A method for increasing the content of natural fragrance substance linalool in osmanthus petal by applying ofnac2 gene

Abstract

The present invention discloses an application OfNAC2 A method for genetically increasing the content of linalool, a natural aroma substance in osmanthus petals, wherein the OfNAC2 The gene is a NAC transcription factor gene that contains the gene OfNAC2 The overexpression vector was transformed into osmanthus petals for transient expression. The results showed that OfNAC2 The gene expression level increased significantly, and the content of linalool in the petals increased significantly compared with the control group, indicating that OfNAC2 It plays an important role in promoting the synthesis of linalool in osmanthus and can be used in osmanthus genetic engineering to improve the natural aroma quality of osmanthus and the breeding of excellent varieties.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of plant genetic engineering, and particularly relates to a method for increasing the content of natural linalool in Osmanthus fragrans petals by using OfNAC2 gene. BACKGROUND

[0002] Linalool is a chain terpene alcohol compound extracted from plants, which is a colorless and easily flowing liquid at room temperature, has lily fragrance, wood green fragrance and fruit fragrance. It is one of the main components of linalyl leaf oil, linalyl oil, rosewood oil, rosewood oil, coriander seed oil, white orchid leaf oil, lavender oil, damasene leaf oil, bergamot oil, and numerous flower (jasmine, rose, damasene, orange flower, ylang ylang, etc.) essential oils. Linalool is also one of the main components of green tea aroma. Linalool is commonly used for the synthesis of fragrances and spices, and can be used as a medicine for sedation, antibiosis and insect killing. In addition, linalool can be used as a deodorant with strong ability to cover up bad odors.

[0003] Osmanthus fragrans Lour. is an important fragrant flower plant with high economic and health care value. Linalool, a monoterpenoid in Osmanthus fragrans, is widely distributed in various varieties and is a key active substance in Osmanthus fragrans. The NAC transcription factor family is a plant-specific transcription factor family, which has more than 100 members and consists of two domains: a highly conserved N-terminal NAC binding domain that participates in specific binding of cis-acting elements, and a variable C-terminal domain that is responsible for the regulation of transcriptional activation. NAC transcription factors mainly participate in plant growth and development, anti-aging, stress resistance and other biological processes. Therefore, it is of great significance to study and explore the related genes in the genome of Osmanthus fragrans that promote the synthesis of linalool and its oxides and various types of ionones in Osmanthus fragrans. In the present application, the gene that promotes the synthesis of linalool in Osmanthus fragrans is screened by using bioinformatics technology, and the function is verified in the petals of Osmanthus fragrans. SUMMARY

[0004] In view of the above problems existing in the prior art, the present application provides an OfNAC2 gene related to the synthesis of linalool in Osmanthus fragrans. Overexpression of the gene can promote the synthesis of linalool in Osmanthus fragrans, and can be used for improving the natural fragrance quality of Osmanthus fragrans and breeding excellent varieties in the genetic engineering of Osmanthus fragrans.

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

[0006] The OfNAC2 gene related to the synthesis of linalool in Osmanthus fragrans, the CDS sequence of the OfNAC2 gene is shown as SEQ ID NO. 1, and the amino acid sequence of the encoded protein is shown as SEQ ID NO. 2.

[0007] Biological material containing OfNAC2 gene, including expression cassette, expression vector, recombinant bacteria.

[0008] Application of OfNAC2 gene, including:

[0009] (1) Application in osmanthus breeding;

[0010] (2) Application in promoting the content of key aroma substance linalool in osmanthus natural extract.

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

[0012] (1) Constructing an overexpression vector containing 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 with the recombinant agrobacterium to overexpress OfNAC2 gene in osmanthus;

[0015] Preferably, in the step (3), the osmanthus petals are transformed with the recombinant bacteria, and the agrobacterium is agrobacterium GV3101.

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

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

[0018] Figure 1 It is the agarose gel electrophoresis diagram of the OfNAC2 gene amplification product.

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

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

[0021] The technical solutions of the present application are described in detail below in combination with the drawings and specific examples, which are only used to explain the present application and are not intended to limit the protection scope of the present application.

[0022] Example 1 Screening and cloning of target gene

[0023] (1) Obtaining the target gene: according to the Weighted correlation network analysis (WGCNA) of the transcriptome of Osmanthus fragrans at different time periods after methyl jasmonate treatment, 1 gene sequence with the strongest correlation with linalool synthesis in Osmanthus fragrans was screened, and homologous sequence alignment was performed on the NCBI online website. The gene has the closest genetic relationship with OeNAC2 of Olea europaea and has very high homology, so it is named OfNAC2.

[0024] (2) Designing primers: specific primers OfNAC2-CDS-F and OfNAC2-CDS-R for amplifying the full-length CDS of OfNAC2 gene were designed by Primer5.0 software (see Table 1), and the amplification primers were synthesized by Beijing Qikang Biotechnology Co., Ltd.

[0025] Table 1 Primer list

[0026]

[0027] (3) Target gene amplification: cDNA of Osmanthus fragrans was used as a template to amplify the CDS of the target gene according 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 procedure

[0031]

[0032] (4) Gel electrophoresis detection and gene sequencing: 50ml TAE was added with 0.5g agarose powder, and after melting in a microwave oven, 1.5μL 10000x nucleic acid dye was added, poured into a gel plate, and after solidification, sample was spotted. After electrophoresis at 120V, 150mA for 28min, the band was observed in a Gel-Logie200 gel scanning imager, and the target band was 888bp (as shown in Figure 1The target band was recovered and ligated to a T-vector, and then transformed into E. coli for sequencing. The sequence of the OfNAC2 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 liquid, and named OfNAC2-CDS-Plasmid, which was used as a template for constructing a vector.

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

[0034] (1) Cloning of the target gene

[0035] ① First round of PCR reaction: The OfNAC2 gene CDS sequence containing attB sites at both ends was amplified using the plasmid OfNAC2-CDS-Plasmid as a template and referring to the Phanta High-Fidelity Enzyme instruction manual. The PCR reaction system and procedure are shown in Table 4 and Table 3. After the PCR reaction, part of the PCR product was run on a gel to detect whether there was a clear target band. If so, 2 μL of the remaining PCR product was used as a template for the second round of PCR reaction.

[0036] Table 4: First round of PCR reaction system

[0037]

[0038]

[0039] ② Second round of PCR reaction: The OfNAC2 gene CDS sequence containing Adapter-attB sites at both ends was amplified using the first round of PCR product as a template and referring to the Phanta High-Fidelity Enzyme instruction manual. The PCR reaction system and procedure are shown in Table 5 and Table 3. After the PCR reaction, the entire PCR product was run on a gel and gel recovery was performed. The recovered product was named attB-OfNAC2-ox.

[0040] Table 5: Second round of PCR reaction system

[0041]

[0042] (2) BP reaction: The BP reaction system was prepared (as shown in Table 6), mixed and centrifuged briefly, and then incubated at 25°C overnight. The reaction product can be stored at -20°C. The reaction product was transformed into E. coli, and plated on LB solid medium (containing 50 μg / ml kanamycin). After transformation, the single colonies were cultured at 37°C until they grew. PCR identification was performed using OfNAC2-CDS-F and OfNAC2-CDS-R. The positive single colonies were expanded and sequenced. After sequencing, the plasmid was extracted and used for LR reaction. The plasmid was named pDONR221-OfNAC2-ox.

[0043] Table 6 BP reaction system

[0044]

[0045] (3) LR reaction: The LR reaction system (Table 7) was prepared, mixed gently, centrifuged briefly, and then reacted overnight at 25°C. The operation method was consistent with that of the BP reaction. When E. coli was transformed, LB solid medium (containing 50 μg / ml spectinomycin) was coated. After sequencing, the plasmid was extracted, and the constructed plasmid was named PK7WG2D-OfNAC2. The positive bacteria liquid was stored in 50% glycerol at the same volume.

[0046] Table 7 LR reaction system

[0047]

[0048] Example 3 Transformation of Agrobacterium GV3101

[0049] (1) The Agrobacterium GV3101 competent cells stored in a -80°C ultra-low temperature refrigerator were taken out and thawed on ice. 1 μL of plasmid PK7WG2D-OfNAC2 was added to 100 μL of competent cells, mixed gently, and then sequentially subjected to ice bath for 5 min, liquid nitrogen freezing for 5 min, 37°C water bath for 5 min, and ice bath for 5 min.

[0050] (2) 500 μL of LB liquid medium without resistance was added, and the mixture was cultured at 28°C and 200 rpm on a shaker for 1 h.

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

[0052] (4) Bacterial detection and backup: PCR identification was performed using OfNAC2-CDS-F and OfNAC2-CDS-R. If the target band in the bacterial detection was correct and had consistent brightness, the corresponding colonies in the backup plate were picked into LB liquid medium (containing 50 μg / ml spectinomycin) and shaken. The bacterial liquid and 50% glycerol were mixed at the same volume ratio, frozen in liquid nitrogen, and stored in a -80°C ultra-low temperature refrigerator.

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

[0054] (1) Primary shaking culture: The Agrobacterium positive transformation bacterial 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 on a shaker.

[0055] (2) Secondary shaking: Take the overnight bacteria liquid and inoculate 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 (so that its final concentration is 20 μmol / L) and 2 mL of 1 mol / L MES (so that its final concentration is 20 mmol / L), and cultivate at 28°C on a shaking table until OD 600 = 0.6-0.8 (at this time, the activity of Agrobacterium is the highest).

[0056] (3) Preparation of infection liquid: sequentially add 1 mL of 10 mmol / L AS (so that its final concentration is 20 μmol / L), 5 mL of 1 mol / L magnesium chloride solution (so that its final concentration is 10 mmol / L), and 5 mL of 1 mol / L MES (so that its final concentration is 10 mmol / L), add distilled water to 500 ml, and adjust the pH value to 5.6.

[0057] (4) Collection of bacteria, resuspension: the bacteria liquid with OD 600 = 0.6-0.8 is loaded into a 50 mL sterilized centrifuge tube, centrifuged at 4°C and 4000 r / min for 10 min, and the supernatant is discarded. The bacteria are suspended in the infection liquid to form a uniform turbid liquid, and the OD 600 = 0.5-0.6 is adjusted, and the bacteria are placed in the dark at room temperature for 2 h.

[0058] (5) Infection: take fresh Osmanthus fragrans petals, remove the peduncle, and only leave the petal part; place the Osmanthus fragrans petals into a 50 mL centrifuge tube containing the positive Agrobacterium infection liquid, and the process should be rapid. Perform vacuum infiltration at 0.06 MPa for about 10 minutes, and slowly release the gas; take out the Osmanthus fragrans petals, and wash the residual bacteria liquid on the petals with sterile water.

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

[0060] (7) GC-MS detection of the content of linalool in the transiently transformed Osmanthus fragrans petals: seal 0.2 g of petals and 2 μL of 10,000-fold methyl nonoate in a 20 mL extraction bottle for 10 min, then insert a 2 cm extraction head (50 / 30 μm, DVB / Carboxen / PDMS, Supelco, USA) into a 55°C water bath for 30 min, finally insert the gas chromatograph sample inlet into a 230°C desorption for 5 min, and then perform GC-MS.

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

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

[0063] The conditions of MS 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] The content of linalool in the transiently transformed Osmanthus petals by the super-expression vector PK7WG2D-OfNAC2 was significantly increased compared with the control group Control, as detected by GC-MS. Figure 2 ).

[0065] Example 5 RT-qPCR verification of transiently transformed Osmanthus petals

[0066] The total RNA of the transiently transformed Osmanthus petals was extracted by Trizol super-pure RNA extraction kit (Beijing Kangwei Century Biotechnology Co., Ltd.). The extracted RNA was reverse transcribed into cDNA by TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix (Quanxi Gold, Beijing) reverse transcription kit. The obtained 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, and the 2 x SYBR Green qPCR Mix kit of Beijing Aidley Biotechnology Co., Ltd. was used to prepare the RT-qPCR reaction system. The reaction system is shown in Table 8. The PCR reaction program adopts a three-step method: 95°C for 2 min; 95°C for 15 s, 58°C for 15 s, and 72°C for 20 s, with 40 cycles. After the fluorescence quantitative PCR program was completed, the results were analyzed by Roche LC96 software, and the relative expression amount of OfNAC2 gene was calculated with the internal reference gene OfActin as a reference by Excel tool. ΔΔCt The results showed that the expression amount of OfNAC2 in the transiently transformed Osmanthus petals by the super-expression vector PK7WG2D-OfNAC2 was significantly increased compared with the control group CK. Figure 3 ).

[0067] Table 8 RT-qPCR reaction system

[0068]

Claims

1. Related to the synthesis of linalool from Osmanthus fragrans OfNAC2 A gene characterized by described OfNAC2 The CDS sequence of the gene is shown in SEQ ID NO.

1.

2. The method according to claim 1 OfNAC2 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 OfNAC2 Genetic biomaterial, 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 biomaterial 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 linalool from osmanthus fragrans, characterized in that: Overexpression of the method of claim 1 in Osmanthus fragrans OfNAC2 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. 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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