Mint gene as well as cloning method and application thereof

Abstract

The invention provides a mint gene as well as a cloning method and application thereof. The nucleotide sequence of the mint gene is shown as SEQ ID NO.1. According to the invention, MTPS1 gene cloning is carried out from mint; expression sequences in leaves are assembled by using mint transcriptome sequencing; then designing a PCR (Polymerase Chain Reaction) specific primer by utilizing primer design software Primer Premier, extracting total RNA (Ribonucleic Acid) from mint leaves, carrying out reverse transcription PCR, and cloning to obtain the MTPS1 gene. When MTPS1 is expressed in tomatoes, synthesis of volatile aromatic substances in fruits can be changed, and it can be seen that the mint gene can change volatile aroma components of the plant fruits.

Description

Technical Field

[0001] The invention belongs to the technical field of plant genetic engineering, and specifically relates to a mint gene and a cloning method and application thereof. Background Art

[0002] Mint (Mentha spp.) belongs to the genus Mentha of the Lamiaceae family. There are abundant wild plant resources in East China and Northeast my country. The cultivation and consumption of Mentha plants has a long history in my country. At the same time, it is also the most important medicinal plant and spice crop. As a natural plant resource for extracting volatile terpene compounds, mint is used in daily chemicals, food, medicine and other fields for its unique aroma and excellent medicinal value, and has important economic value. Similar to other Lamiaceae plants, mint also shows excellent environmental adaptability. It is cultivated in most parts of my country and used for the extraction of mint oil, making China, the United States and India the world's three largest mint oil producing countries.

[0003] The wide application of mint plants is mainly due to their naturally synthesized volatile substances, mainly monoterpenes and sesquiterpenes, including dozens of terpenes, derived alcohols, ketones, esters and their isomers. Among them, the volatile components with higher content include menthol, menthone, pulegone, eucalyptol, linalool, piperitone, and L-carvone. At present, the framework of the biosynthetic pathway of terpenoids in plants has been speculated, mainly mevalonate pathway (MVA pathway) and methylerythritol-4-phosphate pathway (MEP pathway). The synthesis of mint terpenes is believed to be mainly based on the MEP pathway. MEP is converted into geranyl diphosphate (GPP) through multi-step reactions, and then converted into various terpenoid components through hydroxylation, methylation, demethylation or isomerization by monoterpene synthase (TPS). The catalytic reaction of TPS determines the final product and rate of the synthesis of mint volatile components.

[0004] Therefore, cloning and analyzing the TPS gene of mint is not only of great significance for understanding the biosynthesis and composition differences of volatile substances in mint plants with rich aroma types, but also provides a possibility for obtaining new gene resources and using them for improving plant genetic traits. Summary of the invention

[0005] The invention aims to provide a mint gene and a cloning method thereof, clone a gene sequence from mint and name it Mint Terpene Synthase 1 (MTPS1 for short); clone the MTPS1 gene from mint: assemble and annotate the highly abundant expression sequences in mint leaves by transcriptome sequencing; then design PCR specific primers by using primer design software PrimerPremier, extract total RNA from mint leaves, perform reverse transcription-PCR (RT-PCR), and clone and obtain the MTPS1 gene.

[0006] Another object of the present invention is to provide the use of the mint gene in improving the synthesis of volatile substances in fruits, wherein the mint gene can change the volatile aroma components of plant fruits.

[0007] Another object of the present invention is to provide an expression vector containing the mint gene and a genetically engineered bacterium containing the expression vector.

[0008] The technical solution adopted by the present invention is as follows:

[0009] A mint gene, the mint gene is referred to as MTPS1, and its nucleotide sequence is shown as SEQ ID NO.1.

[0010] The amino acid sequence encoded by the mint gene is shown in SEQ ID NO.2.

[0011] The invention also provides an expression vector containing the mint gene.

[0012] Furthermore, the expression vector is obtained by inserting the mint gene into a plant expression vector pBI121.

[0013] The invention also provides a genetic engineering bacterium containing the expression vector.

[0014] Furthermore, the genetically engineered bacteria are obtained by transferring the expression vector into Escherichia coli or Agrobacterium and culturing the bacteria.

[0015] The present invention also provides a method for cloning the mint gene, which comprises the following steps:

[0016] 1) RNA extraction from mint leaf tissue;

[0017] 2) Reverse transcription to obtain cDNA;

[0018] 3) Design primer sequences and use the obtained cDNA to clone the MTPS1 gene.

[0019] In step 3), the primer sequences are specifically designed as follows: MTPS1F, whose nucleotide sequence is shown in SEQ ID NO.3; MTPS1R, whose nucleotide sequence is shown in SEQ ID NO.4.

[0020] The reagents used in step 3) are: place a 200 μL EP tube on ice and add the following reagents: 0.5 μL high-fidelity DNA polymerase; 10 μL 5× Buffer; 4 μL dNTP; 1 μL cDNA; 1 μL MTPS1F; 1 μL MTPS1R; and 32.5 μL ddH2O.

[0021] In step 3), the amplification program is: pre-denaturation at 95°C for 1 min; denaturation at 95°C for 20 s, annealing at 55°C for 20 s, extension at 72°C for 70 s, wherein 30 cycles of denaturation-annealing-extension are performed; and total extension at 72°C for 5 min.

[0022] The invention also provides application of the mint gene in improving the synthesis of volatile substances in fruits.

[0023] The present invention cloned the MTPS1 gene from mint. When MTPS1 is expressed in tomatoes through genetic transformation, the volatile substance components in tomato fruits are changed. Compared with the wild type, eucalyptol is synthesized in the tomato fruits transformed with MTPS1 expression. The gene with the nucleotide sequence shown in SEQ ID NO.1 plays a role in the synthesis of volatile components of fruits. The MTPS1 gene cloned from mint in the present invention can be used to change the flavor of fruits.

[0024] Compared with the prior art, the mint gene MTPS1 of the present invention provides a new gene resource for plant trait improvement and can be used to change the volatile aroma components of plant fruits. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Colony PCR identification of MTPS1 gene connected to pEASY-Blunt cloning vector, in the figure: M is DNA marker; 1-5 are amplified bands of single Escherichia coli colonies transformed with MTPS1.

[0026] Figure 2 Colony PCR identification of Agrobacterium transformed with plant expression vector pBI121-MTPS1, in the figure: M is DNA marker; 1-8 are amplification bands of single Agrobacterium colonies transformed with pBI121-MTPS1; + is the positive control amplification band of pEASY-MTPS1 plasmid; - is the negative amplification control of untransformed Agrobacterium.

[0027] Figure 3Quantitative PCR identification of MTPS1 gene tomato genetic transformation plants. In the figure: WT is wild-type tomato; MTPS1-OE is MTPS1 gene transformed tomato.

[0028] Figure 4 The expression of the MTPS1 gene changes the volatile aroma components of tomato fruit. Upper figure: purge and trap chromatogram of wild-type tomato fruit; lower figure: purge and trap chromatogram of MTPS1-transformed tomato fruit. DETAILED DESCRIPTION

[0029] The present invention is described in detail below with reference to the embodiments.

[0030] In the following examples, where no specific experimental conditions are specified, they are based on conventional conditions well known to those skilled in the art, such as the conditions described in Sambrook J. and Russell, DW Molecular Cloning Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 2001), or the conditions recommended by the manufacturer.

[0031] Example 1 Detection of Volatile Components in Mint Leaves

[0032] 1. Plant materials

[0033] The mint material ‘M1’ was obtained from its native habitat in Lu’an, Anhui Province, and was planted in an artificial climate chamber at 25°C, 12 h light / 20°C, 12 h dark.

[0034] 2. Methods

[0035] 1) Sample preparation: Collect fresh leaves of mint plants with the same growth status, quickly cut them into cubes of about 0.5 cm in size, mix them, randomly take 0.5 g and place them in a 40 mL purge bottle, add 10 mL of pure water, seal it and then inject it for testing.

[0036] 2) Purge and trap conditions

[0037] The prepared sample was injected through an automatic sampler. High-purity nitrogen was used for purging, with a pre-purge time of 0.2 min, a purge time of 13 min, a purge temperature of 20 ° C, and a purge flow rate of 40 mL / min. After the volatile components were adsorbed on the adsorbent, the trap was heated to 200 ° C, the trapped components were desorbed by helium for 0.5 min, and then directly transferred to the gas chromatography system.

[0038] 3) Chromatographic conditions

[0039] Chromatographic column: HP-5MS capillary column (30m×250μm, 0.24μm); injection port temperature 250℃, carrier gas helium flow rate 1mL / min, split ratio 15:1; temperature program: 40℃ for 3min, 8℃ / min to 100℃, then 10℃ / min to 280℃ for 10min.

[0040] 4) Mass spectrometry conditions

[0041] Electron bombardment ion source, electron energy 70eV, ion source temperature 230℃, transfer line temperature 280℃, quadrupole temperature 150℃; scanning range 33-500m / z, full scan mode.

[0042] 5) The mass spectrometry results were searched using the standard mass spectrometry database NIST to identify the chemical composition of each test sample.

[0043] 3. Results

[0044] The volatile components of fresh leaves of mint 'M1' were detected and analyzed by purge and trap-gas chromatography-mass spectrometry. 19 terpenoids were synthesized, with a relative content of 21.83%, including 5.89% of myrcene, 7.10% of pinene, 4.31% of caryophyllene, and 1.28% of ocimene. In addition, mint leaves also contain a high content of monoterpene alcohol eucalyptol, with a relative content of 26.68%. This shows that mint 'M1' contains a high content of terpenes and their derived volatile substances, suggesting that its terpene synthase may have high activity.

[0045] Example 2 Transcriptome Sequencing of Mint Leaf Tissue

[0046] In order to obtain functional gene expression sequences, leaf tissues of mature mint plants were taken for transcriptome sequence determination and assembly annotation.

[0047] 1. Reagents

[0048] Plant RNA extraction kit was purchased from Beijing Quanshijin Biotechnology Co., Ltd., DNase I was purchased from Sangon Biotechnology (Shanghai) Co., Ltd., and RNA library preparation kit (Illumina Truseq TM RNA sample prep Kit) was from Meiji Biotechnology Co., Ltd., and the remaining reagents were imported or domestic analytical grade products.

[0049] 2. Plant materials

[0050] Plants of mint 'M1' were grown in an artificial climate chamber at 25°C, 12 h light / 20°C, 12 h dark.

[0051] 3. Methods

[0052] 3.1 RNA extraction

[0053] Mint leaf tissue was collected for RNA extraction, and the experimental procedures were carried out according to the kit manufacturer's instructions.

[0054] 1) Grind the mint tissue into powder in liquid nitrogen quickly, take 100 mg of tissue and add 500 μL of BB6 solution (add 10 μL of β-mercaptoethanol to every 1 mL of BB6, prepare it before use, and vortex vigorously to mix). Incubate at room temperature for 3 minutes;

[0055] 2) Centrifuge at 12000g for 2-5 minutes, and transfer the supernatant from the centrifuge tube to an RNase-free centrifuge tube;

[0056] 3) Add 0.5 times the volume of anhydrous ethanol to the supernatant and mix well;

[0057] 4) Vortex thoroughly to mix and disperse the precipitate;

[0058] 5) Add the obtained solution and precipitate to the centrifuge column, centrifuge at 12000g for 30s, and discard the effluent;

[0059] 6) Add 500 μL CB6 solution, centrifuge at 12000 g for 30 s at room temperature, and discard the flow-through;

[0060] 7) Add 80 μL of DNase I working solution to the center of the centrifuge column (DNase I working solution preparation: take 70 μL of Reaction Buffer into an RNase-free tube, then add 30 U of DNase I and mix well), leave at room temperature for 15 minutes, and repeat step 6);

[0061] 8) Add 500 μL WB6 solution (add anhydrous ethanol before use), centrifuge at 12000 g for 30 s, and discard the flow-through;

[0062] 9) Repeat step 8);

[0063] 10) Centrifuge at 12000g for 2 min at room temperature to completely remove residual ethanol;

[0064] 11) Add 50 μL RNase-free ddH2O to the center of the centrifuge column and let stand at room temperature for 1 min;

[0065] 12) Centrifuge at 12000g for 2 min at room temperature to elute RNA;

[0066] 13) Check the purity and concentration of RNA samples and store the RNA at -80°C.

[0067] 3.2 Transcriptome sequencing and assembly annotation

[0068] Transcriptome sequencing using Illumina TruSeq TM RNA sample prep Kit, using Illumina Novasek 6000 sequencing platform, follow the steps below:

[0069] 1) Enrich mRNA using magnetic beads with Oligo dT and randomly shear the mRNA;

[0070] 2) Synthesize cDNA chain using mRNA as template and purify cDNA using beads;

[0071] 3) The purified double-stranded cDNA is then end-repaired and connected to sequencing adapters, and then the fragment size is selected using micro beads, and the cDNA library is obtained by PCR enrichment;

[0072] 4) Detect the concentration and insert size of the library;

[0073] 5) Sequence the cDNA library using the Illumina Novaseq 6000 high-throughput sequencing platform;

[0074] 6) Remove the adapter sequence, non-A / C / T / G bases at the 5' end, and low-quality sequences from the original sequence of the sequencing fragments (reads) to obtain high-quality sequencing data;

[0075] 7) Use Trinity (https: / / github.com / trinityrnaseq / trinityrnaseq / wiki) to assemble the filtered raw sequence data;

[0076] 8) All transcripts obtained by transcriptome sequencing assembly were compared with NR (NCBI Non-redundant Protein Sequences Database, https: / / www.ncbi.nlm.nih.gov / public / ), Swiss-Prot (Swiss-Prot Protein Sequences Database, http: / / www.geneontology.org), Pfam (Protein Family, http: / / pfam.xfam.org / ), eggNOG (Evolutionary Genealogy of Genes, Non-supervised Orthologous Groups, http: / / eggnogdb.embl.de / # / app / home), GO (Gene Ontology, http: / / www.geneontology.org) and KEGG (Kyoto Encyclopedia of Genes and Genomes, http: / / www.genome.jp / kegg / ) databases using BLAST+ software to obtain annotation information of expressed sequences;

[0077] 9) Estimate gene expression levels using FPKM (Fragments Per Kilobase of exon model per Million mapped fragments) values.

[0078] 4. Results

[0079] Through the above steps, RNA was extracted, library was constructed, transcriptome sequencing was performed on mint leaf tissue, and then the sequencing sequence was assembled. Quantitative analysis of the expression level of assembled genes showed that the transcription level of MTPS1 gene was high in the three mint leaf libraries, with an average expression of 76.85, suggesting that it may play a role in the synthesis of mint volatile terpenes.

[0080] Example 3: Cloning of the peppermint MTPS1 gene

[0081] According to the assembled sequence of the mint MTPS1 gene in Example 2, PCR specific primers were designed using the primer design software Primer Premier, total RNA was extracted from leaves of mature mint plants, and the MTPS1 gene was cloned by reverse transcription-PCR (RT-PCR).

[0082] 1. Reagents

[0083] Plant RNA extraction kit, reverse transcription kit, and high-fidelity DNA polymerase were purchased from Beijing Quanshijin Biotechnology Co., Ltd., primers were synthesized by Sangon Biotechnology (Shanghai) Co., Ltd., and the remaining reagents were imported or domestic analytical grade products.

[0084] 2. Vectors and strains

[0085] The cloning vector pEASY-Blunt Simple Cloning Vector and Escherichia coli strain DH5α were purchased from Beijing Quanshijin Biotechnology Co., Ltd.

[0086] 3. Culture medium and reagents

[0087] LB liquid medium: 10 g / L tryptone, 5 g / L yeast powder, 10 g / L NaCl. Adjust pH to 7.0 with NaOH and sterilize by high pressure.

[0088] LB solid medium: Add 1% agar powder to the above LB liquid medium and sterilize by high pressure to prepare LB solid medium.

[0089] 100×Mg 2+ Solution: Weigh 20.33 g MgCl2·6H2O and 24.65 g MgSO4·7H2O into 100 mL deionized water and sterilize by high pressure.

[0090] SOC medium: tryptone 20g / L, yeast powder 5g / L, NaCl 0.58g / L, KCl 0.19g / L, 100×Mg 2+ 10 mL, adjust pH to 7.0 with NaOH, autoclave, and then add 2 mL of filter-sterilized 1 mol / L glucose.

[0091] 1000× Kanamycin: 50 mg / mL, dissolved in sterile deionized water, stored at -20°C.

[0092] 4. Methods

[0093] 4.1 RNA extraction from peppermint leaf tissue

[0094] The operation steps were carried out as described in 3.1 of Example 2.

[0095] 4.2 RT-PCR

[0096] 4.2.1RT

[0097] The total RNA extracted from peppermint leaves in 4.1 was used for reverse transcription, and the experimental steps were carried out according to the kit manufacturer's instructions.

[0098] 1) Take 1 μg of total RNA and 1 μL of polyT 18 (0.5 μg / μL) primer mix, make up to 8 μL with RNase-free ddH2O, and mix gently;

[0099] 2) Keep at 65°C for 5 min, then immediately transfer to an ice bath and place for 2 min;

[0100] 3) Add 10 μL of 2× reaction buffer, 1 μL of gDNA Remover, and 1 μL of reverse transcriptase, react at 42°C for 30 min to synthesize the first-strand cDNA;

[0101] 4) Heat at 85°C for 5 seconds to inactivate the reverse transcriptase and terminate the reaction.

[0102] 4.2.2PCR

[0103] According to the inferred sequence of the MTPS1 gene obtained in Example 2, the primer sequences designed by using Primer Premier software are shown as SEQ ID NO.3 and SEQ ID NO.4, respectively. The specific sequences are as follows:

[0104] MTPS1F:5'cttcgtgggatcatattttttg 3', SEQ ID NO.3;

[0105] MTPS1R:5'taattcgataggcggacatag 3', SEQ ID NO.4;

[0106] The mint leaf tissue cDNA obtained in step 4.2.1 of this example was used to clone the MTPS1 gene.

[0107] Place the 200 μL EP tube on ice and add the following reagents:

[0108] High-fidelity DNA polymerase, 0.5 μL;

[0109] 5× Buffer, 10 μL;

[0110] dNTPs, 4 μL;

[0111] cDNA, 1 μL;

[0112] MTPS1F, 1 μL;

[0113] MTPS1R, 1 μL;

[0114] ddH2O, 32.5 μL;

[0115] Amplification was performed according to the following program: 95°C for 1 min (pre-denaturation); 95°C for 20 s (denaturation), 55°C for 20 s (annealing), 72°C for 70 s (extension), 30 cycles of denaturation-annealing-extension; 72°C for 5 min (total extension).

[0116] Through the above operations, the PCR amplification product of the MTPS1 gene was obtained.

[0117] 4.3 Ligation of PCR amplification products with pEASY-Blunt vector

[0118] The MTPS1 gene PCR amplification product obtained in step 4.2 of this example was ligated with the cloning vector pEASY-BluntSimple Cloning Vector at a molar ratio of 1:4 (25° C., 15 min). The ligation system was as follows:

[0119] pEASY-Blunt Simple Cloning Vector (50μg / μL), 4μL;

[0120] PCR product (~150 μg / μL), 1 μL;

[0121] 4.4 E. coli transformation

[0122] 1) Take out the frozen Escherichia coli strain DH5α competent cells and thaw them in an ice bath;

[0123] 2) Gently mix the ligation product described in 4.3 with the competent E. coli cells and place on ice for 30 minutes;

[0124] 3) Heat shock at 42°C for 45 seconds, then immediately ice bath for 1-2 minutes;

[0125] 4) Add 0.8 mL of SOC, mix well, and culture with gentle shaking at 37°C for 1 h;

[0126] 5) Centrifuge at 4000 rpm for 1 min at room temperature, discard part of the supernatant, keep about 100 μL of the supernatant, mix the supernatant and cells with a pipette, apply to LB solid medium containing kanamycin (50 μg / mL), and culture at 37°C for 12 h.

[0127] 4.5 Colony PCR Identification

[0128] The E. coli monoclonal clone described in step 4.4 of this example was subjected to colony PCR identification to confirm that the inserted fragment was the target gene. The reaction system and amplification procedure were the same as in step 4.2.2 above, and the results were as follows: Figure 1 As shown, the results showed that E. coli transformed with MTPS1 showed positive amplification bands.

[0129]

[0130] The amino acid sequence encoded by the MTPS1 gene is shown in SEQ ID NO.2, specifically:

[0131] MSTIISVHHHHHHVPKPQLNIVHTKNKRASINLPWSLSPSSAASRLMPQSISSKLDNEKPPNETIRRRSGNY

[0132] GPSLWDFDYIQSLNTYHYKEEKQLNWEEELIVQVKKMLMGKKMEAVKQLELIDDLKNLGLSYFFQGEIK

[0133] NILNSIYNEHNFSQNNKVGDLYFTALGFRLLRHHGFDVSQEIFDSFKDDKGSSDDTKGMLQLYEASFLLR

[0134] QGEDTLELAKQISTKFLEEKLEAGIDDDNLTSSIRHSLEIPLHWRIQRLEARWFLDAYSMRKDMNPIIFELA

[0135] KLDFNIVQATLQEELKEVSRWWWNSRLPQKLPFVRDRLVESYYWAIGMFDAHKFGYERKTAAKIITLITA

[0136] LDDVYDIYGTLDELQQFTHVIRRWDTESATQLPYYLQLFYFVLYNFVSEVAYDILKEEGFISIPFLRRAWVD

[0137] LVEGYLQEAKWYHKKHTPNMEEYLDNASITIGAPAVITQVYFMLPKSKEKPVIESYDEIIRLSGMLVRLPD

[0138] DLGTSPFEMKRGDVAKSIQIYMKEQNVTREEAEKHVRFLIWEAWKKMNTVMAPLRDDLVMAAANLGRDAQFMYLDGDGNHSQLHHHIANLLLFNQYV.

[0139] Example 4: Construction of MTPS1 gene plant expression vector

[0140] The pEASY-MTPS1 plasmid obtained in Example 3 was used as a template for PCR amplification, and Xba I and Sal I restriction sites were introduced. The PCR product and the plant expression vector pBI121 were digested with Xba I and Sal I, respectively, and the digested products were recovered by gel ligation and transformed into Escherichia coli and Agrobacterium.

[0141] 1. Reagents

[0142] Plasmid extraction kit and gel recovery kit were purchased from Tiangen Biochemical Technology Co., Ltd.; high-fidelity DNA polymerase and T4 DNA ligase were purchased from Beijing Quanshijin Biotechnology Co., Ltd.; restriction endonucleases Xba I and Sal I were purchased from NewEngland Biolabs. All other reagents were imported or domestic analytically pure products.

[0143] 2. Escherichia coli and Agrobacterium strains

[0144] Escherichia coli strain DH5α was purchased from Beijing Quanshijin Biotechnology Co., Ltd., and Agrobacterium tumefaciens strain EHA105 was preserved in the laboratory.

[0145] 3. Culture medium and antibiotics

[0146] The preparation methods of LB liquid culture medium, LB solid culture medium and SOC culture medium are as described in Example 3.

[0147] 1000× Kanamycin: The preparation method is as described in Example 3.

[0148] 1000× Rifampicin: 20 mg / mL, dissolved in sterile deionized water, stored at -20°C.

[0149] 4. Methods

[0150] 4.1 Plasmid extraction

[0151] Plasmids were extracted from the pEASY-MTPS1 vector and the plant expression vector pBI121 obtained in Example 3, and the experimental steps were carried out according to the kit manufacturer's instructions.

[0152] 1) Column equilibration: Add 500 μL of equilibration solution BL to the adsorption column, centrifuge at 12000 rpm for 1 min, discard the waste liquid and set aside;

[0153] 2) Centrifuge at 12000 rpm for 1 min, collect the bacterial precipitate, and discard the supernatant; add 250 μL P1 solution (with RNase A added), and mix by pipetting until the bacterial precipitate is completely suspended;

[0154] 3) Add 250 μL of P2 solution and gently invert the centrifuge tube upside down to fully lyse the bacteria;

[0155] 4) Add 350 μL of P3 solution, gently invert the centrifuge tube upside down, and then centrifuge at 12000 rpm for 10 min;

[0156] 5) Aspirate the supernatant into a new centrifuge tube and centrifuge at 12000 rpm for 5 min;

[0157] 6) Carefully transfer the supernatant to the adsorption column, centrifuge at 12000 rpm for 1 min, and discard the waste liquid;

[0158] 7) Add 500 μL PD solution, centrifuge at 12000 rpm for 1 min, and discard the waste liquid;

[0159] 8) Add 600 μL PW solution (with anhydrous ethanol), centrifuge at 12000 rpm for 1 min, discard the waste liquid, and repeat the operation once;

[0160] 9) Centrifuge at 12000 rpm for 2 min to remove the remaining PW solution;

[0161] 10) Transfer the adsorption column to a new centrifuge tube, add 50 μL of sterile deionized water to the center of the column, leave at room temperature for 2 minutes, then centrifuge at 12,000 rpm for 2 minutes to elute the DNA.

[0162] 4.2PCR

[0163] The obtained pEASY-MTPS1 plasmid was used as a template for PCR amplification, and Xba I and Sal I restriction sites were introduced to amplify the coding sequence. The primer sequences are shown in SEQ ID NO.5 and SEQ ID NO.6, respectively, and the specific sequences are as follows:

[0164] MTPS1F2:5'ggtctagacttcgtgggatcatattttttg 3', SEQ ID NO.5;

[0165] MTPS1R2:5'gcgtcgactaattcgataggcggacatag 3', SEQ ID NO.6;

[0166] 4.3 Enzyme Digestion

[0167] The PCR amplification product of the expected size was purified, and then the pBI121 plasmid and MTPS1 gene amplification product obtained in 4.1 and 4.2 were digested with restriction endonucleases Xba I and Sal I.

[0168] 4.4 Rubber Recycling

[0169] The plasmid and MTPS1 gene amplification product after restriction digestion in 4.3 were recovered by gel recovery, and the experimental steps were carried out according to the manufacturer's instructions.

[0170] 1) Add 500 μL of equilibrium solution BL to the gel recovery adsorption column, centrifuge at 12000 rpm for 1 min, and discard the waste liquid at the bottom of the column;

[0171] 2) Wearing plastic gloves, recover the fragments separated by electrophoresis on the UV gel cutting instrument and place the cut fragments into the pre-prepared EP tube;

[0172] 3) Determine the volume of sol solution PC according to the mass of the glue, and add it in a 1:1 volume ratio. Put the EP tube into the heater and dissolve it at 50℃ for 10-15 minutes until the glue is completely dissolved;

[0173] 4) After the sol is completely cooled to room temperature, the dissolved liquid is transferred to the gel recovery adsorption column and allowed to stand for 3 minutes;

[0174] 5) Centrifuge at 12000rpm for 1min, pour off the waste liquid at the bottom of the collection tube. Add 600μL of washing solution PW to the adsorption tube;

[0175] 6) Centrifuge at 12000rpm for 1min, and discard the waste liquid at the bottom of the collection tube. Repeat the washing once;

[0176] 7) Place the adsorption column in a centrifuge and centrifuge at 12000 rpm for 3 min, then leave it for 15 min;

[0177] 8) Add 30 μL of eluent EB to the adsorption membrane in the center of the adsorption column, let it stand for 3 minutes, and centrifuge at 12000 rpm for 3 minutes to obtain the gel recovery product.

[0178] 4.5 Ligation of digested fragments

[0179] The MTPS1 gene fragment recovered by the above 4.4 digestion and the pBI121 plasmid fragment were ligated with T4 ligase. The ligation reaction system was as follows, 25°C, 3h:

[0180] MTPS1 gene fragment, 4 μL;

[0181] pBI121, 3 μL;

[0182] 5× reaction buffer, 2 μL;

[0183] T4 ligase, 1 μL;

[0184] 4.6 E. coli transformation

[0185] The experimental steps are as described in 4.4 of Example 3.

[0186] 4.7 Identification of recombinant plasmid by colony PCR

[0187] The method steps are as described in 4.5 of Example 3.

[0188] The recombinant vector (named pBI121-MTPS1) identified by colony PCR was sequenced, and the correctly sequenced recombinant E. coli was stored at -80°C.

[0189] 4.8 Agrobacterium transformation

[0190] 1) Extract the pBI121-MTPS1 plasmid according to the steps described in 4.1 of this example;

[0191] 2) Add pBI121-MTPS1 plasmid to 50 μL of Agrobacterium strain EHA105 competent cells, gently stir to mix, and place on ice for 30 min;

[0192] 3) Place in liquid nitrogen for 1 min;

[0193] 4) Place the EP tube on a 37°C constant temperature heater and heat for 5 minutes;

[0194] 5) Add 800 μL of SOC culture medium and culture in a shaker at 28°C and 200 rpm for 4-5 h;

[0195] 6) Centrifuge the bacterial solution at 4000 rpm for 5 min;

[0196] 7) Aspirate the supernatant in a clean bench, leaving about 100 μL, and gently pipette the bacteria to suspend and mix;

[0197] 8) The bacterial solution was evenly spread on LB solid medium containing kanamycin (50 μg / mL) and rifampicin (20 μg / mL), and cultured in a constant temperature incubator at 28°C for 48 h;

[0198] 9) Colony PCR identification was performed in the same manner as in 4.5 of Example 3. The results are as follows: Figure 2 As shown, the Agrobacterium clone transformed with pBI121-MTPS1 showed an amplified band of the same size as the plasmid positive control. The positive Agrobacterium transformed with the pBI121-MTPS1 recombinant plasmid was stored at -80°C.

[0199] Example 5: Genetic transformation of tomato with MTPS1 gene

[0200] The MTPS1 gene was genetically transformed into tomatoes through Agrobacterium-mediated method, and the MTPS1 gene of genetically transformed tomato lines was quantitatively identified.

[0201] 1. Reagents

[0202] Acetosyringone, 6-benzylaminopurine (6-BA), indoleacetic acid (IAA), 2,4-dichlorophenoxyacetic acid (2,4-D), and kinetin (KT) were purchased from Sigma, USA; RNA extraction kit, reverse transcription kit, and real-time quantitative PCR reagent TransStart Green qPCR SuperMix were purchased from Beijing Quanshijin Biotechnology Co., Ltd.; primers were synthesized by Sangon Biotechnology (Shanghai) Co., Ltd., and the remaining reagents were imported or domestic analytical grade products.

[0203] 2. Plant materials

[0204] Tomatoes (Solanum lycopersicum variety Ailsa Craig) were grown in an artificial climate chamber at 25°C, 12 h light / 20°C, 12 h dark.

[0205] 3. Solutions and culture media

[0206] 20×AB salt solution: Weigh 2g NaCl, 0.3g KCl, 0.005g FeSO4, 0.06g MgSO4·7H2O, 0.3g CaCl2·2H2O, respectively, add deionized water to fully dissolve, and make up to 200mL. Sterilize at 121℃ for 20min and store at 4℃.

[0207] 0.5M 2-morpholineethanesulfonic acid (MES): Weigh 4.88g MES, first add deionized water to fully dissolve it, then adjust the pH to 5.6-6.0 with 0.5M NaOH solution, finally make up to 50mL, filter and sterilize with a 0.22μm filter membrane, and store at 4℃.

[0208] 20% glucose solution: Weigh 20 g of glucose, dissolve it in 100 mL of deionized water, filter and sterilize it through a 0.22 μm filter membrane, and store it at 4°C.

[0209] 50× phosphate buffer: Prepare 0.078 g / mL NaH2PO4 solution and 0.087 g / mL K2HPO4 solution respectively, then mix the two evenly at a ratio of 5:1 (v / v), filter and sterilize through a 0.22 μm filter membrane, and store at 4°C.

[0210] 250× acetosyringone (ACE): Weigh 0.5 g ACE, dissolve it in 50 mL 70% ethanol, filter it with a 0.22 μm filter membrane to sterilize it, and dispense it into 1.5 mL sterile EP tubes and store it at -20°C.

[0211] 500×6-Benzylaminopurine (6-BA): Weigh 50 mg of 6-BA and dissolve it fully in 5 mL of 1 M KOH solution. Then dilute to 50 mL with deionized water, filter and sterilize with a 0.22 μm filter membrane, and store at -20°C.

[0212] 500× Indoleacetic acid (IAA): Weigh 50 mg of IAA, dissolve it fully in anhydrous ethanol, then dilute to 50 mL with deionized water, filter and sterilize with a 0.22 μm filter membrane, and store at -20°C.

[0213] 5000×2,4-dichlorophenoxyacetic acid (2,4-D): Weigh 50 mg of 2,4-D, first dissolve it fully in 1 M KOH solution, then make up to 50 mL with deionized water, filter and sterilize with a 0.22 μm filter membrane, and store at -20°C.

[0214] 10000× Kinetin (KT): Weigh 50 mg of KT, first add 1M HCl solution to fully dissolve it, then dilute to 50 mL with deionized water, filter and sterilize with a 0.22 μm filter membrane, and store at -20°C.

[0215] 500×Timentin: Weigh 5 g of Timentin, dissolve it in 50 mL of deionized water, filter and sterilize it with a 0.22 μm filter membrane, and store it at -20°C.

[0216] Tissue culture infection solution (50 mL): In an ultra-clean workbench, add 20×AB salt solution 2.5 mL, 0.5M MES 1 mL, 20% glucose solution 2.5 mL, 50×phosphate buffer 1 mL, and 250×ACE 200 μL into a sterilized container in sequence, mix well and set aside.

[0217] 1 / 2MS medium: Weigh 10g sucrose, 1.5g MS medium powder, and 3.5g agar powder, add deionized water first, stir to fully dissolve, then adjust the pH to 5.6-6.0 with 1M KOH solution, finally make up to 500mL with deionized water, sterilize at 121℃ for 20min and set aside.

[0218] Preculture medium: When 1 / 2MS medium (500 mL) is cooled to about 60°C after sterilization, add 500 μL of 500×6-BA and 20 μL of 500×IAA, mix well, and dispense into sterilized culture dishes for later use.

[0219] Co-culture medium: When the MS medium (500 mL) is sterilized and cooled to about 60°C, add 735 μL of 250×ACE, 100 μL of 5000×2,4-D, and 50 μL of 10000×KT to the culture medium, mix well, and dispense into sterilized culture dishes for later use.

[0220] Screening medium: After sterilization and cooling, add 500×6-BA 1mL, 500×IAA 100μL, 500×Timentin1mL, and 1000×Kanamycin to the MS medium (500mL), mix thoroughly, and dispense into sterilized culture dishes or tissue culture bottles for later use. According to the final concentration of kanamycin, there are three screening mediums: 60μg / mL, 65μg / mL, and 70μg / mL.

[0221] Rooting medium: When the MS medium (500 mL) is cooled to about 60°C after sterilization, add 1 mL of 500×IAA, 1 mL of 500×Timentin, and 700 μL of 1000×Kanamycin to the medium and mix thoroughly. Dispense into sterilized tissue culture bottles for later use.

[0222] 4. Methods

[0223] 4.1 Tomato genetic transformation

[0224] 1) Tomato sterile seedling culture: Take an appropriate amount of wild-type tomato seeds and place them in a 50 mL centrifuge tube. Treat the seeds with 75% ethanol for 5 minutes in a clean bench and wash with sterile water. Then treat the seeds with 15% sodium hypochlorite solution for 15 minutes and wash with sterile water. Spread the seeds on sterile filter paper and then use sterile tweezers to evenly plant them on the surface of 1 / 2 MS culture medium. Culture at 25°C in the dark for about a week until the seeds turn white, and then transfer them to light conditions to culture and grow sterile seedlings.

[0225] 2) Pre-culture of tomato explants: Use sterile tweezers to pick up sterile seedlings in a clean bench, cut off the roots, cotyledons and stems into callus segments of about 5 mm in length, place in a pre-culture medium, and culture at 25°C for 2 days.

[0226] 3) Agrobacterium infection: Take out the Agrobacterium strain that has been transferred into the MTPS1 gene plant expression vector (pBI121-MTPS1) from the -80℃ refrigerator, streak it on LB medium containing kanamycin (50μg / mL) and rifampicin (20μg / mL), and culture it at 28℃ for 2 days. Pick a single clone and place it in 3mL liquid medium containing the same antibiotics, and culture it at 28℃ for 12h. Take 2mL of bacterial liquid and add it to LB medium containing the same antibiotics to expand the culture until the OD 600 When the OD is about 0.5, centrifuge at 5000 rpm for 5 min. Resuspend the bacteria in the infection solution to a final concentration of OD 600 =0.1. Transfer the tomato cotyledons and stem segments pre-cultured for 2 days into the infection solution and soak for 15 minutes. Then take out the explants and place them evenly on sterile filter paper, dry the bacterial solution, transfer them to the co-culture medium, and culture them at 25℃ for 2 days.

[0227] 4) Callus screening and cultivation: Leaves and stem segments infected with Agrobacterium in the co-culture medium were placed in a screening medium with a kanamycin concentration of 60 mg / mL and cultured at 25°C for about 30 days. They were then transferred to screening medium with antibiotic concentrations of 65 mg / mL and 70 mg / mL for subculture.

[0228] 5) Rooting culture: Take out the differentiated seedlings with a growth height of about 5 cm from the screening medium, place them in the rooting medium, and culture them at 25°C for about 30 days until the root system is formed. Then take out the differentiated seedlings, rinse the root agar, and move them into the soil culture pot and grow them in the artificial culture room.

[0229] 4.2 Identification of transgenic plants

[0230] 1) According to the full-length sequence of the MTPS1 gene cloned in Example 3, the primer design software PrimerPremier was used to design quantitative PCR primers. The UBI stably expressed in tomato tissue was used as the internal reference gene to perform quantitative PCR analysis of the MTPS1 expression level. The MTPS1 primers are MTPS1RTF and MTPS1RTR, and the UBI primers are UBIF and UBI6R. The primer sequences are shown in SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, and SEQ ID NO.10, respectively, and the specific sequences are as follows:

[0231] MTPS1RTF:5'tggggagagatgcacagtttatgt 3', SEQ ID NO.7;

[0232] MTPS1RTR:5'gtacgtgactagaaacacgaaaatg 3', SEQ ID NO.8;

[0233] UBIF:5'aggttgatgacactggaaaggt 3', SEQ ID NO.9;

[0234] UBIR: 5'aatcgcctccagccttgttgta 3', SEQ ID NO. 10.

[0235] 2) The fruits of wild-type and genetically transformed tomato plants were collected respectively, and RNA was extracted according to the operation steps described in 3.1 of Example 2. The extracted total RNA was reverse transcribed to obtain cDNA according to the operation steps described in 4.2 of Example 3.

[0236] 3) Using the cDNA obtained above as a template and UBI as an internal reference gene, quantitative PCR analysis of MTPS1 expression levels was performed.

[0237] The quantitative PCR reaction system is as follows:

[0238] forward primer (10 μM), 0.5 μL;

[0239] Reverse primer (10 μM), 0.5 μL;

[0240] 2×TransStart Green qPCR SuperMix, 10 μL;

[0241] cDNA 9 μL;

[0242] Reaction conditions: 95°C for 30s; 95°C for 5s, 60°C for 15s, 72°C for 10s, 40 cycles. The cDNA was the cDNA template described in Example 4.2 and diluted 30 times for quantitative PCR. After amplification, 65°C for 5s, increasing by 0.5°C for each cycle, 60 cycles, and melting curve analysis was performed. Each sample was repeated three times.

[0243] 5. Results

[0244] Quantitative PCR results ( Figure 3 ) showed that the expression of MTPS1 gene in positive transformed plants (MTPS1-OE) was significantly increased compared with that in untransformed wild-type tomatoes (WT) (** indicates P<0.01). Genetically transformed tomato lines expressing MTPS1 gene were obtained by Agrobacterium-mediated method.

[0245] Example 6: Overexpression of the MTPS1 gene alters the synthesis of volatile components in tomato fruit

[0246] The tomato strains into which the MTPS1 gene was introduced obtained in Example 5 were planted in parallel with wild-type plants, and the volatile components in their fruits were detected and analyzed.

[0247] 1. Materials

[0248] Tomato plants were grown in an artificial climate chamber at 25°C, 12 h light / 20°C, 12 h dark.

[0249] 2. Methods

[0250] 1) Sample preparation: fresh fruits of wild-type and MTPS1 gene transformed tomatoes at the same growth stage were taken and quickly ground into a paste. 0.4 g was placed in a 40 mL purge bottle, which was sealed and sampled for detection.

[0251] 2) Purge and trap conditions

[0252] The prepared sample was injected through an automatic sampler. High-purity nitrogen was used for purging, with a pre-purge time of 0.2 min, a purge time of 15 min, a purge temperature of 35 ° C, and a purge flow rate of 40 mL / min. After the volatile components were adsorbed on the adsorbent, the trap was heated to 200 ° C, the trapped components were desorbed by helium for 0.5 min, and then directly transferred to the gas chromatography system.

[0253] 3) Chromatographic conditions

[0254] Chromatographic column: HP-5MS capillary column (30m×250μm, 0.24μm); injection port temperature 250℃, carrier gas helium flow rate 1mL / min, split ratio 15:1; temperature program: 40℃ for 3min, 8℃ / min to 100℃, then 10℃ / min to 280℃ for 10min.

[0255] 4) Mass spectrometry conditions

[0256] Electron bombardment ion source, electron energy 70eV, ion source temperature 230℃, transfer line temperature 280℃, quadrupole temperature 150℃; scanning range 33-500m / z, full scan mode.

[0257] 5) The mass spectrometry results were searched using the standard mass spectrometry database NIST to identify the chemical composition of each test sample.

[0258] 4. Results

[0259] The volatile components of tomato fruit were detected and analyzed by purge and trap-gas chromatography-mass spectrometry. Figure 4 The results showed that compared with the wild type, the fruits of MTPS1 gene-transformed tomatoes contained eucalyptol, which was not detected in the wild type. Figure 4 As shown in the figure above, the introduction of the MTPS1 gene enables tomatoes to synthesize more volatile aromatic substances, such as Figure 4 As shown in the figure below.

[0260] Therefore, it can be seen from the results of this example that the mint gene with the nucleotide sequence shown in SEQ ID NO.1 plays a role in the synthesis of volatile terpenoids in fruits, and the gene MTPS1 cloned from mint can be used to improve the aroma components of plant fruits.

[0261] The detailed description of a mint gene, its cloning method and application with reference to the above-mentioned embodiments is illustrative rather than restrictive, and several embodiments can be listed according to the limited scope. Therefore, changes and modifications without departing from the overall concept of the present invention should fall within the scope of protection of the present invention.

Claims

1. A mint gene, characterized in that: The nucleotide sequence of the mint gene is shown in SEQ ID NO.

1.

2. The mint gene according to claim 1, characterized in that The amino acid sequence encoded by the mint gene is shown in SEQ ID NO.

2.

3. An expression vector containing the mint gene according to claim 1 or 2.

4. The expression vector according to claim 3, characterized in that The expression vector is obtained by inserting the mint gene as claimed in claim 1 or 2 into the plant expression vector pBI121.

5. A genetically engineered bacterium containing the expression vector as claimed in claim 3 or 4.

6. The genetically engineered bacterium according to claim 5, characterized in that: The genetically engineered bacteria are obtained by transferring the expression vector described in claim 3 or 4 into Escherichia coli or Agrobacterium for cultivation.

7. The method for cloning a mint gene according to claim 1 or 2, characterized in that: The cloning method comprises the following steps: 1) RNA extraction from mint leaf tissue; 2) Reverse transcription to obtain cDNA; 3) Design primer sequences and use the obtained cDNA to clone the MTPS1 gene.

8. The method for cloning a mint gene according to claim 7, characterized in that: In step 3), the primer sequences are specifically designed as follows: MTPS1F, whose nucleotide sequence is shown in SEQ ID NO.3; MTPS1R, whose nucleotide sequence is shown in SEQ ID NO.

4.

9. Use of the mint gene as claimed in claim 1 or 2 in improving the synthesis of volatile substances in fruits.

Images