Application of Santalum album AREB6 gene
By expressing the sandalwood AREB6 gene in sandalwood tissues, the problem of insufficient α-sandalwood alcohol production in sandalwood in the prior art was solved, and the effect of significantly improving the biosynthesis yield of α-sandalwood alcohol was achieved, which has important economic benefits.
Patent Information
- Application Number
- CN202410621449.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2024-05-20
- Publication Date
- 2025-03-21
- Estimated Expiration
- 2044-05-20
AI Technical Summary
There is a lack of effective methods in the prior art to increase the biosynthesis yield of α-sandalwood alcohol in sandalwood, resulting in poor economic benefits.
Through genetic engineering operations, the sandalwood AREB6 gene is highly expressed in sandalwood tissue to improve the biosynthesis yield of α-sandalwood alcohol.
High expression of sandalwood AREB6 gene can significantly increase the content of α-sandalwood alcohol in sandalwood tissues and increase the biosynthesis yield of sandalwood alcohol, which has great economic benefits.
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Figure CN118546944B_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the field of plant genetic engineering, relates to the application of sandalwood AREB6 gene, and particularly relates to the application of sandalwood AREB6 gene in sandalwood to improve the biosynthesis yield of santalol. Background Art
[0002] Sandalwood (Santalum album L.) is a slow-growing evergreen tree native to tropical mountainous areas of India, Sri Lanka and the Indonesian archipelago. Sandalwood is a world-renowned top-grade carving material, and the essential oil extracted from its heartwood is known as "liquid gold". Therefore, sandalwood is one of the most precious woody plants in the world and has good economic benefits. Studying the formation mechanism of sandalwood essential oil is of great significance for the molecular breeding of sandalwood and the synthesis of sandalwood essential oil through metabolic engineering.
[0003] Santalol is the main component of sandalwood essential oil. α-Santalol accounts for the majority of santalol. In recent years, the upstream genes SaAACT, SaHMGS, SaMK, SaPMK and the downstream key enzyme genes SaSSY and SaCYP736A167 of the mevalonic acid (MVA) biosynthesis pathway of santalol have been identified, but the molecular regulatory mechanisms of these key genes are rarely reported, and there are no reports on the application of genes whose high expression can significantly increase the amount of santalol synthesis.
[0004] The CAS number of α-santalol is: 115-71-9.
[0005] The structural formula of α-santalol is:
[0006] If the production of santalol in sandalwood can be increased through transgenic breeding or gene regulation, it will have great economic benefits. Summary of the invention
[0007] In order to solve the problems existing in the prior art, the first aspect of the present invention provides a method for increasing the content of α-santalol in sandalwood tissue, wherein the method comprises: using genetic engineering to highly express the sandalwood AREB6 gene in the sandalwood tissue.
[0008] In some embodiments, the variety of sandalwood is Santalum album.
[0009] In some embodiments, the protein sequence encoded by the Santalum album AREB6 gene is shown as SEQ ID NO.2.
[0010] In some embodiments, the method comprises the steps of:
[0011] Step 1: Transfer the sandalwood AREB6 gene into the Agrobacterium gene expression plasmid to obtain a recombinant plasmid;
[0012] Step 2: transferring the recombinant plasmid into Agrobacterium to obtain recombinant Agrobacterium capable of expressing the sandalwood AREB6 gene;
[0013] Step 3: transforming a sandalwood plant with the recombinant Agrobacterium to increase the content of α-santalol in the sandalwood plant; or
[0014] The recombinant Agrobacterium is used to transform sandalwood callus tissue, so that the content of α-santalol in the sandalwood callus tissue is increased.
[0015] The second aspect of the present invention provides a use of a biological material in preparing a preparation for increasing the content of α-santalol in sandalwood tissue, wherein the biological material is any one of the following B1, B2, B3, B4, B5, B6 and B7;
[0016] B1: protein encoded by the Santalum album AREB6 gene;
[0017] B2: a fusion protein, the amino acid sequence of which contains the amino acid sequence of the protein encoded by the Santalum album AREB6 gene described in B1 and the amino acid sequence of a functional protein fragment or an inert protein fragment;
[0018] B3: a gene, the coding sequence of which can encode the protein encoded by the Santalum album AREB6 gene described in B1 or the fusion protein described in B2;
[0019] B4: a gene expression cassette, wherein the gene product in the gene expression cassette is the protein encoded by the Santalum AREB6 gene described in B1 or the fusion protein described in B2;
[0020] B5: a genetic engineering vector, wherein the genetic engineering vector contains the gene expression cassette described in B4;
[0021] B6: a cell, wherein the cell contains the genetic engineering expression vector described in B5, and the encoded protein in the gene expression cassette of the genetic engineering vector is constitutively expressed, tissue-specifically expressed, or artificially induced; and
[0022] B7: A composition comprising the protein encoded by the Santalum album AREB6 gene described in B1, the fusion protein described in B2, the genetically engineered expression vector described in B5 or the cell described in B6.
[0023] In some embodiments, the functional protein fragment is a tag peptide or a fluorescent protein tag used to separate and purify the protein.
[0024] In some embodiments, the protein sequence encoded by the Santalum album AREB6 gene is shown as SEQ ID NO.2.
[0025] In some embodiments, the variety of sandalwood is Santalum album.
[0026] The third aspect of the present invention provides a qPCR primer combination for evaluating the ability of sandalwood tissue to synthesize α-santalol. In the primer combination, the upstream primer sequence is shown in SEQ ID NO.5, and the downstream primer sequence is shown in SEQ ID NO.6.
[0027] In some embodiments, the variety of sandalwood is Santalum album. BRIEF DESCRIPTION OF THE DRAWINGS
[0028] Figure 1 This is a PCR gel photo of the SaAREB6 gene amplification results.
[0029] Figure 2 This is a statistical diagram of the tissue expression characteristics of the SaAREB6 gene.
[0030] Figure 3 This is a map of plasmid SaAREB6-OE.
[0031] Figure 4 This is the map of plasmid SaAREB6-Anti.
[0032] Figure 5 This is a graph showing the changes in α-santalol content in sandalwood plants treated with plasmid SaAREB6-OE.
[0033] Figure 6 These are morphological photos of sandalwood callus transformed with the control plasmid, plasmid SaAREB6-OE and plasmid SaAREB6-Anti.
[0034] Figure 7 This is a comparison chart of the SaAREB6 gene expression levels in sandalwood callus transformed with the control plasmid, plasmid SaAREB6-OE and plasmid SaAREB6-Anti.
[0035] Figure 8 This is a comparison chart of the fresh weight of sandalwood callus transformed with the control plasmid, plasmid SaAREB6-OE and plasmid SaAREB6-Anti.
[0036] Fig. 9 This is a comparison chart of the α-santalol content in sandalwood callus transformed with the control plasmid, plasmid SaAREB6-OE and plasmid SaAREB6-Anti. DETAILED DESCRIPTION
[0037] In order to make the objectives, technical solutions and advantages of the present invention more clear, the embodiments of the present invention will be further described in detail below with reference to the accompanying drawings.
[0038] Example 1: Cloning of Santalum album AREB6 gene
[0039] The sandalwood variety used in the present invention is Indian sandalwood (Old Mountain Sandalwood).
[0040] The genome of Santalum album was sequenced and assembled using the second generation sequencing technology, and the genome sequence was not made public. According to the naming rules of homologous genes, the Santalum album AREB6 gene (SaAREB6 gene) was annotated.
[0041] The coding sequence of the Santalum album AREB6 gene obtained by the aforementioned sequencing is as follows (SEQ ID NO.1):
[0042]
[0043] The amino acid sequence of the protein encoded by the Santalum album AREB6 gene is as follows (SEQ ID NO.2):
[0044] MNLRNFGNEPPLPPIEGGGGGGGGGSRVGGGGGRPVGNFPLTRQPSVYSLTFDEFQSTMGGIGKDFGSMNMDELLKNIWSAEETQQTMGPSGSRQEGGGGVIPGGNL QRQGSLTLPRTLSQKTVDEVWKDISREDGGGKDGNGNSRPSLQQQRQQTLGEMTLEEFLVRAGVVREDTNLAGRPNSINVLGLGFQQIDGRSGLMGNRILESNNHFA MQSLNLPLNVNGVTPSQQQQQQQLFSQQNPTYTSPMSLPTSAQLGSPGIRGGIVGIPDPSINSNSIQGSTLQGGAIGMAGLGAGAVTVAAGSPSNQLSSDAVGKSNG DTSSVSPPYMFNGGMRGRKRSGAVDKVVERRQRRMIKNRESAARSRARKQAYTMELEAEIAKLKEDNQELRKKQAQMEMQKNQVLEMMQSQQGVKKRCLRRTQTGPW
[0045] According to the aforementioned Santalum AREB6 gene sequence, the upstream primer F1 and the downstream primer R1 of Santalum AREB6 gene were designed. The total RNA was extracted from the 16-year-old Santalum leaf tissue, and the total RNA was reverse transcribed into cDNA. The cDNA was amplified by PCR using primer F1 and primer R1 as the primer pair to obtain the Santalum AREB6 gene amplification product.
[0046] F1 (SEQ ID NO.3):
[0047] 5'-ATGAATTTGAGGAATTTCGGGA
[0048] R1 (SEQ ID NO.4):
[0049] 5'-CCATGGCCCCGTCTGTGTCCG
[0050] See the agarose gel electrophoresis photos of the above PCR amplification products for details. Figure 1Among them, lanes 1-2 are PCR amplification products, and lane 3 is a molecular weight marker. It can be seen that the length is consistent with the length of the Santalum AREB6 gene coding sequence of 1290bp. Based on this preliminary judgment, PCR successfully amplified the Santalum AREB6 gene.
[0051] The amplified product was connected to a sequencing vector, and the inserted sequence was sequenced using the Sanger method, and the result was the same as the aforementioned SEQ ID NO. 1. Thus, a recombinant plasmid containing the Santalum AREB6 gene coding sequence was obtained.
[0052] Example 2: Study on tissue expression characteristics of SaAREB6
[0053] Six tissues of 16-year-old Santalum album were collected, including roots, haustoria, leaves, phloem, sapwood, and heartwood. The transition zone of the heartwood was sampled. The RNA of the aforementioned tissues was reverse transcribed into cDNA, and fluorescent quantitative PCR was performed using cDNA as a template.
[0054] qPCR primers F2 and R2 were designed according to the Santalum album AREB6 gene sequence, and qPCR internal reference primers F3 and R3 were designed according to the Santalum album Actin gene sequence.
[0055] F2 (SEQ ID NO.5):
[0056] 5'-TCAACATTACAAGGTGGCGC
[0057] R2 (SEQ ID NO.6):
[0058] 5'-TGTATGGAACCGGTGACACA
[0059] F3 (SEQ ID NO.7):
[0060] 5'-TGCTCTTCCCCATGCCAT
[0061] R3 (SEQ ID NO.8):
[0062] 5'-AACAATTTCCCGCTCAGCAG
[0063] All reactions were performed with three biological replicates and four technical replicates.
[0064] The reaction system (20 μL) was as follows: 2×SYBR Green qPCR Mix 10 μL, RNase Free delH 2 O 8μL, cDNA 1μL, upstream and downstream primers 0.5μL each.
[0065] The expression level of SaAREB6 gene was normalized by using Actin gene of Santalum album as internal reference gene. -ΔΔCt Finally, SPSS Statistics 27 software was used to analyze whether there were significant differences in gene expression.
[0066] The expression level of AREB6 gene in the root was normalized, and the relative expression levels of AREB6 gene in the root, haustoria, leaf, phloem, sapwood and heartwood were calculated.
[0067] The statistical results can be found in Figure 2 It can be seen that the expression level of AREB6 gene in sapwood and heartwood is significantly higher than that in other tissues.
[0068] Heartwood is the main santalol-producing tissue, and it is speculated that the AREB6 gene may regulate the biosynthesis of santalol.
[0069] Example 3: Preparation of SaAREB6 gene high expression vector
[0070] The empty vector was pBWA(V)HS-GFP expression vector, which was purchased from Wuhan Boyuan Biotechnology Co., Ltd. with the catalog number REC02-I.
[0071] The recombinant sequencing vector containing the SaAREB6 coding sequence obtained in Example 1 was used as a template, and the following F4 and R4 were used as primers to perform a PCR reaction. The reaction system (20 μL) was as follows: 2×Phanta Max Master Mix 10 μL, RNase Free ddH 2 O 8μL, cDNA 1μL, upstream and downstream primers 0.5μL each.
[0072] F4 (SEQ ID NO.9):
[0073]
[0074] R4 (SEQ ID NO.10):
[0075]
[0076] In primer F4, the sequence shown in the box is the restriction endonuclease XbaΙ recognition site, the downstream of the box is a sequence complementary to the template, and the box and its upstream sequence are homologous to the restriction endonuclease XbaΙ recognition site and its upstream sequence on pBWA(V)HS-GFP.
[0077] In primer R4, the sequence shown in the box is the recognition site of the restriction endonuclease SmaΙ, and the sequence downstream of the box is complementary to the template. The box and its upstream sequence are homologous to the restriction endonuclease SmaΙ recognition site and its downstream sequence on pBWA(V)HS-GFP.
[0078] The obtained PCR product and the pBWA(V)HS-GFP expression vector double-digested with XbaΙ and SmaΙ were subjected to agarose gel electrophoresis to verify the correctness of the sequence and purified.
[0079] The one-step seamless cloning kit (CV19-One Step Seamless Cloning kit, Aidelai Biotechnology Co., Ltd. Note: This one-step seamless cloning kit does not rely on T4 ligase and is not restricted by the restriction sites of the vector and the target fragment. Instead, it directly uses the overlapping fragment recombination method to directional recombine and connect the vector linearized by any method and the PCR fragment with 16-25bp overlapping regions at both ends. It can achieve efficient directional seamless cloning of blunt-end PCR product fragments into the vector within 30 minutes) according to the instructions of the kit manual. The above-mentioned purified PCR product and the restriction-cut pBWA(V)HS-GFP vector were homologously recombined to obtain a recombinant vector. The homologous recombination system is as follows: 5×CEⅡBuffer 4μL, linearized vector 2μL, target gene fragment 6μL, ExnaseⅡ2μL, ddH 2 O 6μL. Mix the reaction system and centrifuge briefly. The reaction conditions are 37℃, 30min. After the reaction, take 0.1-10ng of the ligation product to transform E. coli, and apply the bacterial solution to LB solid medium (containing 50μg / mL kanamycin), put it in a 37℃ incubator and invert it for overnight culture, pick a single colony and put it in LB liquid medium (containing 50μg / mL kanamycin) and shake the bacteria. The culture conditions are 37℃, 180rpm. The turbid bacterial solution cultured overnight is PCR-treated with primers F1 and R1 in Example 1, and the positive clones that are successfully connected are screened by electrophoresis of the amplified products for plasmid extraction. Thus, SaAREB6 is constructed on the pBWA(V)HS expression vector to obtain the recombinant vector pBWA(V)HS-SaAREB6, and the plasmid is named SaAREB6-OE. The plasmid expresses SaAREB6 and GFP in fusion.
[0080] For details on plasmid transformation in this example, see Figure 3 The structure between LB and TR in the empty vector pBWA(V)HS-GFP can be found in Figure 3 A, the structure between LB and TR in the recombinant vector can be found in Figure 3 B, the complete structure of the recombinant plasmid SaAREB6-OE can be found in Figure 3 C.
[0081] Example 4: Preparation of SaAREB6 gene low expression vector
[0082] For details on plasmid transformation in this example, see Figure 4 The structure between LB and TR in the empty vector pBWA(V)HS-ccdb-RNAi can be found in Figure 4 A. The structure between LB and TR in the recombinant vector can be found in Figure 4 B, the order of the inserted sequence is tgtF1 (partial sequences)-Loop-tgtF2 (partial sequences), and the linker sequence of the enzyme ligation is not shown. The complete structure of the recombinant plasmid SaAREB6-Anti is shown in Figure 4 C, the linker sequence of the enzyme ligation is not shown. The relevant sequence information is described below.
[0083] The empty vector was pBWA(V)HS-ccdb-RNAi, which was purchased from Wuhan Boyuan Biotechnology Co., Ltd. with the catalog number REC20-I.
[0084] tgtF1 (SEQ ID NO.11, 250 bp):
[0085] 5'-aagacctaattctatcaatgttttaggccttgggtttcaacagattgatgggaggtctggattgatgggcaat cgaatattggagagtaataatcattttgctatgcaatctcttaatttaccattgaatgtcaatggggtaacaccttcccagcagcagcagcaacagcaattgttttcccagcaaaatcccacttacacatctcccatgtctttaccaactagtgctcaactgggcagtcctggaatt
[0086] Loop (SEQ ID NO.12, 200 bp):
[0087] 5'-cctgcaggtctagtttttctccttcattttcttggttaggacccttttctctctttttatttttttgagctttgatctttcttta aactgatctattttttaattgattggttatggtgtaaatattacatagctttaactgataatctgattactttatttcgtgtgtctat gatgatgatgatagttacagagcccgggc
[0088] tgtF2 (SEQ ID NO.13, 250 bp):
[0089] 5'-aattccaggactgcccagttgagcactagttggtaaagacatgggagatgtgtaagtgggattttgctggg aaaacaattgctgttgctgctgctgctgggaaggtgttaccccattgacattcaatggtaaattaagagattgcatagcaaaatgattattactctccaatattcgattgcccatcaatccagacctcccatcaatctgttgaaacccaaggcctaaaacattgatagaattaggtctt
[0090] tgtF1 is a part of the SaAREB6 coding sequence shown in SEQ ID NO.1.
[0091] Loop is a circular structure in which the double strands of antisense RNA are connected.
[0092] tgtF2 is a part of the reverse complementary sequence of the SaAREB6 coding sequence shown in SEQ ID NO.1.
[0093] The following three primer pairs were synthesized.
[0094] 1: tgtF1 primer pair
[0095] F5 (SEQ ID NO.14):
[0096] cagtGGTCTCacaacaagacctaattctatcaatgttttaggccttggg
[0097] R5 (SEQ ID NO.15):
[0098] cgatGGTCTCacaggaattccaggactgcccagttgagc
[0099] 2:Loop Primer Pair
[0100] F6 (SEQ ID NO.16):
[0101] cgatGGTCTCacctgcaggtctagtttttctccttcattttc
[0102] R6 (SEQ ID NO.17):
[0103] cgatGGTCTCagcccgggctctgtaactatcatc
[0104] 3: tgtF2 primer pair
[0105] F7 (SEQ ID NO.18):
[0106] cagtGGTCTCagggcaattccaggactgcccagttgagc
[0107] R7 (SEQ ID NO.19):
[0108] cagtGGTCTCatacaaagacctaattctatcaatgttttaggccttggg
[0109] The purified tgtF1 amplification product, the purified loop amplification product, the purified tgtF2 amplification product and the empty vector pBWA(V)HS-ccdb-RNAi were mixed, and restriction endonuclease Eco31I and T4 DNA ligase were added to perform a one-step enzyme digestion and ligation reaction.
[0110] The enzyme digestion and ligation reaction products were transformed into competent E. coli, and then the plasmid was extracted and PCR was performed using the universal primers F8 and R8 on the vector. After gel electrophoresis, the recombination was confirmed to be correct. The recombinant plasmid was called SaAREB6-Anti.
[0111] The sequence of F8 (SEQ ID NO.20) is as follows:
[0112] 5'-GCTATCTGTCACTTTATTGTG
[0113] The sequence of R8 (SEQ ID NO.21) is as follows:
[0114] 5'-AACTTTATTGCCAAATGTTTG
[0115] Example 5: Promoting the synthesis of santalol in sandalwood plants
[0116] SaAREB6-OE (experimental group) and empty vector (control group, pBWA(V)HS-GFP) were respectively transferred into Agrobacterium GV3101, and positive single colonies were picked and inoculated into YEB liquid medium and cultured at 28°C for 48 h. The supernatant was discarded after centrifugation. The Agrobacterium culture was resuspended in 10 mM MgCl 2 , 10 mM MES, 5% sucrose, 0.05% Silwet L-77 and 200 mM acetosyringone (pH 5.8) buffer, and cultured with shaking until OD 600 =1.0. One-month-old sandalwood plants with uniform growth were placed in a sealed glass container (10 L) and vacuum pumped for 15 min with a vacuum degree of 10 kPa. Then, the infiltration solution (10 mM MgCl 2 , 10 mM MES, 5% sucrose, 0.05% Silwet L-77 and 200 mM acetosyringone, pH 5.8) for 3-5 min, and then transferred to Hoagland nutrient solution for dark culture for 3 d.
[0117] The roots of 30 plants in each group were combined into a mixed sample and frozen in liquid nitrogen. The same method as in Example 2 was used to determine the expression levels of the SaAREB6 gene in the roots of the experimental group and the control group.
[0118] Take 1.5-2g of transformed sandalwood root, place it in an envelope, and dry it naturally at room temperature for 2 weeks. The dried sample is ground into powder in liquid nitrogen. Weigh the weight of the empty sample bottle, divide the powder into sample bottles, about 500μL per bottle, and weigh it again. Add 1mL of n-hexane and mix at room temperature for 24h. Put the sample bottle directly into a 5mL centrifuge tube, centrifuge at 2000rpm, room temperature for 5min. Transfer the supernatant to a new sample bottle, add 1mL of n-hexane to the original sample bottle, mix at room temperature for 24h, and perform a second extraction. Put the sample bottle directly into a 5mL centrifuge tube, centrifuge at 2000rpm, room temperature for 5min. Transfer the supernatant to the same sample bottle, combine the liquids from the two extractions, and the total volume is about 2mL. Concentrate 2mL to about 500μL with nitrogen blowing, and determine the content of α-santalol in the root tissue by GC-MS method.
[0119] Results Figure 5 , wherein A shows the control of SaAREB6 gene expression, B shows the α-santalol content of the experimental group and the control group. Compared with the control, the santalol content of the strain overexpressing SaAREB6 increased by about 40%, and C shows the curve of the α-santalol content of the experimental group and the control group determined by the GC-MS method. The α-santalol content in B is calculated by the method in C.
[0120] It can be seen that high expression of SaAREB6 gene in adult sandalwood tissue can significantly increase the content of α-santalol.
[0121] Example 6: Promoting the synthesis of sandalwood in sandalwood callus
[0122] SaAREB6-OE (experimental group), SaAREB6-Anti and empty vector (control group, pBWA(V)HS-GFP) were respectively transferred into Agrobacterium GV3101, and 4-week-old sandalwood callus was co-cultured with soil Agrobacterium GV3101 for 20 min, and then transferred to MS solid medium (pH = 5.8) supplemented with 1.0 mg / L 2,4-D for co-culture for 2 days at 22°C. Then, the sandalwood callus was transferred to MS solid medium supplemented with 40 mg·L -1 Screening was performed in MS solid medium (pH=5.8) containing hygromycin, and then the positive callus was transferred to MS solid medium (pH=5.8) containing 1.0 mg / L 2,4-D and subcultured for 4 weeks.
[0123] Figure 6 Wild-type callus (WT) not transformed with exogenous genes, callus transformed with SaAREB6-OE (OE), and callus transformed with SaAREB6-Anti (Anti) are shown.
[0124] It can be seen that the wild-type callus and SaAREB6-Anti transformed callus (Anti) were loose white, while the SaAREB6-OE transformed callus was dark brown, which may be speculated to have a higher content of secondary metabolites such as santalol.
[0125] The same method as in Example 2 was used to detect the expression of the SaAREB6 gene in the untransformed wild-type callus (WT), the SaAREB6-OE transformed callus, and the SaAREB6-Anti transformed callus. The results are shown in Figure 7 Among them, the expression levels of SaAREB6 gene in SaAREB6 overexpressing callus and SaAREB6 silencing callus were 3.83 and 0.41 times that of the wild type, respectively.
[0126] The fresh weights of wild-type callus (WT), SaAREB6-OE transformed callus, and SaAREB6-Anti transformed callus were measured to be 0.59 g, 0.32 g, and 0.56 g, respectively. Figure 8 The results are similar to Figure 6 It is possible that SaAREB6-OE promotes the synthesis of santalol and consumes nutrients, which affects the growth rate of callus tissue.
[0127] The method of Example 5 was used to determine the concentrations of α-santalol in wild-type callus (WT), callus transformed by SaAREB6-OE, and callus transformed by SaAREB6-Anti. The concentrations were 40.63 ng / g, 111.36 ng / g, and 19.30 ng / g, respectively. Fig. 9 When this content is compared with Figure 8 The α-santalol content in each callus was obtained by multiplying the weight of the callus tissues shown, which were 23.97 ng, 36.28 ng, and 10.81 ng, respectively. The concentration of α-santalol in the callus tissues and the actual content in each callus tissue had the same variation pattern.
[0128] Summary: The abundance of SaAREB6 gene is high in the heartwood producing α-santalol. High expression of SaAREB6 gene can increase the yield of α-santalol, while silencing SaAREB6 can reduce α-santalol. SaAREB6 gene can promote the biosynthesis of α-santalol, increase the yield of α-santalol, and improve the efficiency of sandalwood in producing α-santalol, which has application prospects.
[0129] It is known from common technical knowledge that the present invention can be implemented by other embodiments that do not deviate from its spirit or essential features. Therefore, the above disclosed embodiments are only illustrative in all respects and are not exclusive. All changes within the scope of the present invention or within the scope equivalent to the present invention are included in the present invention.
Claims
1. A method for increasing the content of α-santalol in sandalwood tissue, the method comprising: using genetic engineering to highly express the sandalwood AREB6 gene in the sandalwood tissue; the protein sequence encoded by the sandalwood AREB6 gene is shown in SEQ ID NO.
2.
2. The method according to claim 1, characterized in that The variety of sandalwood is old mountain sandalwood.
3. The method according to claim 1 or 2, characterized in that The method comprises the following steps: Step 1: Transfer the sandalwood AREB6 gene into the Agrobacterium gene expression plasmid to obtain a recombinant plasmid; Step 2: transferring the recombinant plasmid into Agrobacterium to obtain recombinant Agrobacterium capable of expressing the sandalwood AREB6 gene; Step 3: transforming sandalwood plants with the recombinant Agrobacterium to increase the content of α-santalol in the sandalwood plants; or The recombinant Agrobacterium is used to transform sandalwood callus tissue, so that the content of α-santalol in the sandalwood callus tissue is increased.
4. Use of a biological material in preparing a preparation for increasing the content of α-santalol in sandalwood tissue, wherein the biological material is any one of the following B1, B2, B3, B4, B5, B6 and B7; B1: protein encoded by the Santalum album AREB6 gene; the protein sequence encoded by the Santalum album AREB6 gene is shown in SEQ ID NO.2; B2: a fusion protein, the amino acid sequence of which contains the amino acid sequence of the protein encoded by the Santalum AREB6 gene described in B1 and a tag peptide or fluorescent protein tag for separating and purifying the protein; B3: a gene, the coding sequence of which can encode the protein encoded by the Santalum album AREB6 gene described in B1 or the fusion protein described in B2; B4: a gene expression cassette, wherein the gene product in the gene expression cassette is the protein encoded by the Santalum AREB6 gene described in B1 or the fusion protein described in B2; B5: a genetic engineering vector, wherein the genetic engineering vector contains the gene expression cassette described in B4; B6: a cell, wherein the cell contains the genetic engineering expression vector described in B5, and the encoded protein in the gene expression cassette of the genetic engineering vector is constitutively expressed, tissue-specifically expressed, or artificially induced; as well as B7: A composition comprising the protein encoded by the Santalum album AREB6 gene described in B1, the fusion protein described in B2, the genetically engineered expression vector described in B5 or the cell described in B6.
5. The use according to claim 4, characterized in that The variety of sandalwood is old mountain sandalwood.