Method for increasing natural fragrance substance linalool of osmanthus petal by applying ofwrky33 gene

Abstract

This invention discloses a method for promoting the synthesis of linalool, a natural aroma compound in osmanthus. OfWRKY33 Genes and their applications, as described OfWRKY33 The gene is a WRKY transcription factor gene, which will contain the gene OfWRKY33 The overexpression vector was transformed into osmanthus petals for transient expression. The results showed that the transiently transformed osmanthus petals contained... OfWRKY33 Gene expression levels were significantly increased, and the content of linalool in the petals was significantly higher than that in the control group. The content of linalool oxide was also increased, indicating that... OfWRKY33 The gene plays an important role in promoting the synthesis of linalool and its oxides in osmanthus, and can be used in osmanthus genetic engineering to improve the aroma quality of natural osmanthus extracts and to select superior varieties.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, specifically relating to the OfWRKY33 gene related to the synthesis of linalool in osmanthus and its application. Background Technology

[0002] Linalool is a chain-like terpene alcohol compound 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 numerous flowers (jasmine, rose, bitter orange, orange blossom, ylang-ylang, etc.), and is also a key component of the aroma of green tea. Linalool is commonly used in the synthesis of fragrances and perfumes. It also has medicinal uses for its sedative, antibacterial, and insecticidal properties. Furthermore, it serves as a deodorant, possessing a strong ability to mask unpleasant odors. Linalool has various oxides. Linalool oxides are naturally found in essential oils of coriander, camphor, lavender, mixed lavender, lily, and cinnamon. They can be used in daily fragrances and in the formulation of essential oils such as mixed lavender. Very small amounts are used in edible flavorings such as peach, mango, and passion fruit.

[0003] Osmanthus fragrans Lour. is an important fragrant flowering plant with high economic and health value. Osmanthus is rich in monoterpenoid linalool and its derivatives, which are widely distributed across various varieties and are key aromatic active substances in osmanthus. The WRKY transcription factor family is a plant-specific family of transcription factors that participate in multiple plant signal transduction pathways, such as plant growth and development, and stress responses to biotic and abiotic stresses. Related studies have shown that WRKY transcription factors can enhance plant resistance to stress and disease. Therefore, researching and identifying genes in the osmanthus genome that promote the synthesis of linalool and its oxides is of great significance. This invention utilizes bioinformatics techniques to screen for genes in osmanthus that promote the synthesis of linalool and its oxides, 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 OfWRKY33 gene related to the synthesis of linalool and its oxides in osmanthus. Overexpression of this gene can promote the synthesis of linalool and its oxides in osmanthus, and can be used in osmanthus genetic engineering to improve the natural fragrance quality of osmanthus 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 OfWRKY33 gene, which is associated with the synthesis of linalool and its oxides, has a CDS sequence as shown in SEQ ID NO.1 and an amino acid sequence of the protein it encodes as shown in SEQ ID NO.2.

[0007] Biological materials containing the OfWRKY33 gene include expression cassettes, expression vectors, and recombinant bacteria.

[0008] Applications of the OfWRKY33 gene include:

[0009] (1) Application in Osmanthus breeding;

[0010] (2) Application in promoting the content of linalool and linalool oxide, key aroma substances in osmanthus.

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

[0012] (1) Construct an overexpression vector containing the OfWRKY33 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 OfWRKY33 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 OfWRKY33 gene, which promotes the synthesis of linalool and its oxides in osmanthus. OfWRKY33 is a WRKY transcription factor gene. Transient expression of an overexpression vector containing the OfWRKY33 gene was performed on osmanthus petals. Results showed a significant increase in OfWRKY33 gene expression in the transiently transformed osmanthus petals, and a significant increase in the content of linalool and linalool oxides compared to the control group. This indicates that the OfWRKY33 gene plays an important role in promoting the synthesis of linalool and its oxides in osmanthus and can be used in osmanthus genetic engineering to improve ornamental traits and genetic quality. Attached Figure Description

[0019] Figure 1 Agarose gel electrophoresis image of the amplified product of the target gene OfWRKY33.

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

[0021] Figure 3 The expression level of the OfWRKY33 gene in transiently transformed osmanthus petals. 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 transcriptome data analysis of five Osmanthus varieties from the research group of Zheng Riru at Huazhong Agricultural University, one gene sequence was screened and compared with the sequence of the model plant Arabidopsis thaliana. It was determined that the gene belongs to the WRKY gene family and was named OfWRKY33 based on the position of the gene family member on the chromosome.

[0025] (2) Primer design: Specific primers of OfWRKY33-CDS-F and OfWRKY33-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: Add 0.3g agarose powder to 30ml TAE, melt it in a microwave oven, add 1.5μL of 10000× nucleic acid dye, pour into a gel casting plate, and load the sample after solidification. After electrophoresis at 120V and 150mA for 25min, observe the bands using a Gel-Logie200 gel scanning imager. The target band is 1467bp (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 OfWRKY33 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 OfWRKY33-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 OfWRKY33 gene

[0035] (1) Cloning of the target gene

[0036] ① First round of PCR reaction: Using plasmid OfWRKY33-CDS-Plasmid as a template, the CDS sequence of the OfWRKY33 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 PCR reaction: Using the first round PCR product as a template, the CDS sequence of the OfWRKY33 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-OfWRKY33-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 OfWRKY33-CDS-F and OfWRKY33-CDS-R. Select positive single colonies, amplify and sequence them. If the sequencing is correct, extract the plasmid for LR reaction. The plasmid name is pDONR221-OfWRKY33-ox.

[0043] Table 6 BP Reaction System

[0044]

[0045] (3) LR reaction: Prepare the LR reaction system (Table 7), gently pipette 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-OfWRKY33. 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-OfWRKY33 to every 100 μL of competent cells, mix well by pipetting, and then successively incubate on ice for 20 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 OfWRKY33-CDS-F and OfWRKY33-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 and linalool oxide content 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 LMES (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 and linalool oxide content in osmanthus petals in transient transformation: 0.2 g of petals and 2 μL of methyl nonanoate (10,000 times dilution) 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 head was inserted into the gas chromatograph injection port and desorbed at 230 °C for 5 min. 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 content of linalool, the target product, in osmanthus petals transiently transformed by the hyperexpression PK7WG2D-OfWRKY33 was significantly increased compared to the control group (CK), and the content of linalool oxide also increased. Figure 2 ).

[0065] Example 5: qRT-PCR verification 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 qRT-PCR 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 analyzed using Excel. -△△Ct Analysis was conducted, and the relative expression level of the OfWRKY33 gene was calculated using the internal reference gene OfActin as a reference. The results showed that the expression level of OfWRKY33 in Osmanthus petals transiently transformed with the overexpression vector PK7WG2D-OfWRKY33 was significantly increased compared to the control group (CK). Figure 3 ).

[0067] Table 8 qRT-PCR reaction system

[0068]

Claims

1. Related to the synthesis of linalool and its oxides from osmanthus OfWRKY33 Genes, characterized by, The OfWRKY33 The CDS sequence of the gene is shown in SEQ ID NO.

1.

2. The claim 1 OfWRKY33 The gene encodes a protein characterized by, The amino acid sequence of the protein is shown in SEQ ID NO.

2.

3. Containing the contents of claim 1 OfWRKY33 Gene-based biomaterials, characterized in that, The biological materials are expression cassettes, expression vectors, and recombinant bacteria.

4. The claim 1 OfWRKY33 The application of the gene or the protein encoded by claim 2 or the biomaterial described in claim 3 in promoting the synthesis of linalool and linalool oxide in osmanthus petals.

5. The claim 1 OfWRKY33 The application of the gene or the protein encoded by claim 2 or the biological material described in claim 3 in Osmanthus breeding, characterized in that, The petals of the osmanthus flower have a high content of linalool and linalool oxide.

6. A method for promoting the synthesis of linalool and linalool oxide in osmanthus petals, characterized in that, Overexpression of the substance described in claim 1 in Osmanthus fragrans OfWRKY33 Gene.

7. The method according to claim 6, characterized in that, Includes the following steps: (1) Construct a structure containing the following as shown in SEQ ID NO. 1 OfWRKY33 Gene overexpression vectors; (2) Transform the overexpression vector into Agrobacterium to obtain recombinant Agrobacterium; (3) Osmanthus fragrans was transformed with the recombinant Agrobacterium described above, and its expression was overexpressed in Osmanthus fragrans. OfWRKY33 Gene.

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