Method for increasing natural aroma substance linalool of sweet osmanthus petals by applying OfWRKY33 gene

Abstract

The invention discloses a related OfWRKY33 gene for promoting synthesis of linalool serving as a natural aroma substance of osmanthus fragrans and application of the OfWRKY33 gene, the OfWRKY33 gene is a WRKY transcription factor gene, an overexpression vector containing the gene OfWRKY33 is converted into osmanthus fragrans petals for transient expression, and results show that the expression quantity of the OfWRKY33 gene in the osmanthus fragrans petals subjected to transient expression is remarkably increased, so that the OfWRKY33 gene can be used for promoting synthesis of linalool serving as a natural aroma substance of osmanthus fragrans. The content of linalool in petals is remarkably increased compared with that of a control group, the content of linalool oxide is increased, it is indicated that the OfWRKY33 gene plays an important role in promoting synthesis of linalool and oxide of osmanthus fragrans, and the OfWRKY33 gene can be used for improving the aroma quality of osmanthus fragrans natural extracts, breeding good varieties and the like in osmanthus fragrans genetic engineering.

Description

Technical Field

[0001] The invention belongs to the technical field of plant genetic engineering, and particularly relates to an OfWRKY33 gene related to osmanthus linalool synthesis and an application thereof. 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. Linalool has a variety of oxides. Linalool oxide is naturally present in essential oils such as coriander, camphor, lavender, lavender, lily of the valley, and cinnamon. It can be used in daily flavors and in the preparation of essential oils such as lavender. A very small amount is used in edible flavors such as peach, mango, and passion fruit.

[0003] Sweet osmanthus (Osmanthus fragrans Lour.) is an important fragrant flower plant with extremely high economic and health value. The monoterpenoid linalool and its derivatives are rich in sweet osmanthus and widely distributed in various varieties. They are the key aroma active substances in sweet osmanthus. The WRKY transcription factor family is a type of transcription factor family unique to plants. WRKY transcription factors are involved in multiple signal transduction pathways in plants, such as: plant growth and development, stress response to biotic stress and abiotic stress, etc. Related studies have shown that WRKY transcription factors can enhance plant stress resistance and disease 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. The present invention uses bioinformatics technology to screen out genes that promote the synthesis of sweet osmanthus linalool and its oxides 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 OfWRKY33 gene related to the synthesis of linalool and its oxides from osmanthus. Overexpression of the gene can promote the synthesis of linalool and its oxides 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 OfWRKY33 gene related to the synthesis of osmanthus linalool and its oxides, the CDS sequence of the OfWRKY33 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 OfWRKY33 gene include expression cassette, expression vector and recombinant bacteria.

[0008] Applications of OfWRKY33 gene include:

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

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

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

[0012] (1) constructing an overexpression vector containing the OfWRKY33 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 OfWRKY33 gene in Osmanthus fragrans;

[0015] Preferably, in step (3), recombinant bacteria are used to transform sweet osmanthus 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 an OfWRKY33 gene related to promoting the synthesis of linalool and its oxides in osmanthus, which is a WRKY transcription factor gene. An overexpression vector containing the gene OfWRKY33 is transformed into osmanthus petals for transient expression. The results show that the expression level of the OfWRKY33 gene in the transiently transformed osmanthus petals is significantly increased, and the contents of linalool and linalool oxides in the petals are significantly increased compared with the control group, indicating that the OfWRKY33 gene plays an important role in promoting the synthesis of linalool and its oxides 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

[0019] Figure 1 This is the agarose gel electrophoresis diagram of the amplified product of the target gene OfWRKY33.

[0020] Figure 2 This is the result of GC-MS detection of the content of linalool and linalool oxide in instantaneous conversion osmanthus petals.

[0021] Figure 3 is the expression level of OfWRKY33 gene in transiently transformed Osmanthus fragrans petals. DETAILED DESCRIPTION

[0022] 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 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 varieties conducted by Zheng Riru's research group at 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 belonged to the WRKY gene family and was named OfWRKY33 according to the location of the gene family member on the chromosome.

[0025] (2) Primer design: Specific primers OfWRKY33-CDS-F and OfWRKY33-CDS-R (see Table 1) for amplifying the full-length CDS of the OfWRKY33 gene were designed using Primer5.0 software, and 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 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.

[0029] Table 2 PCR reaction system

[0030]

[0031] Table 3 PCR reaction program

[0032]

[0033] (4) Gel electrophoresis detection and gene sequencing: 0.3 g agarose powder was added to 30 ml TAE, and 1.5 μL 10000× nucleic acid dye was added after melting in a microwave oven. The sample was then spotted after solidification. 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 1467 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 OfWRKY33 gene CDS 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 correct sequencing and named OfWRKY33-CDS-Plasmid, which was used as a template for constructing the vector later.

[0034] Example 2 Construction of an overexpression vector of the Osmanthus fragrans OfWRKY33 gene

[0035] (1) Target gene cloning

[0036] ① The first round of PCR reaction: Using the plasmid OfWRKY33-CDS-Plasmid as a template, refer to the instructions of Phanta high-fidelity enzyme to amplify the CDS sequence of the OfWRKY33 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.

[0037] Table 4 First round PCR reaction system

[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 OfWRKY33 gene containing Adapter-attB sites at both ends. The PCR reaction system and procedures are shown in Table 5 and Table 3. After the PCR reaction, all PCR products were run on the gel for detection and gel cutting and recovery. 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, 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 OfWRKY33-CDS-F and OfWRKY33-CDS-R for PCR identification, select positive single clones for expansion and sequencing, and extract plasmids after sequencing is correct 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 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-OfWRKY33. 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-OfWRKY33 to every 100μL of competent cells, pipette and mix well, then place in an ice bath for 20 minutes, quick freeze in liquid nitrogen for 5 minutes, place in a 37℃ water bath for 5 minutes, and place in an ice bath for 5 minutes.

[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: OfWRKY33-CDS-F and OfWRKY33-CDS-R were used for PCR identification. If the target bands in the bacterial inspection were correct and of the same brightness, the corresponding colonies in the backup plate were picked into LB liquid culture medium (containing 50 μg / ml spectinomycin) and shaken. 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 refrigerator.

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

[0054] (1) Primary shaking: The Agrobacterium-positive transformation culture liquid 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 and linalool oxide 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 and extracted in a 55 °C water bath for 30 min. Finally, the head was inserted into the gas chromatography injection port for desorption 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 content of the target product linalool in the osmanthus petals transiently transformed by the super-surface carrier PK7WG2D-OfWRKY33 was significantly higher than that in the control group CK, and the content of linalool oxides increased ( Figure 2 ).

[0065] Example 5 qRT-PCR 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 gDNA Removal and cDNA Synthesis SuperMix (Quanshijin, Beijing) reverse transcription kit. The cDNA 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 qRT-PCR 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, 60℃ 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 Excel tools are used for 2 -△△Ct The relative expression of OfWRKY33 gene was calculated by using the internal reference gene OfActin as a reference. The results showed that the expression of OfWRKY33 in the petals of Osmanthus fragrans transiently transformed with the overexpression vector PK7WG2D-OfWRKY33 was significantly increased compared with that in the control group CK ( Figure 3 ).

[0067] Table 8 qRT-PCR reaction system

[0068]

Claims

1. Synthesis of linalool and its oxides OfWRKY33 A gene characterized by Said OfWRKY33 The CDS sequence of the gene is shown in SEQ ID NO.

1.

2. The method according to claim 1 OfWRKY33 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 OfWRKY33 Genetic biological material, characterized in that The biological materials include expression cassettes, expression vectors and recombinant bacteria.

4. The method according to claim 1 OfWRKY33 Use of the gene or the encoded protein according to claim 2 or the biomaterial according to claim 3 in promoting the synthesis of osmanthus linalool and linalool oxide.

5. The method according to claim 1 OfWRKY33 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 and linalool oxide, characterized in that: Super-expression of the method of claim 1 in sweet osmanthus OfWRKY33 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. OfWRKY33 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 OfWRKY33 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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