A method for improving vinblastine content of catharanthus roseus based on ein3 / eils transcription factor creil1 and a method for determining vinblastine content

Abstract

The application discloses a method for improving vinblastine content in Catharanthus roseus based on EIN3 / EILs transcription factor CrEIL1 and a method for determining the vinblastine content. According to the method, the CrEIL1 gene of the Catharanthus roseus is cloned, a pLB-CrEIL1 plasmid vector is constructed, the CrEIL1 overexpression vector is constructed by using the pLB-CrEIL1 plasmid vector, and the CrEIL1 overexpression vector is used to mediate the overexpression of CrEIL1 in the Catharanthus roseus by means of Agrobacterium tumefaciens, so that the vinblastine yield in the Catharanthus roseus can be improved at low cost, in an environment-friendly and reliable manner.

Description

Technical Field

[0001] This invention relates to the field of bioengineering, and in particular to a method for increasing the vincristine content in periwinkle based on the EIN3 / EILs transcription factor CrEIL1, and a method for determining the vincristine content. Background Technology

[0002] *Catharanthus roseus* (L.) G. Don, as one of the model plants for studying terpenoid indole alkaloids (TIAs), produces nearly 200 kinds of TIAs. TIAs are a class of secondary metabolites produced by plants during long-term adaptation to their ecological environment, used to protect themselves. Some of these have strong pharmacological activity and are mainly used in the clinical treatment of various diseases. The terpenoid indole alkaloids in *Catharanthus roseus* include ajmalicine, vindoline, catharanthine, serpentine, vinblastine, and vincristine. The bisindole alkaloids vincristine and vinblastine play an indispensable role in most chemotherapy treatments for Hodgkin's lymphoma, lymphosarcoma, neuroblastoma, breast cancer, lung cancer, and childhood leukemia. Currently, the United States has approved a number of commercial drugs using vincristine as a raw material. For example, vinorelbine, the sulfate of vincristine, is highly effective in treating cancers such as acute leukemia, breast cancer, neuroblastoma, ovarian cancer, and chronic lymphocytic leukemia. In addition, studies have shown that amantadine and serpentine are used to treat circulatory system diseases, especially relieving cerebrovascular mental disorders and allergic reactions. Ventolin and vincristine have some effect in lowering blood lipids and can be used to treat diseases such as diabetes.

[0003] Currently, periwinkle is the only plant source for producing vincristine and vinblastine, and the dry weight of periwinkle plants contains only 0.01% of bisindole alkaloids (approximately 1.0 g of vincristine is produced from 500-750 kg of dried leaves), resulting in high extraction costs. Synthesizing alkaloids in cultured cells in vitro is difficult; without specific induction, it is hard to produce TIAs. While there are reports of semi-synthetic artificial synthesis, the substrate still needs to be extracted from periwinkle, and industrial production efficiency is very low. To date, there are no other more economical methods to obtain bisindole TIAs, and metabolic engineering is currently the most effective way to increase TIA yield. Therefore, in-depth research into the molecular regulatory mechanisms of periwinkle alkaloid synthesis is fundamental to improving TIA yield.

[0004] All TIAs in periwinkle originate from the intermediate precursor 3α(S)-stritosidine. The upstream biosynthesis of TIAs is divided into the iridoid pathway (multi-step enzymatic reaction to synthesize secologanin) and the shikimic acid pathway (multi-step enzymatic reaction to synthesize tryptamine). Sesologanin is derived from isopentenyl diphosphate (IPP). In the presence of IPP, indole alkaloids are mainly synthesized in plastids via the MEP pathway. Further, secologanin and tryptamine undergo a condensation reaction with isostigmosiderin synthase (STR) to generate isostigmosiderin. Subsequently, in the downstream pathway, under the action of isostigmosiderin β-D-glucosidase (SGD), 3α(S)-stritosidine is converted to strictosidine aglycone, which can be used to synthesize various TIAs. Further, through multiple enzymatic reactions, dihydroprecondylcarpine acetate is formed. Then, under the action of catharanthine synthase (CS) and tabersonine synthase (TS), vinblastine and tabersonine are formed. Tabersonine undergoes a seven-step reaction to form vindolin. Finally, vinblastine and vindolin are reacted by peroxidase 1 (PRX1) to form vinblastine, generating the intermediate α-3',4'-anhydrovinblastine. This intermediate then undergoes multiple steps to generate anhydrovinblastine, vinblastine, and vincristine. DXS1, TDC, 7-DLGT, 7DLH, LAMT, SLS, Asα, and STR are upstream synthetic genes, while SGD, REDOX2, SAT, HL1, HL2, DAT, and PRX1 are downstream synthetic genes.

[0005] EIN3 / EILs transcription factors are important nuclear transcription factors in the ethylene signaling pathway. They have been isolated from various higher plants and belong to a small family of transcription factors. These transcription factors are highly conserved at the N-terminus of their amino acid sequences and regulate the expression of related genes by directly binding to the primary ethylene responder element (PERE). They participate not only in important life activities such as fruit ripening, flower senescence, salt tolerance, frost tolerance, and resistance to mechanical stress, but also in regulating the synthesis of plant secondary metabolites.

[0006] CrEIL1 is an EIN3 / EILs transcription factor isolated from periwinkle. Transient expression in periwinkle petals significantly increased the content of vincristine, indicating that it is a potential transcription factor that promotes vincristine biosynthesis. Therefore, this transcription factor is of great significance for promoting efficient vincristine synthesis. In known studies, the CrERF5 transcription factor in periwinkle, which responds to ethylene signaling, has a regulatory role in the synthesis of terpenoid indole alkaloids, while the gene functions of EIN3 / EIL1-like transcription factors in the ethylene signaling pathway of periwinkle are rarely reported.

[0007] Therefore, those skilled in the art are dedicated to developing a low-cost, environmentally friendly, and reliable method to increase the yield of vinblastine in periwinkle. Summary of the Invention

[0008] In view of the above-mentioned deficiencies of the prior art, the technical problem to be solved by the present invention is to provide a low-cost, environmentally friendly and reliable method for increasing the yield of vinblastine in periwinkle.

[0009] To achieve the above objectives, this invention provides a method for increasing the vincristine content in periwinkle based on the EIN3 / EILs transcription factor CrEIL1, characterized in that the method includes the following steps:

[0010] Step 1: Clone the CrEIL1 gene from periwinkle and construct the pLB-CrEIL1 plasmid vector;

[0011] Step 2: Construct a CrEIL1 overexpression vector using the pLB-CrEIL1 plasmid vector;

[0012] Step 3: Using the CrEIL1 overexpression vector, CrEIL1 was overexpressed in periwinkle via Agrobacterium tumefaciens-mediated expression.

[0013] In a preferred embodiment of the present invention, step 1 further includes:

[0014] Using the extracted total RNA from periwinkle as a template, the dosage was calculated based on the RNA concentration, and total cDNA was obtained under the action of PowerScript reverse transcriptase.

[0015] Based on the nucleotide sequence of the CrEIL1 gene, i.e. SEQ ID NO.1, gene-specific primers were designed, and the CrEIL1 gene was amplified from total cDNA by PCR. After the PCR product was recovered and purified, it was ligated into a blunt-ended pLB vector to obtain the pLB-CrEIL1 plasmid vector, and the plasmid was extracted.

[0016] The specific primer sequences are as follows:

[0017] Forward primer SEQ ID NO.31: GATATCCTTTGCGTAAATCC;

[0018] Reverse primer SEQ ID NO.32: CCTCCTTGTTCATGAACTGC.

[0019] In another preferred embodiment of the present invention, step 2 further includes:

[0020] The CrEIL1 gene was amplified from the correctly sequenced blunt-ended vector pLB-CrEIL1 and constructed into the plant expression vector p2300.

[0021] In another preferred embodiment of the present invention, when the CrEIL1 gene is constructed on the expression vector p2300, the forward primer introduces the BcuI restriction site, and the reverse primer introduces the Eco91I restriction site. The primer sequences are as follows: SEQ ID NO.5 Forward primer p2300-CrEIL1-FP:GTAGATCTGACTAGTATGGTGAAGTTTGATGAA; SEQ ID NO.6 Reverse primer p2300-CrEIL1-RP:TTCGAGCTGGTCACCTTGGATCTCCTTGGTGAAAAT.

[0022] In another preferred embodiment of the present invention, step 3 further includes:

[0023] Step 3.1: Construct an engineered Agrobacterium strain using the CrEIL1 overexpression vector;

[0024] Step 3.2: Transform periwinkle petals using the Agrobacterium engineered strain constructed in Step 3.1;

[0025] Step 3.3: Extract periwinkle petals.

[0026] In another preferred embodiment of the present invention, step 3.1 specifically includes:

[0027] The overexpression vector was transformed into the pSoup19 helper plasmid Agrobacterium tumefaciens GV3101 using the freeze-thaw method to obtain an engineered Agrobacterium strain containing the target vector.

[0028] In another preferred embodiment of the present invention, step 3.2 specifically includes:

[0029] Positive strains of the constructed Agrobacterium engineered strain were inoculated into 10 ml of liquid culture medium at a ratio of 1:100 and cultured overnight at 28°C. The cells were collected by centrifugation at 4500 rpm for 10 min. The cells were resuspended in MS liquid culture medium, and the concentrations of each bacterial suspension were diluted to OD600 of 0.6 with MS liquid culture medium. Acetylsyringone (AS) with a final concentration of 200 μmol / L and MES with a pH of 5.7 (10 mmol / L) were added, and the mixture was incubated at room temperature for 3 h for later use.

[0030] In another preferred embodiment of the present invention, step 3.2 further includes:

[0031] The engineered strain of Agrobacterium was injected into the petals of a periwinkle plant in a planted state using a 1 mL needleless syringe. The plant was then cultured in the dark for 1 day and then in the light for 3 days.

[0032] In another preferred embodiment of the present invention, step 3.3 specifically includes:

[0033] Petals of transformed and cultured periwinkle were cut, flash-frozen in liquid nitrogen, and ground into powder. The powder sample was placed in an EP tube, 800 μL of methanol solution was added, and the tube was shaken for 2 min to mix thoroughly until no more sample could be dissolved. The powder sample was sonicated for 45 min at a power of 55 W. The sonicated sample was centrifuged at 12000 rpm for 10 min at 4 °C. The supernatant was collected, filtered through a 0.22 μm filter, and placed in a new centrifuge tube, which was then sealed with sealing film.

[0034] The present invention also provides a method for detecting the vincristine content in periwinkle overexpressing CrEIL1 as described above, characterized in that LC-MS is used for detection.

[0035] Technical effect

[0036] 1. Current methods for extracting vinblastine face challenges of high cost and low yield, making it difficult to meet market demand. By constructing a CrEIL1 overexpression vector, the expression of this gene in periwinkle petals was promoted, enhancing its regulatory ability on the vinblastine synthesis pathway, thereby promoting the expression of key enzymes and significantly increasing the content of vinblastine, amantadine, and loganic acid in the petals. Experiments showed that the content of vinblastine, amantadine, and loganic acid was significantly increased in transgenic periwinkle overexpressing CrEIL1, indicating that this method can effectively improve the synthesis of the target product. CrEIL1 has a significant promoting effect on 14 key enzymes and transcription factors CrORCA3 and CrWRKY1 in the vinblastine biosynthesis pathway. Dual-luciferase reporter assays and transient tobacco conversion significantly activated the activity of CrORCA3 and CrWRKY1 gene transcription factors through CrEIL1. Enhancing the activity of key enzymes and optimizing the biosynthesis process of periwinkle alkaloids thus increases the yield of the final product.

[0037] 2. In terms of technological advantages, overexpressing CrEIL1 through transgenic technology can significantly increase the synthesis of vincristine and its precursors, while reducing production costs in traditional extraction processes, thereby enhancing the drug's market competitiveness. Furthermore, as a key transcription factor, the mechanism of action of CrEIL1 has been clarified, helping to better understand the complex regulatory network of alkaloid synthesis in plants and providing an important theoretical foundation for subsequent research. Compared with chemical synthesis methods, this technology produces vincristine through a biosynthetic pathway, reducing chemical pollution and aligning with the trends of sustainable development and environmental protection.

[0038] 3. In terms of performance indicators, the transgenic technology significantly increased the yield of vincristine in periwinkle compared to the control group, reaching several times that of traditional methods, ensuring a stable supply of raw materials. Simultaneously, CrEIL1 expression did not negatively impact the normal growth of periwinkle, ensuring a balance between high yield and plant growth, and improving production reliability. Furthermore, the application of a dual-luciferin reporter vector enabled more precise evaluation of the regulatory effect of CrEIL1, providing data support for further optimization of genetic engineering strategies and enhancing the operability of the technology.

[0039] 4. In terms of promotion and application: Through Agrobacterium-mediated transformation, this technology can not only be applied to the genetic engineering of periwinkle but also extended to other plants, providing new possibilities for the production of various plant-based drugs. Furthermore, the CrEIL1 transgenic strategy can be integrated into periwinkle breeding programs, ensuring the long-term, sustainable, and efficient production of vinblastine to meet the growing demand of the pharmaceutical industry. With the continued rise in global demand for anti-tumor drugs, the production efficiency and cost of vinblastine will directly impact market supply capacity; therefore, this technology shows promising prospects in meeting market demand.

[0040] The following will further explain the concept, specific structure, and technical effects of the present invention in conjunction with the accompanying drawings, so as to fully understand the purpose, features, and effects of the present invention. Attached Figure Description

[0041] Figure 1 This is a schematic diagram illustrating the activation activity of the promoter of a key enzyme gene in the vincristine synthesis pathway by the transient conversion of tobacco CrEIL1, according to a preferred embodiment of the present invention.

[0042] Figure 2 This is a schematic diagram illustrating the significant activation of the transcription factors CrORCA3 and CrWRKY1 by the transient transformation of tobacco.

[0043] Figure 3 This is a schematic diagram illustrating how the CrEIL1 transcription factor regulates the expression levels of vincristine and intermediates in the synthetic pathway in periwinkle.

[0044] Figure 4 Standard curves of periwinkle TIAs and some intermediate products. Detailed Implementation

[0045] The following description, with reference to the accompanying drawings, illustrates several preferred embodiments of the present invention to make its technical content clearer and easier to understand. The present invention can be embodied in many different forms, and the scope of protection of the present invention is not limited to the embodiments mentioned herein.

[0046] Experimental methods not specifically described in the following examples are generally performed under standard conditions, such as those described in Sambrook et al. Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer.

[0047] Example 1: Cloning of the CrEIL1 gene in periwinkle

[0048] 1. Extraction of total RNA from the periwinkle genome

[0049] Using total RNA extracted from periwinkle (purchased from Suzhou Limei Horticulture Co., Ltd.) as a template, the amount of RNA used was calculated based on the RNA concentration, and cDNA was obtained under the action of PowerScript reverse transcriptase. The nucleotide sequence of the CrEIL1 gene (SEQ ID NO. 1), obtained from periwinkle genome sequencing results, was used. Gene-specific primers were designed.

[0050] The specific primer sequences are as follows:

[0051] Forward primer SEQ ID NO.31: GATATCCTTTGCGTAAATCC;

[0052] Reverse primer SEQ ID NO.32: CCTCCTTGTTCATGAACTGC.

[0053] The CrEIL1 gene was amplified from total cDNA by PCR. After the PCR product was recovered and purified, it was ligated into a blunt-terminated pLB vector (a product of Tiangen Biotech Co., Ltd.) and sequenced to obtain the pLB-CrEIL1 plasmid vector. The plasmid was then extracted and sequenced.

[0054] Through the above steps, the coding sequence (SEQ ID NO:1) of the CrEIL1 transcription factor in periwinkle was obtained and its protein coding sequence (SEQ ID NO:2) was deduced, wherein the start codon is ATG and the stop codon is TAA.

[0055] Example 2: Construction of a plant expression vector containing the CrEIL1 gene

[0056] 1. Construction of the overexpression vector pHB-CrEIL1-GFP

[0057] The CrEIL1 gene was amplified from the correctly sequenced blunt-terminated vector pLB and constructed into the plant expression vector pHB-GFP. To facilitate the construction of the expression vector, the forward primer introduced the BamHI restriction site, and the reverse primer introduced the SpeI restriction site. The primer sequences are as follows:

[0058] Forward primer pHB-CrEIL1-GFP-FP: CTCAAGCTTGGATCCATGGTGAAGTTTGATGAAGA, i.e., SEQ ID NO.3

[0059] Reverse primer pHB-CrEIL1-GFP-RP: GCTCACCATACTAGTTGGATCTCCTTGGTGAAAAT, i.e., SEQ ID NO.4

[0060] Example 3: Construction of dual-luciferin reporter vectors for promoters of key enzyme genes 7-DLGT, 7DLH1, LAMT, SLS1, ASα, REDOX2, SAT, HL1, HL2, and DAT, and promoters of transcription factors CrORCA3 and CrWRKY1.

[0061] 1. PCR amplification of the promoters of key enzyme genes 7-DLGT, 7DLH1, LAMT, SLS1, ASα, REDOX2, SAT, HL1, HL2, and DAT, and the promoters of transcription factors CrORCA3 and CrWRKY1 for vincristine biosynthesis.

[0062] Based on the sequence information of key enzyme genes for vincristine biosynthesis in the NCBI database, specific primers for promoter amplification of 7-DLGT, 7DLH1, LAMT, SLS1, ASα, REDOX2, SAT, HL1, HL2, DAT, CrORCA3, and CrWRKY1 were designed. Amplification was performed using periwinkle genomic DNA as a template, with HindIII and PstI restriction sites added upstream and downstream of the primers, respectively.

[0063] Table 1. Specific primers for promoter amplification

[0064]

[0065]

[0066] 2. Link the promoter fragment into the dual-luciferase reporter vector.

[0067] Using homologous recombination, the amplified promoter sequences of 7-DLGT, 7DLH1, LAMT, SLS1, ASα, REDOX2, SAT, HL1, HL2, DAT, CrORCA3, and CrWRKY1 were cloned via ClonExpress II One Step Cloning. The kit (Novizan, Nanjing) was constructed onto the pGreenII0800-LUC vector to obtain the plant dual-luciferin detection reporter vectors pGreenII0800-Pro7-DLGT, pGreenII0800-Pro7DLH1, pGreenII0800-ProLAMT, pGreenII0800-ProSLS1, pGreenII0800-ProASα, pGreenII0800-ProREDOX2, pGreenII0800-ProSAT, pGreenII0800-ProHL1, pGreenII0800-ProHL2, pGreenII0800-ProDAT, pGreenII0800-ProCrORCA3, and pGreenII0800-ProCrWRKY1.

[0068] Example 4: Detection of the activation effect of transcription factor CrEIL1 on the promoters of key enzyme genes DXS1, TDC, STR, SGD, PRX1, 7-DLGT, 7DLH1, LAMT, SLS1, ASα, REDOX2, SAT, HL1, HL2, DAT and transcription factors CrORCA3 and CrWRKY1 by transient transformation of tobacco.

[0069] 1. Obtaining Agrobacterium engineered strains

[0070] The empty pHB-GFP vector and the plant expression vector pHB-CrEIL1-GFP containing CrEIL1 from Example 2 were transformed into Agrobacterium tumefaciens GV3101 using the freeze-thaw method with the pSoup19 helper plasmid. The plant dual-luciferase detection reporter vectors pGreenII0800-Pro7-DLGT, pGreenII0800-Pro7DLH1, pGreenII0800-ProLAMT, pGreenII0800-ProSLS1, pGreenII0800-ProASα, and pGreen... from Example 3 were also transformed into the vectors. Agrobacterium tumefaciens strains II0800-ProREDOX2, pGreenII0800-ProSAT, pGreenII0800-ProHL1, pGreenII0800-ProHL2, pGreenII0800-ProDAT, pGreenII0800-ProCrORCA3, and pGreenII0800-ProCrWRKY1 were transformed into Agrobacterium tumefaciens strain GV3101 carrying the pSoup19 helper plasmid using the freeze-thaw method, resulting in Agrobacterium tumefaciens engineered strains containing an empty vector, the CrEIL1 gene, and a promoter, respectively.

[0071] 2. Instantaneous conversion of tobacco

[0072] Positive strains of the above-mentioned Agrobacterium engineered strains were inoculated into 10 ml of liquid culture medium at a ratio of 1:100 and cultured overnight at 28°C. The cells were collected by centrifugation at 4500 rpm for 10 min. The cells were resuspended in MS liquid culture medium, and the concentration of each bacterial suspension was diluted to OD600 of 0.6 with MS liquid culture medium. Acetylsyringone (AS) and MES (pH 5.7) were added to a final concentration of 200 μmol / L and incubated at room temperature for 3 h for later use.

[0073] Agrobacterium engineered strains containing pHB-CrEIL1-GFP and empty vectors were mixed with Agrobacterium engineered strains containing pGreenII0800-Pro7-DLGT, pGreenII0800-Pro7DLH1, pGreenII0800-ProLAMT, pGreenII0800-ProSLS1, pGreenII0800-ProASα, pGreenII0800-ProREDOX2, pGreenII0800-ProSAT, pGreenII0800-ProHL1, pGreenII0800-ProHL2, pGreenII0800-ProDAT, pGreenII0800-ProCrORCA3, and pGreenII0800-ProCrWRKY1 plant dual-luciferin detection report vectors at a 1:1 ratio. The mixtures were then injected into tobacco leaves that had grown for 4-5 weeks using a needleless syringe. The leaves were first cultured in the dark for 1 day, followed by culture in the light for 1 day.

[0074] 3. Dual-Luciferase detection

[0075] Tobacco leaves cultured for 2 days were flash-frozen in liquid nitrogen and then ground into powder. Fluorescence intensity was detected using the Promega Dual-Luciferase Reporter Assay System kit and a GloMax 20 / 20 Luminometer fluorescence detector, following the kit instructions. Results are as follows: Figure 1 As shown, Dual-LUC analysis of candidate transcription factors on the activation of the promoters of TIAs synthase genes (A) 7-DLGT, (B) 7DLH1, (C) LAMT, (D) SLS1, (E) ASα, (F) REDOX2, (G) SAT, (H) HL1, (I) HL2, and (J) DAT was performed; the pHB-GFP empty vector served as a control. The 7-DLGT, 7DLH1, LAMT, SLS1, ASα, REDOX2, SAT, HL1, and DAT promoters were significantly activated. The transcription factor CrEIL1 significantly activated the activity of the 7-DLGT, 7DLH1, LAMT, SLS1, ASα, REDOX2, SAT, HL1, and DAT promoters. Figure 2 As shown, Dual-LUC analysis showed that the CrEIL1 transcription factor activated the promoters of TIAs synthase genes (A)CrORCA3 and (B)CrWRKY1; with the pHB-GFP empty vector as a control, the transcription factor CrEIL1 significantly activated the promoter activity of the transcription factors CrORCA3 and CrWRKY1.

[0076] Example 5: Construction of CrEIL1 overexpression vector

[0077] The CrEIL1 gene was amplified from the correctly sequenced blunt-terminated vector pLB and constructed into the plant expression vector p2300. To facilitate the construction of the expression vector, the forward primer introduced the BcuI restriction site, and the reverse primer introduced the Eco91I restriction site. The primer sequences are as follows:

[0078] The forward primer p2300-CrEIL1-FP:GTAGATCTGACTAGT ATGGTGAAGTTTGATGAA, i.e., SEQ ID NO. 5

[0079] The reverse primer p2300-CrEIL1-RP:TTCGAGCTGGTCACCTTGGATCTCCTTGGTGAAAAT is SEQ ID NO. 6.

[0080] Example 6: Agrobacterium tumefaciens-mediated overexpression of CrEIL1 promotes vincristine synthesis in periwinkle.

[0081] 1. Obtaining Agrobacterium engineered strains

[0082] The overexpression vector p2300-CrEIL1 was transformed into the pSoup19 helper plasmid Agrobacterium tumefaciens GV3101 (purchased from Shanghai Weidi Biotechnology Co., Ltd.) using the freeze-thaw method to obtain an engineered Agrobacterium strain containing the target vector.

[0083] 2. Instantly transform periwinkle petals

[0084] Positive strains of the above-mentioned Agrobacterium engineered strains were inoculated into 10 ml of liquid culture medium at a ratio of 1:100 and cultured overnight at 28°C. The cells were collected by centrifugation at 4500 rpm for 10 min. The cells were resuspended in MS liquid culture medium, and the concentration of each bacterial suspension was diluted to OD600 of 0.6 with MS liquid culture medium. Acetylsyringone (AS) and MES (pH 5.7) were added to a final concentration of 200 μmol / L and incubated at room temperature for 3 h for later use.

[0085] The engineered strain of Agrobacterium was injected into the petals of a periwinkle plant in a planted state using a 1 mL needleless syringe. The plant was then cultured in the dark for 1 day and then in the light for 3 days.

[0086] 3. Extracting periwinkle petals

[0087] Petals of the cultured periwinkle were cut, flash-frozen in liquid nitrogen, and then ground into powder. The powder sample was placed in an EP tube, 800 μL of methanol solution was added, and the tube was shaken for 2 min to mix thoroughly until no more sample could be dissolved. The powder sample was then sonicated for 45 min (sonicator setting 55W). The sonicated sample was then centrifuged at 12000 rpm for 10 min at 4℃. The supernatant was collected, filtered through a 0.22 μm filter, and placed in a new centrifuge tube, which was then sealed with sealing film.

[0088] Example 7: LC-MS determination of TIAs content in periwinkle petals with transient overexpression

[0089] 1. LC-MS conditions and preparation of standard solutions

[0090] LC-MS experiments were performed using a Waters BEH C18 (2.1 mm * 100 mm, 1.7 μm) column in an ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometer. Mobile phases were A: formic acid / water 1 / 1000 (v) and B: formic acid / acetonitrile 1 / 1000 (v). The detection wavelength was 254 nm, and the flow rate was 0.35 mL / min for both phases, with an injection volume of 2 μL. Ionization was performed using ESI+.

[0091] Table 2 Standard Product Concentration Gradient Configuration

[0092]

[0093] 2. Construction of Standard Curve

[0094] Record the chromatograms and chromatographic parameters, and perform regression analysis on the peak area (Y) against the standard content (X, μg), such as... Figure 4 The linear regression line for the standard sample is shown. Figure 4 The figures show the standard curves for loganic acid, vendolidine, and amantadine, respectively. * indicates a significant difference from the control, and ** indicates an extremely significant difference from the control.

[0095] 3. Determination of the content of vincristine and intermediate products in the synthetic pathway

[0096] The accumulation of TIAs (amadine, vindolin, vincristine, vinblastine, and dehydrated vincristine) and their intermediates (tryptophan, tryptophan, loganic acid, and schizolognoside) was determined by LC-MS. The content of artemisinin in the sample (mg) was calculated by substituting the peak area into the linear regression equation, and then divided by the dry weight of periwinkle petals (g) to calculate the content of amadine, vindolin, vincristine, vinblastine, dehydrated vincristine, tryptophan, tryptophan, loganic acid, and schizolognoside in periwinkle petals.

[0097] In this embodiment, LC-MS was used to determine the contents of amantadine, vinblastine, vincristine, vinblastine, dehydrated vincristine, tryptophan, tryptophan, loganic acid, and schizolognoside in periwinkle petals. Using a metabolic engineering strategy of transforming the CrEIL1 overexpression vector, it was found that overexpression of the CrEIL1 gene can significantly increase the contents of two important alkaloids (vinblastine and amantadine) and one precursor (loganic acid) in periwinkle petals. This provides strong experimental evidence for using this gene to conduct transcriptional regulation studies and thereby increase the content of terpenoid indole alkaloids in periwinkle.

[0098] The EIN3 / EILs transcription factor CrEIL1 of periwinkle, which relates to this invention, can increase the vincristine content in periwinkle. The coding sequence of this transcription factor is linked to a plant expression regulatory vector to construct a plant expression vector containing the coding sequence. The expression vector is then transformed into Agrobacterium, and the Agrobacterium is further transformed into periwinkle petals. The vincristine content in periwinkle petals obtained by this invention is significantly regulated, as shown in the following results. Figure 3 As shown, compared with the control group, the content of loganic acid, vendolidine, and amantadine was significantly increased by 51.32%, 40.10%, and 86.44% in periwinkle petal samples overexpressing CrEIL1. Among them, periwinkle petals overexpressing CrEIL1 significantly increased the content of loganic acid, vendolidine, and amantadine.

[0099] The preferred embodiments of the present invention have been described in detail above. It should be understood that those skilled in the art can make numerous modifications and variations based on the concept of the present invention without creative effort. Therefore, all technical solutions that can be obtained by those skilled in the art based on the concept of the present invention through logical analysis, reasoning, or limited experimentation on the basis of existing technology should be within the scope of protection defined by the claims.

Claims

1. The application of the CrEIL1 gene in increasing the content of loganic acid, vendolidine, and amantadine in periwinkle petals, characterized in that... The application includes the following steps: Step 1: Clone the periwinkle CrEIL1 gene and construct the pLB-CrEIL1 plasmid vector; the nucleotide sequence of the CrEIL1 gene is shown in SEQ ID NO.1; Step 2: Construct a CrEIL1 overexpression vector using the pLB-CrEIL1 plasmid vector; Step 3: Using the CrEIL1 overexpression vector, CrEIL1 was overexpressed in periwinkle petals via Agrobacterium tumefaciens.

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