Transcription factor ca dreb12 encoding gene, protein and recombinant vector of centella asiatica and application thereof

Abstract

The application discloses a Centella asiatica transcription factor CaDREB12 coding gene, protein, recombinant carrier and application thereof, and belongs to the technical field of molecular biology and plant genetic engineering. In the prior art, there is a lack of transcription factor gene resources capable of positively regulating biosynthesis of a main active component, i.e., hydroxyl asiatic acid, of Centella asiatica. The nucleotide sequence of the CaDREB12 coding gene provided in the application is shown as SEQ ID NO. 1. By using the gene to construct a recombinant expression carrier and transform Centella asiatica, transgenic hairy roots with greatly increased content of hydroxyl asiatic acid can be obtained, and the application provides important gene resources and technical foundations for metabolic engineering directional improvement of medicinal active components of Centella asiatica, innovation of germplasm resources and research of synthetic biology.

Description

Technical Field

[0001] This invention belongs to the fields of molecular biology and plant genetic engineering technology. More specifically, this invention relates to a gene, protein, recombinant vector, and application of the Centella asiatica transcription factor CaDREB12. Background Technology

[0002] Centella asiatica (L.) Urb. is a traditional medicinal and edible plant. Its main active ingredients include triterpenoids such as asiaticoside, asiaticoside, asiatic acid, and asiaticoside, which possess anti-inflammatory, anti-fibrotic, and anti-tumor pharmacological activities. However, the natural content of these active ingredients in Centella asiatica is low, making it difficult to meet medicinal needs. Regulating the biosynthetic pathways of active ingredients through genetic engineering is an effective strategy to increase their content. Transcription factors can regulate the expression of multiple key enzyme genes in secondary metabolic pathways and play an important role in the accumulation of plant active ingredients. The AP2 / ERF transcription factor family is widely involved in the regulation of plant secondary metabolism, but in Centella asiatica, AP2 / ERF transcription factor genes that can positively regulate the biosynthesis of major active ingredients such as asiaticoside have not yet been reported, which restricts the research progress of improving the quality of Centella asiatica using molecular breeding or synthetic biology techniques. Summary of the Invention

[0003] One object of the present invention is to address at least the aforementioned deficiencies and to provide at least the advantages that will be described later.

[0004] Another objective of this invention is to provide a gene, protein, recombinant vector, and application of the Centella asiatica transcription factor CaDREB12 encoding gene, which can positively regulate the biosynthetic pathway of asiatic acid by overexpressing the CaDREB12 transcription factor gene. Under non-tissue culture conditions, using Agrobacterium rhizogenes K599-mediated hairy root induction technology, the content of asiatic acid in Centella asiatica hairy roots can be significantly increased, providing new gene resources and technical means for the metabolic engineering improvement of active ingredients in Centella asiatica.

[0005] This invention discloses a gene encoding the Centella Asiatica transcription factor CaDREB12, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0006] The protein expressed by the CaDREB12 encoding gene of this invention belongs to the AP2 / ERF transcription factor family. This protein can specifically recognize and bind to cis-acting elements in the promoters of downstream target genes, thereby initiating or enhancing the transcription of a series of enzyme genes in related metabolic pathways, ultimately achieving positive regulation of the biosynthesis of asiatic acid and promoting the increase of the content of this active ingredient in Centella asiatica.

[0007] This invention discloses a recombinant expression vector for the above-mentioned genes.

[0008] This invention discloses a method for constructing the above-mentioned recombinant expression vector, which includes the following steps: Using Centella asiatica leaf cDNA as a template, PCR amplification was performed using primers CaDREB12-F (nucleotide sequence shown in SEQ ID NO.3) and CaDREB12-R (nucleotide sequence shown in SEQ ID NO.4) to obtain the CaDREB12 gene fragment (nucleotide sequence shown in SEQ ID NO.1). Using the CaDREB12 gene fragment as a template, the first round of PCR amplification was performed using primers CaDREB12-attB1-F (nucleotide sequence as shown in SEQ ID NO.5) and CaDREB12-attB2-R (nucleotide sequence as shown in SEQ ID NO.6) to obtain the first round of PCR products. Using the first round of PCR products as templates, a second round of PCR amplification was performed using primer Adapter attB1 with the nucleotide sequence shown in SEQ ID NO.7 and primer Adapter attB2 with the nucleotide sequence shown in SEQ ID NO.8 to obtain recombinant PCR products with attB sites at both ends; The recombinant PCR product was cloned into the intermediate vector pDONR221 via BP recombination reaction to obtain the intermediate vector pDONR221-CaDREB12. The intermediate vector pDONR221-CaDREB12 was homologously recombinated with the overexpression vector pK7WG2D via LR recombination to obtain the recombinant expression vector pK7WG2D-CaDREB12.

[0009] This invention utilizes a multi-step recombination assembly based on the Gateway cloning technology system. First, the coding sequence of the CaDREB12 gene is obtained from the cDNA of Centella asiatica leaves via PCR amplification. Then, two rounds of PCR reactions are performed to add attB recombination sites to both ends of this sequence, resulting in a gene fragment suitable for recombination. This fragment is cloned into the intermediate vector pDONR221 via a BP recombination reaction, forming an intermediate vector containing the target gene. Subsequently, the CaDREB12 gene sequence is homologously recombinated from the intermediate vector into the corresponding site of the overexpression vector pK7WG2D using an LR recombination reaction, ultimately obtaining a plant overexpression recombinant vector containing the CaDREB12 coding gene. The entire construction process utilizes site-specific recombination to ensure correct gene insertion direction and a complete vector backbone, providing a reliable tool for the subsequent efficient expression of this transcription factor in plants.

[0010] This invention discloses the application of the gene encoding the Centella asiatica transcription factor CaDREB12 in increasing the content of asiatic acid in the hairy roots of Centella asiatica, which includes the following steps: a. The recombinant expression vector pK7WG2D-CaDREB12 was constructed using the above method. The recombinant expression vector contains the gene encoding the Centella asiatica transcription factor CaDREB12. The nucleotide sequence of the gene encoding CaDREB12 is shown in SEQ ID NO.1. b. Transform the overexpression vector obtained in step a into Agrobacterium rhizogenes K599 to obtain recombinant Agrobacterium; c. Inoculate the recombinant Agrobacterium obtained in step b into LB liquid medium and culture at 28°C until the OD600 is 0.8-1.0. After centrifugation, discard the supernatant, resuspend the bacterial cells in an equal volume of 1 / 2 MS liquid medium, and then add 100 μM acetylsyl syringone to obtain the infection solution. d. Cut the leaves of Centella asiatica to create infection wounds, and then immerse the treated Centella asiatica leaves in the infection solution obtained in step c, and shake at 22-28℃ and 90r / min for 10-30 minutes. e. Take out the Centella asiatica leaves treated in step d, absorb the bacterial solution on the surface of the leaves, transfer them to nutrient-rich moist vermiculite, and culture them at 22-28℃ for 20-30 days to induce hairy roots; f. Irradiate the hairy roots obtained in step e with a fluorescent protein excitation light source, screen out the hairy roots with green fluorescence, cut them off and continue to culture them in nutrient-rich moist vermiculite for 25-35 days to obtain Centella asiatica hairy roots overexpressing CaDREB12.

[0011] This invention combines Agrobacterium rhizogenes-mediated genetic transformation with the positive regulation of secondary metabolism by the CaDREB12 transcription factor, thereby increasing the content of asiatic acid in the hairy roots of Centella asiatica. Specifically, the method employs a CDB (cut-soak-germination) strategy to directly treat Centella asiatica leaves under non-tissue culture conditions. A recombinant expression vector carrying the CaDREB12 encoding gene is introduced into Agrobacterium rhizogenes K599. The exogenous gene is integrated into the genome of leaf cells using the Ri plasmid of this strain, inducing transgenic hairy roots at the leaf cut site. Positive hairy roots are selected by irradiation with a fluorescent protein excitation light source to ensure stable expression of the CaDREB12 gene in the hairy roots. This transcription factor, as a member of the AP2 / ERF family, can recognize and activate promoter elements of related enzyme genes in the asiatic acid biosynthetic pathway, driving the enhancement of the entire metabolic flux, ultimately resulting in a significant accumulation of the target active ingredient, asiatic acid, in the harvested hairy roots.

[0012] In the application described in this invention, the centrifugation speed in step c is 8000 rpm and the centrifugation time is 5 minutes.

[0013] In the application described in this invention, after the green fluorescent hairy roots in step f are cut off, PCR identification is also included. The integration of the CaDREB12 gene is confirmed by PCR identification, wherein the primers used for PCR identification are the 35S promoter forward primer and the CaDREB12 gene reverse primer.

[0014] After screening for hairy roots exhibiting green fluorescence, this invention further confirms the integration of the CaDREB12 gene at the molecular level using PCR identification. This identification step uses a 35S promoter forward primer and a CaDREB12 gene reverse primer for amplification. Since the 35S promoter is located at the site in the expression vector that drives the transcription of the exogenous gene, and the reverse primer specifically binds to the CaDREB12 gene coding sequence, a specific band of the expected size can only be amplified if the CaDREB12 gene is successfully integrated into the Centella asiatica genome and the vector structure is intact. This identification step complements the fluorescence screening, providing a reliable guarantee of positive lines for subsequent gene expression analysis and metabolite content determination.

[0015] This invention provides a Centella Asiatica transcription factor CaDREB12 protein, the amino acid sequence of which is shown in SEQ ID NO.2.

[0016] The CaDREB12 protein of this invention belongs to the AP2 / ERF transcription factor family. This protein can specifically recognize and bind to cis-acting elements in the promoter regions of downstream target genes in the asiatic acid biosynthesis pathway. By activating the transcriptional expression of related enzyme genes, it positively drives the entire metabolic pathway toward the target product, thereby achieving the directional accumulation and increased content of asiatic acid in Centella asiatica.

[0017] The present invention has at least the following beneficial effects: This invention marks the first time that the encoding gene and protein of the transcription factor CaDREB12 have been cloned from Centella asiatica, filling the gap in the resources of AP2 / ERF family transcription factors in Centella asiatica capable of positively regulating the biosynthesis of asiaticotropic acid. This provides a key gene element for subsequent targeted improvement of the content of active ingredients in Centella asiatica using genetic engineering techniques. Based on the recombinant expression vector constructed using this gene and the Agrobacterium rhizogenes-mediated genetic transformation method, a non-tissue culture strategy of direct induction on vermiculite substrate was employed. This eliminates the need for a dedicated tissue culture room and complex callus differentiation and subculture steps, requiring only basic aseptic techniques. The operation is simple, short-cycle, and low-cost, significantly lowering the technical threshold for inducing hairy roots and establishing transgenic systems in Centella asiatica. The content of asiaticotropic acid in the hairy roots of Centella asiatica overexpressing the CaDREB12 gene was more than 2-fold higher than the control, verifying the potent positive regulatory ability of this transcription factor on the target metabolic pathway and providing a practical technical route for high-yield production of asiaticotropic acid through metabolic engineering. The combination of genes, vectors, proteins, and methods provided by this invention offers a complete toolkit and new ideas for the innovation of germplasm resources, large-scale production of active ingredients, and synthetic biology research of Centella asiatica, an important plant with both medicinal and edible uses.

[0018] Other advantages, objectives and features of the present invention will become apparent in part from the following description, and in part from those skilled in the art through study and practice of the invention. Attached Figure Description

[0019] Figure 1 This is a schematic diagram of electrophoresis detection after PCR amplification of the CaDREB12 gene. M is the DNA molecular weight marker, and the arrow indicates that the target band is located between 750-1000 bp.

[0020] Figure 2 This is a structural diagram of the pK7WG2D carrier.

[0021] Figure 3 This is a PCR electrophoresis image of colonies of the pK7WG2D-CaDREB12 recombinant plasmid. M is the DNA molecular weight marker, and 1-6 are randomly selected single colonies, all of which amplified bands of the expected size.

[0022] Figure 4 This is the genetic transformation process for hairy roots. a) Hairy roots induced by Centella asiatica leaves as explants (bright field); b) Hairy roots induced by Centella asiatica leaves under the same field of view (GFP fluorescence), showing green fluorescence as positive transgenic hairy roots.

[0023] Figure 5 Electrophoresis diagram for identifying the rolB gene in hairy root systems. M is the DNA molecular weight marker, and 1-6 represent the CaDREB12 overexpressing root systems that survived after screening and culture. Five of the lines showed specific bands at around 500 bp.

[0024] Figure 6 Electrophoresis diagram of CaDREB12 gene identification in hairy root systems. M is the DNA molecular weight marker, and 1-6 represent the CaDREB12 overexpressing root systems that survived after screening and culture. Five of these lines amplified the target band of the expected size.

[0025] Figure 7 This is a schematic diagram of the expression analysis of the CaDREB12 gene. EV represents the unexpressed hairy root, and #1 and #2 are the overexpression lines #1 and #2, respectively. The vertical axis represents the relative expression level, and **** represents P < 0.0001.

[0026] Figure 8 The relative content of asiatic acid in the hairy roots of *Centella asiatica* overexpressing CaDREB12 is shown. EV represents the empty vector hairy root, and #1 and #2 are overexpression lines #1 and #2, respectively. The ordinate represents the relative content of asiatic acid, and **** represents P < 0.0001. Detailed Implementation

[0027] The present invention will be further described in detail below with reference to embodiments, so that those skilled in the art can implement it based on the description.

[0028] It should be noted that, unless otherwise specified, the experimental methods described in the following implementation plan are all conventional methods, and the materials can be obtained commercially unless otherwise specified.

[0029] Example 1: Cloning of the CaDREB12 gene and construction of an overexpression vector Centella asiatica ( Centella asiatica Using fresh leaves of *L.) Urb. as material, total RNA was extracted using the conventional Trizol method, and cDNA was obtained by reverse transcription. Specific primers were designed based on the CDS sequence of the CaDREB12 gene in the *Centella asiatica* genome. The primers were synthesized by Wuhan Qingke Biotechnology Co., Ltd., and their sequences are as follows: CaDREB12-F (SEQ ID NO.3): 5′-ATGGAAATGATGCAAGAAACA-3′; CaDREB12-R (SEQ ID NO.4): 5′-TTAATTAGCAAGAACTTTCCAGATC-3′; PCR amplification was performed using cDNA from Centella asiatica leaves as a template. The amplification system was 50 μL: 25 μL 2×PhantaMax Buffer, 1 μL dNTPMix (10 mmol / L), 2 μL CaDREB12-F (10 μmol / L), 2 μL CaDREB12-R (10 μmol / L), 1 μL PhantaMax Super-Fidelity DNA Polymerase, 2 μL cDNA, and 17 μL ddH2O. The amplification program was: 95℃ pre-denaturation for 3 min; 94℃ denaturation for 30 s, 60℃ annealing for 30 s, and 72℃ extension for 30 s, for a total of 32 cycles; final extension at 72℃ for 10 min, and storage at 4℃.

[0030] After PCR amplification, 5 μL of the product was analyzed by 1% agarose gel electrophoresis. The results showed that the amplified target fragment was between 750 bp and 1000 bp in size, consistent with the expected size of the CaDREB12 gene. Figure 1 The schematic diagram of electrophoresis detection after PCR amplification of the CaDREB12 gene is shown. M is the DNA molecular weight marker, and the arrow indicates that the target band is located between 750-1000 bp. The target fragment was recovered using a gel extraction kit from Novizan to obtain the purified CaDREB12 gene fragment.

[0031] The attB site was added to both sides of the CDS sequence of CaDREB12 through two rounds of PCR reactions.

[0032] The primer sequences for the first round of PCR are as follows: CaDREB12-attB1-F (SEQ ID NO.5): 5′-AAAAAGCAGGCTCCATGGAAATGATGCAAGAAAACA-3′; CaDREB12-attB2-R (SEQ ID NO.6): 5′-AGAAAGCTGGGTTTTTAATTAGCAAGAACTTTCCAGATC-3′; First round of PCR (with partial attB site addition): Using the recovered and purified CaDREB12 gene fragment as a template, amplification was performed using the primers CaDREB12-attB1-F and CaDREB12-attB2-R. The amplification system was 50 μL: 25 μL 2×Phanta Max Buffer, 1 μL dNTP Mix (10 mmol / L), 2 μL CaDREB12-attB1-F (10 μmol / L), 2 μL CaDREB12-attB2-R (10 μmol / L), 1 μL Phanta Max Super-Fidelity DNA Polymerase, 2 μL gene fragment template, and ddH2O to a final volume of 50 μL. Amplification program: 95℃ pre-denaturation for 3 min; 94℃ denaturation for 30 s, 60℃ annealing for 30 s, 72℃ extension for 30 s, for a total of 32 cycles; final extension at 72℃ for 10 min. The PCR products were recovered by 1% agarose gel electrophoresis to obtain the first round of PCR products.

[0033] The primer sequences for the second round of PCR are as follows: Adapter attB1 (SEQ ID NO.7): 5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTC-3′; Adapter attB2 (SEQ ID NO.8): 5′-GGGGACCACTTTGTACAAGAAAGCTGGGT-3′; Second-round PCR (with complete attB sites): Using the first-round PCR product as a template, amplification was performed using the aforementioned primers Adapter attB1 and Adapter attB2. The amplification system and procedure were the same as in the first-round PCR. The PCR product was detected by 1% agarose gel electrophoresis, and the recombinant PCR product with complete attB sites at both ends, approximately 913 bp in size, was recovered.

[0034] The BP recombinant reaction system was prepared on ice: 0.6 μL of intermediate vector pDONR221, 1 μL of recovered recombinant PCR product, 0.4 μL of BP clonase, and 1 μL of ddH2O, for a total volume of 3 μL. The mixture was gently aspirated and mixed, briefly centrifuged, and incubated at 25°C for 4 h. After the reaction, the mixture was cooled on ice. The recombinant product was transformed into *E. coli* DH5α competent cells: 3 μL of the recombinant product was added to the thawed DH5α competent cells, gently aspirated, incubated on ice for 30 min, heat-shocked at 42°C for 1 min, incubated on ice for 2 min, and then 500 μL of LB liquid medium was added. The cells were incubated at 37°C with shaking at 200 rpm for 45 min. 200 μL of the bacterial culture was spread onto LB solid medium containing 50 mg / L kanamycin and incubated upright at 37°C for 1 h, followed by inverted incubation for 12 h. Single colonies were picked for colony PCR verification using Adapter attB1 and Adapter attB2 primers. Positive clones were sent to a gene company for sequencing. Sequencing results showed that the intermediate vector pDONR221-CaDREB12 was successfully constructed. The nucleotide sequence of the CaDREB12 gene is shown in SEQ ID NO.1, and the amino acid sequence of the protein it encodes is shown in SEQ ID NO.2.

[0035] The CDS sequence of CaDREB12 was assembled into the pK7WG2D overexpression vector using an LR recombination reaction employing Gateway technology. Figure 2 The LR recombinant reaction system was prepared on ice: 1.0 μL of the intermediate vector pDONR221-CaDREB12 containing the target fragment, 0.6 μL of the pK7WG2D overexpression vector, 0.4 μL of LR clonase, and 1 μL of ddH2O, for a total volume of 3 μL. The mixture was gently pipetted and incubated at 25°C for 4 h. After the reaction, the bacteria were transformed into *E. coli* DH5α, and the bacterial culture was plated on LB agarose gel containing 50 mg / L spectinomycin. Single colonies were picked for PCR identification using Adapter attB1 and Adapter attB2 primers. The PCR products were detected by 1% agarose gel electrophoresis. Figure 3 The PCR electrophoresis image of the pK7WG2D-CaDREB12 recombinant plasmid colonies is shown. M is the DNA molecular weight marker, and 1-6 are randomly selected single colonies. All colonies amplified the expected size bands, and positive clones were sent to a gene company for sequencing. After successful sequencing, the recombinant plasmid was extracted to obtain the recombinant expression vector pK7WG2D-CaDREB12.

[0036] The nucleotide sequence of the CaDREB12 gene is shown in SEQ ID NO.1, specifically as follows: ATGGAAATGATGCAAGAAAACATTATGCCTTTTGGGCATTACGAGGGTACTGATCTAAGGTCTTCCCTGTCGAATCTAATAATATCAAGCGGCACAAACACCTTGGATTCAATCTTCTCTCACTGCTCATCATCAACCCCCTATACCAACCCTGTTTTTGAGCCATTAGGCTCTTCAGTGTATCTCAAACAAAGAGATTTACTTCAAAAATTCTGCGAAGAAAACCCCTCAAGAAACCAGATTTCAAGGCCAGTTCTTTCTAACCCACTTCAGAATTCCCTATTTAAGACCAGTTATACTGCTCCACCAAAGAAAAAGCTGTACAGAGGAGTGAGACAGAGGCACTGGGGAAAATGGGTAGCCGAGATTAGACTCCCACAGAACAGAATGAGAGTTTGGTTGGGAACTTATGAAACAGCTGAGGTCGCAGCCTATGCCTATGATCGTGCTGCCTATAAGCTACGTGGAGAGTATGCGCGCTTGAACTTCCCAAATCTTCGTGATCCAAGCAAACTAGGATTTGGAGATGGTTCAAGATTGAATGCTTTGAAGATTTCTGTGGATGCTAAAATCCAAGCAATTTGTCAGAAAGTTAAGAGAGAGAAGGCTAATAAGAACAGTAGAAAAACTGGTGGTAGTGCTGCTGGTGATAGTATTGGTAATGCGTGTGAAAAAGAACCAAGAATCAATTCTAGTTCTCCATCTTCATTAATTGGTAATGACAGTTTATTCAGTAATGAAATGGTTTCTTCAAGTGTTTCGGAAGATGGGTTATGGAAGGACGAGAACTCGCCATATTCTGTTTCCGGGGCTTGTGCAGCAGCGCCGGAGGAATTGGAGTTTGAGGGTTGCTCTCTTGCACGCATGCCATCATATGACCCAGAGTTGATCTGGAAAGTTCTTGCTAATTAA The encoded protein amino acid sequence is shown in SEQ ID NO.2, specifically: MEMMQENIMPFGHYEGTDLRSSLSNLIISSGTNTLDSIFSHCSSSTPYTNPVFEPLGSSVYLKQRDLLQKFCEENPSRNQISRPVLSNPLQNSLFKTSYTAPPKKKLYRGVRQRHWGKWVAEIRLPQNRMRVWLGTYETAEVAAYAYDRAAY KLRGEYARLNFPNLRDPSKLGFGDGSRLNALKISVDAKIQAICQKVKREKANKNSRKTGGSAAGDSIGNACEKEPRINSSSPSSLIGNDSLFSNEMVSSSSEDGLWKDENSPYSVSGACAAAPEELEFEGCSLARMPSYDPELIWKVLAN*.

[0037] Example 2: Agrobacterium rhizogenes-mediated induction of hairy roots in Centella asiatica and screening for overexpression of hairy roots Agrobacterium rhizogenes K599 was selected as the infecting strain. The recombinant expression vector pK7WG2D-CaDREB12 constructed in Example 1 was transformed into K599 competent cells by heat shock and plated on LB solid medium containing 50 mg / L spectinomycin, and cultured upside down at 28°C for 48 h. Single colonies were picked and inoculated into 1.5 mL of LB liquid medium (containing 50 mg / L spectinomycin), and cultured at 28°C with shaking at 220 rpm for 16 h. The inoculum was transferred to 50 mL of LB liquid medium (containing 50 mg / L spectinomycin) at a rate of 0.8%, and cultured at 28°C with shaking at 220 rpm until the OD600 of the bacterial culture reached 0.8-1.0. The bacterial culture was centrifuged at 8000 rpm for 5 min, the supernatant was discarded, and the cells were resuspended in an equal volume of fresh 1 / 2 MS liquid medium. 100 μmol / L acetylsyleugenone was added, and the culture was incubated at room temperature for 1 h to prepare the infection solution.

[0038] Select healthy Centella asiatica leaves, rinse them with running water to remove surface dust, then wipe the leaf surface with 75% ethanol for 10 seconds, rinse twice with sterile water, and blot dry with sterile filter paper. Using sterile surgical scissors, make 3-4 incisions approximately 0.5 cm long along both sides of the leaf's main vein (only cutting the epidermis and mesophyll tissue, without cutting the entire leaf) to create uniform infection wounds. Completely immerse the leaves in the above infection solution and place them on a shaker at 28℃ and 90 rpm for 20 minutes. After infection, blot away excess bacterial solution from the leaf surface with sterile paper towels, and transfer the leaves to nutrient-rich, moist vermiculite for incubation at 25℃. After 25 days of incubation, white hairy roots begin to grow from the leaf incision sites.

[0039] When the hairy roots reached 30 days of growth, they were irradiated with a LUYOR-3415RG dual-wavelength portable fluorescent protein excitation light source to screen for transgenic hairy roots exhibiting green fluorescence, such as... Figure 4 The genetic transformation process for hairy roots is shown in Figure a. Hairy roots induced from Centella asiatica leaves as explants (bright field). Figure b. Hairy roots induced from Centella asiatica leaves under the same field of view (GFP fluorescence), showing green fluorescence as positive transgenic hairy roots. The green fluorescence-positive hairy roots were cut off and cultured in nutrient-rich moist vermiculite for 30 days to obtain Centella asiatica hairy root lines overexpressing CaDREB12. Simultaneously, the K599 strain transformed with the empty vector pK7WG2D was treated using the same method to obtain empty vector hairy roots (EV) as a control.

[0040] The specific method for obtaining nutrient-rich moist vermiculite is as follows: the vermiculite is sterilized by high-pressure steam at 121℃ for 20 minutes, cooled and placed in a sterilized container, and 1 / 2 MS nutrient solution that has been sterilized by high pressure is added until the vermiculite is completely moistened and no free water flows out, thus obtaining nutrient-rich moist vermiculite.

[0041] Example 3: PCR identification of hairy roots overexpressing CaDREB12 Approximately 0.1 g each of overexpressing CaDREB12 hairy roots and empty vector hairy roots cultured for 30 days in Example 2 were used to extract genomic DNA using the CTAB method. An upstream primer 35S-F was designed based on the 35S promoter sequence contained in the vector, and a downstream primer CaDREB12-det-R was designed based on the CaDREB12 gene coding sequence. Simultaneously, using the rolB gene on the Agrobacterium rhizogenes Ri plasmid as an internal control, rolB-F and rolB-R primers were designed. The primer sequences are as follows: 35S-F (SEQ ID NO.9): 5′-GACGCACAATCCCACTATCC-3′; CaDREB12-det-R (SEQ ID NO. 10): 5′-TTAATTAGCAAGAACTTTCCAGATC-3′; rolB-F (SEQ ID NO. 11): 5′-AGTCGCCGAGGTTTCTTTCTT-3′; rolB-R (SEQ ID NO. 12): 5′-GCGAGAAGATGCAGAAAGTACTG-3′; The PCR amplification system consisted of 20 μL: 10 μL of 2×PCR mix, 0.5 μL of forward primer (10 μmol / L), 0.5 μL of reverse primer (10 μmol / L), 2 μL of DNA template, and ddH2O to a final volume of 20 μL. The amplification program was as follows: 95℃ pre-denaturation for 3 min; 94℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 1 min, for a total of 32 cycles; final extension at 72℃ for 10 min, and storage at 4℃.

[0042] PCR products were detected by 1% agarose gel electrophoresis. Amplification using rolB-F and rolB-R primers showed that five of the six tested hairy root lines exhibited specific bands around 500 bp, consistent with the expected size of the rolB gene. Figure 5 Electrophoresis images of hairy root lines for rolB gene identification are shown. M represents the DNA molecular weight marker, and 1-6 represent the CaDREB12 overexpressing roots that survived after screening and culture. Five lines showed specific bands around 500 bp, confirming that these hairy roots were induced by Agrobacterium K599. Amplification using primers 35S-F and CaDREB12-det-R showed that all five rolB-positive lines amplified bands of approximately 1200 bp, consistent with the theoretical length from the vector promoter to the end of the gene coding region. The overall transgene positivity rate among the six tested lines was calculated to be 83.33%. Figure 6 Electrophoresis diagram of CaDREB12 gene identification in hairy root systems. M is the DNA molecular weight marker, and #1 to #6 represent the CaDREB12 overexpressing root systems that survived after screening and culture. Five of these lines amplified the expected target band of approximately 1200 bp. Two positive lines were randomly selected and labeled as overexpressing line #1 and overexpressing line #2 for subsequent experiments.

[0043] Example 4: Detection of CaDREB12 overexpressing hairy roots by real-time PCR Approximately 0.1 g each of the overexpression lines #1 and #2 obtained in Example 3, and empty vector hairy roots (EV), were used to extract total RNA using the Trizol method, and cDNA was synthesized by reverse transcription. Ca-actin was used as an internal control gene, and the expression level of the CaDREB12 gene was detected by qRT-PCR. The primer sequences are as follows: CaDREB12-qRT-F (SEQ ID NO.13): 5′-ACGAGGGTACTGATCTAAGGTCTTC-3′; CaDREB12-qRT-R (SEQ ID NO. 14): 5′-ATAGGGGGTTGATGATGAGCAGTGA-3′; Ca-actin-F (SEQ ID NO. 15): 5′-TGGTTGCACCTCCAGAGAGA-3′; Ca-actin-R (SEQ ID NO. 16): 5′-CAGACTCGTCGTATTCCCC-3′.

[0044] The qRT-PCR reaction system consisted of 10 μL: 5 μL SYBR Green qPCR Mix (2×), 0.3 μL forward primer (10 μmol / L), 0.3 μL reverse primer (10 μmol / L), 0.5 μL cDNA template, and 3.9 μL RNase-free ddH2O. Reaction conditions were: 95℃ pre-denaturation for 30 s; 95℃ denaturation for 10 s, 60℃ annealing for 30 s, and 72℃ extension for 1 min, for a total of 40 cycles. Three technical replicates were set up for each sample. Relative expression levels were calculated using the 2^-ΔΔCt method.

[0045] The results show that ( Figure 7 In overexpression lines #1 and #2, the expression level of the CaDREB12 gene was significantly increased compared to the empty vector hairy root EV, increasing by approximately 3.2-fold and 3.8-fold, respectively, with highly significant differences (P < 0.0001). This indicates that the CaDREB12 gene has been successfully overexpressed in the overexpression lines.

[0046] Example 5: Determination of hydroxyasiatic acid content in the hairy roots of Centella asiatica The overexpression lines #1 and #2, as well as the empty-vector hairy root EV verified in Example 4, were cut from the fluorescently screened samples and transferred to moist vermiculite. After 30 days of continued culture under the same conditions, hairy root samples were collected. The samples were freeze-dried using vacuum freeze-drying technology and then ground into a homogeneous powder using a grinder (MM400, Retsch) at 30 Hz for 1.5 min. 50 mg of the sample powder was accurately weighed and added to 1200 μL of pre-cooled 70% methanol aqueous solution (containing internal standard). The mixture was vortexed for 30 s every 30 min for a total of 6 times. The mixture was centrifuged at 12000 rpm for 3 min, and the supernatant was filtered through a 0.22 μm microporous membrane. The filtrate was placed in a sample vial for UPLC-MS / MS analysis.

[0047] Liquid chromatography conditions: Column: Agilent SB-C18 (1.8 μm, 2.1 mm × 100 mm); Mobile phase A: pure water containing 0.1% formic acid; Mobile phase B: acetonitrile containing 0.1% formic acid; Elution gradient: 95% A, 5% B at 0 min; within 9 min, the proportion of phase B linearly increases to 95%, maintained for 1 min; within 10.00-11.10 min, the proportion of phase B linearly decreases to 5%, and is maintained at 5% B for 14 min; Flow rate: 0.35 mL / min; Column temperature: 40 °C; Injection volume: 2 μL.

[0048] Mass spectrometry conditions: Electrospray ionization (ESI) source, temperature 550℃; ion spray voltage 5500V (positive ion mode) and -4500V (negative ion mode); ion source gas I, gas II, and curtain gas set to 50, 60, and 25 psi, respectively; collision ionization parameter set to high. Multiple reaction monitoring (MRM) mode was used for scanning.

[0049] The asiatic acid standard was determined under the conditions described above, and a standard curve was plotted. The content of asiatic acid in each sample was calculated based on the standard curve. Three biological replicates were set up for each sample. Results are expressed as mean ± standard deviation, and t-tests were used to analyze the significance of differences.

[0050] Experimental results are as follows Figure 8 As shown, the relative contents of asiatic acid in overexpression lines #1 and #2 were 2.3 and 2.5 times that of the empty vector hairy root EV, respectively, with highly significant differences (P < 0.0001). This indicates that overexpression of the CaDREB12 encoding gene can significantly increase the content of asiatic acid in the hairy roots of Centella asiatica.

[0051] Although embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. It can be applied to various fields suitable for the present invention. Further modifications can be readily implemented by those skilled in the art.

Claims

1. A gene encoding the Centella asiatica transcription factor CaDREB12, characterized in that... Its nucleotide sequence is shown in SEQ ID NO.

1.

2. A recombinant expression vector comprising the gene of claim 1.

3. A method for constructing a recombinant expression vector as described in claim 2, characterized in that... Includes the following steps: Using Centella asiatica leaf cDNA as a template, PCR amplification was performed using primers CaDREB12-F (nucleotide sequence as shown in SEQ ID NO.3) and CaDREB12-R (nucleotide sequence as shown in SEQ ID NO.4) to obtain the CaDREB12 gene fragment (nucleotide sequence as shown in SEQ ID NO.1). Using the CaDREB12 gene fragment as a template, the first round of PCR amplification was performed using primers CaDREB12-attB1-F (nucleotide sequence as shown in SEQ ID NO.5) and CaDREB12-attB2-R (nucleotide sequence as shown in SEQ ID NO.6) to obtain the first round of PCR products. Using the first round of PCR products as templates, a second round of PCR amplification was performed using primer Adapter attB1 with the nucleotide sequence shown in SEQ ID NO.7 and primer Adapter attB2 with the nucleotide sequence shown in SEQ ID NO.8 to obtain recombinant PCR products with attB sites at both ends; The recombinant PCR product was cloned into the intermediate vector pDONR221 via BP recombination reaction to obtain the intermediate vector pDONR221-CaDREB12. The intermediate vector pDONR221-CaDREB12 was homologously recombinated with the overexpression vector pK7WG2D via LR recombination to obtain the recombinant expression vector pK7WG2D-CaDREB12.

4. The application of a gene encoding the Centella asiatica transcription factor CaDREB12 in increasing the content of asiatic acid in the hairy roots of Centella asiatica, characterized in that... Includes the following steps: a. The recombinant expression vector pK7WG2D-CaDREB12 was constructed using the method described in claim 3. The recombinant expression vector contains the gene encoding the Centella asiatica transcription factor CaDREB12, and the nucleotide sequence of the gene encoding CaDREB12 is shown in SEQ ID NO.

1. b. Transform the recombinant expression vector obtained in step a into Agrobacterium rhizogenes K599 to obtain recombinant Agrobacterium; c. Inoculate the recombinant Agrobacterium obtained in step b into LB liquid medium and culture at 28°C until the OD600 is 0.8-1.

0. After centrifugation, discard the supernatant, resuspend the bacterial cells in an equal volume of 1 / 2 MS liquid medium, and then add 100 μM acetylsyl syringone to obtain the infection solution. d. Cut and wound the leaves of Centella asiatica to create infection wounds, and then immerse the treated Centella asiatica leaves in the infection solution obtained in step c, and shake at 22-28℃ and 90r / min for 10-30 minutes. e. Take out the Centella asiatica leaves treated in step d, absorb the bacterial solution on the surface of the leaves, transfer them to nutrient-rich moist vermiculite, and culture them at 22-28℃ for 20-30 days to induce hairy roots; f. Irradiate the hairy roots obtained in step e with a fluorescent protein excitation light source, screen out the hairy roots with green fluorescence, cut them off and continue to culture them in nutrient-rich moist vermiculite for 25-35 days to obtain Centella asiatica hairy roots overexpressing CaDREB12.

5. The application according to claim 4, characterized in that... The centrifugation speed in step c is 8000 rpm and the centrifugation time is 5 minutes.

6. The application according to claim 4, characterized in that... After the green fluorescent hairy roots described in step f are cut off, PCR identification is also included. The integration of the CaDREB12 gene is confirmed by PCR. The primers used for PCR identification are the 35S promoter forward primer and the CaDREB12 gene reverse primer.

7. A Centella asiatica transcription factor CaDREB12 protein, characterized in that... Its amino acid sequence is shown in SEQ ID NO.2.

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