Application of pachysandene alcohol enzyme PcENO3 in preparation of product for regulating pachysandene alcohol biosynthesis

Abstract

The application relates to the technical field of plant genetic engineering, and particularly discloses application of a patchouli alcohol synthase PcENO3 in preparation of a product for regulating patchouli alcohol biosynthesis. The application first discovers that the patchouli alcohol synthase PcENO3 is an interaction protein of the patchouli alcohol synthase, and proves that the patchouli alcohol synthase PcENO3 interacts with the patchouli alcohol synthase PcPTS, and can effectively improve the in-vitro enzyme catalytic activity of the PcPTS and the content of the patchouli alcohol. Cloning of the protein coding gene and biological function verification have important theoretical and practical significance for the molecular mechanism research of the interaction protein in regulating patchouli alcohol biosynthesis, and particularly for biosynthesis of the patchouli alcohol through a metabolic pathway or yeast fermentation engineering.

Description

Technical Field

[0001] This application relates to the field of plant genetic engineering technology, and in particular to the application of a patchouli enolase PcENO3 in the preparation of products that regulate the biosynthesis of patchouli alcohol. Background Technology

[0002] Patchouli is one of the ten most famous traditional Chinese medicinal herbs from Guangdong. Its dried above-ground parts are used medicinally, possessing aromatic and purifying properties, stimulating appetite and relieving nausea, and dispelling summer heat. As a traditional Chinese medicinal plant, patchouli is not only used in formulations but is also a major ingredient in many traditional Chinese medicine preparations, such as antiviral oral liquids, Huoxiang Zhengqi Water, and Huoxiang Rhinitis Capsules. Patchouli oil, separated from patchouli leaves by steam distillation, is the main active ingredient of patchouli and has a distinctive aroma, widely used in the perfume and cosmetics industries. To date, the highest yield of patchouli oil is approximately 2.6%. Due to the lack of other artificial substitutes, the market demand for patchouli oil is increasing annually. The world uses approximately 1,600 tons of patchouli essential oil annually, indicating a very large market demand.

[0003] Patchouli alcohol is the main component of patchouli oil, accounting for approximately 20-60% of its content. It is a tricyclic sesquiterpene secondary metabolite compound and has been used as an indicator component in all editions of the Chinese Pharmacopoeia for evaluating the quality of patchouli medicinal materials and patchouli oil. Patchouli alcohol is also a key component in maintaining the aroma of patchouli and is an important composite indicator for evaluating the characteristics of patchouli. Patchouli is the only commercial source of patchouli alcohol, and limited natural resources cause its price to fluctuate between US$30 and US$200 per kilogram. Although patchouli is widely cultivated in tropical and subtropical regions of Asia, such as India, Malaysia, China, and Vietnam, the patchouli oil content extracted from the patchouli plant is low (dry weight ratio: 0.54-5.2%) and unstable, making it difficult to meet the huge global market demand for patchouli medicinal materials. Therefore, it is necessary to systematically study the molecular mechanism of its synthesis and accumulation. This not only provides a basis for the metabolic engineering of patchouli alcohol, but also has important significance for increasing the yield of patchouli oil.

[0004] Patchoulol synthase (PTS) is a rate-limiting enzyme in the biosynthesis of patchouli alcohol, directly catalyzing the formation of patchouli alcohol and other sesquiterpenes from farnesyl diphosphate (FDP). Therefore, studying how PTS regulates patchouli alcohol synthesis and increasing its content is of significant economic importance. In recent years, numerous reports have been published on the cloning, expression patterns, enzyme activity and function, and metabolic engineering applications of the PTS gene. Furthermore, due to the considerable influence of transcription factors on the accumulation of secondary metabolites, the transcriptional regulation of their core enzyme genes has received widespread attention. For example, many transcription factors, mainly from the MYB, AP2 / ERF, and bZIP families, regulate PTS transcription alone or in synergy, thereby promoting patchouli biosynthesis. However, there is currently limited literature on the multifaceted mechanisms regulating patchouli biosynthesis outside of the transcriptional level. In particular, research on protein-protein interactions in the patchouli biosynthetic pathway is severely lacking. Summary of the Invention

[0005] The purpose of this application is to overcome the shortcomings of the prior art and provide an application of patchouli enolase PcENO3 in the preparation of products that regulate the biosynthesis of patchouli alcohol. The patchouli enolase PcENO3 provided in this application can bind to PcPTS, promoting its enzyme activity and facilitating the biosynthesis of patchouli alcohol, thus contributing to the development of patchouli alcohol biosynthesis technology through its own metabolic pathway or yeast fermentation engineering.

[0006] To achieve the above objectives, the technical solution adopted in this application is as follows:

[0007] In a first aspect, this application provides a patchouli enolase PcENO3, wherein the patchouli enolase PcENO3 is the amino acid sequence shown in SEQ ID NO:1 or a derived amino acid sequence obtained by substituting, deleting or adding at least one amino acid.

[0008] In some preferred embodiments, the nucleotide sequence of the patchouli enolase PcENO3 is shown in SEQ ID NO:2.

[0009] In our previous study, we used GST-PTS as a bait protein to perform a GST pull-down experiment on the total protein extract of patchouli leaves. We pulled down candidate interacting proteins of PTS and analyzed them using liquid chromatography-tandem mass spectrometry (LC-MS / MS), screening out 93 candidate interacting proteins. Through literature and database searches, we selected 8 candidate proteins involved in protein-protein interactions or secondary metabolic regulation. These proteins may play important roles in regulating PcPTS enzyme activity and patchouli alcohol biosynthesis. Therefore, in this application, we used a yeast two-hybrid assay to find that only patchouli enolase PcENO3 interacts with PcPTS among the 8 candidate proteins. Further, by combining GST pull-down and BiFC experiments, we confirmed that patchouli enolase PcENO3 interacts with patchouli alcohol synthase PcPTS both in vivo and in vitro.

[0010] As a preferred embodiment of the patchouli enolase PcENO3 described in this application, the patchouli enolase PcENO3 as described in claim 1 is characterized in that the amino acid sequence of the patchouli enolase PcENO3 is as shown in SEQ ID NO:1.

[0011] Secondly, this application provides a nucleic acid molecule encoding the above-mentioned patchouli enolase PcENO3.

[0012] Thirdly, this application provides an expression vector containing the aforementioned nucleic acid molecules.

[0013] As a preferred embodiment of the expression vector described in this application, the expression vector includes at least one of the expression vectors pGBKT7, pGADT7, pET-32a, pGEX-6P-1, pSPYNE, pSPYCE and pTRV2.

[0014] Fourthly, this application provides the host cell for the aforementioned nucleic acid molecule or expression vector.

[0015] Fifthly, this application provides the application of the above-mentioned patchouli enolase PcENO3 in the preparation of products that regulate the catalytic activity of PcPTS enzymes.

[0016] This application promotes the synthesis of patchouli alcohol by adding patchouli enolase PcENO3 to the in vitro enzymatic reaction of PcPTS to enhance the catalytic activity of PcPTS enzyme.

[0017] Specifically, in some specific embodiments, the gene encoding patchouli enolase PcENO3 was inserted into the pET-32a vector and transformed into the Rosseta(DE3) strain to obtain a prokaryotic expression strain expressing His-PcENO3. The gene encoding patchouli synthase PcPTS was inserted into the pGEX-6P-1 vector and transformed into BL21 to obtain a prokaryotic expression strain expressing GST-PcPTS.

[0018] The constructed prokaryotic expression strain was activated and cultured to OD. 600 IPTG was added to final concentrations of 0.2 mM and 0.5 mM, respectively, between 0.4 and 0.6, and His-PcENO3 and GST-PcPTS fusion proteins were induced to express for 18 h at 16 °C and 120 rpm. The GST-PcPTS and His-PcENO3 fusion proteins were purified using glutathione resin (Genscript, China) and Ni-NTA purification resin (Qiagen, USA), respectively. After purity and concentration were determined by 10% SDS-PAGE gel electrophoresis and the Bradford assay, the purified proteins were subjected to in vitro enzymatic reactions in reaction buffer (50 mM Tris-HCl, 10 mM MgCl2, pH 7.4). Compared with GST-PcPTS alone, the co-incubation of the mixture of GST-PcPTS and His-PcENO3 resulted in a higher yield of sesquiterpenes. Among them, the content of patchouli generated by catalysis (0.53 ± 0.09 μg / mL) increased by 55.8% (0.34 ± 0.00 μg / mL) compared with PcPTS alone, thus showing stronger catalytic activity.

[0019] Sixthly, this application provides the application of the above-mentioned patchouli enolase PcENO3 in the preparation of products that regulate the biosynthesis of patchouli alcohol.

[0020] The patchouli enolase PcENO3 of this application can interact with patchouli synthase PcPTS, thereby increasing the patchouli alcohol content in plant leaves.

[0021] Seventhly, this application provides the use of a silencing agent for patchouli enolase PcENO3 or the expression vector as described in claim 4 or 5 in the preparation of products with regulated patchouli content.

[0022] Specifically, in some specific embodiments, a partial sequence (SEQ ID NO:3) of patchouli enolase PcENO3 was inserted into the pTRV2 vector and introduced into recipient plants via Agrobacterium tumefaciens to obtain virus-mediated gene silencing (VIGS) PcENO3-silenced plants. Compared with the original plants, the patchouli alcohol content in the leaves of VIGS-induced PcENO3-silenced plants was significantly reduced. The patchouli enolase PcENO3 silencing sequence is shown in SEQ ID NO:3.

[0023] Eighthly, this application provides the application of primers for detecting the above-mentioned patchouli enolase PcENO3 in the preparation of a kit for detecting patchouli alcohol content, wherein the nucleotide sequences of the primers include any pair shown in SEQ ID NO:6-7, SEQ ID NO:10-11, SEQ ID NO:14-15, and SEQ ID NO:18-19.

[0024] Ninthly, this application provides a method for increasing the patchouli alcohol content, comprising the following steps:

[0025] The patchouli enolase PcENO3 encoding gene was inserted into an expression vector and transformed into host cells to obtain a prokaryotic expression strain expressing His-PcENO3.

[0026] The patchouli synthase PcPTS encoding gene was inserted into an expression vector and transformed into host cells to obtain a prokaryotic expression strain expressing GST-PcPTS.

[0027] Prokaryotic expression strains expressing His-PcENO3 and GST-PcPTS were activated and cultured, and IPTG was added to induce the expression of His-PcENO3 and GST-PcPTS fusion proteins. The His-PcENO3 and GST-PcPTS fusion proteins were then purified and subjected to in vitro enzymatic reactions.

[0028] Compared with the prior art, this application has the following beneficial effects:

[0029] This application is the first to discover that patchouli enolase PcENO3 is an interacting protein with patchouli alcohol synthase, and demonstrates that this patchouli enolase PcENO3 interacts with patchouli alcohol synthase PcPTS, effectively increasing the in vitro enzymatic catalytic activity of PcPTS and the content of patchouli alcohol. To further clarify the effect of PcENO3 on patchouli alcohol biosynthesis, a VIGS silencing vector for the target gene was constructed, and VIGS experiments were conducted on patchouli leaves. The results showed that silencing the PcENO3 gene led to a decrease in patchouli alcohol content. This confirms that PcENO3 can effectively increase the content of patchouli alcohol. The cloning and biological function verification of this protein-encoding gene have important theoretical and practical significance for studying the molecular mechanism of interacting proteins in regulating patchouli alcohol biosynthesis, especially for the biosynthesis of patchouli alcohol through its own metabolic pathway or yeast fermentation engineering. Attached Figure Description

[0030] Figure 1 Schematic diagrams of prokaryotic expression vectors pET-32a-PcENO3 and pGEX-6P-1-PcPTS, and plant VIGS vector (A is prokaryotic expression vector pET-32a-PcENO3, B is prokaryotic expression vector pGEX-6P-1-PcPTS, and C is plant VIGS vector pTRV2-PcENO3);

[0031] Figure 2 Figure 1 shows the results of a yeast double hybridization experiment on the interaction between patchouli enolase PcENO3 and PcPTS in yeast.

[0032] Figure 3 Figure 1 shows the results of the GST pull-down assay for the direct in vitro binding of patchouli enolase PcENO3 and PcPTS.

[0033] Figure 4 The figure shows the results of the BiFC experiment on the interaction between patchouli enolase PcENO3 and PcPTS in Arabidopsis protoplasts.

[0034] Figure 5 Figure showing the comparison of the content of patchouli alcohol synthesized by patchouli enolase PcENO3 and the control pair patchouli alcohol synthase PcPTS.

[0035] Figure 6 The image shows the expression level of patchouli enolase PcENO3 after VIGS-induced silencing.

[0036] Figure 7 The figure shows the results of detecting patchouli alcohol content after patchouli enolase PcENO3 was silencing by VIGS. Detailed Implementation

[0037] To better illustrate the purpose, technical solution, and advantages of this application, the following description will be provided in conjunction with the accompanying drawings and specific embodiments.

[0038] In the following embodiments, unless otherwise specified, the experimental methods used are conventional methods, and the materials and reagents used are commercially available unless otherwise specified.

[0039] The amino acid sequence of patchouli enolase PcENO3 involved in this application is shown in SEQ ID NO: 1.

[0040] SEQ ID NO: 1 for MSVQEYLEKHTLSRKIEDAVNAAVRAKTPDPVLFISNHMKKSVPSVITNVKARQILDSRGIPTVEVDLYTNKGVFRASAPSGSTSGIYEATELRDGDRGTYLGKGVSRAVKNINE KISEALVGMDPTLQVQIDQAMIDLDKTDKKGELGANAILAVSMAACRAGAAEKEVPLYKHIADLAGKTNYHLPVPAITLITGGKHAGNNLAIQEIMVLPIGAKKFEEALQIGSETYHHLK AVITEKYGSHGCSVGEDGGFAPNISSLKEGLDFVKEAIGRTGYNEKIKISIGVAATEFCIGTKYDLDYKSPNKSGQNFKSGEDMIEMYKELCEVYPIVSIEDPFDKEDWEHSKYFSSLGI CQVVGADMLMSNPKRIERAVQEAACSSLLLKVNQMGTVTEAIEAVKLAKDANWSVVISQRSGESEDSFLADLAVGLATGQIKAGAPCRGERLAKYNQLLRIEEELGDQAVYVGDDWRHW.

[0041] The nucleic acid sequence of patchouli enolase PcENO3 is shown in SEQ ID NO: 2.

[0042]

[0043] The silencing sequence of PcENO3 is shown in SEQ ID NO: 3:

[0044] SEQ ID NO: 3 is:

[0045] TACTCTTCAAGTTCAAATTGATCAAGCCATGATAGACTTGGACAAGACAGATAAAAAGGGTGAACTTGGGGCAAATGCAATTTTAGCAGTCTCAATGGCTGCTTGCAGAGCAGGGGCAGCTGAGAAAGAGGTTCCACTGTACAAACACATTGCTGATCTTGCTGGCAAAACGAATTACCATCTTCCTGTACCAGCTATCACTCTCATAACTGGTGGAAAACATGCTGGGAAC AATCTGGCGATTCAGGAAATTATGGTACTTCCAATTGGGGCAAAAAAATTTGAGGAAGCATTGCAAATAGGATCTGAGACATATCATCATCTAAAGGCTGTGATTACAGAAAAATATGGTTCGCATGGATGTAGTGTTGGCGAAGATGGTGGTTTTGCCCCGAATATATCCAGCTTGAAAGAAGGATTGGATTTTGTAAAGGAAGCTATTGGCAGAACAGGATACAATGAG.

[0046] Example 1: Cloning of the PcENO3 gene and construction of its expression vector

[0047] 1. Select leaves from healthy patchouli plants and extract total RNA from the leaves using the Hippie Plant RNA Mini Kit C (Magen, China). Purity was determined using agarose gel electrophoresis and ultra-micro UV spectrophotometry. 260 / A 280 The ratio is generally between 2.0 and 2.2, and the concentration is determined. Then, 1 μg of RNA is reverse transcribed into cDNA using the Vazyme First-Strand cDNA Synthesis Kit. (Based on Vazyme's...) The Max Super-Fidelity DNA Polymerase kit was used to amplify gene fragments using reverse transcription products as templates and primers (PcENO3-F:ATGTCTGTACAGGAATATCTGGAGA and PcENO3-R:TCACCAGTGCCTCCAGTC, SEQ ID NO:4-5). Fragments consistent with the expected bands, as detected by agarose gel electrophoresis, were recovered using a Vazyme gel extraction kit. The recovered fragments were then ligated into a pLB vector using the TIANGEN pLB zero-background rapid cloning kit, transformed into *E. coli* DH5α, and positive bacterial cultures were identified by colony PCR and sequenced. The results showed the target band was 1425 bp.

[0048] 2. The expression vectors constructed in this experiment were all designed using CE Design V1.04 with specific primers containing a 15-20 bp homologous sequence (excluding restriction enzyme sites) at the 5' end. The target fragments were then amplified using Vazyme's high-fidelity enzymes. The vectors were double-digested with restriction endonucleases, and the digestion reaction solution was incubated at 37℃ for 5 h. The amplification products and digestion products were detected by agarose gel electrophoresis and purified using a gel extraction kit (Magen, China). Vazyme's [technology / technology / etc.] was used to [achieve specific results / etc.]. The IIOne Step Cloning Kit (C112) performs a recombination reaction between the insert fragment with homologous sequence and the linearized vector, then transforms DH5α, selects single clones for bacterial culture PCR verification, sends positive clones to Tianyi Huiyuan Company for sequencing, analyzes the sequencing results, and extracts the plasmid from the positive bacterial culture to obtain the bait recombinant plasmid.

[0049] The restriction enzyme sites for each expression vector are as follows: pGBKT7 (NdeI and NotI), pGADT7 (EcoRI and BamHI), pET-32a (HindIII and EcoRV), pGEX-6P-1 (EcoRI and NotI), pSPYNE and pSPYCE (BamHI and SalHI), and pTRV2 (EcoRI and BamHI). The following recombinant expression vectors were obtained: pGADT7-PcENO3, pGBKT7-PcPTS, PcENO3-pSPYNE, PcPTS-pSPYCE, pET-32a-PcENO3, pGEX-6P-1-PcPTS, and pTRV2-PcENO3.

[0050] The primers used to construct the expression vector are shown in Table 1 below.

[0051] Table 1

[0052]

[0053]

[0054] Example 2: Interaction verification experiment between patchouli enolase interacting protein PcENO3 and PTS

[0055] 1. Transform Y2HGold competent cells with pGBKT7-PcPTS and PGADT7-PcENO3 according to the manufacturer's instructions, and culture them on SD / -Trp-Leu (DDO) medium for 3 days. Pick single colonies, dilute them 10-fold, and spot them onto SD / -Trp-Leu (DDO), SD / -Trp-Leu-Ade-His (QDO), SD / -Trp-Leu-Ade-His+X-α-Gal (QDO / X), and SD / -Trp-Leu-Ade-His+X-α-Gal+AbA (QDO / X / A) deficient media. Incubate at 30°C inverted for 3 days and observe the growth of yeast colonies. Use Y2H gold [pGADT7] × [pGBKT7-PcPTS] as a negative control and Y2H gold [pGADT7-T] × [pGBKT7-p53] as a positive control.

[0056] The results showed that [pGBKT7-PcPTS]+[pGADT7-PcENO3] and the positive control could grow on DDO and QDO selection plates and turn blue on QDO / X and QDO / X / A selection plates, while the negative control and other experimental groups could not grow normally on QDO series media, indicating that PcENO3 and PcPTS interact in yeast. Figure 2 As shown.

[0057] 2. The plasmids pET-32a-PcENO3 and pGEX-6P-1-PcPTS were introduced into two different expression strains, Rosseta(DE3) and BL21, respectively, using a heat shock method. The plasmids were inoculated onto LB agar containing 50 μg / Amp + 34 μg / mL Chl and 50 μg / Amp, respectively, and incubated overnight at 37°C with inverted incubator. Single colonies were picked for colony PCR verification. Positive colonies were inoculated into LB agar containing the corresponding antibiotics and incubated overnight at 37°C with shaking at 220 rpm. The next day, 3 mL of the bacterial culture was inoculated into 300 mL of LB agar containing 50 μg / mL Amp and incubated at 37°C with shaking at 220 rpm until OD (Organic Dysplasia). 600IPTG was added to final concentrations of 0.2 mM and 0.5 mM, respectively, between 0.4 and 0.6, and the fusion proteins were induced for 16 h at 120 rpm in a shaker at 16 °C. The GST and His fusion proteins were purified using glutathione resin (Genscript, China) and Ni-NTA agarose gel (Qiagen, USA), respectively. The purified proteins were analyzed by 10% SDS-PAGE gel electrophoresis, and protein concentration was assessed using the Bradford assay. Schematic diagrams of the prokaryotic expression vectors pET-32a-PcENO3 and pGEX-6P-1-PcPTS, and the plant VIGS vector are shown below. Figure 1 As shown, A is the prokaryotic expression vector pET-32a-PcENO3, B is the prokaryotic expression vector pGEX-6P-1-PcPTS, and C is the plant VIGS vector pTRV2-PcENO3.

[0058] 3. Mix 2 μg of purified protein (GST-PcPTS and His-PcENO3, with GST at the N-terminus of PcPTS and His at both ends of PcENO3) in GST pull-down binding buffer (50 mM Tris-HCl, 0.2 M NaCl, 1 mM EDTA, 1 mM DTT, 1 mM EDTA, 10 mM MgCl2, 1 mM DTT, pH 8.0) to a total volume of 400 μL. Incubate at 4 °C for 4 h. Then, take 20 μL of the mixture as input and transfer the remaining mixture to a glutathione resin chromatography column (GenScript, China). Incubate at 4 °C for 1 h to allow the GST and GST-PcPTS proteins to bind to the glutathione resin. One hour later, the GST column was washed five times with GST pull-down binding buffer, and the bound protein was eluted with GST pull-down elution buffer. The elution buffer was then used for Western blot detection with anti-MBP, anti-His, or anti-GST monoclonal antibodies.

[0059] The results showed that His-PcENO3 and GST-PcPTS bands could be detected using His and GST in the His-PcENO3 and GST-PcPTS experimental groups, while no His-PcENO3 target band was found in the control group. This indicates that His-PcENO3 protein and GST-PcPTS protein can interact in vitro. Figure 3 As shown, PcENO3 and PcPTS can bind directly in vitro.

[0060] 4. Protoplasts were isolated from the mesophyll tissue of 4-week-old Arabidopsis thaliana col-0 plants. The constructed PcPTS-SPYCE and PcENO3-SPYNE plasmids were transformed into protoplasts using 40% PEG solution. Co-transfection with AP2CL-nYFP and MPK4-cYFP served as a positive control, while co-transfection with PcPTS-SPYCE and pUC-SPYNE vectors, SPYCE and PcENO3-SPYNE vector, or SPYNE and SPYCE served as negative controls. YFP fluorescence signals were visualized using a laser scanning confocal microscope (FV1000, Olympus, Tokyo, Japan). The excitation wavelength for YFP fluorescence was 514 nm, and the receiving range was 500-550 nm. The excitation wavelength for chloroplast autofluorescence was 640 nm, and the receiving wavelength was 663–738 nm.

[0061] The results showed that abundant yellow fluorescence was observed in the cytoplasm of Arabidopsis protoplasts in both the positive control group and the experimental group (PcPTS-SPYCE+PcENO3-SPYN), indicating that PcPTS and PcENO3 interact in the cytoplasm of Arabidopsis protoplasts. Figure 4 As shown.

[0062] Example 3: Effect of patchouli enolase PcENO3 on the in vitro enzyme catalytic activity of PcPTS

[0063] 1. Add substrate (50 μM E, E-FPP) and a mixture of 50 μg GST-PcPTS and His-PcENO3 to the reaction buffer (50 mM Tris-HCl, 10 mM MgCl2, pH 7.4), with a total volume of 1 mL. Use substrate without FPP as a control. Cover with 400 μL of n-hexane and incubate at 30 °C for 7 h. Vortex for 5 min to stop the reaction and extract the product. Then centrifuge at 14000 rpm at room temperature for 5 min to separate the organic and inorganic phases. Transfer 300 μL of the organic phase containing the product to a 1.5 mL GC flask with a liner tube (200 μL) and analyze using an HP7890B / 5977A gas chromatography-mass spectrometry (GC-MS) (Agilent Technologies, USA).

[0064] 2. GC-MS was performed in full scan mode, with an m / z range of 40-550. For separation, an Agilent HP-5MS column (30m × 0.25mm × 0.25μm) was used. Helium was used as the carrier gas at a rate of 1 mL / min, with an injection volume of 1 μL in splitless mode. The instrument temperature was initially set at 50℃ and held for 2 min, then increased to 130℃ at a rate of 20℃ / min, followed by an increase to 150℃ at a rate of 2℃ / min and held for 5 min, and finally increased to 230℃ at a rate of 20℃ / min. The obtained data were integrated chromatographically, and mass spectra were extracted. Mass spectra were retrieved from the NIST14 / Wiley275 mass spectral library for compound analysis. The patchouli alcohol content in the samples was calculated by comparison with patchouli alcohol standards. Patchouli and other terpenoid products were identified by comparing the retention index (RI) of the samples with the RI values ​​of the mass spectrometry and mass spectrometry library (NIST14 / Wiley275) and the mass spectrometry of the patchouli alcohol standard (Solarbio, China). Patchouli alcohol was quantified using the standard curve of patchouli alcohol, and the enzyme catalysis experiment was repeated three times. Results are expressed as mean ± standard deviation (SD). Statistical significance was evaluated using analysis of variance; *P < 0.05, **P < 0.01 were considered statistically significant, and ns indicated no significant difference.

[0065] The results showed that only three sesquiterpenes were detected in the reaction products: α-guaiacol (Match = 829, RI = 1442), α-brucene (Match = 879, RI = 1510), and patchouli alcohol (Match = 880, RI = 1655). Patchouli alcohol in the enzymatic reaction products was quantitatively analyzed using a standard calibration curve with a linearization range of 0.146–5.84 μg / mL. The results showed that the mixture of GST-PcPTS and His-PcENO3 produced higher yields of sesquiterpenes, with the catalytically generated patchouli content (0.53 ± 0.09 μg / mL) increasing by 55.8% (0.34 ± 0.00 μg / mL) compared to PcPTS alone, thus exhibiting stronger catalytic activity. In conclusion, the interaction with PcENO3 enhanced the catalytic activity of PcPTS, such as… Figure 5 As shown.

[0066] Example 4: Application of patchouli enolase PcENO3 in the biosynthesis of patchouli alcohol

[0067] 1. Using the reverse transcription product as a template, a partial coding sequence (463 bp, SEQ ID NO: 3) of PcENO3 was amplified using primers pTRV2-PcENO3-F and pTRV2-PcENO3-R. The amplification was performed using Vazyme's [technology / method / technology - missing from original text]. The IIOne StepCloning Kit (C112) was used to recombine the amplified fragment with the linearized pTRV2 vector to obtain the recombinant vector pTRV2-PcENO3. The pTRV2-PcENO3, pTRV1, and pTRV2 vectors were then transformed into Agrobacterium strain GV3101. The strains were cultured to OD... 600 Agrobacterium cells with a concentration of 1 were resuspended in a solution containing infection medium (10 mM MES, 10 mM MgCl2 and 200 μM acetyl ketone, pH 5.7), and OD was adjusted. 600 1. Subsequently, cell suspensions of pTRV2 (as a control) or pTRV2-PcENO3 were mixed with pTRV1 cell suspension at a 1:1 ratio and incubated in the dark at room temperature for 3 hours. Then, the Agrobacterium suspension was injected into the patchouli leaves from the back using a 1 mL sterile syringe until the entire leaf was completely infiltrated by the solution. After infection, the cells were grown under controlled conditions (24°C for 16 h, 55% relative humidity). 14 days after injection, leaf tissues were collected and frozen at -80°C for RNA and patchouli alcohol extraction.

[0068] 2. Patchouli leaves were rapidly ground into a fine powder under liquid nitrogen. 0.2 g of each sample was accurately weighed, then extracted with 1.5 mL of n-hexane using ultrasound for 30 min, followed by a 1 h water bath at 56 °C. After micro-centrifugation, the supernatant was collected and filtered through a 0.22 μM organic filter membrane. The supernatant was diluted 5-fold with n-hexane and transferred to a 1.5 mL sample vial for GC-MS analysis. The GC-MS detection method was slightly modified from Example 3: a split injection mode with a split ratio of 10:1 was used. The instrument's initial temperature was 50 °C and held for 2 min, then increased to 130 °C at a rate of 20 °C / min, followed by an increase to 150 °C at a rate of 2 °C / min and held for 1 min. Student's t-test was used to evaluate statistical significance; *P < 0.05 and **P < 0.01 were considered statistically significant.

[0069] RT-qPCR results show (e.g.) Figure 6 Compared with the control group, the transcriptional level of PcENO3 in pTRV2-PcENO3 plants was significantly reduced by 56%, indicating that PcENO3 was effectively silenced (the nucleic acid sequence of PcENO3 used for silencing is shown in SEQ ID NO: 3), which can be used for further research.

[0070] A standard calibration curve for patchouli alcohol with a linearization range from 5.84 μg / mL to 116.8 μg / mL was used (R). 2 =0.99) was used to quantify the patchouli alcohol content in n-hexane, and the results showed (e.g. Figure 7The patchouli alcohol content in the pTRV2-PcENO3 group (0.65±0.02 mg / g fresh weight) was significantly lower than that in the control group (1.05±0.11 mg / g fresh weight), a decrease of 38.1%. These results indicate that virus-induced PcENO3 silencing affects the biosynthesis of patchouli alcohol.

[0071] The above experiments show that patchouli enolase PcENO3 interacts with patchouli alcohol synthase in vivo and in vitro. PcENO3 promotes the in vitro enzyme catalytic activity of patchouli alcohol synthase PcPTS. Silencing its encoding gene PcENO3 can significantly inhibit the content of patchouli alcohol in patchouli leaves.

[0072] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of this application and are not intended to limit the scope of protection of this application. Although this application has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of this application without departing from the substance and scope of the technical solutions of this application.

Claims

1. A patchouli enolase PcENO3, characterized in that, The amino acid sequence of the patchouli enolase PcENO3 is shown in SEQ ID NO:

1.

2. A nucleic acid molecule encoding patchouli enolase PcENO3 as described in claim 1.

3. An expression vector comprising the nucleic acid molecule as described in claim 2.

4. The expression vector as described in claim 3, characterized in that, The expression vector includes at least one of the expression vectors pGBKT7, pGADT7, pET-32a, pGEX-6P-1, pSPYNE, pSPYCE and pTRV2.

5. The application of patchouli enolase PcENO3 as described in claim 1 in the preparation of products with enhanced PcPTS enzyme catalytic activity.

6. The application of patchouli enolase PcENO3 as described in claim 1 in the preparation of products with enhanced patchouli alcohol biosynthesis; characterized in that, The patchouli enolase PcENO3 interacts with patchouli synthase PcPTS, enhancing the in vitro enzymatic catalytic activity of PcPTS and thus improving the biosynthesis of patchouli alcohol.

7. The application of the expression vector according to claim 3 or 4 in the preparation of products with increased patchouli content; characterized in that, The patchouli enolase PcENO3 expressed by the expression vector interacts with patchouli synthase PcPTS, enhancing the in vitro enzymatic catalytic activity of PcPTS and thus improving the biosynthesis of patchouli alcohol.

8. A method for increasing the patchouli alcohol content, characterized in that, Includes the following steps: The patchouli enolase PcENO3 encoding gene as described in claim 1 was inserted into the expression vector and transformed into host cells to obtain a prokaryotic expression strain expressing His-PcENO3. The patchouli synthase PcPTS encoding gene was inserted into an expression vector and transformed into host cells to obtain a prokaryotic expression strain expressing GST-PcPTS. Prokaryotic strains expressing His-PcENO3 and GST-PcPTS were activated and cultured, and IPTG was added to induce the expression of His-PcENO3 and GST-PcPTS fusion proteins. The His-PcENO3 and GST-PcPTS fusion proteins were then purified. Substrate E, E-FPP and a mixture of GST-PcPTS and His-PcENO3 were added to the reaction buffer, and then an in vitro enzymatic reaction was carried out.

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