Application of Osmanthus fragrans OfTPS6 gene in synthesis of important terpenoid aroma substance linalool and oxide thereof

Abstract

The invention discloses application of an OfTPS6 gene in synthesis of an important terpenoid aroma substance linalool and an oxide thereof, a plant overexpression vector containing the OfTPS6 gene is used for instantaneously converting OfTPS6 gene into OfTPS6 petals and detecting volatile matters of infected petals, compared with a control group, the expression quantity of OfTPS6 is increased by 3.42 times, the content of linalool is increased to 460.12 mu g / gFW from 182.25 mu g / gFW, and the content of linalool is increased to 460.12 mu g / gFW. And the content of linalool oxide is increased from 452.09 [mu] g / g FW to 2919.65 [mu] g / g FW. The OfTPS6 gene overexpressed in the petals can significantly improve the content of linalool and oxides thereof in the petals, which proves that the OfTPS6 is the linalool synthetase gene and can be used for improving the natural aroma quality of osmanthus fragrans, breeding excellent 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 specifically relates to the application of osmanthus fragrans OfTPS6 gene in synthesizing important terpene aroma substance linalool and its oxides. Background Art

[0002] Osmanthus fragrans Lour. is an evergreen woody flowering plant of the genus Oleaceae and is one of the ten famous traditional flowers in my country. It is widely planted for urban greening and ornamental purposes due to its rich fragrance. It is also widely used in the food, cosmetics and essential oil industries. Previous studies have shown that the petals of osmanthus contain 94 volatile aromatic substances and 146 free aromatic substances, mainly including linalool and its oxides, β-ionone, α-ionone, γ-decalactone, etc. Among them, linalool and its four oxides are aroma-active substances that are widely present in all varieties and have rich content and high aroma vitality values. They give the odor attributes of osmanthus flowers. Their content and components play an important role in the fragrance intensity and fragrance type of osmanthus.

[0003] At present, the release law and metabolic pathway of linalool have been clarified. Linalool synthase is the key enzyme in the last step of converting geranyl diphosphate (GPP) into linalool. At present, there are few reports on linalool synthase genes. The present invention aims to explore the key linalool synthase genes of osmanthus fragrans, and provide a method for improving the natural aroma quality and the variety improvement of osmanthus fragrans in genetic engineering. Summary of the invention

[0004] In view of the above problems existing in the prior art, the present invention provides a key linalool synthase gene OfTPS6 of osmanthus fragrans, which can be used in osmanthus fragrans genetic engineering to improve the natural aroma quality of osmanthus fragrans and breed excellent varieties.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions:

[0006] The osmanthus fragrans linalool synthase gene OfTPS6, the CDS sequence of the OfTPS6 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 OfTPS6 gene, including expression cassette, expression vector and recombinant bacteria.

[0008] Applications of the OfTPS6 gene include:

[0009] (1) Application in the synthesis of linalool and linalool oxide.

[0010] (2) Application in Osmanthus fragrans breeding.

[0011] A method for increasing the content of linalool and linalool oxide in osmanthus, comprising overexpressing the OfTPS6 gene in osmanthus, specifically:

[0012] (1) constructing an overexpression vector containing the OfTPS6 gene (SEQ ID NO.1);

[0013] (2) transforming the expression vector into Agrobacterium to obtain recombinant expression Agrobacterium;

[0014] (3) Using the recombinant Agrobacterium to transform Osmanthus fragrans, and overexpressing the OfTPS6 gene in Osmanthus fragrans. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The gene expression and volatile substance content detection of OfTPS6 transient transformation in sweet osmanthus petals. In the figure, A is the gene expression detection of OfTPS6 overexpression system, and B is the linalool and linalool oxide content detection of OfTPS6 overexpression system.

[0016] Figure 2 The expression level and volatile substance content of the transiently silenced OfTPS6 gene in sweet osmanthus petals are detected. A is the gene expression level detection of OfTPS6 silencing system, and B is the content detection of linalool and linalool oxide in OfTPS6 silencing system. DETAILED DESCRIPTION

[0017] The technical solution of the present invention is described 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.

[0018] Example 1 Screening and cloning of target genes

[0019] The inventors identified a highly expressed linalool synthase gene from the genome and transcriptomes of 'Boye Yingui' at different flowering stages, and the gene was named OfTPS6.

[0020] 1. RNA extraction from petals of 'Boye Yingui'

[0021] (1) Weigh 0.2 g of sample, grind it thoroughly in liquid nitrogen and place it in a 2 mL enzyme-free centrifuge tube. Quickly add 1 mL of TRlzon Reagen and mix well to fully lyse the sample.

[0022] (2) Leave at room temperature for 5 min to allow the protein-nucleic acid complex to completely separate;

[0023] (3) Add chloroform at a ratio of 200 μL chloroform per 1 ml TRizon Reagent, cap the tube, shake vigorously for 15 seconds, and leave at room temperature for 2 minutes;

[0024] (4) Centrifuge at 4°C and 12,000 rpm for 10 min. The sample is then divided into three layers: a red organic phase, an intermediate layer, and an upper colorless aqueous phase. The RNA is mainly in the upper aqueous phase. Transfer the upper aqueous phase to a new RNase-Free centrifuge tube.

[0025] (5) Add an equal volume of 70% ethanol (prepared with RNase-free water) to the aqueous solution obtained, invert and mix; add the entire solution to the RNA adsorption column;

[0026] (6) Centrifuge at 12000 rpm for 20 seconds, discard the waste liquid in the collection tube, and put the adsorption column back into the collection tube;

[0027] (7) Add 700 μL of Buffer RW1 to the adsorption column, centrifuge at 12,000 rpm for 20 s, discard the waste liquid in the collection tube, and put the adsorption column back into the collection tube;

[0028] (8) Add 500 μL of Buffer RW2 to the adsorption column, centrifuge at 12,000 rpm for 20 s, discard the waste liquid in the collection tube, and put the adsorption column back into the collection tube;

[0029] (9) Repeat step 8 and place the adsorption column at room temperature for several minutes to dry thoroughly;

[0030] (10) Place the adsorption column in a new enzyme-free centrifuge tube, add 30-50 μL RNase-Free Water, leave at room temperature for 1 min, centrifuge at 12000 rpm for 1 min, collect the RNA solution, and store at -80°C.

[0031] 2. Design primers and amplify target genes

[0032] According to the sequence information of the linalool synthase gene OfTPS6 in the transcriptome of 'Boye Yingui', specific primers (upstream primer 5'-ATGGAGTGTACAATGAGGAGTATTT-3', downstream primer 5'-TCAATTAATAGGAGTGAAGAACAAA-3') were designed using Primer5.0, and cloning was performed using the cDNA of 'Boye Yingui' as a template. The PCR reaction system and procedure are shown in Table 1. The PCR product was purified using 1.5% gel agar, and the product was recovered. The experimental method was referred to the agarose gel DNA recovery kit. The recovered product was connected to the pTOPO-TA vector to form a circle, and the connection product was transformed into Escherichia coli DH5α by heat shock, cultured at 37°C overnight, and the M13F and M13R vector universal primers were used to detect the positive monoclonal clones. The positive monoclonal clones were sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing, and the monoclonal clones with correct sequencing were shaken and preserved in a -80°C refrigerator. The CDS sequence of the OfTPS6 gene is shown in SEQ ID NO.1, and the amino acid sequence of the encoded protein is shown in SEQ ID NO.2.

[0033] Table 1 PCR reaction system and procedure

[0034]

[0035] Example 2 Verification of the Function of the OfTPS6 Gene in Osmanthus Fragrance Petals

[0036] 1. Construction of overexpression vector PK7WG2D-OfTPS6

[0037] In the first step, PCR amplification and BP enzyme were used to construct the entry vector. In the second step, LR enzyme was used to recombinate the entry vector containing the OfTPS6 gene and the final vector PK7WG2D. The BP reaction and LR reaction systems and procedures are shown in Table 2. The recombinant plasmid was transformed into Escherichia coli DH5α, and the plasmid was extracted after sequencing was correct. The constructed plasmid was named PK7WG2D-OfTPS6.

[0038] Table 2 BP and LR reaction systems

[0039]

[0040] 2. Transformation of Agrobacterium GV3101

[0041] Take the GV3101 competent cell stored at -80℃ to room temperature, add 1μl PK7WG2D-OfTPS6 plasmid when it is in an ice-water mixing state, beat to mix, let stand on ice for 5 minutes, then place in liquid nitrogen for 5 minutes, 37℃ water bath for 5 minutes, ice bath for 5 minutes, add 500μl LB medium, culture at 28℃ shaking for 2-3 hours, centrifuge at 5000r / min for 5 minutes, then apply to the corresponding antibiotic LB plate, invert and culture in a 28℃ incubator for 2-3 days.

[0042] Single clones were picked for positive detection using universal vector primers. The fragment length was the length of the target gene OfTPS6 plus part of the vector. The positive single clones were shaken and then added with 50% sterile glycerol (final concentration was 20%), quickly frozen in liquid nitrogen, and then stored in a -80°C refrigerator.

[0043] 3. Instant transformation of osmanthus petals

[0044] (1) Activate Agrobacterium containing the PK7WG2D-OfTPS6 plasmid by streaking, select a single clone for positive detection, and transfer the positive bacteria into a liquid medium containing spectinomycin for overnight culture;

[0045] (2) Take the overnight cultured bacteria and add them to LB liquid medium at a ratio of 1:50 and culture until OD 600 The value was 0.6-0.8; the bacterial solution was collected and infected with infection solution (MES10mmol / L, MgCl 2 10mmol / L, AS20μmol / L) to readjust OD 600 =0.6-0.8, place in a 28°C incubator in the dark for 2h;

[0046] (3) Pick osmanthus flower petals in full bloom, remove the pedicels, place the petals in Agrobacterium culture solution, make the petals completely immersed in the culture solution, perform vacuum penetration at 0.06 MPa for 5 minutes, and then slowly introduce air; take out the petals, wash the Agrobacterium culture solution remaining on the surface of the petals with sterile water, soak them in 5% sterile sucrose solution, and place them in the dark for 60 hours before detecting volatiles in the infected petals.

[0047] 4. Real-time fluorescence quantification

[0048] use The RNA of transiently transformed 'Boye Yingui' petals was reverse transcribed into cDNA using the One-Step gDNA Removal and cDNA Synthesis SuperMix reverse transcription kit. Quantitative primers (5'-AAAGGAGGAGGAAGAACGAG-3', 5'-TATCGACGCTGGAGAAGTATGAATC-3') were designed using Primer5.0. β-actin was used as the internal reference gene for fluorescence quantitative analysis. The experimental operation was protected from light throughout. The fluorescence quantitative system is shown in Table 3. The procedure was referred to the kit manual, specifically: 95°C pre-denaturation for 30 s, 95°C denaturation for 5 s, 60°C annealing for 30 s, 72°C extension for 10 s, 40 cycles, followed by 95°C for 15 s, 60°C for 1 min, 95°C for 15 s, and ABI Amplification was performed on a 7500 fluorescence quantitative PCR instrument. Three biological replicates and three mechanical replicates were set for each sample. The relative expression level was analyzed by the method.

[0049] Table 3 Fluorescence quantitative reaction system

[0050]

[0051] 5. GC-MS detection of volatile products

[0052] Gas chromatography-mass spectrometry conditions: DB-5MS column (30 m × 0.25 mm × 0.25 μm, Themo Scientific, Bellefonte, PA, USA), carrier gas was high-purity helium (99.999%), split ratio 20:1, flow rate 1 ml / min. The temperatures of the ion source and injection port were 280 °C and 230 °C, respectively, and the temperature of the transfer line was 250 °C.

[0053] Temperature program: 40°C for 3 min, then heated to 120°C at a rate of 3°C / min and kept for 3 min, then heated to 220°C at a rate of 8°C / min and kept for 2 min. MS conditions are as follows: EI (electron impact) ion source, electron impact energy 70 eV, positive ion scan mode, mass scan range m / z 40-450 amu.

[0054] Substance identification method: GC-MS raw data were processed and exported using Xcalibur software. Volatile substance identification was based on standard samples and the NIST mass spectrum database (NIST 2017). The identification results were based on a mass spectrum data of each component with a matching degree of >80% with the mass spectrum data of the standard substances provided in the spectrum library NIST (2017). At the same time, the Kovats' Retention Indices (RI) calculated using the normal alkane standard sample (C8–C30) under the same heating program were used for further qualitative confirmation. The RI value calculation formula is as follows: RI = (R TX -R T ) / (R T+1 -R T )×100+C RT ×100. Where: R TX is the retention time of the substance X to be tested / min; R T and R T+1 is the retention time of the normal alkanes immediately before and after the aroma substance X / min; C RT is the number of carbon atoms in normal alkane corresponding to the time of the substance to be tested.

[0055] Substance quantification method: internal standard quantification method is used. The content of each aroma component (μg / g FW) = peak area of ​​each component / peak area of ​​internal standard × content of internal standard (μg) / sample amount (g) × f (f is the correction factor of each component to the internal standard, f=1).

[0056] The OfTPS6 coding region sequence was constructed in the plant overexpression vector PK7WG2D and transiently transformed into Osmanthus fragrans petals. After 60 h of dark culture, headspace solid phase microextraction combined with GCMS technology was used to detect the volatiles of the infected petals. Figure 1 As shown, compared with the control group, the expression level of OfTPS6 was upregulated by 3.42 times, the content of linalool increased from 182.25 μg / gFW to 460.12 μg / gFW, an increase of 1.52 times, and the content of linalool oxide increased from 452.09 μg / gFW to 2919.65 μg / gFW, an increase of 5.46 times.

[0057] To further verify the function of OfTPS6, we used VIGS technology to transiently silence OfTPS6 in osmanthus petals. Figure 2 As shown in the figure, compared with the control group, the linalool content of the OfTPS6 silenced strain decreased from 604.65 μg / gFW to 411.625 μg / gFW, and the linalool oxide content decreased from 61.40 μg / gFW to 8.45 μg / gFW. Transformation of OfTPS6 in petals can significantly affect the content of linalool and its oxides in petals, proving that OfTPS6 is a linalool synthase gene.

Claims

1. Osmanthus fragrans linalool synthase gene OfTPS6 , characterized in that, Said OfTPS6 The CDS sequence of the gene is shown in SEQ ID NO.

1.

2. The method according to claim 1 OfTPS6 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 OfTPS6 Genetic biological material, characterized in that The biological materials include expression cassettes, expression vectors and recombinant bacteria.

4. The method according to claim 1 OfTPS6 Use of the gene or the encoded protein according to claim 2 or the biomaterial according to claim 3 in the synthesis of osmanthus linalool and linalool oxide.

5. The method according to claim 1 OfTPS6 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 increasing the content of linalool and linalool oxide in osmanthus fragrans, characterized in that: Super-expression of the method of claim 1 in sweet osmanthus OfTPS6 Gene.

7. The method according to claim 6, characterized in that The following steps are involved: (1) Constructing a protein containing SEQ ID NO.1 OfTPS6 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 OfTPS6 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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