A method for increasing natural fragrance substances linalool and its oxides and ionones of osmanthus petal by applying ofnac72 gene

Abstract

The present invention discloses an application Of NAC72 A method for genetically increasing the natural aroma substances linalool and its oxides and ionones in osmanthus petals, the Of NAC72 The gene is a NAC transcription factor gene that contains the gene Of NAC72 The overexpression vector was transformed into osmanthus petals for transient expression. The results showed that Of NAC72 The gene expression level increased significantly, and the contents of linalool, trans-linalool oxide, and various ionones (α-ionone, β-ionone, and dihydro-β-ionone) in the petals increased significantly compared with the control group, indicating that Of NAC72 The gene plays an important role in promoting the synthesis of linalool, its oxides and ionone in osmanthus, and can be used in osmanthus genetic engineering to improve the aroma quality of osmanthus and breed excellent varieties.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant genetic engineering, and particularly relates to a method for increasing the natural aroma substances linalool and its oxides and ionones in osmanthus petals by using the OfNAC72 gene. Background Art

[0002] Terpenes are important aroma-active substances, and linalool and its oxides, β-ionone, and dihydro-β-ionone are key aroma-active substances in osmanthus. Linalool is a chain-like terpene alcohol compound extracted from plants. It is a colorless, free-flowing liquid at room temperature and has the aroma of lily of the valley, woody green, and fruity. It is a major component of essential oils such as camphor leaf oil, camphor oil, kyara oil, rosewood oil, coriander seed oil, brandy leaf oil, lavender oil, citronella leaf oil, bergamot oil, and clary perilla oil, and is also a major component of the aroma of green tea. Linalool is commonly used in the synthesis of flavors and fragrances. It is also used medicinally for sedative, antibacterial, and insecticidal effects. It can also be used as a deodorant, with 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 the flowers their unique fragrance. β-ionone, as the most important aromatic substance in osmanthus, contributes greatly to the fragrance and also has a series of pharmacological effects, such as anti-cancer, chemoprevention, anti-inflammatory and antibacterial.

[0003] Sweet osmanthus (Osmanthus fragrans Lour.) is an important fragrant plant with high economic and health value. Fragrance is a key indicator of its ornamental and economic value. The monoterpenoid linalool and its derivatives are abundant in osmanthus and widely distributed across varieties, making them key aroma-active substances in osmanthus. The NAC transcription factor family is a large family of transcription factors unique to plants, with over 100 members. It consists of two domains: a highly conserved N-terminal NAC-binding domain that specifically binds to cis-acting elements; and a variable C-terminal domain that regulates transcriptional activation. NAC transcription factors are primarily involved in biological processes such as plant growth and development, anti-aging, and stress resistance. However, genes within the NAC family that promote the synthesis of linalool, its oxides, and various ionones in osmanthus have not been reported. Therefore, it is of great significance to study and discover the related genes in the osmanthus genome that promote the synthesis of linalool, its oxides and various ionones. The present invention uses bioinformatics technology to screen out genes that promote the synthesis of linalool, its oxides and various ionones in osmanthus, and conducts functional verification in osmanthus petals. Summary of the Invention

[0004] In response to the above-mentioned problems existing in the prior art, the present invention provides an OfNAC72 gene related to the synthesis of linalool, its oxides and ionones from osmanthus. Overexpression of the gene can promote the synthesis of linalool, trans-linalool oxides and various ionones from osmanthus, and can be used in osmanthus genetic engineering to improve the aroma quality of osmanthus and breed excellent varieties.

[0005] In order to solve the above technical problems, the technical solutions adopted by the present invention are as follows:

[0006] The OfNAC72 gene is related to the synthesis of osmanthus linalool, its oxides and ionones. The CDS sequence of the OfNAC72 gene is shown in SEQ ID NO.1, and the amino acid sequence of the encoded protein is shown in SEQ ID NO.2.

[0007] Biological materials containing the OfNAC72 gene, including expression cassettes, expression vectors, and recombinant bacteria.

[0008] Applications of the OfNAC72 gene include:

[0009] (1) Application in Osmanthus fragrans breeding;

[0010] (2) Its application in promoting the synthesis of linalool, trans-linalool oxide and ionone (including α-ionone, β-ionone and dihydro-β-ionone), the key aroma substances of osmanthus.

[0011] A method for promoting the synthesis of osmanthus linalool, trans-linalool oxide and ionone, comprising the following steps:

[0012] (1) constructing an overexpression vector containing the OfNAC72 gene as shown in SEQ ID NO.1;

[0013] (2) transforming the expression vector into Agrobacterium to obtain recombinant expression Agrobacterium;

[0014] (3) transforming Osmanthus fragrans with the recombinant Agrobacterium to overexpress the OfNAC72 gene in Osmanthus fragrans;

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

[0016] Preferably, the Agrobacterium is Agrobacterium GV3101.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] The present invention provides a gene OfNAC72 related to promoting the synthesis of linalool, its oxides, and ionones in osmanthus. It is a NAC transcription factor gene. An overexpression vector containing the gene OfNAC72 was transformed into osmanthus petals for transient expression. The results showed that the expression level of the OfNAC72 gene in the transiently transformed osmanthus petals was significantly increased, and the contents of linalool, trans-linalool oxide, and various ionones (α-ionone, β-ionone, dihydro-β-ionone) in the petals were significantly increased compared with the control group, indicating that the OfNAC72 gene plays an important role in promoting the synthesis of linalool, its oxides, and various ionones in osmanthus. It can be used in breeding work such as improving ornamental traits and genetic quality in osmanthus genetic engineering. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is the agarose gel electrophoresis diagram of the amplified product of the target gene OfNAC72.

[0020] Figure 2 This is the result of GC-MS detection of the contents of linalool, trans-linalool oxide and various ionones in the petals of Osmanthus fragrans by instantaneous conversion.

[0021] Figure 3 is the expression level of the OfNAC72 gene in transiently transformed Osmanthus fragrans petals. DETAILED DESCRIPTION

[0022] The technical solution of the present invention is described in detail below with reference to the accompanying drawings and specific embodiments. The examples given are only used to explain the present invention and are not used to limit the scope of the present invention.

[0023] Example 1 Screening and cloning of target genes

[0024] (1) Obtaining the target gene: Based on the transcriptome data analysis of five Osmanthus fragrans varieties by the research group of Zheng Riru of Huazhong Agricultural University, a gene sequence was screened and compared with the sequence of the model plant Arabidopsis thaliana. It was determined that the gene belongs to the NAC gene family. Since the members of this gene family are most closely related to GaNAC72 of Australian cotton in the evolutionary tree and have extremely high homology, it was named OfNAC72.

[0025] (2) Primer design: Specific primers OfNAC72-CDS-F and OfNAC72-CDS-R (see Table 1) for amplifying the full-length CDS of the OfNAC72 gene were designed using Primer 5.0 software. The amplification primers were synthesized by Beijing Qingke Biotechnology Co., Ltd.

[0026] Table 1 Primer list

[0027]

[0028] (3) Target gene amplification: Using Osmanthus fragrans cDNA as a template, the OfNAC72 gene CDS was amplified according to the instructions of Phanta high-fidelity enzyme. The PCR reaction system and procedure are shown in Tables 2 and 3.

[0029] Table 2 PCR reaction system

[0030]

[0031]

[0032] Table 3 PCR reaction program

[0033]

[0034] (4) Gel electrophoresis detection and gene sequencing: 0.3 g agarose powder was added to 30 ml TAE, and after melting in a microwave oven, 1.5 μL 10000× nucleic acid dye was added and poured into a gel plate. After solidification, the sample was spotted. After electrophoresis at 120 V and 150 mA for 25 min, the bands were observed on a Gel-Logie200 gel scanning imager. The target band was 999 bp (e.g. Figure 1 The target band was recovered, ligated to a T-vector, and transformed into E. coli for sequencing. The CDS sequence of the OfNAC72 gene is shown in SEQ ID NO. 1, and the amino acid sequence of the encoded protein is shown in SEQ ID NO. 2. A plasmid was extracted from the correctly sequenced bacterial solution and named OfNAC72-CDS-Plasmid, which served as a template for subsequent vector construction.

[0035] Example 2 Construction of an overexpression vector for the Osmanthus fragrans OfNZC72 gene

[0036] (1) Target gene cloning

[0037] ① First-round PCR: Using the OfNAC72-CDS-Plasmid plasmid as a template, amplify the CDS sequence of the OfNAC72 gene containing attB sites at both ends, according to the instructions for the Phanta high-fidelity enzyme. 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 detect the presence of a clear target band. If a clear target band was present, 2 μL of the remaining PCR product was used as a template for the second-round PCR reaction.

[0038] Table 4 First round PCR reaction system

[0039]

[0040]

[0041] Second-round PCR: Using the first-round PCR product as a template, amplify the CDS sequence of the OfNAC72 gene containing Adapter-attB sites at both ends using 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 and recovered. The recovered product was designated attB-OfNAC72-ox.

[0042] Table 5 Second round PCR reaction system

[0043]

[0044] (2) BP reaction: Prepare the BP reaction system (as shown in Table 6), gently pipette to mix, briefly centrifuge, and react overnight at 25°C. The reaction product can be stored at -20°C. Transform the reaction product into E. coli and spread on LB solid medium (containing 50 μg / ml kanamycin). After transformation, culture at 37°C until a single colony grows. Perform PCR identification using OfNAC72-CDS-F and OfNAC72-CDS-R. Select a positive single colony, expand it, and sequence it. After sequencing, extract the plasmid for LR reaction. The plasmid is named pDONR221-OfNAC72-ox.

[0045] Table 6 BP reaction system

[0046]

[0047]

[0048] (3) LR reaction: Prepare the LR reaction system (Table 7), gently pipette to mix, briefly centrifuge, and react overnight at 25°C. The procedure is the same as for the BP reaction. For E. coli transformation, apply LB solid medium (containing 50 μg / ml spectinomycin). After sequencing, extract the plasmid. The constructed plasmid is named PK7WG2D-OfNAC72, and an equal volume of the positive bacterial suspension is stored in 50% glycerol.

[0049] Table 7LR reaction system

[0050]

[0051] Example 3 Transformation of Agrobacterium GV3101

[0052] (1) Remove the competent Agrobacterium GV3101 cells stored in a -80°C freezer and thaw on ice. Add 1 μL of plasmid PK7WG2D-OfNAC72 to every 100 μL of competent cells, pipette to mix thoroughly, and then place on ice for 20 minutes, freeze in liquid nitrogen for 5 minutes, bathe in 37°C water for 5 minutes, and then place on ice for 5 minutes.

[0053] (2) Add 500 μL of LB liquid medium without antibiotics, and cultivate at 28°C for 1 h on a 200 rpm shaker.

[0054] (3) After cultivation, centrifuge the bacterial solution at 6000 rpm for 1 min, discard part of the supernatant, and leave 100 μL to uniformly spread on LB solid medium (containing 50 μg / mL spectinomycin), seal with a sealing film, and invert in a 28°C incubator for 40-48 h.

[0055] (4) Bacterial detection and backup: perform PCR identification with OfNAC72-CDS-F and OfNAC72-CDS-R, and if the target band in the bacterial detection is correct and has consistent brightness, then pick the corresponding colony in the backup plate into LB liquid medium (containing 50 μg / mL spectinomycin) to shake the bacteria, and then preserve the bacteria in the same volume ratio of bacterial solution and 50% glycerol, freeze quickly in liquid nitrogen, and store in a -80°C ultra-low temperature refrigerator.

[0056] Example 4: Infection of Osmanthus fragrans petals and determination of linalool, trans-linalool oxide and various ionones by GC-MS

[0057] (1) Primary shaking of bacteria: inoculate the Agrobacterium positive transformation bacterial solution into LB liquid medium (containing 50 μg / mL spectinomycin) at a volume ratio of 1:50, and cultivate overnight at 28°C on a shaker.

[0058] (2) Secondary shaking of bacteria: take the overnight bacterial solution 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 a final concentration of 20 μmol / L) and 2 mL of 1 mol / L MES (to a final concentration of 20 mmol / L), and cultivate at 28°C on a shaker until the OD 600 = 0.6-0.8 (at this time, the Agrobacterium has the highest activity).

[0059] (3) Preparation of infection solution: sequentially add 1 mL of 10 mmol / L AS (to a final concentration of 20 μmol / L), 5 mL of 1 mol / L magnesium chloride solution (to a final concentration of 10 mmol / L), and 5 mL of 1 mol / L MES (to a final concentration of 10 mmol / L), add distilled water to a total volume of 500 mL, and adjust the pH to 5.6.

[0060] (4) Collection of bacterial cells and resuspension: take the bacterial solution with an OD 600 = 0.6-0.8, centrifuge at 4°C and 4000 r / min for 10 min, and discard the supernatant. Gently suspend the bacterial cells in the infection solution to obtain a uniform turbid solution, adjust the OD 600 = 0.5-0.6, and stand in the dark at room temperature for 2 h.

[0061] (5) Infection: Take fresh osmanthus petals, remove the pedicels, and leave only the petals; place the osmanthus petals in a 50mL centrifuge tube containing positive Agrobacterium infection solution. This process should be rapid, perform vacuum infiltration at 0.06Mpa for about 10 minutes, and slowly release the air; remove the osmanthus petals and wash the remaining bacterial solution on the petals with sterile water.

[0062] (6) Cultivation: Place the washed petals in a 50 mL centrifuge tube containing 5% sucrose solution, cover the top with sterile gauze so that the petals are completely immersed in it, and place it in a dark room at room temperature for 60 hours.

[0063] (7) GC-MS detection of the contents of linalool, trans-linalool oxide and various ionones in transiently converted osmanthus petals: 0.2 g of petals and 2 μL of 10,000-fold methyl nonanoate were sealed in a 20 mL extraction bottle and equilibrated for 10 min. Then, a 2 cm extraction head (50 / 30 μm, DVB / Carboxen / PDMS, Supelco, USA) was inserted and extracted in a 55 °C water bath for 30 min. Finally, the extraction head was inserted into the gas chromatography injection port and desorbed at 230 °C for 5 min, followed by GC-MS.

[0064] Detection conditions: A DB-5MS column (30 m × 0.25 mm × 0.25 μm, Themo Scientific, Bellefonte, PA, USA) was used, and the carrier gas was high-purity helium (99.999%) with a split ratio of 20:1 and a flow rate of 1 mL / min. The ion source and inlet temperatures were 280°C and 230°C, respectively, and the transfer line temperature was 250°C.

[0065] The GC temperature program was as follows: 40 °C for 3 min, then heated to 120 °C at a rate of 3 °C / min and held for 3 min, then heated to 220 °C at a rate of 8 °C / min and held for 2 min.

[0066] The MS conditions were as follows: EI (electron impact) ion source, electron impact energy 70 eV, positive ion scan mode, mass scan range m / z 40-450 amu.

[0067] GC-MS analysis showed that the contents of target products linalool and trans-linalool oxide and various ionones (α-ionone, β-ionone, dihydro-β-ionone) in the osmanthus petals transiently transformed by the super-surface carrier PK7WG2D-OfNAC72 were significantly increased compared with those in the control group CK ( Figure 2 ).

[0068] Example 5 qRT-PCR Verification of Transient Transformation of Osmanthus Fragrance Petals

[0069] The total RNA of the instant transformed Osmanthus fragrans petals was extracted by Trizol super pure RNA extraction kit (Beijing Kangwei Century Biotechnology Co., Ltd.). The extracted RNA was reversely transcribed into cDNA by TransScript One-Step gDNA Removal and cDNASynthesis SuperMix (Quanson, Beijing) reverse transcription kit. The obtained cDNA was diluted 5 times with water, and 1 μL thereof was taken as a template. The Osmanthus fragrans OfActin gene was used as an internal reference. The 2×SYBR Green qPCR Mix kit of Beijing Aidley Biotechnology Co., Ltd. was used to prepare a qRT-PCR reaction system. The reaction system was as shown in Table 8. The PCR reaction procedure was a three-step method: 95 ℃ reaction for 2 min; 95 ℃ reaction for 15 s, 60 ℃ reaction for 15 s, and 72 ℃ reaction for 20 s; and the cycle number was 40. After the completion of the fluorescence quantitative PCR procedure, the results were analyzed by using the Roche LC96 software, and the relative expression amount of the OfNAC72 gene was calculated by taking the internal reference gene OfActin as a reference by using the Excel tool. ΔΔCt The results showed that the expression amount of OfNAC72 in the instant transformed Osmanthus fragrans petals of the overexpression vector PK7WG2D-OfNAC72 was significantly increased compared with the control group CK. Figure 3

[0070] Table 8 qRT-PCR reaction system

[0071] ​

Claims

1. Related to the synthesis of linalool, its oxides and ionones from Osmanthus fragrans OfNAC72 A gene characterized by described OfNAC72 The CDS sequence of the gene is shown in SEQ ID NO.

1.

2. The method according to claim 1 OfNAC72 The protein encoded by the gene is characterized in that The amino acid sequence of the protein is shown in SEQ ID NO.

2.

3. Containing the composition according to claim 1 OfNAC72 Genetic biomaterial, characterized in that The biological materials are expression cassettes, expression vectors and recombinant bacteria.

4. The method according to claim 1 OfNAC72 Use of the gene or the encoded protein according to claim 2 or the biomaterial according to claim 3 in promoting the synthesis of linalool, trans-linalool oxide, α-ionone, β-ionone, and dihydro-β-ionone in osmanthus.

5. The method according to claim 1 OfNAC72 Use of the gene or the coded protein according to claim 2 or the biological material according to 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 in osmanthus fragrans, characterized in that: Overexpression of the method of claim 1 in Osmanthus fragrans OfNAC72 Gene.

7. The method according to claim 6, characterized in that The following steps are involved: (1) Construct the protein containing the protein shown in SEQ ID NO. OfNAC72 Gene overexpression vector; (2) transforming the overexpression vector into Agrobacterium to obtain recombinant Agrobacterium; (3) Transforming Osmanthus fragrans with the recombinant Agrobacterium to overexpress OfNAC72 Gene.

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

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

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