Sandalwood SaJA2L protein and / or SaJA2L gene and application thereof in promoting synthesis of sandalwood essential oil

Abstract

The invention discloses sandalwood SaJA2L protein and / or SaJA2L gene and application of the sandalwood SaJA2L protein and / or SaJA2L gene in promoting synthesis of sandalwood essential oil. The amino acid sequence of the sandalwood SaJA2L protein is shown as SEQ ID NO. 2. According to the invention, the SaJA2L gene is obtained in sandalwood for the first time, the regulation effect of the gene in synthesis of natural sandalwood essential oil is disclosed, and the synthesis of the sandalwood essential oil can be obviously promoted by overexpressing the SaJA2L gene in sandalwood. The invention provides a new technical choice for increasing the yield of the natural sandalwood essential oil, and has important popularization and application values in the aspect of cultivating new sandalwood varieties with high essential oil synthesis efficiency.

Description

Technical Field

[0001] The present invention relates to the technical field of plant breeding, and in particular to a sandalwood SaJA2L protein and / or a SaJA2L gene and an application thereof in promoting the synthesis of sandalwood essential oil. Background Art

[0002] sandalwood( Santalum album Sandalwood (Sandalwood L.) is a plant of immense economic value and cultural significance. Sandalwood essential oil, with its unique aroma and diverse bioactivities, including antibacterial, anti-inflammatory, and antidepressant properties, is widely used in high-end perfumes, cosmetics, pharmaceuticals, and traditional fragrances. However, due to its slow growth and demanding ecological requirements, natural sandalwood resources are extremely limited. This has resulted in an inability to meet market demand, keeping its price high and restricting its wider application.

[0003] The main components of sandalwood essential oil are santalene (α-santalene and β-santalene), and santalol (α-santalol and β-santalol). These compounds give sandalwood its signature aroma and play a key role in various biological activities. The synthesis of natural sandalwood essential oil is complex, beginning with the conversion of farnesyl diphosphate (FPP) to santalene by santalene synthase SaSSY (a member of the terpene synthase TPS family), followed by catalysis by the P450 oxidase SaCYP736A167 to santalol. To alleviate the shortage of natural sandalwood essential oil, various chemical synthesis techniques have emerged in recent years, allowing for the production of sandalwood essential oil through artificial synthesis. However, current synthetic techniques struggle to replicate the entire natural essential oil synthesis process, resulting in significant differences in aroma and biological activity. This leads to low market acceptance, high production costs, and environmental pollution, making it a poor substitute for natural sandalwood essential oil. How to increase the yield of natural sandalwood essential oil remains a key problem that technicians in this field urgently need to overcome. Summary of the Invention

[0004] In order to solve the above problems existing in the prior art, the present invention provides a sandalwood SaJA2L protein and / or a SaJA2L gene and their use in promoting the synthesis of sandalwood essential oil.

[0005] The first object of the present invention is to provide sandalwood SaJA2L protein.

[0006] The second object of the present invention is to provide the use of sandalwood SaJA2L protein and / or SaJA2L gene in promoting the synthesis of sandalwood essential oil.

[0007] The third object of the present invention is to provide the use of sandalwood SaJA2L protein and / or SaJA2L gene in promoting the synthesis of santalene and / or santalol.

[0008] The fourth object of the present invention is to provide a biomaterial that promotes the synthesis of sandalwood essential oil.

[0009] A fifth object of the present invention is to provide an application of the biomaterial in promoting the synthesis of sandalwood essential oil.

[0010] A sixth object of the present invention is to provide a product that promotes the synthesis of sandalwood essential oil.

[0011] A seventh object of the present invention is to provide a method for promoting the synthesis of sandalwood essential oil.

[0012] In order to achieve the above object, the present invention is implemented through the following scheme: The present invention discovered the SaJA2L gene in sandalwood, which encodes the SaJA2L protein, and successfully overexpressed the SaJA2L gene in sandalwood through tissue culture and Agrobacterium vacuum infiltration transformation, clarifying its regulatory function in the synthesis of sandalwood essential oil.

[0013] The present invention seeks protection for the following: The amino acid sequence of the sandalwood SaJA2L protein is shown in SEQ ID NO.2.

[0014] Application of the sandalwood SaJA2L protein and / or SaJA2L gene in promoting the synthesis of sandalwood essential oil. The amino acid sequence of the sandalwood SaJA2L protein is shown in SEQ ID NO.2.

[0015] Preferably, the sandalwood essential oil is sandalwood essential oil from Indian sandalwood.

[0016] The use of sandalwood SaJA2L protein and / or SaJA2L gene in promoting the synthesis of santalene and / or santalol, wherein the amino acid sequence of the sandalwood SaJA2L protein is shown in SEQ ID NO.2.

[0017] Preferably, the santalene and / or santalol are santalene and / or santalol from Indian sandalwood.

[0018] More preferably, the santalene includes α-santalene and β-santalene.

[0019] More preferably, the santalol includes α-santalol and β-santalol.

[0020] Use of a reagent for promoting the expression of sandalwood SaJA2L protein and / or SaJA2L gene in promoting the synthesis of sandalwood essential oil, wherein the amino acid sequence of the sandalwood SaJA2L protein is shown in SEQ ID NO.2.

[0021] Preferably, the sandalwood essential oil is sandalwood essential oil from Indian sandalwood.

[0022] Use of an agent for promoting the expression of sandalwood SaJA2L protein and / or SaJA2L gene in promoting the synthesis of santalene and / or santalol, wherein the amino acid sequence of the sandalwood SaJA2L protein is shown in SEQ ID NO.2.

[0023] Preferably, the santalene and / or santalol are santalene and / or santalol from Indian sandalwood.

[0024] More preferably, the santalene includes α-santalene and β-santalene.

[0025] More preferably, the santalol includes α-santalol and β-santalol.

[0026] A biomaterial for promoting the synthesis of sandalwood essential oil, which is any one of the following (1) to (4): (1) a nucleic acid molecule encoding a sandalwood SaJA2L protein, the amino acid sequence of which is shown in SEQ ID NO. 2; (2) an expression cassette containing the nucleic acid molecule described in (1); (3) a recombinant expression vector containing the nucleic acid molecule described in (1); (4) A microorganism containing the recombinant expression vector described in (3).

[0027] Preferably, the nucleotide sequence of the nucleic acid molecule in (1) is as shown in SEQ ID NO.1.

[0028] Preferably, the recombinant expression vector described in (3) uses the PBWA(V)HS-GFP vector as its backbone.

[0029] More preferably, the recombinant expression vector in (3) uses the PBWA(V)HS-GFP vector as a backbone, and the nucleic acid molecule in (1) is located between the SmaI site and the XbaI site.

[0030] The present invention has no special limitation on the source and type of the microorganisms described in (4), including but not limited to Escherichia coli, Agrobacterium and other conventional microorganisms that can carry the recombinant expression vector can achieve the purpose of the present invention.

[0031] Preferably, the microorganism in (4) is Agrobacterium.

[0032] More preferably, the Agrobacterium is Agrobacterium tumefaciens.

[0033] More preferably, the Agrobacterium tumefaciens is strain LBA4404.

[0034] Use of any of the above-mentioned biological materials in promoting the synthesis of sandalwood essential oil.

[0035] Preferably, the sandalwood essential oil is sandalwood essential oil from Indian sandalwood.

[0036] Use of any of the above biological materials in promoting the synthesis of santalene and / or santalol.

[0037] Preferably, the sandalwood essential oil is santalene and / or santalol of Indian sandalwood.

[0038] More preferably, the santalene includes α-santalene and β-santalene.

[0039] More preferably, the santalol includes α-santalol and β-santalol.

[0040] A product for promoting the synthesis of sandalwood essential oil, comprising a biological material for promoting the synthesis of sandalwood essential oil, wherein the biological material is any one of the following (1) to (4): (1) a nucleic acid molecule encoding a sandalwood SaJA2L protein, the amino acid sequence of which is shown in SEQ ID NO. 2; (2) an expression cassette containing the nucleic acid molecule described in (1); (3) a recombinant expression vector containing the nucleic acid molecule described in (1); (4) A microorganism containing the recombinant expression vector described in (3).

[0041] Preferably, the nucleotide sequence of the nucleic acid molecule in (1) is as shown in SEQ ID NO.1.

[0042] Preferably, the recombinant expression vector described in (3) uses the PBWA(V)HS-GFP vector as its backbone.

[0043] More preferably, the recombinant expression vector in (3) uses the PBWA(V)HS-GFP vector as a backbone, and the nucleic acid molecule in (1) is located between the SmaI site and the XbaI site.

[0044] The present invention has no special limitation on the source and type of the microorganisms described in (4), including but not limited to Escherichia coli, Agrobacterium and other conventional microorganisms that can carry the recombinant expression vector can achieve the purpose of the present invention.

[0045] Preferably, the microorganism in (4) is Agrobacterium.

[0046] More preferably, the Agrobacterium is Agrobacterium tumefaciens.

[0047] More preferably, the Agrobacterium tumefaciens is strain LBA4404.

[0048] Application of any of the products in promoting the synthesis of sandalwood essential oil.

[0049] Preferably, the sandalwood essential oil is sandalwood essential oil from Indian sandalwood.

[0050] Use of any of the products in promoting the synthesis of santalene and / or santalol.

[0051] Preferably, the sandalwood essential oil is santalene and / or santalol of Indian sandalwood.

[0052] More preferably, the santalene includes α-santalene and β-santalene.

[0053] More preferably, the santalol includes α-santalol and β-santalol.

[0054] A method for promoting the synthesis of sandalwood essential oil, which increases the expression level of sandalwood SaJA2L protein and / or SaJA2L gene in sandalwood.

[0055] Preferably, any one of the biological materials or any one of the products is used to treat sandalwood to increase the expression level of the sandalwood SaJA2L protein and / or SaJA2L gene in the sandalwood.

[0056] Preferably, the promoting the synthesis of sandalwood essential oil includes promoting the synthesis of santalene and / or santalol.

[0057] More preferably, the santalene includes α-santalene and β-santalene.

[0058] More preferably, the santalol includes α-santalol and β-santalol.

[0059] Preferably, the sandalwood is Indian old mountain sandalwood.

[0060] Compared with the prior art, the present invention has the following beneficial effects: This study, published in Nature Communications, identifies the SaJA2L gene in sandalwood for the first time and reveals its regulatory role in the synthesis of natural sandalwood essential oil. Overexpressing the SaJA2L gene in sandalwood significantly promotes the synthesis of sandalwood essential oil. This study provides a new technical option for increasing the yield of natural sandalwood essential oil and has significant application value in cultivating new sandalwood varieties with high essential oil synthesis efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] Figure 1 This is the result of PCR amplification and electrophoresis detection of the CDS region of the SaJA2L gene (SEQ ID NO. 1) in Example 1. M represents DNA Marker, and 1 to 3 represent PCR products, respectively.

[0062] Figure 2 The electrophoresis detection results of the monoclonal bacterial solution PCR in Example 1 are shown. M represents DNA Marker, and 1 to 3 represent three monoclonal bacterial solutions, respectively.

[0063] Figure 3 This is a map of the sandalwood SaJA2L overexpression vector in Example 1.

[0064] Figure 4 These are microscopic photographs of root tissues of the WT plant and three JA2L plants in Example 2. WT represents the WT plant, and #1, #2, and #3 represent JA2L plant #1, JA2L plant #2, and JA2L plant #3, respectively.

[0065] Figure 5 The statistical results of the relative mRNA expression levels of the SaJA2L gene in the WT plant and three JA2L plants in Example 2 are shown. WT represents the WT plant, #1, #2, and #3 represent JA2L plant #1, JA2L plant #2, and JA2L plant #3, respectively. Different lowercase letters indicate significant differences among the groups (p<0.05), and the same lowercase letters indicate no significant differences among the groups (p≥0.05).

[0066] Figure 6 The statistical results of the relative mRNA expression levels of SaSSY gene and SaCYP736A167 gene in WT plants and JA2L plants in Example 2, WT represents WT plants, #2 represents JA2L plant #2, different lowercase letters indicate significant differences between groups (p < 0.05), and the same lowercase letters indicate no significant differences between groups (p ≥ 0.05).

[0067] Figure 7 The statistical results of the α-santalene content in the WT plant and three JA2L plants in Example 3 are shown. WT represents the WT plant, #1, #2, and #3 represent JA2L plant #1, JA2L plant #2, and JA2L plant #3, respectively. Different lowercase letters indicate significant differences among the groups (p<0.05), and the same lowercase letters indicate no significant differences among the groups (p≥0.05).

[0068] Figure 8 The statistical results of β-santalene content in WT plants and three JA2L plants in Example 3 are shown. WT represents WT plants, #1, #2, and #3 represent JA2L plant #1, JA2L plant #2, and JA2L plant #3, respectively. Different lowercase letters indicate significant differences among the groups (p<0.05), and the same lowercase letters indicate no significant differences among the groups (p≥0.05).

[0069] Figure 9The statistical results of the β-santalol content in the WT plant and three JA2L plants in Example 3 are shown. WT represents the WT plant, #1, #2, and #3 represent JA2L plant #1, JA2L plant #2, and JA2L plant #3, respectively. Different lowercase letters indicate significant differences among the groups (p<0.05), and the same lowercase letters indicate no significant differences among the groups (p≥0.05).

[0070] Figure 10 The statistical results of the α-santalol content in the WT plant and three JA2L plants in Example 3 are shown. WT represents the WT plant, #1, #2, and #3 represent JA2L plant #1, JA2L plant #2, and JA2L plant #3, respectively. Different lowercase letters indicate significant differences among the groups (p<0.05), and the same lowercase letters indicate no significant differences among the groups (p≥0.05). DETAILED DESCRIPTION

[0071] The present invention is further described in detail below with reference to the accompanying drawings and specific examples. The examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods used in the following examples are conventional methods unless otherwise specified; the materials and reagents used are commercially available unless otherwise specified.

[0072] Example 1 Construction of Santalum album SaJA2L overexpression vector 1. Amplification of target fragment

[0073] Genomic cDNA from root tissue of Santalum album (cultivar: Indian old mountain Santalum) was used as a template. PCR amplification was performed using the upstream cloning primer SaJA2L-F (5'-ATGGGTGTTCCGAGATCCGA-3' (SEQ ID NO. 3)) and the downstream cloning primer SaJA2L-R (5'-TCACTGTCGGGTGAACCCGA-3' (SEQ ID NO. 4)) using a gene cloning kit (Novozymes). The PCR reaction system was as follows: 2×Phanta Max Master Mix, 10 μL; ddH2O, 8 μL; SaJA2L-F (SEQ ID NO. 3), 0.5 μL; SaJA2L-R (SEQ ID NO. 4), 0.5 μL; and cDNA, 1 μL.

[0074] The PCR amplification products were collected and tested by agarose gel electrophoresis. Figure 1 As shown, the PCR amplification product has a single band and its size is consistent with the length of the CDS region of the SaJA2L gene (SEQ ID NO. 1).

[0075] 2. Carrier connection Using double enzyme digestion and homologous recombination, the CDS region of the SaJA2L gene (SEQ ID NO. 1) amplified in the previous step was connected to the pBWA(V)HS-GFP vector (expressing green fluorescence) using restriction endonucleases SmaI and XbaI. Then, the gene was transformed into Escherichia coli and plated on LB solid medium. A single clone was picked for expansion culture, and the bacterial liquid was collected for PCR identification. Figure 2 As shown in the figure, each monoclonal clone has a single band that meets the expected size, which is a positive monoclonal clone.

[0076] The plasmid of the positive single clone was extracted and sequenced. The sequencing results showed that the sequence was correct, which means that the construct was successfully obtained. Figure 3 The sandalwood SaJA2L overexpression vector shown is denoted as pBWA(V)HS-SaJA2L vector.

[0077] Example 2 Construction of a Santalum album SaJA2L overexpression strain 1. Construction of Agrobacterium The pBWA(V)HS-SaJA2L vector obtained in Example 1 was introduced into Agrobacterium tumefaciens (LBA4404 strain) by freeze-thaw method to obtain Agrobacterium containing the sandalwood SaJA2L overexpression vector, which was designated as LBA4404-SaJA2L Agrobacterium.

[0078] Following the same method, the pBWA(V)HS-GFP vector was introduced into Agrobacterium tumefaciens (strain LBA4404) to obtain Agrobacterium containing an empty vector, which was designated as LBA4404-control Agrobacterium.

[0079] 2. Agrobacterium Vacuum Infiltration Transformation (1) Preparation of reagents Dissolve 10 mM MgCl2, 200 μM AS (acetosyringone) and 10 mM MES (2-(N-morpholino)ethanesulfonic acid) in water, mix well, adjust the pH to 5.7, and prepare the infection solution. Sterilize it by filtering with a 0.02 μm filter head and set aside.

[0080] (2) Preparation of infection solution Agrobacterium LBA4404-SaJA2L was cultured in LB liquid medium in a constant temperature shaker at 200 rpm at 30°C until the bacterial liquid OD 600 The LBA4404-SaJA2L bacterial solution was 0.6. Take 10 mL of the LBA4404-SaJA2L bacterial solution and centrifuge at 6000 rpm for 5 minutes. Remove the supernatant and resuspend the pellet in 10 mL of infection solution. Centrifuge again at 6000 rpm for 5 minutes. Remove the supernatant and resuspend the pellet in 10 mL of infection solution to obtain the infection solution containing LBA4404-SaJA2L Agrobacterium, recorded as SaJA2L infection solution.

[0081] According to the same method, an infection solution containing LBA4404-empty Agrobacterium was prepared and recorded as the control infection solution.

[0082] (3) Conversion Root tissue from one-year-old wild-type sandalwood plants (cultivar Santalum altissima, deposited at the South China Botanical Garden, Chinese Academy of Sciences; this biological material is publicly available from the applicant) was completely immersed in the SaJA2L infection solution and then placed in a vacuum desiccator (Tianjin Jinteng Experimental Equipment Co., Ltd., model GM-0.33A) under evacuation at 0.08 MPa for 5 minutes. Following transformation, the root tissue was cleaned with the infection solution and then cultured. The resulting sandalwood plants were designated JA2L plant #1, JA2L plant #2, and JA2L plant #3.

[0083] The root tissues of one-year-old wild-type sandalwood plants were completely immersed in the control infection solution and then transformed according to the same method. The resulting sandalwood plants were recorded as WT plants.

[0084] Laser microscope observation showed that Figure 4As shown, under the irradiation of excitation light, the root tissues of WT plant and JA2L plant #1, JA2L plant #2 and JA2L plant #3 all expressed green fluorescence, indicating that all plants had been successfully transformed.

[0085] 3. Identification of transgenic plants Root tissues from three JA2L plants (JA2L plant #1, JA2L plant #2, and JA2L plant #3) and a WT plant were used to extract RNA and reverse transcribe cDNA. Using the cDNA as a template, quantitative PCR was used to detect the mRNA expression of the SaJA2L, SaSSY, and SaCYP736A167 genes using the primers listed in Table 1.

[0086] The fluorescence quantitative PCR reaction system was as follows: 2×SYBR Green qPCR Mix, 10 μL; RNase-Free ddH2O, 8 μL; cDNA, 1 μL; upstream quantitative primer (F), 0.5 μL; downstream quantitative primer (F), 0.5 μL.

[0087] Table 1 Fluorescence quantitative PCR primers

[0088] The mRNA expression of SaJA2L gene is as follows Figure 5 As shown in the figure, compared with the WT plant, the mRNA expression levels of SaJA2L in all three JA2L plants were significantly increased, with JA2L plant #2 showing the highest SaJA2L mRNA expression level, indicating that the SaJA2L overexpression line of sandalwood was successfully constructed.

[0089] The mRNA expression of SaSSY gene and SaCYP736A167 gene is as follows Figure 6 As shown, compared to WT plants, the mRNA expression levels of both SaSSY and SaCYP736A167 genes were significantly increased in JA2L plant #2. Similar results were observed in JA2L plant #1 and JA2L plant #3. This suggests that overexpressing the SaJA2L gene in sandalwood can upregulate the terpene biosynthesis pathway involved in sandalwood essential oil production.

[0090] Example 3 Synthesis of sandalwood essential oil by the SaJA2L overexpression strain 1. Determination of sandalwood essential oil Root tissues of the one-month-old WT plant and three JA2L plants (JA2L plant #1, JA2L plant #2, and JA2L plant #3) obtained in Example 2 were collected, dried, and then ground to obtain sandalwood root powder of each plant.

[0091] 0.1 g of sandalwood root powder from each plant was taken and used with 1 mL of n-hexane to extract metabolites by repeatedly shaking the extract for 3 days. The extract was collected and centrifuged at 2000 r / min for 5 min. The supernatant was taken and evaporated to dryness with nitrogen.

[0092] The dried residues of each plant were collected and dissolved in 50 μL of n-hexane. Dodecane (0.1 mg / mL) was added as an internal standard and detected according to the following method: a GC-2010 gas chromatograph (Shimadzu, Suzhou, China) was operated in electron ionization selected ion monitoring mode. The sandalwood essential oil samples were analyzed on a DB Wax fused silica column (Agilent Technologies) (30 m long, 250 μm inner diameter, 0.25 μm film thickness). The injector was operated in pulsed splitless mode, and the injector temperature was maintained at 250°C. Helium was used as the carrier gas with a flow rate of 0.8 mL / min and a pulse pressure of 25 psi for 0.5 min. The scan range was m / z 40-500. The SIM analysis was performed at m / z 93, 94, 105, 107, 119, 122, and 202 with a dwell time of 50 ms. The oven program consisted of: 50°C for 3 minutes; ramping to 250°C at 5°C / min, holding for 2 minutes; then ramping to 300°C at 5°C / min, holding for 15 minutes. Data acquisition and processing were performed using ChemStation software (Agilent Technologies). Compounds were identified by comparing mass spectra with the NIST / EPA / NIH Mass Spectral Library, version 2.0 (http: / / chemdata.nist.gov / ). The relative abundance of essential oil components was calculated by manual integration of peak areas and normalization using internal standards and the amount of tissue used (dry weight). Analysis was performed using three biological replicates, each containing two technical replicates.

[0093] 2. Measurement results like Figures 7 to 10 As shown in the figure, compared with the WT plants, the contents of α-santalene, β-santalene, β-santalol, and α-santalol in the three JA2L plants were significantly increased, indicating that overexpression of the SaJA2L gene in sandalwood can promote the synthesis of sandalwood essential oil.

[0094] Finally, it should be noted that the above embodiments are intended only to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Those skilled in the art will readily appreciate that other variations or modifications may be made based on the above descriptions and concepts. It is not necessary and impossible to provide an exhaustive list of all possible implementations. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the claims of the present invention.

Claims

1. Sandalwood SaJA2L protein, characterized in that Its amino acid sequence is shown in SEQ ID NO.

2.

2. The use of sandalwood SaJA2L protein and / or SaJA2L gene in promoting the synthesis of sandalwood essential oil, characterized in that: The amino acid sequence of the sandalwood SaJA2L protein is shown in SEQ ID NO.

2.

3. Use of sandalwood SaJA2L protein and / or SaJA2L gene in promoting the synthesis of santalene and / or santalol, characterized in that: The amino acid sequence of the sandalwood SaJA2L protein is shown in SEQ ID NO.

2.

4. A biomaterial for promoting the synthesis of sandalwood essential oil, characterized in that: It is any one of the following (1) to (4): (1) A nucleic acid molecule encoding the sandalwood SaJA2L protein according to claim 1; (2) an expression cassette containing the nucleic acid molecule described in (1); (3) a recombinant expression vector containing the nucleic acid molecule described in (1); (4) A microorganism containing the recombinant expression vector described in (3).

5. The biomaterial according to claim 4, characterized in that The nucleotide sequence of the nucleic acid molecule described in (1) is shown in SEQ ID NO.

1.

6. The biomaterial according to claim 4, characterized in that The microorganism described in (4) is Agrobacterium.

7. Use of the biomaterial according to any one of claims 4 to 6 in promoting the synthesis of sandalwood essential oil.

8. A product for promoting the synthesis of sandalwood essential oil, characterized in that: The biomaterial according to any one of claims 4 to 6.

9. A method for promoting the synthesis of sandalwood essential oil, characterized in that: The expression level of the sandalwood SaJA2L protein and / or SaJA2L gene in sandalwood is increased, wherein the amino acid sequence of the sandalwood SaJA2L protein is shown in SEQ ID NO.

2.

10. The method according to claim 9, characterized in that Sandalwood is treated with the biomaterial according to any one of claims 4 to 6.

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