Application of NAC16 gene in Santalum album

Abstract

The invention discloses a method for improving sandalwood traits. The method comprises: using genetic engineering to highly express sandalwood NAC16 gene in sandalwood tissue; improving sandalwood traits comprises: increasing the content of α-santalol in sandalwood tissue; eliminating or slowing down yellowing of sandalwood leaves caused by drought; eliminating or slowing down the dwarfing of sandalwood plants caused by drought; eliminating or slowing down the carbon dioxide assimilation rate of sandalwood plants caused by drought; eliminating or slowing down the transpiration water loss of sandalwood leaves caused by drought; and eliminating or slowing down the biomass reduction of sandalwood plants caused by drought.

Description

Technical Field

[0001] The invention belongs to the field of plant genetic engineering, relates to the application of sandalwood NAC16 gene, and particularly relates to the application of sandalwood SaNAC16 gene in improving the drought resistance of sandalwood and the biosynthesis yield of santalol. Background Art

[0002] Drought is a major environmental stress factor affecting plant growth and development. Water shortage triggers a series of changes at the morphological, physiological, biochemical and molecular levels, which adversely affect plant growth, health and productivity. To avoid these negative effects, plants have also evolved various strategies to reduce water loss. Among these strategies, plants improve stress resistance by producing secondary metabolites (such as carotenoids, anthocyanins and terpenoids to increase water absorption capacity, reduce transpiration, and produce scavenging substances), which is a widespread and effective way.

[0003] Sandalwood (Santalum album L.) is a species of the genus Santalum (Santalaceae) in the family Santalaceae.

[0004] Sandalwood is an evergreen semi-parasitic tree. The secondary metabolite accumulated in its aromatic heartwood, sandalwood essential oil, is valuable and widely used in traditional Chinese medicine and cosmetics. It is known as "liquid gold". After decades of introduction and cultivation, although sandalwood can be planted, reproduced and produced in Guangdong, Hainan, Fujian, Yunnan and Sichuan, there are few large-scale high-quality artificial forests, and there are generally problems such as slow growth and low oil yield. Therefore, studying the formation mechanism of sandalwood essential oil is of great significance for the selection and breeding of excellent sandalwood varieties and the synthesis of sandalwood essential oil through metabolic engineering.

[0005] Santalols is the main component of sandalwood essential oil. The content of santalols is an important criterion for evaluating the quality of essential oils (ISO, 2002). The content of santalols in high-quality essential oils can reach more than 80%. Therefore, increasing the content of santalols in sandalwood essential oil is the key to essential oil production.

[0006] In recent years, the upstream genes SaAACT, SaHMGS, SaMK, SaPMK and the downstream key enzyme genes SaSSY and SaCYP736A167 in the mevalonic acid (MVA) pathway of santalol biosynthesis have been identified, but the molecular regulatory mechanisms of these key genes have rarely been reported.

[0007] The CAS number of α-santalol is: 115-71-9; the structural formula of α-santalol is:

[0008]

[0009] If the production of santalol in sandalwood can be increased through transgenic breeding or gene regulation, and the stress resistance of sandalwood can be improved at the same time, it will have great economic benefits. Summary of the invention

[0010] In order to solve the problems existing in the prior art, the first aspect of the present invention provides a method for improving the properties of sandalwood, the method comprising: using genetic engineering to highly express the sandalwood NAC16 gene in sandalwood tissue;

[0011] The improved sandalwood properties include:

[0012] Increasing the content of α-santalol in sandalwood tissue; and

[0013] Improve sandalwood's drought resistance.

[0014] In some embodiments, improving the drought resistance of sandalwood comprises:

[0015] Eliminate or slow down the yellowing of sandalwood leaves caused by drought;

[0016] Eliminate or slow down the dwarfing of sandalwood plants caused by drought;

[0017] Eliminate or mitigate the slowdown in carbon dioxide assimilation in sandalwood plants caused by drought;

[0018] Eliminate or slow down the loss of water through transpiration from the stomata of sandalwood leaves caused by drought; and

[0019] Eliminate or mitigate the reduction in sandalwood plant biomass caused by drought.

[0020] In some embodiments, the variety of sandalwood is Santalum album.

[0021] In some embodiments, the protein sequence encoded by the sandalwood NAC16 gene is shown as SEQ ID NO.2.

[0022] In some embodiments, the method comprises the steps of:

[0023] Step 1: Transfer the coding sequence of the sandalwood NAC16 gene into the Agrobacterium gene expression plasmid to obtain a recombinant plasmid;

[0024] Step 2: transferring the recombinant plasmid into Agrobacterium to obtain recombinant Agrobacterium capable of expressing the sandalwood NAC16 gene;

[0025] Step 3: transforming sandalwood plants with the recombinant Agrobacterium to obtain the sandalwood strain with improved traits.

[0026] In some embodiments, the Agrobacterium is Agrobacterium rhizogenes K599.

[0027] The second aspect of the present invention provides a use of a biomaterial in preparing a preparation for improving the properties of sandalwood;

[0028] The improved sandalwood properties include:

[0029] Increasing the content of α-santalol in sandalwood tissue; and

[0030] Improve sandalwood's drought resistance;

[0031] The biological material is any one of the following B1, B2, B3, B4, B5, B6, B7 and B8;

[0032] B1: protein encoded by the NAC16 gene of Santalum album;

[0033] B2: a fusion protein, the amino acid sequence of which contains the amino acid sequence of the protein encoded by the sandalwood NAC16 gene described in B1 and the amino acid sequence of a functional protein fragment or an inert protein fragment;

[0034] B3: RNA, which can be translated to obtain the protein encoded by the sandalwood NAC16 gene described in B1 or the fusion protein described in B2;

[0035] B4: a gene, the coding sequence of which can encode the protein encoded by the sandalwood NAC16 gene described in B1 or the fusion protein described in B2;

[0036] B5: a gene expression cassette, wherein the gene expression product in the gene expression cassette is the RNA described in B3;

[0037] B6: a genetic engineering vector, wherein the genetic engineering vector contains the gene expression cassette described in B5;

[0038] B7: a cell, wherein the cell contains the genetic engineering expression vector described in B6, wherein the encoded protein in the gene expression cassette of the genetic engineering vector is constitutively expressed, tissue-specifically expressed, or artificially induced; and

[0039] B8: A composition comprising the protein encoded by the sandalwood SaNAC16 gene described in B1, the fusion protein described in B2, the RNA described in B3, the genetically engineered expression vector described in B6 or the cell described in B7.

[0040] In some embodiments, selected from any one or a combination of the following C1, C2, C3 and C4;

[0041] C1: Improving the drought resistance of sandalwood includes:

[0042] Eliminate or slow down the yellowing of sandalwood leaves caused by drought;

[0043] Eliminate or slow down the dwarfing of sandalwood plants caused by drought;

[0044] Eliminate or mitigate the slowdown in carbon dioxide assimilation in sandalwood plants caused by drought;

[0045] Eliminate or slow down the loss of water through transpiration from the stomata of sandalwood leaves caused by drought; and

[0046] Eliminate or mitigate the reduction in sandalwood plant biomass caused by drought;

[0047] C2: The functional protein fragment is a tag peptide, a fluorescent protein tag and / or a signal peptide used to separate and purify the protein;

[0048] C3: The protein sequence encoded by the sandalwood NAC16 gene is shown in SEQ ID NO.2;

[0049] C4: The variety of sandalwood is old mountain sandalwood.

[0050] The third aspect of the present invention provides a qPCR primer combination for evaluating the ability of sandalwood tissue to synthesize α-santalol and / or the drought resistance of sandalwood. 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.

[0051] In some embodiments, the variety of sandalwood is Santalum album. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 This is a PCR gel photo of the SaNAC16 gene amplification results.

[0053] Figure 2 This is a statistical diagram of the tissue expression characteristics of the SaNAC16 gene.

[0054] Figure 3 This is a map of plasmid SaNAC16-OE.

[0055] Figure 4 This is the electrophoresis diagram for transgenic line detection.

[0056] Figure 5 Shown are the santalol contents of SaNAC16 transgenic plants and controls under drought stress.

[0057] Figure 6 These are photos of the phenotypes of SaNAC16 transgenic plants and controls under drought stress.

[0058] Figure 7 Shown are the plant heights of SaNAC16 transgenic plants and controls under drought stress.

[0059] Figure 8The graph shows the photosynthetic rate of SaNAC16 transgenic plants and controls under drought stress.

[0060] Fig. 9 Electron microscopic images of leaf stomata of SaNAC16 transgenic plants and controls under drought stress.

[0061] Fig.10 Shown are the biomass of SaNAC16 transgenic plants and controls under drought stress. DETAILED DESCRIPTION

[0062] 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.

[0063] Materials and instruments not described in the present invention are conventional materials and instruments in the art, and operation details not described in the present invention are conventional operations in the art. Unless otherwise specified, the nucleic acid sequences shown in the present invention are written from left to right in the direction of 5' to 3'.

[0064] Example 1: Cloning of the Santalum album NAC16 gene

[0065] The sandalwood variety used in the present invention is Indian sandalwood (Old Mountain Sandalwood).

[0066] The genome of Santalum album L. was sequenced and assembled using the second generation sequencing technology, and the genome sequence was not made public. According to the rules for naming homologous genes, the NAC16 gene of Santalum album L. (Sa is the abbreviation of Santalum album L., and the function of SaNAC16 transcription factor has not been reported yet) was annotated. NAC transcription factors are a family of transcription factors unique to plants, and their names are taken from the first letters of the NAM (no apical meristem) gene of Petunia hybrida, the ATAF1 / 2 gene of Arabidopsis thaliana, and the CUC2 (cup-shaped cotyledon) gene. SaNAC16 represents the 16th member of the NAC gene family of Santalum album L.

[0067] The coding sequence of the SaNAC16 gene obtained by the aforementioned sequencing is as follows (SEQ ID NO.1):

[0068] 5’-ATGGGAGGGGCATCATTGCCGCCCGGATTTCGGTTCCACCCGACCGATGAGGAATTGGTTGGATATTACCTGAAAAGGAAAGTTGAAGGACTGGAGATTGAGCTTGAAGTAATCCCTGTGATTGATCTGTGCAAGTTCGATCCTTGGGAACTGCCAGAACAATCTTTCCTTCCAAATAAAGACATGGAATGGTTCTTTTTCTGCCCCCGTGGCAAAAAGTACCCGAACGGCTTACGTACAAATAGGGCCACCAGATCCGGCTACTGGAAAGTCACTGGAAAAGACCGTAAAGTAGCTTGTCAATCTTCTGTGACTGGGTACCGTAAGACCTTGGTTTTTTATCGGGGACGAGCCCCTTCGGGTAGTCGAACCGATTGGATGATGAATGAGTATCGACTCTGTGATGACTTTTCACGGGGAGCCCCTAGCTTTCAGGGAGCTTTTGCTTTATGCCGAGTTGTCAAAAAGAATGACAATAGTTTCAAAAATGACTGCCATGGAGAATCGAATGCTAAGGGGGCTGGAGGCAG TTCAGGCAATGGAGATTTCAGCTCACGTGGAAGCTTAAATGAGCCTTTGAGCAGCTCCAGTGATGTTCCTACCCCTTCTCCTTGTGAAGTTCCTATTCCTTCTACTTGTGAATTTAATCCAATGGCTGAACTGAATCGAGCCACAATGGTACATGATCATAAAAGCCTATGGGTCTCACCTGATCTGATTCTCGACTCTTCTAAGGCGTACACACAAGGACAAGAAGGCGTATCCGAATGCTT TCCACAGTATGAGTTTCCCAATTCCATGACTCCATGGAGGCCATACACTTATGGGGAAATCTCATCCAGTTTGTCATACTCGAATTTTACAGAGGAAGTTGATACCGCTGAGGATCTTAGCCGAATCGGTTGCATGTCACCTACCAAGGGAATCCCAACTACTTTGGTTGGTGTGGAAATGAAGGGATACCCTATGAAGGTCATGACCAGATTAATTCACTGAGAAATCCAAAACCCTTTTGA

[0069] The amino acid sequence of the protein encoded by the SaNAC16 gene is as follows (SEQ ID NO.2):

[0070] MGGASLPPGFRFHPTDEELVGYYLKRKVEGLEIELEVIPVIDLCKFDPWELPEQSFLPNKDMEWFFFCPRGKKYPNGLRTNRATRSGYWKVTGKDRKVACQSSVTGYRKTLVFYRGRAPSGSRTDWMMNEYRLCDDFSRGAPSFQGAFALCRVVKKNDNSFKNDCHGES NAKGAGGSSSGNGDFSSRGSLNEPLSSSSDVPTPSPCEVPIPSTCEFNPMAELNRATMVHDHKSLWVSPDLILDSSKAYTQGQEGVSECFPQYEFPNSMTPWRPYTYGEISSSLSYSNFTEEVDTAEDLSRIGCMSPYQGNPNYFGWCGNEGIPYEGHDQINSLRNPKPF

[0071] According to the aforementioned SaNAC16 gene sequence, the upstream primer F1 of SaNAC16 gene and the downstream primer R1 of SaNAC16 gene were designed. The total RNA was extracted from the leaf tissue of 16-year-old sandalwood, 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 SaNAC16 gene amplification product.

[0072] F1 (SEQ ID NO.3):

[0073] 5'-ATGGGAGGGGCATCATTGCCGC

[0074] R1 (SEQ ID NO.4):

[0075] 5'-TCAAAAGGGTTTTGGATTTCTC

[0076] See the agarose gel electrophoresis photos of the above PCR amplification products for details. Figure 1 Among 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 SaNAC16 gene coding sequence of 1017bp. Based on this preliminary judgment, PCR successfully amplified the SaNAC16 gene.

[0077] The amplified product was connected to the sequencing vector pMD 19-T Vector (Takara, Dalian, China), and the inserted sequence was sequenced by Sanger method, and the result was the same as the aforementioned SEQ ID NO. 1. Thus, a recombinant plasmid containing the SaNAC16 gene coding sequence was obtained.

[0078] Example 2: Study on tissue expression characteristics of SaNAC16

[0079] 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.

[0080] qPCR primers F2 and R2 were designed according to the SaNAC16 gene sequence, and qPCR internal reference primers F3 and R3 were designed according to the Santalum Actin gene sequence.

[0081] F2 (SEQ ID NO.5):

[0082] 5'-ATTTCAGCTCACGTGGAAGC

[0083] R2 (SEQ ID NO.6):

[0084] 5'-GTGGCTCGATTCAGTTCAGC

[0085] F3 (SEQ ID NO.7):

[0086] 5'-TGCTCTTCCCCATGCCAT

[0087] R3 (SEQ ID NO.8):

[0088] 5'-AACAATTTCCCGCTCAGCAG

[0089] All reactions were performed with three biological replicates and four technical replicates.

[0090] The reaction system (20 μL) was as follows: 2×SYBR Green qPCR Mix 10 μL, RNase Free delH2O 8 μL, cDNA 1 μL, and 0.5 μL of each upstream and downstream primers.

[0091] The expression level of SaNAC16 gene was normalized using the Santalum album Actin gene as an internal reference gene. -ΔΔCtFinally, SPSS Statistics 27 software was used to analyze whether there were significant differences in gene expression.

[0092] The expression level of SaNAC16 gene in the root was normalized, and the relative expression levels of SaNAC16 gene in the root, haustoria, leaf, phloem, sapwood and heartwood were calculated.

[0093] The statistical results can be found in Figure 2 It can be seen that the expression level of SaNAC16 gene in sapwood and heartwood is significantly higher than that in other tissues.

[0094] Heartwood is the main santalol-producing tissue, and it is speculated that the SaNAC16 gene may regulate the biosynthesis of santalol.

[0095] Example 3: Preparation of SaNAC16 gene high expression vector

[0096] 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.

[0097] The recombinant sequencing vector containing the SaNAC16 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: 10 μL of 2×Phanta Max Master Mix, 8 μL of RNase FreeddH2O, 1 μL of cDNA, and 0.5 μL of each of the upstream and downstream primers.

[0098] F4 (SEQ ID NO.9):

[0099]

[0100] R4 (SEQ ID NO.10):

[0101]

[0102] 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.

[0103] 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.

[0104] 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.

[0105] 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, is not restricted by the restriction sites of the vector and the target fragment, and 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, which can achieve efficient directional seamless cloning of the blunt-end PCR product fragment into the vector within 30 minutes) according to the instructions of the kit manual, and the above-mentioned purified PCR product and the restriction-cut pBWA (V) HS-GFP vector are homologously recombined to obtain the recombinant vector. The homologous recombination system is as follows: 5×CEⅡBuffer 4μL, linearized vector 2μL, target gene fragment 6μL, ExnaseⅡ2μL, ddH2O 6μL. The reaction system is mixed and centrifuged briefly. The reaction conditions are 37℃, 30min. After the reaction, 0.1-10 ng of the ligation product was taken to transform Escherichia coli, and the bacterial solution was applied to LB solid medium (containing 50 μg / mL kanamycin), placed in an incubator at 37°C and inverted for overnight culture, and a single colony was picked and placed in LB liquid medium (containing 50 μg / mL kanamycin) and shaken at 37°C and 180 rpm. The turbid bacterial solution cultured overnight was subjected to PCR using primers F1 and R1 in Example 1, and the positive clones that were successfully connected were screened by electrophoresis of the amplified products for plasmid extraction. Thus, SaNAC16 was constructed onto the pBWA(V)HS expression vector to obtain the recombinant vector pBWA(V)HS-SaNAC16, and the plasmid was named SaNAC16-OE, which fused and expressed SaNAC16 and GFP.

[0106] 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 is shown in the upper figure, the structure between LB and TR in the recombinant vector is shown in the middle figure, and the complete structure of the recombinant plasmid SaNAC16-OE is shown in the lower figure.

[0107] Example 4: Transformation of the drought resistance gene SaNAC16 in the roots of sandalwood

[0108] SaNAC16-OE (experimental group) and empty vector (control group, pBWA(V)HS-GFP) were respectively transferred into Agrobacterium rhizogenes K599 (purchased from Shanghai Weidi Biological, catalog number AC1080S), and the positive single colony was picked and inoculated into YEB liquid culture medium, and cultured at 28°C with shaking for 48 hours. Centrifuge and discard the supernatant. Healthy and uniformly growing sandalwood seedlings (6-month-old conventional tissue culture seedlings, plant height 6 cm) were selected, and Agrobacterium infection was performed using the following injection procedure. The colonies carrying the empty vector and SaNAC16-OE were respectively placed in 5mL YEP liquid culture medium, 20 mg / L rifampicin and 50 mg / L kanamycin were added, and cultured at 28°C with shaking at 180 rpm for about 12-14 hours. When OD 600 When the value reaches 0.6-0.8, the culture is centrifuged at 8000rpm for 10 minutes at room temperature and then resuspended in MES buffer (10mM morpholineethanesulfonic acid, pH 5.2, 10mM MgCl2 and 100μM acetosyringone). The Agrobacterium suspension is injected into the base of the stem of the aforementioned sandalwood seedlings, 0.1ml per injection site. 10 days after injection, callus tissue grows at the injection site of the stem. About one month later, the root-inducing Agrobacterium tumefaciens callus naturally grows new roots, and the original root system is cut off to obtain root transgenic sandalwood plants. For 6 transgenic strains, tissue culture seedlings are cultured 3 months later (plant height 9cm), and the aerial parts are cut off along the base of the stem at this time. After 1 month, cluster buds (bud height 2cm) are reborn, and transgenic seedlings of conventional subculture are obtained after root division. The roots of conventional subcultured transgenic sandalwood plants (numbered S1 to S6) and the roots of four wild-type plant tissue culture seedlings (numbered W1 to W4) were taken and tested using PCR (the PCR primers and reaction conditions were the same as those in Example 1).

[0109] PCR product electrophoresis results refer to Figure 4 , where the first lane is the molecular weight marker, and the other lane numbers are sample numbers. It can be seen that the four wild-type plants have no electrophoresis bands, and four of the six transgenic lines (S1, S3, S4, and S6) show bands. Among them, S1 (SaNAC16-OE#1) and S4 (SaNAC16-OE#4) were selected as subsequent experimental materials.

[0110] Example 5: Performance test of root transgenic sandalwood

[0111] 1. Santalol Content

[0112] Take 2g of sandalwood roots of wild type (WT), SaNAC16-OE#1, and SaNAC16-OE#4 conventional subculture root transgenic lines, place them in envelopes, and dry them naturally at room temperature for 2 weeks. The dried samples were 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 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, and 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, and room temperature for 5min. Transfer the supernatant to the same sample bottle, and combine the liquids from the two extractions, with a total volume of about 2mL. The solution was concentrated by nitrogen blowing to about 500 μL from 2 mL, and the α-santalol content in the root tissue was determined by GC-MS.

[0113] Results Figure 5 Compared with the control, the α-santalol content in the roots of the transgenic lines SaNAC16-OE#1 and SaNAC16-OE#4 was significantly higher than that of the wild-type control.

[0114] 2. Drought resistance test of genetically modified sandalwood roots

[0115] For long-term drought treatment, wild-type (WT) and conventional subculture root transgenic lines SaNAC16-OE#1, SaNAC16-OE#4 sandalwood plants were potted in a greenhouse (natural light; day and night temperature: 25 / 20°C; relative humidity: 75%), and the soil water potential (ψsoil) was maintained at -1.52MPa (simulating water shortage conditions, drought state). The soil water potential was measured using the PSYPRO Water Potential System (WESCOR, Utah, USA) for 40 days. Watering was performed at 17:00 every afternoon to maintain the soil water potential of the control and drought treatments.

[0116] The appearance of the three strains mentioned above can be found in Figure 6 .Depend on Figure 6 It can be seen (the three sandalwood seedlings from left to right represent the wild type, #1, and #4, respectively, and part of the plastic pot is retained to indicate the relative size of the photo), after 40 days of drought, the leaves of the wild type sandalwood turned yellow, while the transformed plants SaNAC16-OE#1 and SaNAC16-OE#4 had no obvious effect. This shows that the drought resistance of transgenic sandalwood is significantly enhanced.

[0117] 3. Root system transgenic sandalwood plant height test

[0118] Wild-type (WT) and conventional subculture root transgenic SaNAC16-OE#1, SaNAC16-OE#4 sandalwood plants were potted in a greenhouse (natural light; day and night temperature: 25 / 20℃; relative humidity: 75%), and the soil water potential (ψsoil) was maintained at -1.52MPa (simulating water shortage conditions, drought state) for 40 days. Plant height results refer to Figure 7 , with 6 replicates for each strain.

[0119] Depend on Figure 7 It can be seen that there is no significant difference in plant height between SaNAC16-OE#1 and SaNAC16-OE#4, and both are significantly taller than the wild-type plant, indicating that transgenic sandalwood has a growth advantage under drought stress.

[0120] 4. Photosynthetic capacity test of transgenic sandalwood roots

[0121] Wild-type (WT) and conventional subculture root transgenic SaNAC16-OE#1, SaNAC16-OE#4 sandalwood plants were potted in a greenhouse (natural light; day and night temperature: 25 / 20°C; relative humidity: 75%), and the soil water potential (ψsoil) was maintained at -1.52MPa (simulating water shortage conditions, drought state). The soil water potential was measured using the PSYPRO Water Potential System (WESCOR, Utah, USA) for 40 days. The soil water potential of the control and drought treatments was maintained by watering at 17:00 every afternoon. The photosynthetic capacity was tested every 10 days, and the carbon dioxide assimilation rate was measured using the photosynthetic meter LI-COR 6400 (Lincoln, NE, USA). For the same results, see Figure 8 , 6 replicates, the three columns at each time point are the results of WT, #1, and #4 from left to right.

[0122] Depend on Figure 8 It can be seen that there is no significant difference in the photosynthetic rate between the two strains SaNAC16-OE#1 and SaNAC16-OE#4, but both are higher than the wild-type strain. This shows that after the sandalwood is transformed with the SaNAC16 gene, the carbon dioxide assimilation rate can be significantly increased, which will make the transgenic sandalwood grow faster and the sandalwood seedlings can mature faster. The accelerated growth of the transgenic sandalwood is conducive to faster reduction of the greenhouse effect of carbon dioxide in the atmosphere.

[0123] 5. Test on the stomata opening degree of genetically modified sandalwood roots

[0124] Wild-type (WT) and conventional subculture root transgenic SaNAC16-OE#1, SaNAC16-OE#4 sandalwood plants were potted in a greenhouse (natural light; day and night temperature: 25 / 20°C; relative humidity: 75%), and the soil water potential (ψsoil) was maintained at -1.52 MPa (simulating water shortage conditions, drought state). The soil water potential was measured using the PSYPRO Water Potential System (WESCOR, Utah, USA) for 40 days. Mature leaves (leaf phyllotaxy index = 6-8) of wild-type (WT) and transgenic plants were removed and immediately frozen with liquid nitrogen. The samples were then transferred to a -80°C ultra-low temperature freeze dryer (Biosafer-18A) and dried for 24 hours. After the samples were gold-coated with an ion sputtering coater (Eiko Engineering, Japan, IB-2), stomata were observed using a scanning electron microscope (Hitachi S-4700, Japan) with an accelerating voltage of 10 kV.

[0125] Results Fig. 9 It can be seen that compared with the wild type, the stomata of the transgenic lines are significantly more closed, which is beneficial to reduce water loss caused by transpiration.

[0126] 6. Root biomass test of genetically modified sandalwood

[0127] Wild-type (WT) and conventional subculture root transgenic SaNAC16-OE#1, SaNAC16-OE#4 sandalwood plants were potted in a greenhouse (natural light; day and night temperature: 25 / 20°C; relative humidity: 75%), and the soil water potential (ψsoil) was maintained at -1.52MPa (simulating water shortage conditions, drought state). The soil water potential was measured using the PSYPRO Water Potential System (WESCOR, Utah, USA) for 40 days. Watering was performed at 17:00 every afternoon to maintain the soil water potential of the drought treatment. Six biological replicates were set up for each strain. At this time, the plant height of the wild-type WT plant was 10.58±0.39 cm, the plant height of the transgenic line SaNAC16-OE#1 plant was 14.28±0.37 cm, and the plant height of the transgenic line SaNAC16-OE#4 plant was 13.66±1.41 cm. After the whole plant was harvested, it was dried in an oven at 72°C for 3 days, and the dry weight of sandalwood was determined by a balance, which was the sandalwood biomass.

[0128] Results Fig.10 It can be seen that the biomass of the root transgenic sandalwood strains is significantly higher than that of the wild type, and the transgenic strains grow faster, which is conducive to the rapid development and utilization of sandalwood wood.

[0129] Summary: The abundance of SaNAC16 gene is high in the heartwood producing α-santalol. High expression of SaNAC16 gene can increase the yield of santalol and significantly improve the drought resistance of sandalwood, allowing it to grow normally or near-normally.

[0130] 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 improving the properties of sandalwood, the method comprising: using genetic engineering to highly express the sandalwood NAC16 gene in sandalwood tissue; The protein sequence encoded by the sandalwood NAC16 gene is shown in SEQ ID NO.2; The improved sandalwood properties include: Increase the content of α-santalol in sandalwood tissue; as well as Improve sandalwood's drought resistance.

2. The method according to claim 1, characterized in that The improved drought resistance of sandalwood comprises: Eliminate or slow down the yellowing of sandalwood leaves caused by drought; Eliminate or slow down the dwarfing of sandalwood plants caused by drought; Eliminate or mitigate the slowdown in carbon dioxide assimilation in sandalwood plants caused by drought; Eliminate or slow down the loss of water through transpiration from the stomata of sandalwood leaves caused by drought; and Eliminate or mitigate the reduction in sandalwood plant biomass caused by drought.

3. The method according to claim 1, characterized in that The variety of sandalwood is old mountain sandalwood.

4. The method according to any one of claims 1 to 3, characterized in that The method comprises the following steps: Step 1: The coding sequence of the sandalwood NAC16 gene is transferred into the Agrobacterium gene expression plasmid to obtain a recombinant plasmid; the protein sequence encoded by the sandalwood NAC16 gene is shown in SEQ ID NO.2; Step 2: transferring the recombinant plasmid into Agrobacterium to obtain recombinant Agrobacterium capable of expressing the sandalwood NAC16 gene; Step 3: transforming sandalwood plants with the recombinant Agrobacterium to obtain the sandalwood strain with improved traits.

5. The method according to claim 4, characterized in that The Agrobacterium is Agrobacterium rhizogenes K599.

6. Use of a biomaterial in preparing a preparation for improving the properties of sandalwood; The improved sandalwood properties include: Increase the content of α-santalol in sandalwood tissue; as well as Improve sandalwood's drought resistance; The biological material is any one of the following B1, B2, B3, B4, B5, B6, B7 and B8; B1: protein encoded by the NAC16 gene of Santalum album; the protein sequence encoded by the NAC16 gene of Santalum album 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 sandalwood NAC16 gene described in B1 and the amino acid sequence of a tag peptide, a fluorescent protein tag and / or a signal peptide used for separating and purifying the protein; B3: RNA, which can be translated to obtain the protein encoded by the sandalwood NAC16 gene described in B1 or the fusion protein described in B2; B4: a gene, the coding sequence of which can encode the protein encoded by the sandalwood NAC16 gene described in B1 or the fusion protein described in B2; B5: a gene expression cassette, wherein the gene expression product in the gene expression cassette is the RNA described in B3; B6: a genetic engineering vector, wherein the genetic engineering vector contains the gene expression cassette described in B5; B7: a cell, wherein the cell contains the genetic engineering expression vector described in B6, wherein the encoded protein in the gene expression cassette of the genetic engineering vector is constitutively expressed, tissue-specifically expressed, or artificially induced; and B8: A composition comprising the protein encoded by the sandalwood SaNAC16 gene described in B1, the fusion protein described in B2, the RNA described in B3, the genetically engineered expression vector described in B6 or the cell described in B7.

7. The use according to claim 6, characterized in that Selected from the following C1 and / or C2; C1: Improving the drought resistance of sandalwood includes: Eliminate or slow down the yellowing of sandalwood leaves caused by drought; Eliminate or slow down the dwarfing of sandalwood plants caused by drought; Eliminate or mitigate the slowdown in carbon dioxide assimilation in sandalwood plants caused by drought; Eliminate or slow down the loss of water through transpiration from the stomata of sandalwood leaves caused by drought; and Eliminate or mitigate the reduction in sandalwood plant biomass caused by drought; C2: The variety of sandalwood is old mountain sandalwood.

8. A qPCR primer combination for evaluating the ability of sandalwood tissue to synthesize α-santalol and / or the drought resistance of sandalwood, wherein the upstream primer sequence is shown in SEQ ID NO.5, and the downstream primer sequence is shown in SEQ ID NO.

6.

9. The primer combination according to claim 8, characterized in that The variety of sandalwood is old mountain sandalwood.

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