Application of overexpression of antirrhinum majus AntbHLH16 gene in promoting synthesis of tropane alkaloids and precursors in hairy roots

Abstract

The application belongs to the technical field of biotechnology, and particularly relates to application of overexpression of Anisodus tanguticus AntbHLH16 gene in promoting synthesis of topan alkaloids and precursors thereof by hairy roots, wherein the nucleotide sequence of the Anisodus tanguticus AntbHLH16 gene is shown as SEQ ID NO. 3. The application provides a transcription factor AntbHLH16 derived from Anisodus tanguticus and a coding gene thereof, and overexpression of the transcription factor in hairy roots to promote a breakthrough increase in topan alkaloid biosynthesis.

Description

Technical Field

[0001] This application belongs to the field of biotechnology, specifically relating to the application of overexpression of the anisodamine AntbHLH16 gene in promoting the synthesis of tropane alkaloids and their precursors in hairy roots. Background Technology

[0002] Tropine alkaloids (TAs) are a class of nitrogenous secondary metabolites with important medicinal value, mainly including hyoscyamine, anisodamine, and scopolamine. As anticholinergic drugs, they are widely used for analgesia, anesthesia, relief of smooth muscle spasms, and treatment of Parkinson's disease. Currently, commercially available tropine alkaloids are mainly extracted from Solanaceae plants such as belladonna and datura; however, the natural cultivation cycle of these plants is long, and the content of active ingredients is low, making it difficult to meet market demand.

[0003] Hairy roots, induced by the Ri plasmid of Agrobacterium rhizogenes, possess advantages such as genetic stability, rapid growth, hormone autotrophy, and secondary metabolite synthesis capacity comparable to the parent strain, providing an excellent system for the large-scale in vitro production of tropane alkaloids. However, the content of tropane alkaloids in conventional hairy roots remains low, limiting industrial applications. Utilizing transcription factors to globally upregulate the expression of multiple key enzyme genes in metabolic pathways is a powerful strategy to increase the yield of target secondary metabolites. However, currently identified transcription factors that can significantly promote tropane alkaloid synthesis are few, and most involve weak regulation of individual genes, making it difficult to achieve breakthrough yield increases. For example, Chinese patent CN202510589501.6 screened a *AlbHLH175* gene from *Bellaria spp.*, finding that overexpression of this gene could increase the content of tropane alkaloids and its precursors, but the increase was less than three-fold.

[0004] Therefore, it is necessary to discover novel transcription factors that can efficiently regulate the biosynthesis of tropane alkaloids in order to achieve a breakthrough in the production of tropane alkaloids. Summary of the Invention

[0005] The problem this application aims to solve is to provide a transcription factor AntbHLH16 derived from Scopolamine and its encoding gene, and to overexpress this transcription factor in hairy roots to promote a breakthrough improvement in the biosynthesis of tropane alkaloids.

[0006] To address the above problems, this application provides the following solution: On the one hand, this application provides the application of overexpression of the anisodamine AntbHLH16 gene in promoting the synthesis of tropane alkaloids and their precursors in hairy roots, the nucleotide sequence of which is shown in SEQ ID NO.3.

[0007] Furthermore, the tropane alkaloids and their precursors include at least one of tropinone, tropine, hyoscyamine, anisodamine, and scopolamine.

[0008] On the other hand, this application provides a method for increasing the yield of tropane alkaloids and their precursors in hairy roots by overexpressing the AntbHLH16 gene of claim 1 in the hairy roots of plants with tropane alkaloid synthesis pathways through Agrobacterium rhizogenes-mediated genetic transformation.

[0009] Furthermore, the AntbHLH16 gene of *Anticus henryi* increases the production of tropane alkaloids and their precursors by regulating one of the following genes in hairy roots: AntbHLH16, AntTR1, AntHDH, and AntH6H.

[0010] Furthermore, the plants possessing the tropane alkaloid synthesis pathway include Scopolamine.

[0011] In another aspect, this application provides a plant tissue with a high production capacity of tropane alkaloids and their precursors, the plant tissue comprising hairy root tissue generated by Agrobacterium rhizogenes containing the AntbHLH16 gene of claim 1 infecting a plant with a tropane alkaloid synthesis pathway.

[0012] Furthermore, the plants possessing the tropane alkaloid synthesis pathway include Scopolamine.

[0013] This application has the following beneficial effects: This application successfully screened the AntbHLH16 gene from *Anticoctopus scoparia*, which has an excellent promoting effect on the synthesis of tropane alkaloids and their precursors from hairy roots. After overexpression of this gene, the ability of hairy roots to synthesize tropinone increased by 7.2 times, tropinol by 3.7 times, hyoscyamine by 5.1 times, scopolamine by 3.9 times, and scopolamine by 5.7 times. The overall ability to synthesize tropane alkaloids and their precursors is far superior to that of the existing AlbHLH175 gene from *Berberis vulgaris*, providing a new technical solution for the efficient acquisition of tropane alkaloids. Attached Figure Description

[0014] Figure 1 The sterile seedlings of Scopolamine henryi cultured in Example 1 of this application; Figure 2 The leaves of *Anoectochilus henryi* to be infected in this application; Figure 3 The hairy roots that grow from the leaves of *Scirpus henryi* after infection are the subject of this application; Figure 4 After removing the leaves, hairy roots were obtained for this application; Figure 5The hairy root material obtained in this application; Figure 6 The image shows the electrophoretic detection results of the hairy root gene in this application. Lane M is the DNA marker 2000 bp, lane P is the positive control, lane N is the negative control, and lane C is the blank control. Figure 7 The expression levels of different genes in each positive hairy root of this application; Figure 8 The TAs content of each AntbHLH16 hairy root line overexpressing in this application. Detailed Implementation

[0015] The technical solutions in some embodiments of this application will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments. Based on the embodiments provided in this application, all other embodiments obtained by those skilled in the art are within the scope of protection of this application.

[0016] Unless the context otherwise requires, throughout the specification and claims, the term "comprising" is interpreted as open and encompassing, that is, "including, but not limited to".

[0017] In describing some embodiments, the expressions "at least one of A, B and C" and "at least one of A, B or C" may be used, both of which have the same meaning and include the following combinations of A, B and C: only A, only B, only C, a combination of A and B, a combination of A and C, a combination of B and C, and a combination of A, B and C.

[0018] Materials, equipment and reagents used in this application: (1) Experimental materials: wild Scopolamine ( Anisodus tanguticus (Maxim.) Pascher seeds were provided by Chengdu First Pharmaceutical Co., Ltd. The E. coli strain DH5α used for molecular cloning was obtained from Shanghai Weidi Biotechnology Co., Ltd. The Agrobacterium strain C58C1 used for genetic transformation was obtained from Shanghai Weidi Biotechnology Co., Ltd. The plasmid vector pCAMBIA1305.1 used for the genetic transformation experiment of Scopolamine henryi was obtained from Chengdu University of Traditional Chinese Medicine.

[0019] (2) Experimental instruments: The main experimental instruments used in this application are shown in Table 1. Table 1 Main Experimental Instruments (3) Experimental reagents: The main experimental reagents used in this application are shown in Table 2. Table 2 Main Experimental Reagents Example 1: Aseptic germination of Scopolamine seeds Wild *Scirpus henryi* seeds were soaked in a 400 ppm gibberellin solution at 4°C for one day, then left at room temperature for one day. The seeds were rinsed with sterile water to remove the gibberellin and dried. In a clean bench, the seeds were then soaked sequentially in 75% ethanol for 30 seconds, followed by 20% sodium hypochlorite for 20 minutes. They were then rinsed three times with sterile water, dried with clean paper towels, and placed in MS solid medium. The seeds were cultured in the dark at room temperature for one week. After germination, the seeds were transferred to tissue culture flasks containing 0.2 mg / L IBA and cultured until both cotyledons had fully emerged, yielding the desired result. Figure 1 The image shows sterile seedlings of Scopolamine henryi.

[0020] Example 2 AntbHLH16 Construction of overexpression vectors Specific primers pCAMBIA1305.1-AntbHLH16-F and pCAMBIA1305.1-AntbHLH16-R containing BamHI and SacHI restriction sites were designed, with nucleotide sequences shown in SEQ ID NO.1 and SEQ ID NO.2, respectively. PCR amplification was performed to obtain the target fragment with the nucleotide sequence shown in SEQ ID NO.3. The pCAMBIA1305.1 vector plasmid was then linearized by double digestion with BamHI and SacHI. The target fragment was inserted into the vector using recombinase. 10 μL of the recombinant product was added to competent *E. coli* cells, and positive colonies were screened for culture. The plasmid was extracted and sequenced. The correctly sequenced recombinant plasmid was then transformed into *Agrobacterium* C58C1.

[0021] Example 3 AntbHLH16 Induction of overexpression of hairy roots The positively tested Agrobacterium tumefaciens strain pCAMBIA1305.1-AntbHLH16 C58C1 and the control strain pCAMBIA1305.1 Agrobacterium tumefaciens C58C1 were respectively inoculated into 20 mL of LB liquid medium containing kanamycin and rifampin (the control medium did not contain kanamycin). The medium was then incubated overnight at 200 rpm in a 28°C shaker until the bacterial growth rate reached OD. 600When the OD value reached approximately 0.6, a second PCR test was performed on Agrobacterium. The positively tested bacterial suspension was centrifuged at 4000 rpm for 10 min to collect the cells. After removing the supernatant, the suspension was resuspended in 1 mL of liquid MS medium containing 20 μM AS, and then transferred to a new 1.5 mL EP tube. 400 μL of the bacterial suspension was added to a pre-shaken 50 mL EP tube, and 20 mL of liquid MS medium containing 20 μM AS was added to the EP tube. The OD value of the mixture was measured, and further bacterial suspension was added based on the measured OD value until the OD value reached the target value. 600 The OD value was adjusted to approximately 0.3. The bacterial culture with the adjusted OD value was placed in a shaker at 28°C and cultured at 220 rpm for 30 minutes to obtain the infected bacterial culture.

[0022] Leaves from healthy, sterile *Anisodacna scoparia* seedlings were cut in a clean bench and placed in liquid MS medium containing 20 μM AS. The leaves were poked or cut to a suitable size to increase the wound and improve infection efficiency (e.g., ...). Figure 2 (As shown), then add 20 mL of bacterial inoculum, and co-incubate at 28°C and 100 rpm for 10 min on a shaker. After inoculation, place the infected leaves on sterile paper towels to absorb the bacterial inoculum from the leaf surface, and place them leaf-side up on a solid MS medium containing 20 μM AS lined with filter paper. Incubate in the dark at 28°C for 2 days to obtain the results shown. Figure 3 The leaves are shown. The leaves were then transferred to solid MS medium containing 200 mg / L Timentin and cultured for 10 days. They were then transferred to solid MS medium containing 100 mg / L Timentin, and this process was repeated every 14 days, gradually decreasing the Timentin antibiotic concentration by 50 mg / L each time. Once the hairy roots were over 2 cm long, the hairy roots were cut off and transferred again to new solid MS medium containing 100 mg / L Timentin (e.g., ...). Figure 4 (As shown), and every 14 days, the bacteria were transferred to new solid MS medium containing Timentin, gradually reducing the Timentin antibiotic concentration by 25 mg / L each time, until the antibiotic concentration reached 0, ensuring that Agrobacterium was completely removed. A portion of the Agrobacterium's hairy roots was cut off and transferred to 6,7-V liquid medium, and cultured at 25°C in the dark with shaking at 110 rpm for 30 days, yielding the results shown. Figure 5 The hairy root material shown.

[0023] Example 4: Extraction and positive detection of genomic DNA from hairy roots Genomic DNA was extracted from the hairy roots of *Scirpus spicata* using the VAMNE Magnetic Universal Plant Total DNA Kit (Vazyme, Nanjing, China). A 1 μL sample was used as a template for amplification, and the DNA was amplified using 35S-F (sequence shown in SEQ ID NO. 4) and AntbHLH16-R (sequence shown in SEQ ID NO. 5). Electrophoresis was then performed, and the results are shown below. Figure 6 As shown.

[0024] The results showed that L-1, L-12, L-14, and L-15 were positive hairy roots, which were collected as raw materials for subsequent experiments.

[0025] Example 5: Detection of hairy root gene expression levels Positive hairy roots (L-1, L-12, L-14, L-15) and empty vector control hairy roots were placed in 1.5 mL enzyme-free EP tubes, respectively. RNA was extracted from the hairy roots and reverse transcribed into cDNA. The cDNA was diluted and used as a template for RT-qPCR. Gene expression levels were detected using qPCR primers designed with Primer Premier 5.0 as shown in Table 3. The results are as follows: Figure 7 As shown.

[0026] Table 3 qPCR Primer Table The results showed that the expression levels of AntbHLH16, AntTR1, AntHDH, and AntH6H genes in lines L-1, L-12, L-14, and L-15 were significantly increased compared to the empty vector control hairy root, especially the expression level of AntbHLH16, which was much higher than that of the empty vector control hairy root. This indicates that the present application has successfully constructed hairy root lines L-1, L-12, L-14, and L-15 that overexpress AntbHLH16. These four hairy root lines will be used as experimental samples in the future.

[0027] Example 6: Detection of TAs content in hairy roots overexpressing AntbHLH16 (1) Preparation of reference solution Accurately weigh 1 mg each of tropinone, α-tropinol, scopolamine hydrobromide, hyoscyamine hydrobromide, and hyoscyamine reference standards, and place them in 10 mL volumetric flasks. Add methanol to the mark for each flask, and sonicate to filter, preparing stock solutions of appropriate concentrations. Accurately pipette 0.1 mL, 0.2 mL, 0.4 mL, 0.6 mL, and 1.0 mL of each reference solution into 10 mL volumetric flasks, add methanol to the mark for each, stopper, sonicate for 30 min, and let stand for 30 min. Collect the filtrate to obtain mixed reference solutions of corresponding concentration gradients. Plot a standard curve with concentration on the x-axis. Store the reference solutions at 4℃ and filter through a 0.22 μm microporous membrane before injection.

[0028] (2) Preparation of test solution After aspirating the surface liquid culture medium from hairy roots cultured on a shaker for 30 days, the roots were placed in 50 mL centrifuge tubes and freeze-dried for 48–72 h. The freeze-dried hairy root material was then placed in 10 mL EP tubes and ground into powder using a tissue homogenizer at 60 Hz.

[0029] Accurately weigh 10 mg of hairy root powder and place it in a 2 mL volumetric flask. Add methanol to the mark and sonicate for 30 min. Remove the flask and allow it to cool to room temperature. Add methanol solution to make up the lost mass. Filter the supernatant through a 0.22 μm microporous membrane and use the filtrate as the test solution. Dilute the filtrate 100 times in a volumetric flask. Repeat each test sample three times. Store all test solutions at 4 °C.

[0030] (3) The chromatographic column was an Agilent ZORBAX SB-C18 column (4.6 mm × 150 mm, 5 μm); the mobile phase was 0.1% formic acid aqueous solution (A) - methanol solution (B), and the gradient elution scheme was optimized as follows: 0~2.0 min, 5%~10% B; 2.0~6.0 min, 10%~40% B; 6.0~12.0 min, 40%~95% B, flow rate 0.30 mL / min, injection volume 2 μL, and column temperature 30℃. The operating parameters of the ESI ion source were optimized as follows: nebulizer: 15 psi; capillary voltage: 4000 V; gas temperature: 300℃; gas flow rate: 11 L / min. The MS / MS parameters of each analyte were systematically optimized. Data acquisition was performed using multiple reaction monitoring (MRM) mode. The optimized MRM parameters for each analyte—including precursor ion, product ion transitions, fragment voltage, and collision energy (CE)—are shown in Table 4. The results are as follows: Figure 8 As shown.

[0031] Table 4. Mass spectrometric parameters and retention times of the five compounds. The results showed that the content of TAs (tranquilizers) was significantly increased in the hairy root lines overexpressing AntbHLH16. In the L-1 line, the ability of hairy roots to synthesize tropinone, anisodamine, and scopolamine was significantly increased compared to the empty control hairy roots, by approximately 7.2 times, 3.9 times, and 5.7 times, respectively. In the L-14 and L-15 lines, the ability of hairy roots to synthesize tropinol and anisodamine was significantly increased compared to the empty control hairy roots, by approximately 3.7 times and 5.1 times, respectively. Therefore, overexpression of the AntbHLH16 gene in *Anisodamine sibiricum* hairy roots can successfully enhance the ability of hairy roots to synthesize TAs. Future optimization of hairy root overexpression lines based on the AntbHLH16 gene is expected to yield a line that integrates the efficient synthesis of multiple TAs, greatly increasing TA yield.

[0032] The above description is merely a preferred embodiment of the present invention. It should be understood that the present invention is not limited to the forms disclosed herein and should not be construed as excluding other embodiments. It can be used in various other combinations, modifications, and environments, and can be altered within the scope of the concept described herein through the above teachings or related technologies or knowledge. Modifications and variations made by those skilled in the art that do not depart from the spirit and scope of the present invention should be within the protection scope of the appended claims.

Claims

1. The application of overexpression of the AntbHLH16 gene in promoting the synthesis of tropane alkaloids and their precursors in hairy roots, characterized in that, The nucleotide sequence of the AntbHLH16 gene of Scopolamine is shown in SEQ ID NO.

3.

2. The application according to claim 1, characterized in that, The tropane alkaloids and their precursors include at least one of tropidine, tropine, hyoscyamine, scopolamine, and hyoscyamine.

3. A method for increasing the yield of tropane alkaloids and their precursors in hairy roots, characterized in that, The AntbHLH16 gene of claim 1 was overexpressed in the hairy roots of plants with a tropane alkaloid synthesis pathway by Agrobacterium rhizogenes-mediated genetic transformation.

4. The method according to claim 3, characterized in that, The AntbHLH16 gene of Scopolamine increases the production of tropane alkaloids and their precursors by regulating one of the genes AntbHLH16, AntTR1, AntHDH, and AntH6H in hairy roots.

5. The method according to claim 3, characterized in that, The plants possessing the tropane alkaloid synthesis pathway include Scopolamine.

6. A plant tissue with a high production capacity of tropane alkaloids and their precursors, characterized in that, The plant tissue includes hairy root tissue generated by Agrobacterium rhizogenes containing the AntbHLH16 gene of claim 1 after infecting a plant with a tropane alkaloid synthesis pathway.

7. The plant tissue according to claim 6, characterized in that, The plants possessing the tropane alkaloid synthesis pathway include Scopolamine.

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