Method for increasing content of natural aroma substance linalool in sweet osmanthus petals by applying OfNAC2 gene

Abstract

The invention discloses a method for increasing the content of linalool serving as a natural aroma substance of sweet-scented osmanthus petals by applying an OfNAC2 gene, the OfNAC2 gene is an NAC transcription factor gene, an over-expression vector containing the gene OfNAC2 is converted into the sweet-scented osmanthus petals for transient expression, and results show that the expression quantity of the OfNAC2 gene in the sweet-scented osmanthus petals subjected to transient expression is remarkably increased, so that the content of linalool serving as a natural aroma substance of the sweet-scented osmanthus petals is increased. The content of linalool in petals is remarkably increased compared with a control group, which indicates that OfNAC2 plays an important role in promoting synthesis of linalool in osmanthus fragrans, and can be used for improving natural aroma quality of osmanthus fragrans and breeding excellent varieties in osmanthus fragrans genetic engineering.

Description

Technical Field

[0001] The invention belongs to the technical field of plant genetic engineering, and in particular relates to a method for increasing the content of linalool, a natural aroma substance in osmanthus petals, by using the OfNAC2 gene. Background Art

[0002] Linalool is a chain terpene alcohol compound extracted from plants. It is a colorless, free-flowing liquid at room temperature with the aroma of lily of the valley, woody green and fruity. It is one of the main components of essential oils such as camphor leaf oil, camphor oil, kiang wood oil, rosewood oil, coriander seed oil, white orchid leaf oil, lavender oil, taro leaf oil, bergamot oil, clary perilla oil and many flowers (jasmine, rose, taro flower, orange flower, ylang ylang flower, etc.), and is also one of the main components of green tea aroma. Linalool is often used in the synthesis of flavors and fragrances. At the same time, linalool is used as medicine for sedation, antibacterial and insecticide. In addition, it can be used as a deodorant and has a strong ability to cover up bad odors.

[0003] Sweet osmanthus (Osmanthus fragrans Lour.) is an important fragrant flower plant with extremely high economic and health value. The monoterpenoid linalool in sweet osmanthus is rich and widely distributed in various varieties. It is the key aroma active substance in sweet osmanthus. The NAC transcription factor family is a type of transcription factor family unique to plants. The family is large, with more than 100 members. It consists of two domains: a highly conserved N-terminal NAC binding domain, which is involved in the specific binding of cis-acting elements; the other is a variable C-terminal domain, which is responsible for the regulation of transcriptional activation. NAC transcription factors are mainly involved in biological processes such as plant growth and development, anti-aging, and stress resistance. Therefore, it is of great significance to study and discover the related genes in the sweet osmanthus genome that promote the synthesis of sweet osmanthus linalool and its oxides and various ionones. The present invention uses bioinformatics technology to screen out genes that promote the synthesis of sweet osmanthus linalool in sweet osmanthus, and performs functional verification in sweet osmanthus petals. Summary of the invention

[0004] In view of the above problems existing in the prior art, the present invention provides an OfNAC2 gene related to the synthesis of linalool from osmanthus. Overexpression of the gene can promote the synthesis of linalool from osmanthus, and can be used in osmanthus genetic engineering to improve the natural aroma quality of osmanthus and the breeding of excellent varieties.

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

[0006] The OfNAC2 gene related to the synthesis of osmanthus linalool, the CDS sequence of the OfNAC2 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 OfNAC2 gene include expression cassette, expression vector and recombinant bacteria.

[0008] Applications of the OfNAC2 gene include:

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

[0010] (2) Its application in increasing the content of linalool, a key aroma substance in natural osmanthus extracts.

[0011] A method for promoting the synthesis of osmanthus linalool comprises the following steps:

[0012] (1) constructing an overexpression vector containing the OfNAC2 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 OfNAC2 gene in Osmanthus fragrans;

[0015] Preferably, in the step (3), the recombinant bacteria are used to transform the sweet osmanthus petals, and the Agrobacterium is Agrobacterium GV3101.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] The OfNAC2 gene related to promoting linalool synthesis in osmanthus provided by the present invention is a NAC transcription factor gene. An overexpression vector containing the gene OfNAC2 is transformed into osmanthus petals for transient expression. The results show that the expression level of the OfNAC2 gene in the transiently transformed osmanthus petals is significantly increased, and the linalool content in the petals is significantly increased compared with the control group, indicating that the OfNAC2 gene plays an important role in promoting the synthesis of linalool in osmanthus, and can be used for breeding work such as improving ornamental traits and genetic quality in osmanthus genetic engineering. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is the agarose gel electrophoresis diagram of the amplified product of the target gene OfNAC2.

[0019] Figure 2 The results of GC-MS detection of linalool content in transiently transformed osmanthus petals. Control represents the control group, and OE represents OfNAC2 gene transiently transformed osmanthus petals.

[0020] Figure 3 is the expression level of OfNAC2 gene in transiently transformed sweet osmanthus petals. Control represents the control group, and OE represents OfNAC2 gene transiently transformed sweet osmanthus petals. DETAILED DESCRIPTION

[0021] The technical solution of the present invention is described in detail below in conjunction with the accompanying drawings and specific embodiments. The examples given are only used to explain the present invention and are not used to limit the protection scope of the present invention.

[0022] Example 1 Screening and cloning of target genes

[0023] (1) Obtaining the target gene: Based on the weighted correlation network analysis (WGCNA) of the transcriptome of Osmanthus fragrans at different stages after methyl jasmonate treatment, a gene sequence with the strongest correlation with linalool synthesis in Osmanthus fragrans was screened out. Homologous sequences were compared on the NCBI online website. The gene was most closely related to OeNAC2 of Olea europaea and had extremely high homology, so it was named OfNAC2.

[0024] (2) Primer design: Specific primers OfNAC2-CDS-F and OfNAC2-CDS-R (see Table 1) for amplifying the full-length CDS of the OfNAC2 gene were designed using Primer 5.0 software, and the amplification primers were synthesized by Beijing Qingke Biotechnology Co., Ltd.

[0025] Table 1 Primer list

[0026]

[0027] (3) Target gene amplification: Using Osmanthus fragrans cDNA as a template, the target gene CDS was amplified with reference to the instructions of Phanta high-fidelity enzyme. The PCR reaction system and procedure are shown in Tables 2 and 3.

[0028] Table 2 PCR reaction system

[0029]

[0030] Table 3 PCR reaction program

[0031]

[0032] (4) Gel electrophoresis detection and gene sequencing: 0.5 g agarose powder was added to 50 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 28 min, the bands were observed on a Gel-Logie200 gel scanning imager. The target band was 888 bp (e.g. Figure 1The target band was recovered and connected to a T-vector, and transformed into E. coli for sequencing. The sequence of the CDS of the OfNAC2 gene is shown in SEQ ID NO.1, and the amino acid sequence of the encoded protein is shown in SEQ ID NO.2. The plasmid was extracted from the bacterial solution with the correct sequencing and named OfNAC2-CDS-Plasmid, which was used as a template for constructing the vector later.

[0033] Example 2 Construction of an overexpression vector of the Osmanthus fragrans OfNAC2 gene

[0034] (1) Target gene cloning

[0035] ① The first round of PCR reaction: Using the plasmid OfNAC2-CDS-Plasmid as a template, refer to the instructions of Phanta high-fidelity enzyme to amplify the CDS sequence of the OfNAC2 gene with attB sites at both ends. The PCR reaction system and procedures are shown in Table 4 and Table 3. After the PCR reaction is completed, take part of the PCR product to run a gel to detect whether there is a clear target band. If there is, take 2μL of the remaining PCR product as a template for the second round of PCR reaction.

[0036] Table 4 First round PCR reaction system

[0037]

[0038]

[0039] ② Second round of PCR reaction: Using the first round of PCR product as a template, refer to the instructions of Phanta high-fidelity enzyme to amplify the CDS sequence of the OfNAC2 gene containing Adapter-attB sites at both ends. The PCR reaction system and procedure are shown in Table 5 and Table 3. After the PCR reaction, all PCR products were run on the gel for detection and recovered by gel cutting. The recovered product was named attB-OfNAC2-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, centrifuge briefly, and react overnight at 25°C. The reaction product can be stored at -20°C. Take the reaction product to transform E. coli and apply LB solid medium (containing 50 μg / ml kanamycin). After transformation, culture at 37°C until a single clone grows. Use OfNAC2-CDS-F and OfNAC2-CDS-R for PCR identification, select the positive single clone, expand it and sequence it. After sequencing is correct, extract the plasmid for LR reaction. The plasmid name is pDONR221-OfNAC2-ox.

[0043] Table 6 BP reaction system

[0044]

[0045] (3) LR reaction: Prepare the LR reaction system (Table 7), gently pipette to mix, centrifuge briefly, and react overnight at 25°C. The operation method is the same as the BP reaction. When transforming E. coli, apply LB solid medium (containing 50 μg / ml spectinomycin). After sequencing is correct, extract the plasmid, and the constructed plasmid is named PK7WG2D-OfNAC2. Store the positive bacterial solution in 50% glycerol in equal volume.

[0046] Table 7LR reaction system

[0047]

[0048] Example 3 Transformation of Agrobacterium GV3101

[0049] (1) Take out the competent cells of Agrobacterium tumefaciens GV3101 stored in a -80℃ ultra-low temperature freezer and thaw them on ice. Add 1μL of plasmid PK7WG2D-OfNAC2 to every 100μL of competent cells, pipette and mix well, then place in an ice bath for 5 min, freeze in liquid nitrogen for 5 min, bath in 37℃ water for 5 min, and then place in an ice bath for 5 min.

[0050] (2) Add 500 μL of LB liquid medium without resistance and culture on a shaker at 28°C and 200 rpm for 1 h.

[0051] (3) After the culture was completed, the bacterial solution was centrifuged at 6000 rpm for 1 min, part of the supernatant was discarded, and 100 μL was evenly spread on LB solid medium (containing 50 μg / ml spectinomycin), sealed with a sealing film, and inverted in a 28°C incubator for 40-48 h.

[0052] (4) Bacterial inspection and backup: PCR identification was performed using OfNAC2-CDS-F and OfNAC2-CDS-R. If the target bands in the bacterial inspection were correct and of uniform brightness, the corresponding colonies in the backup plate were picked up and placed in LB liquid culture medium (containing 50 μg / ml spectinomycin) for shaking. The bacterial solution and 50% glycerol were then preserved in equal volume ratios, quickly frozen in liquid nitrogen, and stored in a -80°C ultra-low temperature freezer.

[0053] Example 4 Infection of Osmanthus fragrans petals and determination of linalool content by GC-MS

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

[0055] (2) Secondary shaking: Take the overnight bacterial solution and inoculate it into 100 mL 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), and culture at 28 °C in a shaking incubator until the OD reaches 0. 600 =0.6-0.8 (Agrobacterium activity is highest at this time).

[0056] (3) Prepare 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 to 5.6.

[0057] (4) Collect the cells and resuspend them: OD 600 = 0.6-0.8 of the bacterial solution into a 50mL sterilized centrifuge tube, centrifuge at 4°C, 4000r / min for 10min, and discard the supernatant. Gently suspend the bacteria with the infection solution until the turbid liquid is uniform, and adjust the OD 600 =0.5-0.6, and let stand in dark place at room temperature for 2h.

[0058] (5) Infection: Take fresh osmanthus petals, remove the pedicels, and leave only the petals; place the osmanthus petals in a 50 mL centrifuge tube containing positive Agrobacterium infection solution. This process should be rapid, perform vacuum penetration at 0.06 MPa for about 10 minutes, and slowly release the air; take out the osmanthus petals and wash the remaining bacterial solution on the petals with sterile water.

[0059] (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 h.

[0060] (7) GC-MS detection of linalool content in instantaneous conversion osmanthus petals: 0.2 g of petals and 2 μL of 10,000-fold nonanoic acid methyl ester 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 into the bottle and extracted in a 55 °C water bath for 30 min. Finally, the bottle was inserted into the gas chromatography injection port and desorbed at 230 °C for 5 min, followed by GC-MS.

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

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

[0063] 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.

[0064] GC-MS analysis showed that the linalool content in the osmanthus petals transiently transformed with the super-surface carrier PK7WG2D-OfNAC2 was significantly higher than that in the control group ( Figure 2 ).

[0065] Example 5 RT-qPCR verification of transient transformation of osmanthus petals

[0066] The total RNA of transiently transformed osmanthus petals was extracted using Trizol ultrapure RNA extraction kit (Beijing Kangwei Century Biotechnology Co., Ltd.). The extracted RNA was reverse transcribed into cDNA using TransScript One-Step gDNARemoval and cDNA SynthesisSuperMix (Quanshijin, Beijing) reverse transcription kit. The cDNA obtained was diluted 5 times with water and 1 μL was taken as a template. The osmanthus OfActin gene was used as an internal reference. The RT-qPCR reaction system was prepared using the 2×SYBR Green qPCR Mix kit of Beijing Aidelai Biotechnology Co., Ltd. The reaction system is shown in Table 8. The PCR reaction program adopts a three-step method: 95℃ reaction for 2min; 95℃ reaction for 15s, 58℃ reaction for 15s, 72℃ reaction for 20s, and the number of cycles is 40. After the fluorescence quantitative PCR program is completed, the results are analyzed using Roche LC96 software, and 2- ΔΔCt The relative expression of OfNAC2 gene was calculated by using the internal reference gene OfActin as a reference. The results showed that the expression of OfNAC2 in the petals of Osmanthus fragrans transiently transformed with the overexpression vector PK7WG2D-OfNAC2 was significantly increased compared with that in the control group CK ( Figure 3 ).

[0067] Table 8 RT-qPCR reaction system

[0068]

Claims

1. Related to the synthesis of osmanthus linalool OfNAC2 A gene characterized by Said OfNAC2 The CDS sequence of the gene is shown in SEQ ID NO.

1.

2. The method according to claim 1 OfNAC2 The encoded protein of 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 OfNAC2 Genetic biological material, characterized in that The biological materials include expression cassettes, expression vectors and recombinant bacteria.

4. The method according to claim 1 OfNAC2 Use of the gene or the encoded protein according to claim 2 or the biological material according to claim 3 in promoting the synthesis of linalool from osmanthus fragrans.

5. The method according to claim 1 OfNAC2 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 osmanthus linalool, characterized in that: Super-expression of the method of claim 1 in sweet osmanthus OfNAC2 Gene.

7. The method according to claim 6, characterized in that The following steps are involved: (1) Constructing a protein containing the protein shown in SEQ ID NO. OfNAC2 Gene overexpression vector; (2) transforming the expression vector into Agrobacterium to obtain recombinant expression Agrobacterium; (3) Transforming Osmanthus fragrans with the recombinant Agrobacterium to overexpress OfNAC2 Gene.

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

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

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