression of ApMYC2 and ApHSFB2b followed by methyl jasmonate spraying on andrographolide accumulation.

[0052] (1) Gene expression level analysis

[0053] Total RNA extraction and reverse transcription into cDNA from Andrographis paniculata: Total RNA was extracted from all samples using a kit from Magen Biosciences, following the instructions in the kit's manual. RNA concentration was measured using a NanoDrop2000 microspectrophotometer and recorded at 260 nm (OD). 260 ) and 280nm (OD) 280 The absorbance at a certain point () is used to assess RNA purity. Quality RNA samples are stored at -80°C for subsequent experimental procedures.

[0054] The qualified RNA was reverse transcribed using the Aike Rui AG reverse transcription kit. The procedure was as follows: 1 μg of RNA (10 μL system) was taken and reverse transcribed using the reverse transcription kit. The specific preparation method was performed according to the instructions. The reaction system is as follows:

[0055] Table 3. Reverse Transcription Reaction System

[0056] Reagents for RT-PCR reaction Reaction dosage 5×PrimeScriptRTMasterMix 4μL TotalRNA 1μg <![CDATA[RNaseFreeddH2O]]> Bring the volume to 10 μL

[0057] After gently mixing all the above components, place the mixture on a PCR instrument for reverse transcription. The reaction conditions are as follows: 37℃, 50 min (RT-PCR reaction); 85℃, 10 s (to inactivate reverse transcriptase); 4℃, 10 min (to lower the temperature and maintain a low temperature). The reaction products are then subjected to RT-qPCR in the following reaction system:

[0058] Table 4. qPCR reaction system

[0059]

[0060] Reaction conditions: First, pre-denaturation was performed at 95℃ for 5 minutes; then 40 cycles were performed, each cycle consisting of denaturation at 95℃ for 20 seconds and annealing at 60℃ for 30 seconds (real-time fluorescence data were acquired during this step); finally, a final extension was performed at 72℃ for 20 seconds. After the reaction was completed, the Ct value (cycle threshold) of qRT-PCR was derived for analysis of the reaction results. To standardize gene expression levels, the Actin gene was selected as an internal reference gene. The Ct values ​​of the target gene and the Actin gene were compared. The primers used in qRT-PCR are shown in SEQ ID No. 3 to SEQ ID No. 14, and 2... -ΔΔCt The method is used to calculate the relative expression level of the target gene relative to the internal reference gene.

[0061] (2) Detection of andrographolide

[0062] Preparation of the test solution: Accurately weigh 0.1 g of fresh Andrographis paniculata leaves, place them in a 2 ml centrifuge tube, add steel beads, and grind them into powder using a tissue homogenizer (30 Hz, 60 s). Accurately add 1.6 mL of 75% methanol, seal tightly, weigh, and extract with ultrasonic assistance (power 250 W, frequency 25 kHz, temperature 40 ℃) for 60 min. Remove, cool, weigh again, and replenish the lost weight with 75% methanol. Centrifuge at 12000 rpm for 10 min, and collect 1 mL of the supernatant. Filter using a 0.22 μm microporous membrane to obtain the final product.

[0063] Preparation of reference solutions: Using a 0.01 g / mL balance, accurately weigh the andrographolide standard, place it in a stoppered conical flask, accurately add chromatographic methanol to dissolve it, and dilute to volume. Filter through a 0.22 μm microporous membrane to obtain the andrographolide reference solutions (a series of reference solutions with concentrations of 21.04, 42.08, 84.17, 168.34, 336.675, and 502.5 μg / mL).

[0064] Chromatographic conditions: The column was a DIS-C18 (4.6 mm × 250 mm, 5 μm); the mobile phase was water-acetonitrile; the detection wavelength was 205 nm; the flow rate was 1.0 mL / min; the column temperature was 30 ℃; the injection volume was 5 μL, and gradient elution was performed using the following optimized method.

[0065] Table 5. HPLC Gradient Elution Table

[0066] Time (minutes) Acetonitrile (%) water(%) 0～5 20～25 80～75 5～20 25～26 75～74 20～25 26～32 74～68 25～38 32～40 68～60 38～45 40～85 60～15

[0067] Linearity investigation: Andrographolide standard was accurately weighed and serially diluted with chromatographic methanol to prepare a series of reference solutions containing 21.04, 42.08, 84.17, 168.34, 336.675, and 502.5 μg / mL of andrographolide. 5 μL of each reference solution was injected, and the peak area of ​​andrographolide was measured according to the method in Table 5. The peak area was recorded. A standard curve for andrographolide was plotted with the concentration of the reference standard (X μg / mL) on the x-axis and the peak area (Y) on the y-axis, and linear regression analysis was performed. The results showed that the regression equation for andrographolide was Y = 4.555X - 46.06, R0. 2 =0.9996. The results show that andrographolide exhibits good linearity in the range of 21.04–502.5 μg / mL.

[0068] Andrographolide content detection calculation: Substitute the peak area of ​​the andrographis sample detected according to the method in Table 5 above into the standard curve Y = 4.555X - 46.06 to calculate the concentration of the sample to be tested, and finally convert it to mg / g based on the fresh weight.

[0069] The results of gene expression levels and andrographolide content are as follows: Figure 1 As shown in the figure, a) shows the effect of overexpression of ApMYC2, ApHSFB2b, and ApMYC2-ApHSFB2b on the accumulation of andrographolide (AD); b) shows the effect of the combination of overexpression of ApMYC2, ApHSFB2b, and ApMYC2-ApHSFB2b and methyl jasmonic acid treatment on the accumulation of andrographolide (AD); c) shows the effect of overexpression of ApMYC2, ApHSFB2b, and ApMYC2-ApHSFB2b on the expression of key enzyme genes (ApCPS1, ApCPS2, ApUGT73AU1) involved in the andrographolide biosynthesis pathway; d) shows the effect of overexpression of ApMYC2, ApHSFB2b, and ApMYC2-ApHSFB2b and methyl jasmonic acid treatment on the expression of key enzyme genes (ApCPS1, ApCPS2, ApUGT73AU1) involved in the andrographolide biosynthesis pathway.

[0070] The experimental results showed that overexpression of ApMYC2, ApHSFB2b, or the ApMYC2-ApHSFB2b complex alone had no significant effect on the accumulation of andrographolide (a). However, overexpression of ApMYC2, ApHSFB2b, or the ApMYC2-ApHSFB2b complex alone, combined with treatment with methyl jasmonate for 24 h, increased the andrographolide content by 70.6%, 83.6%, and 101.5%, respectively, compared to the control group (b), and also upregulated the expression levels of enzyme genes related to the andrographolide synthesis pathway (d).

[0071] 3. Verification of the activation of the andrographolide biosynthesis gene ApCPS1 by the ApMYC2 and ApMYC2-ApHSFB2b complex via JA signaling:

[0072] (1) Yeast two-hybrid experiment

[0073] To investigate the relationship between ApMYC2 and ApHSFB2b, a yeast two-hybrid experiment was used to explore whether there is an interaction between them.

[0074] The CDS of ApMYC2 (bait gene) was ligated between the NdeI and NotI sites of the pGBKT7 vector using homologous recombination (primers SEQ ID No. 27 and SEQ ID No. 28) to obtain plasmid BK-ApMYC2; the CDS of ApHSFB2b (prey gene) was ligated between the EcoRI and BamHI sites of the pGADT7 vector (primers SEQ ID No. 17 and SEQ ID No. 18) to obtain plasmid AD-ApHSFB2b. The plasmid construction method was the same as in step 1(2), and positive controls (using plasmids pGBKT7-p53 and pGADT7-T), negative controls (using plasmids pGBKT7-Lam and pGADT7-T), and empty vector control groups were set up. Both vectors were simultaneously transformed into yeast strain Y2HGold and cultured on SD / -TL selective medium for 3 days.

[0075] Pick a single yeast colony and incubate it in SD / -TL liquid medium until OD. 600 The concentration was set to 0.2, and then 4 μL of the bacterial culture was spotted onto SD / -TL and SD / -THLA media. If yeast containing the bait gene and the empty pGADT7 vector failed to grow on SD / -THLA media, it was considered that the bait lacked self-activation activity, thus ensuring the reliability of the results.

[0076] The results are as follows Figure 2 As shown in the figure, pGBKT7-p53+pGADT7-T represents the positive control experiment result, pGBKT7-Lam+pGADT7-T represents the negative control experiment result, BK-ApMYC2+AD represents the experiment result of co-transforming the BK-ApMYC2 plasmid and the empty vector pGADT7 into the yeast strain Y2HGold, and BK-ApMYC2+AD-ApHSFB2b represents the experiment result of co-transforming the BK-ApMYC2 and AD-ApHSFB2b vectors into the yeast strain Y2HGold. The experimental results indicate that the Y2Hgold strain containing the BK-ApMYC2 and AD-ApHSFB2b plasmids can grow on SD / -THLA medium, suggesting an in vitro interaction between these two transcription factors.

[0077] (2) Firefly luciferase complementation experiment

[0078] To further confirm the in vivo interaction between ApMYC2 and ApHSFB2b, a firefly luciferase complementation experiment was conducted in this type of tobacco.

[0079] Expression vectors ApMYC2-nLUC (primers SEQ ID No. 29 and SEQ ID No. 30) and ApHSFB2b-cLUC (primers SEQ ID No. 31 and SEQ ID No. 32) were constructed using homologous recombination (using Vazyme's ClonExpress II One Step Cloning Kit C112). Vectors JW771 (N-LUC) and JW772 (C-LUC) were used, with KpnI and SalI restriction enzyme sites, and were transformed into Agrobacterium strain GV3101, respectively. The plasmid construction method was the same as in step 1 (2). A control group with no empty vector was also set up: vectors JW771 and JW772 were transformed into Agrobacterium strain GV3101 using the same method. The two Agrobacterium suspensions were mixed in equal volumes at a 1:1 ratio. The Agrobacterium resuspension was prepared using 1 / 2 MS containing 150 μmol / LAS and then infiltrated into tobacco leaves. After 40-48 hours of infiltration, the fluorescence of luciferase was imaged using a PlantView 100 imaging system (BLT, China). High-purity D-luciferin potassium salt (ST198-25mg, Beyotime) was used as the substrate for fluorescence detection.

[0080] The results are as follows Figure 3 As shown in the figure, nLUC+cLUC(1) represents the result of soaking tobacco leaves with Agrobacterium containing empty vector JW771 and Agrobacterium containing empty vector JW772, cLUC+ApMYC2-nLUC(2) represents the result of soaking tobacco leaves with Agrobacterium containing empty vector JW772 and Agrobacterium containing ApMYC2-nLUC, nLUC+ApHSFB2b-cLUC(3) represents the result of soaking tobacco leaves with Agrobacterium containing empty vector JW771 and Agrobacterium containing ApHSFB2b-cLUC, and ApMYC2-nLUC+ApHSFB2b-cLUC(4) represents the result of soaking tobacco leaves with Agrobacterium containing ApMYC2-nLUC and Agrobacterium containing ApHSFB2b-cLUC. The results showed that only the nLUC-ApMYC2 and cLUC-ApHSFB2b groups exhibited luminescence, while other groups containing empty vector controls did not. This indicates that ApMYC2 and ApHSFB2b interact in vivo, allowing the NLUC and CLUC of luciferase to be spatially close enough and correctly assembled, thereby exerting luciferase activity, i.e., decomposing the substrate to produce fluorescence.

[0081] (3) Yeast one-hybrid experiment

[0082] To investigate the relationship between the transcription factors ApMYC2 and ApHSFB2b and the promoter of the Andrographolide biosynthesis pathway enzyme gene ApCPS1, a yeast one-hybrid experiment was conducted.

[0083] The CDS of ApMYC2 and ApHSFB2b were ligated to the EcoRI and BamHI sites of the pGADT7 vector via homologous recombination (primers: SEQ ID No. 15-SEQ ID No. 16, SEQ ID No. 17-SEQ ID No. 18). A 1043bp fragment of the ApCPS1 promoter (SEQ ID No. 37) was ligated to the EcoRI and SacI sites of the pHIS2 vector via homologous recombination (primers: SEQ ID No. 19 and SEQ ID No. 20). The plasmid construction method was the same as in step 1(2). The bait vector containing the promoter and the prey vector containing the target gene were co-transferred into yeast strain Y187. A positive control (using plasmids pHIS2-p53 and pGADT7-p53) and an empty vector control group were also set up, and the cultures were cultured on SD / -TL selection medium. Single colonies were screened, and the cultures were shaken in SD / -TL liquid medium for 24 h (30℃, 200 rpm). The OD of the yeast cultures was then diluted with sterile water. 600 Adjust to 0.1 and perform gradient point dilution experiments on SD / -THL+10mM 3-AT plates. The negative control included a decoy promoter with the empty vector pGADT7.

[0084] The results are as follows Figure 4 As shown in the figure, pHIS2-p53+pGADT7-p53 represents the positive control experiment result; ApCPS1pro+AD represents the experiment result of co-transforming the ApCPS1 promoter and the empty vector pGADT7 into yeast strain Y187; ApCPS1pro+ApMYC2 represents the experiment result of co-transforming the ApCPS1 promoter and a vector containing the ApMYC2 gene into yeast strain Y187; and ApCPS1pro+ApHSFB2b represents the experiment result of co-transforming the ApCPS1 promoter and a vector containing the ApHSFB2b gene into yeast strain Y187. The results show that the ApCPS1 promoter and the empty vector pGADT7 do not grow in 10 mM 3-AT SD / -THL medium, indicating that 10 mM 3-AT can inhibit the self-activation of the ApCPS1 promoter. Yeast strains containing the ApCPS1 promoter and ApMYC2 gene can grow in SD / -THL medium containing 10 mM 3-AT, indicating that the ApCPS1 promoter and ApMYC2 interact.

[0085] (4) Dual-luciferase assay

[0086] To investigate the effects of ApMYC2 and ApHSFB2b on the promoter of the andrographolide synthesis pathway enzyme gene ApCPS1 in the presence or absence of MeJA, a dual-luciferase experiment was conducted.

[0087] The ApCPS1 promoter sequence was constructed between the XmaI and NcoI sites of the pGreenII 0800LUC vector using homologous recombination (primers were SEQ ID No. 21 and SEQ ID No. 22). The CDS sequences of ApMYC2 and ApHSFB2b were constructed between the SacI and EcoRI sites of the pGreenII 62-SK vector using homologous recombination (primers were SEQ ID No. 23-SEQ ID No. 24 and SEQ ID No. 25-SEQ ID No. 26, respectively). The plasmid construction method was the same as in step 1(2). Successfully sequenced positive plasmids were transformed into Agrobacterium strain GV3101 (psoup-p19), and an empty vector control group was set up. Single colonies were picked and placed in 3 mL of YEB + 25 μg·mL⁻¹. -1 Rif + 50 μg·mL -1 Kan culture medium was incubated at 28°C with shaking at 200 rpm for approximately 24 hours (the bacterial suspension became turbid and turned pale orange); 1 mL of fresh bacterial suspension was inoculated into 25 mL of LB liquid medium (+25 μg / mL). -1 Rif + 50 μg·mL -1 Expanding cultivation to OD in Kan) 600 The OD value was between 0.6 and 0.8 (4-6 h). The cells were collected by centrifugation at 4000 rpm for 7 min at room temperature. The supernatant was discarded, and the cells were resuspended in 15 mL of sterile tobacco staining solution (2.5 g D-glucose, 1.066 g MES, 0.380 g Na3PO4·12H2O, with ddH2O added to 500 mL, freshly prepared). The cells were centrifuged again at 4000 rpm for 6 min. The supernatant was discarded, and the OD value was adjusted by adding tobacco staining solution to the cells. 600 To 0.6 (to minimize the OD difference between different bacterial cultures) 600 (Differences). A bacterial culture containing the pGreenⅡ62-SK+ target transcription factor and a bacterial culture containing the pGreenⅡ0800LUC+ promoter were mixed 1:1. Acetylsyringone (AS) was added to the above mixed infection solution to achieve a final concentration of 150 μmol·L⁻¹. -1 (The stock solution is 200mM, which is 7.5μL added to 10mL of the inoculum). After standing in the dark for 4 hours, it is used for injecting this type of tobacco.

[0088] The area to be injected into the tobacco plant was circled with an oil-based pen. One side of the same leaf was injected with the control group, and the other side with the experimental group. The lower epidermis of the leaf was gently pricked with a needle (without piercing the leaf). 1 mL of bacterial suspension was drawn up with a sterile syringe and slowly injected into the puncture site until the entire circled area was soaked in the suspension (do not exceed the circled area). The injected plants were covered with black bags and incubated overnight in the dark, then cultured under normal greenhouse conditions. Samples were taken after 48 hours for LUC and REN luciferase activity assays (using Beyotime's dual luciferase reporter gene assay kit, RG027). Each experiment was performed in triplicate. For the MeJA treatment group, 200 μM MeJA was sprayed onto the injected leaves 24 hours before sampling.

[0089] The results are as follows Figure 5 As shown in the figure, "Mock" indicates no methyl jasmonic acid treatment, and "MeJA" indicates methyl jasmonic acid treatment. The results showed that without methyl jasmonic acid treatment, both the ApMYC2 and ApMYC2-ApHSFB2b complexes exhibited weak activation of the ApCPS1 promoter. However, after treatment with 200 μM methyl jasmonic acid, the activation of the ApCPS1 promoter by both the ApMYC2 and ApMYC2-ApHSFB2b complexes was significantly upregulated, with the activation level of the ApMYC2-ApHSFB2b complex increasing nearly threefold under MeJA treatment. These findings indicate that the activation of the ApCPS1 promoter by the ApMYC2 and ApMYC2-ApHSFB2b complexes is dependent on JA signaling.

[0090] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for increasing the content of andrographolide based on methyl jasmonate and the ApMYC2-ApHSFB2b module, characterized in that, The process includes the following steps: constructing overexpression vectors for the ApMYC2 gene and ApHSFB2b gene, respectively, and transforming the two overexpression vectors into Andrographis paniculata plants; after transformation, the Andrographis paniculata plants are treated again with methyl jasmonate. The ApMYC2 gene was inserted between the AgeI and XhoI restriction sites of the pEAQ-HT vector to construct an ApMYC2 gene overexpression vector; the nucleotide sequence of the ApMYC2 gene is shown in SEQ ID No.

1. The ApHSFB2b gene was inserted between the AgeI and XhoI restriction sites of the pEAQ-HT vector to construct an ApHSFB2b gene overexpression vector; the nucleotide sequence of the ApHSFB2b gene is shown in SEQ ID No.

2.

2. The method according to claim 1, characterized in that, Six days before harvesting the Andrographis paniculata plants, two overexpression vectors were transformed into the plants; one day before harvesting, the plants were treated with methyl jasmonate.

3. The method according to claim 1, characterized in that, The overexpression vector was introduced into Agrobacterium tumefaciens and transformed into Andrographis paniculata plants. The Agrobacterium tumefaciens was GV3101, and the transformation method was injection.

4. The method according to claim 3, characterized in that, Agrobacterium containing the ApMYC2 gene overexpression vector and Agrobacterium containing the ApHSFB2b gene overexpression vector were mixed in equal proportions and placed in the dark for 3 hours before injection.

5. The method according to claim 4, characterized in that, The injection is a leaf injection, and the injection area is not less than 80% of the leaf area.

6. The method according to claim 1, characterized in that, The treatment method for methyl jasmonate is spraying; the concentration of methyl jasmonate is 200 μM.

7. The method according to claim 1, characterized in that, The Andrographis paniculata plant was in the 6-8 leaf stage.

8. The application of the method according to any one of claims 1 to 7 in the production of andrographolide.

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