Method for increasing content of linalool serving as natural aroma substance of lilium sibiricum petals by applying OfNAC2 gene

Abstract

The invention discloses a method for increasing the content of linalool serving as a natural aroma substance of 'Siberia' lily petals by applying an OfNAC2 gene, the OfNAC2 gene belongs to an NAC transcription factor family, an over-expression vector containing the OfNAC2 gene is transformed to the 'Siberia' lily petals for transient expression, and the result shows that the linalool content of the natural aroma substance of the 'Siberia' lily petals is increased. The content of linalool in instantly converted Lilium sibiricum petals is obviously increased compared with that of a control group, which indicates that the OfNAC2 gene plays an important role in promoting synthesis of linalool of Lilium sibiricum and can be used for improving the aroma quality of Lilium sibiricum and breeding good varieties in Lilium gene engineering.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, specifically relating to a method for increasing the content of linalool, a natural aroma substance in the petals of the 'Siberian' lily, by using the OfNAC2 gene. 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 sedation, antibacterial properties, and insecticidal effects. Furthermore, it can be used as a deodorant, possessing a strong ability to mask unpleasant odors.

[0003] Lilies (Lilium spp.) are perennial monocotyledonous herbaceous plants. They hold a significant position in the global flower trade, especially in bulb production and cut flower trade. Currently, approximately 10,000 varieties with superior traits have been developed, among which the Oriental lily, Musk lily, and Asiatic lily lines dominate the large-scale commercial market. Oriental lilies are primarily composed of terpenoid volatiles, mainly monoterpenes such as linalool, ocimene, and β-myrcene. The 'Siberian' lily is a type of Oriental lily. The NAC transcription factor family is a plant-specific family of transcription factors. This large family contains over 100 members and consists of two domains: a highly conserved N-terminal NAC-binding domain, which participates in the specific binding of cis-acting elements; and a variable C-terminal domain responsible for regulating transcriptional activation. NAC transcription factors are mainly involved in plant growth, development, anti-aging, and stress resistance processes. Therefore, it is of great significance to study and discover the genes that promote the synthesis of linalool in 'Siberian' lily. This invention uses bioinformatics techniques to screen for genes that promote the synthesis of linalool in 'Siberian' lily and performs functional verification in 'Siberian' lily petals. Summary of the Invention

[0004] To address the aforementioned problems in the existing technology, this invention provides an OfNAC2 gene related to the synthesis of linalool in 'Siberian' lily. Overexpression of this gene can promote the synthesis of linalool in 'Siberian' lily and can be used in 'Siberian' lily genetic engineering to improve the natural aroma quality of 'Siberian' lily 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 application of the OfNAC2 gene, or an expression cassette, expression vector, or recombinant bacteria containing the OfNAC2 gene, in promoting the synthesis of linalool in 'Siberian' lily, wherein the CDS sequence of the OfNAC2 gene is shown in SEQ ID NO.1.

[0007] The application of the protein encoded by the OfNAC2 gene in promoting the synthesis of linalool in 'Siberian' lily, the amino acid sequence of which is shown in SEQ ID NO.2.

[0008] A method for promoting the synthesis of linalool from 'Siberian' lilies includes the following steps:

[0009] (1) Construct an overexpression vector containing the OfNAC2 gene as shown in SEQ ID NO. 1;

[0010] (2) The expression vector was transformed into Agrobacterium to obtain recombinant Agrobacterium expression;

[0011] (3) Transform 'Siberian' lily with the recombinant Agrobacterium described above to overexpress the OfNAC2 gene in 'Siberian' lily;

[0012] Preferably, in step (3), recombinant bacteria are used to transform 'Siberian' lily petals;

[0013] Preferably, the Agrobacterium is Agrobacterium GV3101.

[0014] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0015] This invention provides an OfNAC2 gene, an NAC transcription factor gene, that promotes linalool synthesis in 'Siberian' lily. An overexpression vector containing the OfNAC2 gene was transformed into 'Siberian' lily petals for transient expression. Results showed that the expression level of the OfNAC2 gene was significantly increased in the transiently transformed 'Siberian' lily petals, and the linalool content in the petals was significantly higher than that of the control group. This indicates that the OfNAC2 gene plays an important role in promoting linalool synthesis in 'Siberian' lily and can be used in 'Siberian' lily genetic engineering to improve ornamental traits and genetic quality. Attached Figure Description

[0016] Figure 1 Agarose gel electrophoresis image of the amplified product of the target gene OfNAC2.

[0017] Figure 2 The graph shows the results of GC-MS detection of linalool content in petals of transiently transformed 'Siberian' lily. Control represents the control group, and OE represents lily petals transiently transformed with the OfNAC2 gene.

[0018] Figure 3 The expression level of the OfNAC2 gene in transiently transformed 'Siberian' lily petals is shown. Control represents the control group, and OE represents the transient transformation of lily petals with the OfNAC2 gene. Detailed Implementation

[0019] 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 used to explain the present invention and are not intended to limit the scope of protection of the present invention.

[0020] Example 1: Screening and Cloning of the Target Gene

[0021] (1) Obtaining the target gene: Based on the weighted correlation network analysis (WGCNA) of the transcriptome of Osmanthus fragrans at different stages after treatment with methyl jasmonate, the gene sequence with the strongest correlation with linalool synthesis was screened. Homologous sequence comparison was performed on the NCBI online website. This gene is most closely related to olive OeNAC2 and has extremely high homology, so it was named OfNAC2.

[0022] (2) Primer design: Specific primers of OfNAC2-CDS-F and OfNAC2-CDS-R (see Table 1) were designed using Primer 5.0 software and amplified by Beijing Qingke Biotechnology Co., Ltd.

[0023] Table 1 Primer List

[0024]

[0025] (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.

[0026] Table 2 PCR reaction system

[0027]

[0028] Table 3 PCR reaction procedure

[0029]

[0030] (4) Gel electrophoresis detection and gene sequencing: Add 0.5g agarose powder to 50ml 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 28min, observe the bands using a Gel-Logie200 gel scanning imager. The target band is 888bp (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 sequence of the CDS of the OfNAC2 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 OfNAC2-CDS-Plasmid, which was used as a template for the subsequent construction of the vector.

[0031] Example 2: Construction of an overexpression vector for the Osmanthus fragrans OfNAC2 gene

[0032] (1) Cloning of the target gene

[0033] ① First round of PCR reaction: Using plasmid OfNAC2-CDS-Plasmid as a template, the CDS sequence of the OfNAC2 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.

[0034] Table 4 First-round PCR reaction system

[0035]

[0036] ② Second round of PCR reaction: Using the first round of PCR products as templates, the CDS sequence of the OfNAC2 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-OfNAC2-ox.

[0037] Table 5 Second round PCR reaction system

[0038]

[0039] (2) BP reaction: Prepare the BP reaction system (as shown in Table 6), gently pipette and 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. PCR identification is performed using OfNAC2-CDS-F and OfNAC2-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-OfNAC2-ox.

[0040] Table 6 BP Reaction System

[0041]

[0042] (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-OfNAC2. Store the positive bacterial solution in 50% glycerol in an equal volume.

[0043] Table 7 LR Reaction System

[0044]

[0045] Example 3 Transformation of Agrobacterium GV3101

[0046] (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-OfNAC2 to every 100 μL of competent cells, mix well by pipetting, and then successively incubate on ice for 5 min, quick freeze in liquid nitrogen for 5 min, incubate in water at 37℃ for 5 min, and incubate on ice for 5 min.

[0047] (2) Add 500 μL of non-resistant LB liquid medium and incubate at 28°C and 200 rpm for 1 h on a shaker.

[0048] (3) After the culture is completed, centrifuge the bacterial solution 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.

[0049] (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 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℃.

[0050] Example 4: Infection of 'Siberian' lily petals and determination of linalool content by GC-MS

[0051] (1) One-time shaking: 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 at 28°C on a shaker.

[0052] (2) Secondary shaking: 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).

[0053] (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 MES (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.

[0054] (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 =0.3-0.5, let stand at room temperature in the dark for 3 hours.

[0055] (5) Infection: Take a newly opened 'Siberian' lily and use a 1mL disposable syringe to inject the bacterial solution into the back of the 'Siberian' lily petals.

[0056] (6) Cultivation: Insert the injected 'Siberian' lily into a 250ml conical flask containing water and place it in a dark room at room temperature for 60h.

[0057] (7) GC-MS detection of linalool content in petals of transiently transformed 'Siberian' lily: 0.2 g of petals and 2 μL of 100 times 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 sample was inserted into the gas chromatograph injection port and desorbed at 230 °C for 5 min. GC-MS was then performed.

[0058] 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℃.

[0059] 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.

[0060] 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.

[0061] GC-MS analysis showed that the target product linalool was significantly increased in 'Siberian' lily petals transiently transformed by the hyperexpression PK7WG2D-OfNAC2 compared to the control group. Figure 2 ).

[0062] Example 5: RT-qPCR validation of transient transformation of 'Siberian' lily petals

[0063] Total RNA was extracted from the petals of transiently transformed 'Siberian' lily 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 lily LoActin gene was used as an internal control. The RT-qPCR reaction system was prepared using the 2×SYBR Green qPCR Mix kit from Beijing Adley Biotechnology Co., Ltd. The reaction system is shown in Table 8. The PCR reaction program used 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 quantitative PCR program was completed, the results were analyzed using Roche LC96 software and Excel was used for 2− ΔΔCt Analysis was performed, and the relative expression level of the OfNAC2 gene was calculated using the internal reference gene LoActin as a reference. The results showed that the expression level of OfNAC2 in petals of 'Siberian' lily transiently transformed with the overexpression vector PK7WG2D-OfNAC2 was significantly increased compared to the control group (CK). Figure 3 ).

[0064] Table 8 RT-qPCR reaction system

[0065]

Claims

1. OfNAC2 The application of genes in promoting the synthesis of linalool in 'Siberian' lilies, the aforementioned OfNAC2 The CDS sequence of the gene is shown in SEQ ID NO.

1.

2. OfNAC2 The application of a gene-encoded protein in promoting the synthesis of linalool in 'Siberian' lily, the amino acid sequence of which is shown in SEQ ID NO.

2.

3. Contains OfNAC2 The application of gene expression cassettes, expression vectors, or recombinant bacteria in promoting linalool synthesis in 'Siberian' lilies. OfNAC2 The CDS sequence of the gene is shown in SEQ ID NO.

1.

4. A method for promoting the synthesis of linalool from 'Siberian' lilies, characterized in that, Overexpression of SEQ ID NO.1 in 'Siberian' lily OfNAC2 CDS sequence of the gene.

5. The method according to claim 4, characterized in that, Includes the following steps: (1) Construct a structure containing the one shown in SEQ ID NO.1 OfNAC2 Gene overexpression vectors; (2) The expression vector is transformed into Agrobacterium to obtain recombinant Agrobacterium expression; (3) Transform 'Siberian' lily with the recombinant Agrobacterium described above, and overexpress in 'Siberian' lily. OfNAC2 Gene.

6. The method according to claim 5, characterized in that, In step (3), recombinant bacteria are used to transform 'Siberian' lily petals.

7. The method according to claim 6, characterized in that, The Agrobacterium mentioned is Agrobacterium GV3101.

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