A method for increasing the content of natural fragrance substances linalool and its oxides and ionones in osmanthus petals by applying OfERF111 gene

Abstract

This invention discloses a method for promoting the synthesis of linalool, trans-linalool oxide, and ionone, important natural aroma compounds of osmanthus. OfERF111 Genes and their applications, as described OfERF111 The gene is an ERF transcription factor gene, which will contain the gene OfERF111 The overexpression vector was transformed into osmanthus petals for transient expression. The results showed that the transiently transformed osmanthus petals contained... OfERF111 Gene expression levels were significantly increased, and the contents of linalool, trans-linalool oxide, and various ionones (α-ionone, β-ionone, and dihydro-β-ionone) in the petals were significantly higher than those in the control group, indicating that... OfERF111 It plays an important role in promoting the synthesis of linalool and its oxides as well as ionone in osmanthus, and can be used in osmanthus genetic engineering to improve the quality of the natural fragrance of osmanthus and to select superior varieties.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, and in particular relates to a method for increasing the content of linalool and its oxides and ionone, natural aroma substances in osmanthus petals, by using the OfERF111 gene. Background Technology

[0002] Terpenes are important aroma active substances. Linalool and its oxides, β-ionone, and dihydro-β-ionone are key aroma active substances in osmanthus. Linalool is a chain-like terpene alcohol extracted from plants. It is a colorless, easily flowing liquid at room temperature, possessing aromas of lily of the valley, woody notes, and fruitiness. It is a major component of essential oils from linalool leaf oil, linalool oil, galangal oil, rosewood oil, coriander seed oil, magnolia leaf oil, lavender oil, bitter orange leaf oil, bergamot oil, sage oil, and many flowers (jasmine, rose, bitter orange, orange blossom, ylang-ylang, etc.), and is also a major component of the aroma of green tea. Linalool is commonly used in the synthesis of fragrances and perfumes. It is also used medicinally for its sedative, antibacterial, and insecticidal properties. Furthermore, it can be used as a deodorant, possessing a strong ability to mask unpleasant odors. Linalool has a variety of oxides. Linalool and its oxides are the key substances that are abundant in various varieties and give flowers their characteristic fragrance. β-ionone, as the most important fragrance substance in osmanthus, also contributes significantly to the fragrance and has a series of pharmacological effects, such as anti-cancer, chemopreventive, anti-inflammatory and antibacterial effects.

[0003] Osmanthus fragrans Lour. is an important fragrant flowering plant with high economic and health value. Osmanthus is rich in monoterpenoids, linalool, and their derivatives, which are widely distributed across various varieties and are key aroma-active substances in osmanthus. The ERF gene family is an important transcription factor family in plants, belonging to the AP2 / ERF transcription factor superfamily. It is widely involved in multiple plant signal transduction pathways, stress responses, and processes related to stress and disease resistance. However, research on the involvement of the ERF family in the metabolism of plant secondary metabolites, especially aroma substances, is scarce. Therefore, it is of great significance to investigate and discover genes in the osmanthus genome that promote the synthesis of linalool and its oxides, as well as various ionones. This invention utilizes bioinformatics techniques to screen for ERF family genes in osmanthus that promote the synthesis of linalool and its oxides, as well as ionones, and then performs functional verification in osmanthus petals. Summary of the Invention

[0004] To address the aforementioned problems in the existing technology, this invention provides an OfERF111 gene related to the synthesis of linalool and its oxides and ionones in osmanthus. Overexpression of this gene can promote the synthesis of linalool and its oxides, as well as various ionones in osmanthus, and can be used in osmanthus genetic engineering to improve the aroma quality of natural osmanthus extracts and to select superior varieties.

[0005] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0006] The OfERF111 gene is associated with the synthesis of linalool and its oxides and ionone. The CDS sequence of the OfERF111 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.

[0007] Biological materials containing the OfERF111 gene include expression cassettes, expression vectors, and recombinant bacteria.

[0008] Applications of the OfERF111 gene include:

[0009] (1) Application in Osmanthus breeding;

[0010] (2) Application in promoting the content of key aroma substances linalool, trans-linalool oxide and ionone (including α-ionone, β-ionone and dihydro-β-ionone) in natural osmanthus extract.

[0011] A method for promoting the synthesis of linalool and its oxides and ionone from osmanthus flowers includes the following steps:

[0012] (1) Construct an overexpression vector containing the OfERF111 gene as shown in SEQ ID NO.1;

[0013] (2) The expression vector was transformed into Agrobacterium to obtain recombinant Agrobacterium expression;

[0014] (3) Osmanthus fragrans was transformed with the recombinant Agrobacterium described above, and the OfERF111 gene was overexpressed in Osmanthus fragrans;

[0015] Preferably, in step (3), recombinant bacteria are used to transform osmanthus petals;

[0016] Preferably, the Agrobacterium is Agrobacterium GV3101.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0018] This invention provides an OfERF111 gene, an ERF transcription factor gene, which promotes the synthesis of linalool, its oxides, and ionones in Osmanthus fragrans. An overexpression vector containing the OfERF111 gene was transformed into Osmanthus fragrans petals for transient expression. Results showed that the expression level of the OfERF111 gene was significantly increased in the transiently transformed petals, and the contents of linalool, trans-linalool oxides, and various ionones (α-ionone, β-ionone, and dihydro-β-ionone) in the petals were significantly higher than those in the control group. This indicates that the OfERF111 gene plays an important role in promoting the synthesis of linalool, its oxides, and various ionones in Osmanthus fragrans and can be used in Osmanthus fragrans genetic engineering to improve ornamental traits and genetic quality. Attached Figure Description

[0019] Figure 1 Agarose gel electrophoresis image of the amplification product of the target gene OfERF111.

[0020] Figure 2 The figure shows the results of GC-MS detection of linalool, trans-linalool oxide and various ionones in osmanthus petals after transient transformation.

[0021] Figure 3 The expression level of the OfERF111 gene in osmanthus petals transiently transformed. Detailed Implementation

[0022] The technical solution of the present invention will be described in detail below with reference to the accompanying drawings and specific embodiments. The examples given are only for explaining the present invention and are not intended to limit the scope of the present invention.

[0023] Example 1: Screening and Cloning of the Target Gene

[0024] (1) Obtaining the target gene: Based on the weighted correlation network analysis (WGCNA) of the transcriptomes of five Osmanthus fragrans varieties from the research group of Zheng Riru at Huazhong Agricultural University, and combined with yeast one-hybrid library screening of the linalool key synthase gene OfTPS7, a transcription factor sequence Ofr22340 was obtained. Its amino acid sequence has 69% homology with AtERF111, and it also possesses the conserved AP2 domain of the ERF transcription factor family, hence it was named OfERF111. Subcellular localization in tobacco leaves and yeast transcriptional activation activity experiments jointly proved that it is an ERF transcription factor located in the cell nucleus.

[0025] (2) Primer design: Specific primers of OfERF111-CDS-F and OfERF111-CDS-R (see Table 1) were designed using Primer 5.0 software and synthesized by Beijing Qingke Biotechnology Co., Ltd.

[0026] Table 1 Primer List

[0027]

[0028] (3) Amplification of the target gene: Using Osmanthus cDNA as a template, the target gene CDS was amplified according to the Phanta high-fidelity enzyme instructions. The PCR reaction system and procedure are shown in Table 2 and Table 3.

[0029] Table 2 PCR reaction system

[0030]

[0031] Table 3 PCR reaction procedure

[0032]

[0033] (4) Gel electrophoresis and gene sequencing: 0.3g agarose powder was added to 30ml TAE, melted in a microwave oven, and then 1.5μL of 10000× nucleic acid dye was added. The mixture was poured into a gel casting plate, allowed to solidify, and then loaded with the sample. After electrophoresis at 120V and 150mA for 28min, the bands were observed using a Gel-Logie200 gel scanning imager. The target band was 687bp (e.g., ...). Figure 1 (As shown). After the target band was recovered, it was ligated into a T-vector, transformed into E. coli, and sequenced. The CDS sequence of the OfERF111 gene is shown in SEQ ID NO.1, and the amino acid sequence of its encoded protein is shown in SEQ ID NO.2. The plasmid was extracted from the bacterial culture with the correct sequence and named OfERF111-CDS-Plasmid, which was used as a template for the subsequent construction of the vector.

[0034] Example 2: Construction of an overexpression vector for the Osmanthus fragrans OfERF111 gene

[0035] (1) Cloning of the target gene

[0036] ① First round of PCR reaction: Using plasmid OfERF111-CDS-Plasmid as a template, the CDS sequence of the OfERF111 gene containing attB sites at both ends was amplified according to the Phanta high-fidelity enzyme instructions. The PCR reaction system and procedure are shown in Tables 4 and 3. After the PCR reaction, a portion of the PCR product was run on a gel to check for a clear target band. If present, 2 μL of the remaining PCR product was used as a template for the second round of PCR reaction.

[0037] Table 4 First-round PCR reaction system

[0038]

[0039] ② Second round of PCR reaction: Using the first round of PCR products as templates, the CDS sequence of the OfERF111 gene containing Adapter-attB sites at both ends was amplified according to the Phanta high-fidelity enzyme instructions. The PCR reaction system and procedure are shown in Tables 5 and 3. After the PCR reaction, all PCR products were run on a gel for detection and the gel was cut and recovered. The recovered product was named attB-OfERF111-ox.

[0040] Table 5. Second round PCR reaction system

[0041]

[0042] (2) BP reaction: Prepare the BP reaction system (as shown in Table 6), gently pipette to mix, briefly centrifuge, and react overnight at 25℃. The reaction product can be stored at -20℃. Transform E. coli with the reaction product and spread it on LB solid medium (containing 50 μg / ml kanamycin). Incubate at 37℃ after transformation until single colonies grow. Identify by PCR using OfERF111-CDS-F and OfERF111-CDS-R, select positive single colonies for propagation and sequencing. If the sequencing is correct, extract the plasmid for LR reaction. The plasmid name is pDONR221-OfERF111-ox.

[0043] Table 6 BP Reaction System

[0044]

[0045] (3) LR reaction: Prepare the LR reaction system (Table 7), gently aspirate and mix, briefly centrifuge, and react overnight at 25°C. The operation method is the same as the BP reaction. When transforming E. coli, spread it on LB solid medium (containing 50 μg / ml spectinomycin). After confirming that the sequencing is correct, extract the plasmid. The constructed plasmid is named PK7WG2D-OfERF111. Store the positive bacterial culture in 50% glycerol in an equal volume.

[0046] Table 7 LR Reaction System

[0047]

[0048] Example 3: Transformation of Agrobacterium GV3101

[0049] (1) Take out the Agrobacterium GV3101 competent cells stored in the -80℃ ultra-low temperature freezer and thaw them on ice. Add 1 μL of plasmid PK7WG2D-OfERF111 to every 100 μL of competent cells, mix well by pipetting, and then successively incubate on ice for 5 min, quick freeze in liquid nitrogen for 5 min, incubate in water at 37℃ for 5 min, and incubate on ice for 5 min.

[0050] (2) Add 500 μL of non-resistant LB liquid medium and incubate at 28°C and 200 rpm for 1 h on a shaker.

[0051] (3) After the culture is completed, centrifuge the bacterial culture at 6000 rpm for 1 min, discard part of the supernatant, and leave 100 μL to be evenly spread on LB solid medium (containing 50 μg / ml spectinomycin), seal with sealing film, and invert in an incubator at 28℃ for 40-48 h.

[0052] (4) Bacterial detection and backup: PCR identification was performed using OfERF111-CDS-F and OfERF111-CDS-R. If the target band in the bacterial detection was correct and the brightness was consistent, the corresponding colonies in the backup plate were picked into LB liquid medium (containing 50 μg / ml spectinomycin) and shaken. The bacterial solution and 50% glycerol were then kept in equal volume ratio for preservation. After being quick-frozen in liquid nitrogen, the solution was stored in an ultra-low temperature freezer at -80℃.

[0053] Example 4: Infection of osmanthus petals and determination of linalool, trans-linalool oxide and various ionones by GC-MS.

[0054] (1) One-time shaking culture: Agrobacterium positive transformation culture was inoculated into LB liquid medium (containing 50 μg / ml spectinomycin) at a volume ratio of 1:50 and cultured overnight on a shaker at 28°C.

[0055] (2) Secondary shaking inoculation: Take the overnight bacterial culture and inoculate it into 100 mL of LB liquid medium (containing 50 μg / mL spectinomycin) at a volume ratio of 1:50. Add 200 μl of 10 mmol / L AS (to make the final concentration 20 μmol / L) and 2 mL of 1 mol / L MES (to make the final concentration 20 mmol / L). Incubate at 28°C in a shaker until OD. 600 =0.6-0.8 (at which point Agrobacterium activity is highest).

[0056] (3) Preparation of infection solution: Add 1 mL of 10 mmol / L AS (to make the final concentration 20 μmol / L), 5 mL of 1 mol / L magnesium chloride solution (to make the final concentration 10 mmol / L), and 5 mL of 1 mol / L MES (to make the final concentration 10 mmol / L) in sequence, add distilled water to make up to 500 mL, and adjust the pH value to 5.6.

[0057] (4) Collect bacterial cells and resuspend them: OD 600 Transfer the bacterial culture with an OD value of 0.6-0.8 into a 50 mL sterile centrifuge tube, centrifuge at 4°C and 4000 rpm for 10 min, and discard the supernatant. Gently resuspend the bacterial cells in the infection solution until a uniform turbid solution is formed, and adjust the OD value accordingly. 600 =0.5-0.6, let stand at room temperature in the dark for 2 hours.

[0058] (5) Infection: Take fresh osmanthus petals, remove the flower stems, and leave only the petals; place the osmanthus petals into a 50mL centrifuge tube containing positive Agrobacterium infection solution. This process should be quick. Vacuum permeate at 0.06Mpa for about 10 minutes, and slowly release the gas; take out the osmanthus petals and wash the remaining bacterial solution on the petals with sterile water.

[0059] (6) Cultivation: Place the washed petals into a 50mL centrifuge tube containing 5% sucrose solution, cover the top with sterile gauze to completely immerse the petals, and place in a dark room at room temperature for 60h.

[0060] (7) GC-MS detection of linalool, trans-linalool oxide and various ionones in osmanthus petals of transient transformation: 0.2g of petals and 2μL of methyl nonanoate diluted 10,000 times were sealed in a 20mL extraction bottle and equilibrated for 10min. Then, a 2cm extraction head (50 / 30μm, DVB / Carboxen / PDMS, Supelco USA) was inserted and extracted in a 55℃ water bath for 30min. Finally, the sample was inserted into the gas chromatograph injection port and desorbed at 230℃ for 5min. GC-MS was then performed.

[0061] Detection conditions: DB-5MS column (30m × 0.25mm × 0.25μm, Themo Scientific, Bellefonte, PA, USA), carrier gas high-purity helium (99.999%), split ratio 20:1, flow rate 1mL / min. Ion source and injection port temperatures were 280℃ and 230℃, respectively, and transfer line temperature was 250℃.

[0062] The GC temperature program is as follows: hold at 40°C for 3 min, then increase the temperature to 120°C at a rate of 3°C / min and hold for 3 min, then increase the temperature to 220°C at a rate of 8°C / min and hold for 2 min.

[0063] The MS conditions are as follows: EI (electron impact) ion source, electron impact energy 70 eV, positive ion scanning mode, mass scan range m / z 40-450 amu.

[0064] GC-MS analysis showed that the contents of linalool, trans-linalool oxide, and various ionones (α-ionone, β-ionone, and dihydro-β-ionone) in Osmanthus petals transiently transformed by the hyperexpression PK7WG2D-OfERF111 were significantly increased compared with the control group (CK). Figure 2 ).

[0065] Example 5: RT-qPCR Validation of Transient Transformation of Osmanthus Petals

[0066] Total RNA was extracted from transiently transformed Osmanthus fragrans petals using the Trizol Ultrapure RNA Extraction Kit (Beijing Kangwei Century Biotechnology Co., Ltd.). The extracted RNA was reverse transcribed into cDNA using the TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix (TransGold, Beijing) reverse transcription kit. The resulting cDNA was diluted 5-fold with water, and 1 μL was used as a template. The Osmanthus fragrans OfActin gene was used as an internal control. The RT-qPCR reaction system was prepared using the 2×SYBR Green qPCR Mix kit from Beijing Adley Biotechnology Co., Ltd., as shown in Table 8. The PCR reaction program used a three-step method: 95℃ for 2 min; 95℃ for 15 s; 60℃ for 15 s; 72℃ for 20 s; for 40 cycles. After the quantitative PCR program was completed, the results were analyzed using Roche LC96 software and Excel. ΔΔCt Analysis was performed, and the relative expression level of the OfERF111 gene was calculated using the internal reference gene OfActin as a reference. The results showed that the expression level of OfERF111 in Osmanthus petals transiently transformed with the overexpression vector PK7WG2D-OfERF111 was significantly increased compared to the control group (CK). Figure 3 ).

[0067] Table 8 RT-qPCR reaction system

[0068]

Claims

1. The OfERF111 gene associated with the synthesis of linalool and its oxides and ionone in osmanthus, characterized in that, The CDS sequence of the OfERF111 gene is shown in SEQ ID NO.

1.

2. The protein encoded by the OfERF111 gene according to claim 1, characterized in that, The amino acid sequence of the protein is shown in SEQ ID NO.

2.

3. A biomaterial containing the OfERF111 gene as described in claim 1, characterized in that, The biological materials are expression cassettes, expression vectors, and recombinant bacteria.

4. The application of the OfERF111 gene of claim 1, the encoded protein of claim 2, or the biomaterial of claim 3 in promoting the synthesis of linalool, trans-linalool oxide, α-ionone, β-ionone, and dihydro-β-ionone in Osmanthus fragrans.

5. The application of the OfERF111 gene of claim 1, the encoded protein of claim 2, or the biological material of claim 3 in Osmanthus breeding.

6. A method for promoting the synthesis of linalool, trans-linalool oxide, α-ionone, β-ionone, and dihydro-β-ionone from osmanthus flowers, characterized in that, Overexpression of the OfERF111 gene as described in claim 1 in Osmanthus fragrans.

7. The method according to claim 6, characterized in that, Includes the following steps: (1) Construct an overexpression vector containing the OfERF111 gene as shown in SEQ ID NO. 1; (2) Transform the overexpression vector into Agrobacterium to obtain recombinant Agrobacterium; (3) Osmanthus fragrans was transformed with the recombinant Agrobacterium described above, and the OfERF111 gene was overexpressed in Osmanthus fragrans.

8. The method according to claim 7, characterized in that, In step (3), recombinant Agrobacterium is used to transform osmanthus petals.

9. The method according to claim 7, characterized in that, The Agrobacterium mentioned is Agrobacterium GV3101.

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