Mentha gene, cloning method and application thereof

Abstract

The present application provides a mint gene and a cloning method and application thereof, wherein the nucleotide sequence of the mint gene is shown as SEQ ID NO. 1, and the MTPS1 gene is cloned from the mint in the following manner: the expressed sequences in leaves are assembled by using the transcriptome sequencing of the mint; then, the PCR specific primers are designed by using a primer design software Primer Premier, total RNA is extracted from the mint leaves, reverse transcription PCR is carried out, and the MTPS1 gene is cloned. When the MTPS1 is expressed in tomatoes, the synthesis of volatile aromatic substances in the fruits is changed, and it can be seen that the mint gene can change the volatile aromatic components of the plant fruits.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, specifically relating to a mint gene, its cloning method, and its application. Background Technology

[0002] Peppermint (Mentha spp.) belongs to the genus Mentha in the family Lamiaceae. It has abundant wild plant resources in East and Northeast my country. The cultivation and consumption of peppermint plants in my country have a long history; it is also one of the most important medicinal and spice crops. As a natural plant resource for extracting volatile terpenes, peppermint is used in daily chemicals, food, and medicine due to its unique aroma and excellent medicinal value, possessing significant economic value. Similar to other Lamiaceae plants, peppermint exhibits excellent environmental adaptability and is cultivated in most parts of my country for peppermint oil extraction, making China one of the world's three major peppermint oil producing countries, alongside the United States and India.

[0003] The widespread use of peppermint plants stems primarily from their naturally synthesized volatile compounds, mainly monoterpenes and sesquiterpenes, including dozens of terpenes, derived alcohols, ketones, esters, and their isomers. Among these, the most abundant volatile components include menthol, menthone, phorbolone, eucalyptol, linalool, piperonone, and carvone. Currently, the framework for the biosynthetic pathway of terpenes in plants has been hypothesized, mainly the mevalonate pathway (MVA) and the methylerythritol-4-phosphate pathway (MEP). Peppermint terpene synthesis is believed to primarily occur via the MEP pathway. MEP is converted to geranyl diphosphate (GPP) through multiple reactions, and then further converted into various terpenoid components through hydroxylation, methylation, demethylation, or isomerization by terpene synthases (TPS). The catalytic reaction of TPS determines the final products and rate of volatile component synthesis in peppermint.

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

[0005] The purpose of this invention is to provide a mint gene and its cloning method. The gene sequence is cloned from mint and named Mint Terpene Synthase 1 (MTPS1 for short). The cloning of the MTPS1 gene from mint involves: assembling and annotating the highly abundant expression sequences in mint leaves using transcriptome sequencing; then, using the primer design software PrimerPremier, PCR-specific primers are designed, total RNA is extracted from mint leaves, and reverse transcription PCR (RT-PCR) is performed to clone the MTPS1 gene.

[0006] Another object of the present invention is to provide the application of the peppermint gene in the improvement of fruit volatile substance synthesis, wherein the peppermint gene can alter the volatile aroma components of plant fruits.

[0007] Another objective of this invention is to provide an expression vector containing the peppermint gene and a genetically engineered bacterium containing the expression vector.

[0008] The technical solution adopted in this invention is as follows:

[0009] A peppermint gene, referred to as MTPS1, has the nucleotide sequence shown in SEQ ID NO.1.

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

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

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

[0013] The present invention also provides genetically engineered bacteria containing the expression vector.

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

[0015] The present invention also provides a method for cloning the peppermint gene, the cloning method comprising the following steps:

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

[0017] 2) Obtain cDNA through reverse transcription;

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

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

[0020] The reagents used in step 3) are as follows: 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; 32.5 μL ddH2O.

[0021] In step 3), the amplification program is as follows: 95℃ for 1 min pre-denaturation; 95℃ for 20 s denaturation, 55℃ for 20 s annealing, 72℃ for 70 s extension, with 30 cycles of denaturation-annealing-extension; and 72℃ for 5 min total extension.

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

[0023] This invention cloned the MTPS1 gene from peppermint. When MTPS1 was expressed in tomatoes through genetic transformation, the volatile components of the tomato fruit changed. Compared with the wild type, the tomato fruit expressing MTPS1 showed the synthesis of eucalyptol. The gene with the nucleotide sequence shown in SEQ ID NO.1 plays a role in the synthesis of volatile components in the fruit. The MTPS1 gene cloned from peppermint in this invention can be used to modify the flavor of the fruit.

[0024] Compared with the prior art, the peppermint gene MTPS1 of the present invention provides a new gene resource for improving plant traits and can be used to change the volatile aroma components of plant fruits. Attached Figure Description

[0025] Figure 1 Colony PCR identification of MTPS1 gene ligated into pEASY-Blunt cloning vector. In the figure: M is DNA marker; 1-5 are amplification bands of single E. coli colonies transformed with MTPS1.

[0026] Figure 2 Colony PCR identification of Agrobacterium colonies 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-transformed tomato 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 alters the volatile aroma components of tomato fruit. Top: Purge and trap chromatogram of wild-type tomato fruit; Bottom: Purge and trap chromatogram of MTPS1-transformed tomato fruit. Detailed Implementation

[0029] The present invention will now be described in detail with reference to the embodiments.

[0030] In the following examples, unless otherwise specified, all experimental conditions were performed according to conventional conditions well known to those skilled in the art, such as those described in Sambrook J. and Russell, DW’s Molecular Cloning Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 2001), or according to the manufacturer’s recommendations.

[0031] Example 1: Detection of volatile components in peppermint leaves

[0032] 1. Plant materials

[0033] The 'M1' mint material was obtained from its native habitat in Lu'an, Anhui Province, and was grown in an artificial climate chamber at 25°C for 12 hours of light and 20°C for 12 hours of darkness.

[0034] 2. Method

[0035] 1) Sample preparation: Fresh leaves from peppermint plants with uniform growth were collected, quickly cut into cubes of about 0.5 cm in size, mixed, and 0.5 g were randomly placed in a 40 mL purge bottle. 10 mL of pure water was added, and the bottle was sealed before injection for analysis.

[0036] 2) Purge and trap conditions

[0037] The prepared sample was injected using an autosampler. High-purity nitrogen was used for purging: pre-purging time 0.2 min, purging time 13 min, purging temperature 20℃, and purging flow rate 40 mL / min. Once the volatile components were adsorbed onto the adsorbent, the collector was heated to 200℃, and the collected components were desorbed by helium for 0.5 min, then directly transferred to a 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℃ held for 3min, increased to 100℃ at 8℃ / min, then increased to 280℃ at 10℃ / min and held for 10min.

[0040] 4) Mass spectrometry conditions

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

[0042] 5) Use the NIST standard mass spectrometry database to search the mass spectrometry results and identify the chemical composition of each sample.

[0043] 3. Results

[0044] Volatile components of fresh peppermint 'M1' leaves were detected and analyzed using purge-trap-gas chromatography-mass spectrometry. Nineteen terpenoids were synthesized, accounting for 21.83% of the total content, including myrcene (5.89%), pinene (7.10%), caryophyllene (4.31%), and ocimene (1.28%). In addition, the peppermint leaves also contained a high content of the monoterpene alcohol eucalyptol (26.68%). These findings indicate that peppermint 'M1' contains high levels of terpenoids and their derivative volatile compounds, suggesting that its terpene synthases may possess high activity.

[0045] Example 2: Transcriptome sequencing of peppermint leaf tissue

[0046] To obtain the functional gene expression sequences, transcriptome sequencing and assembly annotation were performed on leaf tissues from mature mint plants.

[0047] 1. Reagents

[0048] The plant RNA extraction kit was purchased from Beijing TransGen Biotech Co., Ltd., DNase I was purchased from Sangon Biotech (Shanghai) Co., Ltd., and the RNA library preparation kit was purchased from Illumina Truseq. TM The RNA sample prep kit was from Meiji Biotechnology Co., Ltd., and the other reagents were either imported and repackaged or domestically produced analytical grade products.

[0049] 2. Plant materials

[0050] Peppermint 'M1' plants were grown in an artificial climate chamber at 25°C with 12 hours of light and at 20°C with 12 hours of darkness.

[0051] 3. Methods

[0052] 3.1 RNA Extraction

[0053] RNA was extracted from peppermint leaf tissue, and the experimental procedures were performed according to the kit manufacturer's instructions.

[0054] 1) Grind the peppermint tissue into powder rapidly in liquid nitrogen. Take 100 mg of the tissue and add it to 500 μL of BB6 solution (add 10 μL of β-mercaptoethanol to each 1 mL of BB6, prepare fresh for use, and vortex vigorously to mix). Incubate at room temperature for 3 min;

[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 mix and disperse the sediment;

[0058] 5) Add the obtained solution and precipitate together into a centrifuge column, centrifuge at 12000g for 30s, and discard the eluent;

[0059] 6) Add 500 μL of CB6 solution, centrifuge at 12000g for 30 seconds at room temperature, and discard the effluent;

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

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

[0062] 9) Repeat step 8);

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

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

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

[0066] 13) Detect the purity and concentration of the RNA sample and store the RNA at -80℃.

[0067] 3.2 Transcriptome Sequencing and Assembly Annotation

[0068] Transcriptome sequencing was performed using Illumina Truseq TM The RNA sample prep kit was used with the Illumina Novaseq 6000 sequencing platform, following these steps:

[0069] 1) Enrich mRNA with magnetic beads containing Oligo dT and then randomly fragment the mRNA;

[0070] 2) Synthesize cDNA strands using mRNA as a template and purify cDNA using microbeads;

[0071] 3) The purified double-stranded cDNA is then repaired at the ends and ligated with sequencing adapters. Fragment size selection is performed using microbeads, and cDNA libraries are obtained by PCR enrichment.

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

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

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

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

[0076] 8) Using BLAST+ software, all transcripts obtained from transcriptome sequencing assembly were compared with the 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 to obtain annotation information for the expressed sequences;

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

[0078] 4. Results

[0079] Following the steps outlined above, RNA was extracted from peppermint leaf tissues, libraries were constructed, and transcriptome sequencing was performed. The sequenced sequences were then assembled. Quantitative analysis of the expression levels of the assembled genes revealed that the MTPS1 gene exhibited high transcriptional levels in all three peppermint leaf libraries, with an average expression level of 76.85, suggesting a potential function in the synthesis of volatile terpenes in peppermint.

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

[0081] Based on the assembly sequence of the peppermint MTPS1 gene in Example 2, PCR-specific primers were designed using the primer design software Primer Premier. Total RNA was extracted from the leaves of mature peppermint 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 TransGen Biotech Co., Ltd. Primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd., and the remaining reagents were either imported and repackaged or domestically produced analytical grade products.

[0084] 2. Vectors and strains

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

[0086] 3. Culture medium and reagents

[0087] LB liquid medium: tryptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L. Adjust pH to 7.0 with NaOH and autoclave.

[0088] LB solid medium: Add 1% agar powder to the above LB liquid medium, autoclave, and prepare LB solid medium.

[0089] 100×Mg 2+ Solution: Weigh 20.33g MgCl2·6H2O and 24.65g MgSO4·7H2O and dilute to 100mL deionized water, then autoclave.

[0090] SOC medium: tryptone 20 g / L, yeast extract 5 g / L, NaCl 0.58 g / L, KCl 0.19 g / L, 100×Mg 2+ 10 mL of NaOH was added to adjust the pH to 7.0 and then autoclaved. Then 2 mL of filtered, sterilized 1 mol / L glucose was added.

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

[0092] 4. Methods

[0093] 4.1 RNA extraction from peppermint leaf tissue

[0094] Perform the operation steps as described in 3.1 of Example 2.

[0095] 4.2 RT-PCR

[0096] 4.2.1RT

[0097] Total RNA extracted from peppermint leaves in section 4.1 was used for reverse transcription, and the experimental procedures were performed according to the kit manufacturer's instructions.

[0098] 1) Take 1 μg of total RNA and 1 μL of polyT 18 Mix the primers (0.5 μg / μL), add RNase-free ddH2O to a final volume of 8 μL, and mix gently.

[0099] 2) Keep warm at 65℃ for 5 minutes, then immediately transfer to an ice bath and let stand for 2 minutes;

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

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

[0102] 4.2.2 PCR

[0103] Based on the inferred MTPS1 gene sequence obtained in Example 2, primer sequences were designed using Primer Premier software as shown in 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 MTPS1 gene was cloned using the cDNA from the peppermint leaf tissue obtained in step 4.2.1 of this embodiment.

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

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

[0109] 5×Buffer, 10μL;

[0110] dNTP, 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 procedure: 95℃ for 1 min (pre-denaturation); 95℃ for 20 s (denaturation), 55℃ for 20 s (renaturation), 72℃ for 70 s (extension), for 30 cycles of denaturation-renaturation-extension; 72℃ for 5 min (total extension).

[0116] The PCR amplification product of the MTPS1 gene was obtained through the above operations.

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

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

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

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

[0121] 4.4 Escherichia 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 competent E. coli cells and incubate on ice for 30 minutes;

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

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

[0126] 5) Centrifuge at 4000 rpm for 1 min at room temperature, discard a portion of the supernatant, leaving about 100 μL of supernatant. Mix the supernatant with the cells using a pipette tip, spread it on LB solid medium containing kanamycin (50 μg / mL), and incubate at 37°C for 12 h.

[0127] 4.5 Colony PCR Identification

[0128] The *E. coli* single clone described in step 4.4 of this embodiment was subjected to colony PCR identification to confirm that the inserted fragment was the target gene. The reaction system and amplification procedure were as described in 4.2.2 above, and the results were as follows. Figure 1 As shown, the results indicate 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 as follows:

[0131] MSTIISVHHHHHHVPKPQLNIVHTKNKRASINLPWSLSPSSAASRLMPQSISSKLDNEKPPNETIRRRSGNY

[0132] GPSLWDFDYIQSLNTYHYKEEKQLNWEEELIVQVKKMLMGKKMEAVKQLELIDDLKNLGLSYFFQGEIK

[0133] NILNSIYNEHNFSQNNKVGDLYFTALGFRLLRHHGFDVSQEIFDSFKDDKGSSDDTKGMLQLYEASFLLR

[0134] QGEDTLELAKQISTKFLEEKLEAGIDDDNLTSSIRHSLEIPLHWRIQRLEARWFLDAYSMRKDMNPIIFELA

[0135] KLDFNIVQATLQEELKEVSRWWWNSRLPQKLPFVRDRLVESYYWAIGMFDAHKFGYERKTAAKIITLITA

[0136] LDDVYDIYGTLDELQQFTHVIRRWDTESATQLPYYLQLFYFVLYNFVSEVAYDILKEEGFISIPFLRRAWVD

[0137] LVEGYLQEAKWYHKKHTPNMEEYLDNASITIGAPAVITQVYFMLPKSKEKPVIESYDEIIRLSGMLVRLPD

[0138] DLGTSPFEMKRGDVAKSIQIYMKEQNVTREEAEKHVRFLIWEAWKKMNTVMAPLRDDLVMAAANLGRDAQFMYLDGDGNHSQLHHHIANLLLFNQYV.

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

[0140] PCR amplification was performed using the pEASY-MTPS1 plasmid obtained in Example 3 as a template, 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. The digested products were recovered from the gel, ligated, and transformed into Escherichia coli and Agrobacterium.

[0141] 1. Reagents

[0142] Plasmid extraction kits and gel extraction kits were purchased from Tiangen Biotech Co., Ltd.; high-fidelity DNA polymerase and T4 DNA ligase were purchased from Beijing TransGen Biotech Co., Ltd.; restriction endonucleases Xba I and Sal I were purchased from New England Biolabs. All other reagents were either imported and repackaged or domestically produced analytical grade products.

[0143] 2. Escherichia coli strains and Agrobacterium strains

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

[0145] 3. Culture medium and antibiotics

[0146] The preparation methods for LB liquid medium, LB solid medium, and SOC 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℃.

[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 performed according to the instructions of the kit manufacturer.

[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 for later use;

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

[0154] 3) Add 250 μL of P2 solution, gently invert the centrifuge tube to allow the cells to fully lyse;

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

[0156] 5) Transfer the supernatant to a new centrifuge tube and centrifuge at 12,000 rpm for 5 minutes;

[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 of PD solution, centrifuge at 12000 rpm for 1 min, and discard the waste liquid;

[0159] 8) Add 600 μL of PW solution (with anhydrous ethanol added), 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 any 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, incubate at room temperature for 2 min, and then centrifuge at 12000 rpm for 2 min to elute the DNA.

[0162] 4.2 PCR

[0163] PCR amplification was performed using the obtained pEASY-MTPS1 plasmid as a template, 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 products of the expected size were purified, and then the pBI121 plasmid and MTPS1 gene amplification products obtained in 4.1 and 4.2 were digested with restriction endonucleases Xba I and Sal I.

[0168] 4.4 Glue Recycling

[0169] The plasmids and MTPS1 gene amplification products after enzyme digestion in section 4.3 were recovered by gel extraction, and the experimental procedures were performed according to the instructions of the kit manufacturer.

[0170] 1) Add 500 μL of equilibration 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, collect the electrophoretically separated fragments on the UV gel cutting instrument and put the cut fragments into the pre-prepared EP tubes;

[0172] 3) Determine the volume of PC solvent to add based on the quality of the adhesive, and add it at a 1:1 volume ratio. Place the EP tube in the heater and dissolve at 50℃ for 10-15 minutes, until the adhesive is completely dissolved;

[0173] 4) After the sol is completely dissolved, let it cool to room temperature, then transfer the dissolved liquid into the sol recovery adsorption column and let it stand for 3 minutes.

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

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

[0176] 7) Place the adsorption column in a centrifuge and centrifuge at 12,000 rpm for 3 minutes, then let it stand for 15 minutes;

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

[0178] 4.5 Ligation of enzyme-digested fragments

[0179] The MTPS1 gene fragment and pBI121 plasmid fragment recovered from the enzyme digestion in 4.4 were ligated using T4 ligase. The ligation reaction system was as follows: 25℃, 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 Escherichia coli transformation

[0185] The experimental procedure is as described in section 4.4 of Example 3.

[0186] 4.7 Colony PCR Identification of Recombinant Plasmids

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

[0188] Colony PCR was used to identify the recombinant vector (named pBI121-MTPS1), which was then sequenced. The correctly sequenced recombinant E. coli were stored at -80°C.

[0189] 4.8 Agrobacterium-mediated transformation

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

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

[0192] 3) Place in liquid nitrogen for 1 minute for cooling shock;

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

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

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

[0196] 7) In the clean bench, aspirate the supernatant, leaving approximately 100 μL. Gently pipette the remaining bacterial cells to suspend and mix them.

[0197] 8) Spread the bacterial culture evenly on LB solid medium containing kanamycin (50 μg / mL) and rifampin (20 μg / mL), and incubate at 28℃ for 48 h.

[0198] 9) Perform colony PCR identification according to the same method steps as in Example 3, 4.5. The results are as follows: Figure 2 As shown, Agrobacterium clones transformed with pBI121-MTPS1 exhibited amplification bands of the same size as the plasmid positive control. Positive Agrobacterium clones transformed with the pBI121-MTPS1 recombinant plasmid were stored at -80°C.

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

[0200] The MTPS1 gene was genetically transformed into tomato using Agrobacterium-mediated transformation, and the MTPS1 gene was quantitatively identified in the genetically transformed tomato lines.

[0201] 1. Reagents

[0202] Acetyleugenol, 6-benzylaminopurine (6-BA), indoleacetic acid (IAA), 2,4-dichlorophenoxyacetic acid (2,4-D), and kinetin (KT) were purchased from Sigma-Aldrich, USA; RNA extraction kit, reverse transcription kit, and TransStart Green qPCR SuperMix real-time quantitative PCR reagent were purchased from TransGen Biotech, Ltd., Beijing; primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd., and all other reagents were either imported and repackaged or domestically produced analytical grade products.

[0203] 2. Plant materials

[0204] Tomatoes (Solanum lycopersicum variety Ailsa Craig) were grown in an artificial climate cultivation chamber at 25°C with 12 hours of light and 20°C with 12 hours of darkness.

[0205] 3. Solutions and culture media

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

[0207] 0.5M 2-morpholinoethanesulfonic acid (MES): Weigh 4.88g of MES, add deionized water to dissolve it completely, then adjust the pH to 5.6-6.0 with 0.5M NaOH solution, and finally bring the volume to 50mL. Sterilize by filtration through a 0.22μm filter membrane and store at 4℃.

[0208] 20% glucose solution: Weigh 20g of glucose, dissolve and dilute to 100mL of deionized water, filter through a 0.22μm filter membrane for sterilization, and store at 4℃.

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

[0210] 250× Acetyleugenone (ACE): Weigh 0.5g of ACE, dissolve and dilute to 50mL of 70% ethanol, filter through a 0.22μm filter membrane for sterilization, dispense into 1.5mL sterile EP tubes, and store at -20℃.

[0211] 500×6-Benzylaminopurine (6-BA): Weigh 50 mg of 6-BA, dissolve it completely in 5 mL of 1 M KOH solution, then bring the volume to 50 mL with deionized water, sterilize by filtration through a 0.22 μm filter membrane, and store at -20 °C.

[0212] 500× Indoleacetic Acid (IAA): Weigh 50 mg of IAA, dissolve it completely in anhydrous ethanol, then bring the volume to 50 mL with deionized water, sterilize by filtration through 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, dissolve it completely in 1 M KOH solution, then bring the volume to 50 mL with deionized water, filter through a 0.22 μm filter membrane for sterilization, and store at -20 °C.

[0214] 10000× Kinetin (KT): Weigh 50 mg KT, add 1 M HCl solution to dissolve it completely, then make up to 50 mL with deionized water, filter through a 0.22 μm filter membrane for sterilization, and store at -20 °C.

[0215] 500×Timentin: Weigh 5g of Timentin, dissolve and dilute to 50mL of deionized water, sterilize by filtration through a 0.22μm filter membrane, and store at -20℃.

[0216] Tissue culture infection medium (50mL): In a clean bench, add 2.5mL of 20×AB salt solution, 1mL of 0.5M MES, 2.5mL of 20% glucose solution, 1mL of 50× phosphate buffer, and 200μL of 250×ACE to the sterilized vessel in sequence, mix well and set aside.

[0217] 1 / 2MS medium: Weigh 10g sucrose, 1.5g MS medium powder, and 3.5g agar powder. First, add deionized water and stir to dissolve it completely. Then, adjust the pH to 5.6-6.0 with 1M KOH solution. Finally, bring the volume to 500mL with deionized water and sterilize at 121℃ for 20min.

[0218] Pre-medium: After sterilization, cool 1 / 2 MS medium (500 mL) to about 60°C, add 500 μL of 500×6-BA and 20 μL of 500×IAA, mix thoroughly, and dispense into sterile petri dishes for later use.

[0219] Co-culture medium: After sterilizing MS medium (500mL) and cooling it to about 60℃, add 250×ACE 735μL, 5000×2,4-D 100μL and 10000×KT 50μL to the medium, mix thoroughly, and dispense into sterile petri dishes for later use.

[0220] Screening media: After sterilization and cooling, MS medium (500 mL) is mixed with 1 mL of 500×6-BA, 100 μL of 500×IAA, 1 mL of 500×Timentin, and 1000×Kanamycin. The mixture is then dispensed into sterile petri dishes or tissue culture flasks for later use. Based on the final kanamycin concentration, three screening media are prepared: 60 μg / mL, 65 μg / mL, and 70 μg / mL.

[0221] Rooting medium: After sterilization, cool MS medium (500mL) to about 60℃, add 1mL of 500×IAA, 1mL of 500×Timentin, and 700μL of 1000×Kanamycin to the medium and mix thoroughly. Dispense into sterile tissue culture bottles for later use.

[0222] 4. Methods

[0223] 4.1 Genetic transformation of tomatoes

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

[0225] 2) Pre-culture of tomato explants: Sterile seedlings were picked up with sterile forceps in a clean bench, the roots were cut off, and the cotyledons and stems were cut into callus tissue segments of about 5 mm in length. They were placed in pre-culture medium and cultured for 2 days under 25°C light conditions.

[0226] 3) Agrobacterium infection: Agrobacterium strain transformed with the MTPS1 gene plant expression vector (pBI121-MTPS1) was removed from a -80℃ freezer and streaked onto LB medium containing kanamycin (50 μg / mL) and rifampin (20 μg / mL), and cultured at 28℃ for 2 days. Single colonies were picked and placed in 3 mL of liquid medium containing the same antibiotics, and cultured at 28℃ for 12 h. 2 mL of the bacterial culture was added to LB medium containing the same antibiotics to expand the culture to OD. 600 When the concentration is approximately 0.5, centrifuge at 5000 rpm for 5 min. Resuspend the bacterial cells in the infection solution to a final concentration of OD0.5. 600 =0.1. The tomato cotyledons and stem segments, pre-cultured for 2 days, were transferred to the infection solution and soaked for 15 minutes. Then, the explants were removed, evenly placed on sterile filter paper, the bacterial solution was blotted dry, and transferred to a co-culture medium, incubated at 25°C for 2 days.

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

[0228] 5) Rooting culture: Take out the differentiated seedlings that have grown to a height of about 5 cm from the screening medium and place them in the rooting medium. Incubate at 25℃ for about 30 days until roots are formed. Then remove the differentiated seedlings, rinse the roots with agar, and transfer them to soil culture pots for growth in an artificial culture room.

[0229] 4.2 Identification of transgenic plants

[0230] 1) Based on the full-length MTPS1 gene sequence cloned in Example 3, quantitative PCR primers were designed using the primer design software PrimerPremier. UBI, stably expressed in tomato tissues, was used as an internal control gene for quantitative PCR analysis of MTPS1 expression levels. The MTPS1 primers were MTPS1RTF and MTPS1RTR, and the UBI primers were 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. 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) Collect fruits from wild-type and genetically transformed tomato plants respectively, and extract RNA as described in step 3.1 of Example 2. The extracted total RNA is then reverse transcribed to obtain cDNA as described in step 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 level 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] 9 μL of cDNA;

[0242] Reaction conditions: 95℃ for 30s; 95℃ for 5s, 60℃ for 15s, 72℃ for 10s, 40 cycles. The cDNA used was the cDNA template described in Example 4.2 diluted 30-fold for quantitative PCR. After amplification, the temperature was increased by 0.5℃ per cycle for 60 cycles, followed by melting curve analysis. Each sample was tested three times.

[0243] 5. Results

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

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

[0246] The tomato lines transfected with the MTPS1 gene 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 cultivation chamber at 25°C with 12 hours of light and at 20°C with 12 hours of darkness.

[0249] 2. Method

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

[0251] 2) Purge and trap conditions

[0252] The prepared sample was injected using an autosampler. High-purity nitrogen was used for purging: pre-purging time 0.2 min, purging time 15 min, purging temperature 35℃, and purging flow rate 40 mL / min. Once the volatile components were adsorbed onto the adsorbent, the collector was heated to 200℃, and the collected components were desorbed by helium for 0.5 min, then directly transferred to a 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℃ held for 3min, increased to 100℃ at 8℃ / min, then increased to 280℃ at 10℃ / min and held for 10min.

[0255] 4) Mass spectrometry conditions

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

[0257] 5) Use the NIST standard mass spectrometry database to search the mass spectrometry results and identify the chemical composition of each sample.

[0258] 4. Results

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

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

[0261] The above detailed description of a peppermint gene, its cloning method, and its application, with reference to the embodiments, is illustrative rather than limiting. Several embodiments may be listed within the defined scope. Therefore, variations and modifications that do not depart from the overall concept of the present invention should be within the protection scope of the present invention.

Claims

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

1.

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

2.

3. An expression vector containing the peppermint gene as described in claim 1 or 2.

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

5. Genetically engineered bacteria containing the expression vector as described 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 and culturing it.

7. The method for cloning the peppermint gene as described in claim 1 or 2, characterized in that, The cloning method includes the following steps: 1) RNA extraction from peppermint leaf tissue; 2) Obtain cDNA through reverse transcription; 3) Design primer sequences and use the obtained cDNA to clone the peppermint gene.

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

4.

9. The application of the peppermint gene as described in claim 1 or 2 in improving the synthesis of volatile substances in fruits, characterized in that, The fruit is a tomato fruit; the volatile substance is eucalyptol; and the peppermint gene is genetically transformed in tomatoes using an Agrobacterium-mediated transformation method.

Images