CN116555300A - Ginger lotus terpene synthase gene CaTPS2 and application thereof - Google Patents
Ginger lotus terpene synthase gene CaTPS2 and application thereof Download PDFInfo
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- CN116555300A CN116555300A CN202310615112.7A CN202310615112A CN116555300A CN 116555300 A CN116555300 A CN 116555300A CN 202310615112 A CN202310615112 A CN 202310615112A CN 116555300 A CN116555300 A CN 116555300A
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- catps2
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- terpene
- linalool
- terpene synthase
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
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- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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- C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
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- C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
- C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12Y204/01015—Alpha,alpha-trehalose-phosphate synthase (UDP-forming) (2.4.1.15)
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Abstract
The invention discloses a ginger lotus terpene synthase gene CaTPS2 and application thereof. The coding sequence of the gingerol terpene synthase gene CaTPS2 provided by the invention is shown as SEQ ID NO. 1, and the protein amino acid sequence coded by the gene is shown as SEQ ID NO. 2; researches show that the CaTPS2 gene has the highest expression level in flowers, has a small amount of expression in the fertility bracts, hardly expresses in the sterile bracts and leaves, and is basically consistent with the expression of nerolidol and linalool which are the terpene floral substances of the gingers; after the exogenous recombinant proteins of the CaTPS2 catalyze the substrates FPP and GPP, terpenoid nerolidol and linalool can be respectively generated, and the CaTPS2 is shown to belong to a bifunctional synthetase gene for controlling terpenoid nerolidol and linalool in the gingers. The invention provides technical support for improving the content of the terpene components in the gingers and preparing the nerolidol and the linalool.
Description
Technical Field
The invention belongs to the technical field of plant genetic engineering. More specifically, relates to a ginger lotus terpene synthase gene CaTPS2 and application thereof.
Background
The ginger lotus is perennial bulb flower of Curcuma of Zingiberaceae, and is mainly distributed in south Asia and southeast Asia, and a few species extend to China. The lotus bud slices are exactly like lotus, have the characteristics of gorgeous flower color, unique flower shape, long flower period, long bottle insertion service life and the like, are popular in domestic and foreign markets at present, and are deeply favored by people. Sesquiterpenes and monoterpenes contained in the gingers have strong fragrance and biological activity, and are important raw materials in the industries of medicine, food and cosmetics. For example, monoterpene linalool can be used for making perfumes, deodorants and sedatives, and has good antibacterial activity; sesquiterpene taxol can be used for treating tumors; the sesquiterpene nerolidol not only has certain anti-inflammatory, antifungal, antiparasitic and other effects, but also has good anti-tumor activity.
Terpene synthases (TPS) are key enzymes catalyzing the synthesis of terpenes, play an important role in the diversity of terpenes structures, and are important for the growth and development of plants and the regulation of resistance. TPS is classified, according to the products that it catalyzes the formation, into monoterpene synthase (MonoTPS), sesquiterpene synthase (SesquiTPS) and diterpene synthase (DiTPS), which catalyze the formation of the corresponding monoterpene, sesquiterpene and diterpene products from substrates GPP, FPP and GGPP, respectively. Further studies have shown that part of TPS has bifunctional enzymatic properties, which can react with GPP to form monoterpene species as well as with FPP to form sesquiterpene species. For example, lobelia lily LoTPS3 reacts with GPP to form linalool, while FPP can react to form nerol and α -farnesene (Abbas et al, 2019); both clematis CfTPS1 and CfTPS2 can catalyze GPP to generate linalool, and can catalyze FPP to generate nerolidol (Jiang et al 2020); hosta plantaginea HsTPS1 catalyzes GPP to produce linalool, citronella, and nerol, and FPP to produce small amounts of (E, E) -farnesol and (E, E) -farnesal (Cui et al 2022).
The cloned terpene synthase gene is a precondition for researching the formation mechanism of the lotus terpene components of the ginger, lays a theoretical foundation for comprehensively explaining the formation and regulation mechanism of the lotus terpene components of the ginger and improving the content of the lotus terpene components of the ginger through a genetic engineering means, however, the research of the lotus terpene components of the ginger and the synthase thereof in China is not reported at present, and the biosynthesis way of the lotus terpene components of the ginger is not clear.
Disclosure of Invention
The invention aims to overcome the defects of researches on terpene synthase genes of the gingers and provides a synthase gene CaTPS2 for controlling terpene components such as nerolidol and linalool in the gingers.
The first object of the invention is to provide a ginger lotus terpene synthase gene CaTPS2.
The second object of the invention is to provide a ginger lotus terpene synthase CaTPS2.
The third object of the invention is to provide the application of the gingerol terpene synthase gene CaTPS2 or the gingerol terpene synthase CaTPS2.
It is a fourth object of the present invention to provide a recombinant vector, a recombinant bacterium comprising the recombinant vector and a cell line comprising the recombinant bacterium.
A fifth object of the present invention is to provide a method for growing terpene floral plants.
It is a sixth object of the present invention to provide a process for preparing nerolidol and/or linalool.
The above object of the present invention is achieved by the following technical scheme:
the invention provides a ginger lotus terpene synthase gene CaTPS2, wherein the coding region (CDS) of the gene is 1737bp, the nucleotide sequence of the gene is shown as SEQ ID NO. 1, the gene codes 579 amino acids, the amino acid sequence of the gene is shown as SEQ ID NO.2, the protein molecular weight of the gene is estimated to be 66.96kDa, the isoelectric point (pI) is 6.00, and the gene sequence comprises a DDXXD conserved sequence.
According to the sequence information of the CaTPS2 gene provided by the invention, a person skilled in the art can easily obtain the gene equivalent to CaTPS2 by the following method: (1) obtained by database retrieval; (2) Screening a genomic library or a cDNA library of the ginger lotus or other plants by taking the CaTPS2 gene fragment as a probe; (3) Designing an oligonucleotide primer according to the CaTPS2 gene sequence information, and obtaining the oligonucleotide primer from the genome, mRNA and cDNA of the gingers or other plants by using a PCR amplification method; (4) The gene is obtained by modifying a CaTPS2 gene sequence by a genetic engineering method; (5) obtaining the gene by chemical synthesis.
The invention provides a primer pair for amplifying a ginger lotus terpene synthase gene CaTPS2, and the primer sequence is shown in SEQ ID NO. 3-4.
The research of the invention shows that the expression of the gingerol terpene synthase gene CaTPS2 is related to the development process of flowers, the expression level of the CaTPS2 gene in flowers is highest, a small amount of the gene is expressed in the fertility bracts, and the gene is hardly expressed in the sterile bracts and leaves; after the exogenous recombinant proteins of the CaTPS2 catalyze the substrates FPP and GPP, terpenoid nerolidol and linalool can be respectively generated, and the CaTPS2 is shown to belong to a synthetase gene for controlling terpenoid nerolidol and linalool in the rhizoma zingiberis, so that a novel bioengineering method is provided for the preparation of the nerolidol and the linalool.
Therefore, the invention protects the application of the gingerol terpene synthase gene CaTPS2 or the gingerol terpene synthase CaTPS2 in preparing nerol and/or linalool and cultivating terpene floral plants.
Further, the terpenoid is nerol and/or linalool.
Further, the plant is a ginger lotus.
The invention provides a recombinant vector, which comprises a ginger lotus terpene synthase gene CaTPS2.
The invention provides a recombinant bacterium comprising the recombinant vector.
The invention provides a cell line comprising the recombinant bacterium.
The invention also provides a method for cultivating the terpene floral plants, which comprises the steps of connecting the gingerol terpene synthase gene CaTPS2 or the gingerol terpene synthase CaTPS2 into a plant transformation vector, and then introducing the plant transformation vector into gingerol or other plant cells to obtain the transgenic CaTPS2 gene variety.
The ginger lotus terpene synthase gene CaTPS2 provided by the invention has important application value. One of the applications is to connect the CaTPS2 gene sequence to any plant transformation vector, and to introduce the CaTPS2 gene into the lotus or other plant cells by any transformation method, so as to obtain transgenic plants expressing the gene, thereby being applied to production. When the gene is constructed into a plant transformation vector, the gene or a regulatory sequence thereof can be modified appropriately, and other promoters can be used for replacing the original promoters of the gene before transcription initiation codons, so that the capability of generating terpene nerolidol and linalool and enhancing resistance of plants is widened and enhanced.
Preferably, the recombinant vector or recombinant bacterium is transformed into a plant, and the terpene floral plants are cultivated.
The invention also provides a method for preparing nerolidol and/or linalool, which takes farnesyl pyrophosphate (FPP) and geranyl pyrophosphate (GPP) as substrates and generates nerolidol and/or linalool by the catalysis of a gingerol synthase CaTPS2.
The invention has the following beneficial effects:
the invention clones and obtains a bifunctional enzyme gene CaTPS2 for controlling terpene components nerolidol and linalool in the rhizoma zingiberis and lotus for the first time, and the gene can catalyze FPP and GPP to form terpene compounds nerolidol and linalool, thereby playing an important role in improving the content and resistance of plant terpene components. The terpene synthase gene CaTPS2 studied by the invention can be used for preparing nerolidol and linalool, and further preparing essential oil, essence and medicine containing the nerolidol and linalool. The gene segment of the terpene synthase gene CaTPS2 is constructed on a plant expression vector, and other plant materials can be transformed through an external source, so that a transgenic material containing terpene floral genes is obtained, and an effective method is provided for cultivating fragrance and medicinal plants.
Drawings
FIG. 1 shows the analysis of the homology of the amino acid sequence of the CaTPS2 gene clone and the CaTPS2 of the invention (A: agarose gel electrophoresis of the CaTPS2 clone; B: analysis of the homology of the amino acid sequence of the CaTPS 2; C: analysis of the conserved domain of the CaTPS 2).
FIG. 2 shows the expression specificity of the CaTPS2 gene in the lotus of ginger (A: the phenotypic characteristics of the lotus of ginger; B: the expression level of CaTPS2 in different lotus parts of ginger).
FIG. 3 shows prokaryotic expression of the CaTPS2 gene and in-vitro enzyme catalysis reaction of the recombinant protein (A: SDS-PAGE gel electrophoresis of the CaTPS2 recombinant protein, M is Marker, C is precipitated after breaking recombinant protein bacteria, S is supernatant after breaking recombinant protein bacteria, E is recombinant protein eluent, B: in-vitro catalysis of FPP and GPP to produce nerolidol and linalool).
Detailed Description
The invention is further illustrated in the following drawings and specific examples, which are not intended to limit the invention in any way. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art.
Reagents and materials used in the following examples are commercially available unless otherwise specified.
The ginger lotus samples used in the following examples were all collected from the flower research center ginger lotus germplasm resource nursery of agricultural university in south China.
EXAMPLE 1CaTPS2 Gene cloning
1. Extraction of total RNA of lotus flowers of ginger
The ginger lotus flower preserved in an ultralow temperature refrigerator is used as a material for extracting RNA. The gun head and Eppendorf tube used for RNA extraction are soaked in 0.1% DEPC at 37 ℃ overnight, sterilized at 121 ℃ for 25min, glassware is prepared, the mortar is wrapped with aluminum foil and then is subjected to dry heat treatment at 180 ℃ for 3h, and the mortar is cooled for standby. Extracting total RNA of rhizome of the lotus rhizome by adopting a Trizol method according to the specification of Trizol (TaKaRa). The integrity of the RNA was checked by electrophoresis on a 1% agarose gel, and its concentration and purity were determined by a micro-spectrophotometer method. Storing at-80deg.C for use.
2. PCR amplification and recovery purification
The total RNA of the lotus flowers of the gingers is used as a template, and the Evo M-MLV reverse transcriptase of the Aicory is used for synthesizing single-stranded cDNA. According to the related annotation gene sequence of the ginger lotus transcriptome database, designing a primer, and an upstream primer F1:5'-ATGGCTCCCGCCTCTGCT-3' (SEQ ID NO: 3). Downstream primer R1:5'-TTGTTTAAACAACAATAAGTTTATATACTCCTT-3' (SEQ ID NO: 4). And is synthesized by Shanghai bioengineering company. PCR amplification was performed using the synthesized cDNA as a template and using Phanta high fidelity enzyme, and the specific method was as described in the specification. Pre-denaturation at 94℃for 2min, denaturation at 94℃for 15s, annealing at 60℃for 15s, extension at 72℃for 90s,35 cycles, and then complete extension at 72℃for 5min. Preserving at-20deg.C for use.
After the completion of the PCR reaction, the PCR product was preliminarily detected by 1.0% agarose gel electrophoresis as to whether the target fragment band was contained. After the PCR amplification product is detected by 1% agarose gel electrophoresis, a gel block containing the target fragment is cut out by a scalpel under an ultraviolet lamp, and is recovered by a DNA gel recovery kit (SanPrep column type DNA gel recovery kit, manufacturing engineering), and the recovery method basically refers to the instruction of the kit. The recovered product was then subjected to 1% agarose electrophoresis to see its recovery effect and approximate concentration, in order to ensure the performance of subsequent experiments.
3. Cloning vector ligation
According to the size and effective concentration of the recovered target fragment, a proper amount of recovered and purified product is taken to be connected with a cloning vector, the vector is selected from TaKaRa pMD19-T vector, and the molar ratio of the target DNA to the cloning vector is controlled at 3: about 1, and the specific operation is performed according to the instruction. And the constant temperature connection is carried out for 3 to 6 hours at the temperature of 16 ℃, and the connection time depends on the length of the target fragment. Taking out competent cells DH5 alpha (TaKaRa) from a refrigerator at-80 ℃ in advance, placing the mixture in an ice box for natural melting, adding 10 mu L of connecting liquid into a centrifuge tube of the competent cells in full, carrying out heat shock for 90s in a water bath at 42 ℃ after ice bath for 30min, rapidly placing the mixture on ice for 2-5 min, adding 1mL of LB liquid culture medium, and carrying out shaking culture at 180rpm at 37 ℃ for 1h after uniform mixing. The surface of a plate of LB solid medium containing 100. Mu.g/mL ampicillin was coated with 30. Mu.L of X-gal (20 mg/mL) and 30. Mu.L of IPTG (20 mg/mL), then a proper amount of the conversion solution was coated, and after the conversion solution was completely absorbed, the plate was inverted and cultured overnight in an incubator at 37℃for about 16 hours, and after observation, white colonies were screened by X-gal/IPTG blue-white spots, and recombinant plasmids were preliminarily identified, and the plate was kept at 4 ℃.
After preliminary screening by blue and white spots, white single colonies are picked from an LB plate medium by a sterilized gun head, inoculated into an LB liquid medium containing 100 mug/mL ampicillin, subjected to shaking culture for 3-6 hours at 37 ℃ and 180rpm in a temperature-controlled shaking table, subjected to PCR, and subjected to 1% agarose gel electrophoresis detection on PCR products by using pMD19-T carrier universal primers M13-47 and M13-48. And (3) selecting a bacterial solution containing the target fragment for DNA sequence determination, and completing sequencing work by Shanghai Biotechnology Co.
The sequence obtained by sequencing and the original sequence information of the transcriptome are compared and analyzed, and compared and homology analysis is carried out at NCBI, so that the gene sequence obtained by the invention is determined to be the full-length sequence of TPS family, and the gene is named as CaTPS2 gene, and the protein sequence is deduced according to the gene coding sequence.
The result of agarose gel electrophoresis of the clone of the CaTPS2 gene is shown in FIG. 1A, and shows that the clone obtains a single band which accords with the estimated size; sequencing and comparing to obtain a coding region (CDS) sequence of the CaTPS2 gene, wherein the coding region (CDS) sequence is shown as SEQ ID NO. 1, and is 1737bp in total; it is presumed that the amino acid sequence is shown as SEQ ID NO.2, the molecular weight of the protein is presumed to be 66.96kDa, and the isoelectric point (pI) is presumed to be 6.00. Analysis of amino acid sequence homology of calps 2 as shown in fig. 1B, the gene sequence contained DDXXD conserved sequences, and analysis of calps 2 conserved domain by SMART showed that it contained two conserved sequences of N-terminal domain (PF 01397) and C-terminal active domain (PF 03936) (fig. 1C), so that calps 2 was considered a member of the terpene synthase family.
EXAMPLE 2 analysis of expression of the CatPS2 Gene
Extracting total RNA from different organs, different development periods and different parts of the flowers (figure 2A) of the ginger lotus by using a Trizol method (TaKaRa) and adopting a Hieff qPCR SYBR Green Master Mix (low Rox) method for fluorescence quantitative PCR, wherein the specific principle of the method is shown in the specification. Real-time fluorescent quantitative PCR primers are designed by using Primer Premier 5.0 software, the primers are respectively designed by using Primer Premier 5.0 according to the design principle of the fluorescent quantitative PCR primers, whether the Primer has mismatch or Primer dimer and the amplification efficiency are detected by fluorescent quantitative PCR, a pair of optimal primers are selected, and P1:5'-ATGGCTCCCGCCTCTGCTTATCAAAC-3' (SEQ ID NO: 5). P2:5'-TTGCTTCTGCAACTGAAGCGGCCT-3' (SEQ ID NO: 6). The Primer is designed by Primer premier 5.0 according to the design principle of Real-time PCR Primer, and the action-P1 is: 5'-GAGCATGGAATTGTCAGCAA-3' (SEQ ID NO: 7), action-P2: 5'-AGGGGCTTCAGTGAGCAATA-3' (SEQ ID NO: 8).
Detection was performed by Real-time PCR and a standard curve was made to determine whether the amplification efficiency (E) was in the range of 90-110% for screening. The cDNA of each sample was used as a template, and a fluorescent quantitative PCR reaction was performed on an ABI 7500 fluorescent quantitative PCR apparatus. Each sample was subjected to 3 biological replicates and 3 technical replicates, with ddH2O as a negative control. The reaction system was Hieff qPCR SYBR Green Master Mix (Low Rox) 10.0. Mu.L, the upstream primer (10. Mu.M) 0.4. Mu.L, the downstream primer (10. Mu.M) 0.4. Mu.L, cDNA 2.0. Mu.L, ddH 2 O7.2. Mu.L. The detection procedure was 95℃for 5min,95℃for 10s,55℃for 20s,72℃for 20s,40 cycles. After the reaction is finished, 2 is adopted －△△Ct Data analysis was performed by the method (Livak et al, 2001) to calculate the expression of the ginger lotus CaTPS2 in different samples.
As shown in FIG. 2B, the results of the gene expression analysis showed that the expression level of CaTPS2 was highest in the flowers among different organs of the lotus, and also expressed in small amounts in the fertile bracts, and hardly expressed in the sterile bracts and leaves, consistent with the release levels of nerolidol and linalool. In different parts of the flower, the expression quantity of the CaTPS2 in petals is highest, the expression quantity of the CaTPS2 in the petals is inferior to that of the lips, the expression quantity of the CaTPS2 in the side petals and the core column is extremely low, the release rule of the CaTPS2 is basically consistent with that of linalool, the release rule of the CaTPS2 in the core column is slightly different from that of nerolidol, the expression quantity of the CaTPS2 in the full bloom stage is highest, the expression quantity of the CaTPS2 in the half bloom stage is lower, and the expression quantity of the CaTPS2 in the bud stage and the aging stage is lower, and the CaTPS2 in the full bloom stage and the core column is consistent with that of the nerolidol. The above results indicate that CaTPS2 is a related gene involved in regulating and controlling the synthesis of the terpene substances of the lotus in ginger.
Example 3 prokaryotic expression of CaTPS2 and in vitro functional analysis
1. Vector construction
According to the coding region of the obtained CaTPS2 gene, a homologous recombination primer F5'-gccatggctgatatcggatccATGGCTCCCGCCTCT GCT-3' (shown as SEQ ID NO: 9) containing BamH I and HindIII cleavage sites is used; r:5'-ctcgagtgcggccgcaagcttTTGTTTAAACAA CAATAAGTTTATATACTCCTT-3' (SEQ ID NO: 10), PCR amplification was performed.
The pET-32a prokaryotic expression vector was double digested with BamHI and HindIII restriction enzymes and a 1% agarose gel was used to recover large fragments. UsingII homologous recombination of the gene fragment with the vector was carried out, the amount of the vector was adjusted to 0.03pmol, and the amount of the insert was adjusted to 0.06pmol, and the specific method was as described in the specification. And (3) transforming the connection product into E.coli (E.coli) DH5 alpha competent cells, and obtaining the recombinant prokaryotic expression vector after bacterial liquid PCR and sequencing identification.
2. Recombinant protein expression
Rosetta (DE 3) competent cells were transformed with the identified recombinant plasmid DNA, single colonies were picked and inoculated into fresh 5mL LB (containing 100mg/L Apm) liquid medium, cultured overnight at 37℃at 180 rpm. Transfer 100. Mu.L seed solution into fresh 100mL LB (containing 100mg/L Apm) liquid medium, culture at 37℃and 180rpm to OD 600 The value is 0.4-0.6, 10. Mu.L of IPTG (1M) is added, and the mixture is induced for 20h at 16 ℃. Another control group was also taken without IPTG induction. Centrifuging to collect cells, suspending cells with 5mL of lysis buffer, cooling the cells, and placing the cells on iceThe cells were disrupted by sonication. Centrifuge at 12000rpm at 4℃for 10min, transfer the supernatant to a new centrifuge tube, wash the pellet once with double distilled water, and suspend the pellet with 5mL lysis buffer. The supernatant and the pellet were each taken at 16. Mu.L and stored at-20℃for SDS-PAGE analysis. A 12.5% SDS polyacrylamide gel was prepared and loaded sequentially. The concentrated and the separation gels were electrophoresed with voltages of 80V and 130V, respectively. After electrophoresis, coomassie brilliant blue is dyed for 30min, decolorized by a decolorizing solution for 24h, and the test result is observed and recorded.
3. Purification of recombinant proteins
After the resin was precipitated, the internal liquid was drained and 5mL of ddH was added to the column 2 And O, repeatedly flushing for 3 times, adding 5mL Wash buffer, repeatedly flushing for 3 times, and precooling at 4 ℃.5ml of the cell lysis supernatant was added to a pre-chilled chromatography column, thoroughly mixed and combined on a low speed shaker at 4℃for 1h. The liquid was drained and collected in a centrifuge tube and marked. 20. Mu.L of the effluent was analyzed by SDS-PAGE. 2mL Wash buffer was added, the column was eluted 3 times, and each fraction of the eluate was collected and analyzed by SDS-PAGE at 16. Mu.L each. The column was washed four times with 0.5mL of each of the solution buffers, and each fraction was collected in sequence with a separate collection tube, and then 16. Mu.L of each fraction was analyzed by SDS-PAGE. The eluted protein was added to the ultrafiltration tube and centrifuged at 5000rpm at 4℃for 10min. Then, the mixture was centrifuged at 5000rpm at 2mL Reaction buffer,4 ℃for 10min and repeated three times. The ultrafiltered solution is collected and transferred to a precooled centrifuge tube, added with equal volume of pure glycerol for absorbing and beating, and split charging is carried out by 200 mu L of each tube, 16 mu L of the solution is taken for SDS-PAGE detection, and the rest of the solution is stored at the temperature of minus 80 ℃ for standby.
4. Enzyme-catalyzed functional identification
20. Mu.L of 30mM HEPES (pH 7.5), 50mM DTT,25mM MgCl 2 20. Mu.L each, and 20. Mu.L of protein extract, 1. Mu.L of GPP/FPP, plus ddH 2 O119 mu L, sealing in a sample bottle, reacting at 28deg.C for 1 hr, inserting 75 μm polydimethyl oxolane (PMDS) extraction fiber head into a glass bottle, performing solid-phase microextraction for 1 hr, and placing the extraction fiber head into a gas chromatograph-mass spectrometerThe analysis, gas chromatography conditions were: the chromatographic column is HP-1NNOWAX column (30 m×0.25 mm); the carrier gas is high-purity helium, and the split ratio is 20:1, the pre-column pressure is 50Pa, and the flow is 1mL/min; sampling time is 2min; programming temperature: the column starting temperature was 45℃for 2min, raised to 80℃at a rate of 5℃per min for 1min, and then raised to 250℃at a rate of 10℃per min for 5min. The mass spectrum conditions are as follows: the interface temperature of the GC-MS is 220 ℃, and the electron bombardment source EI is 350V; the ion source temperature is 170 ℃; electron energy 70eV; scanning the mass range of 35-335 aum, and analyzing the acquired mass spectrogram by using a WILLEY/MAINLIB library.
The prokaryotic expression result and in-vitro enzyme activity identification of the CaTPS2 gene are shown in figure 3, and the result of SDS-PAGE gel electrophoresis of the CaTPS2 recombinant protein shows that the expression size of the CaTPS2 recombinant protein is about 67kDa and is close to the expected size (figure 3A). When FPP is used as a substrate, the in-vitro enzyme catalysis reaction product of pET-32a-CaTPS2 is identified as sesquiterpene substance nerolidol through mass spectrum, and when GPP is used as a substrate, monoterpene substance linalool is formed in a catalysis mode (figure 3B). The enzyme coded by the CaTPS2 gene is a terpene synthase gene which can catalyze FPP and GPP to generate terpenoid nerolidol and linalool.
In conclusion, the novel terpene synthase gene CaTPS2 is cloned in the gingers for the first time, and can catalyze FPP and GPP to form terpene compounds nerolidol and linalool, so that the novel terpene synthase gene CaTPS2 plays an important role in improving the content and resistance of plant terpenoid components. The terpene synthase gene CaTPS2 studied by the invention can be used for preparing nerolidol and linalool, and further preparing essential oil, essence and medicine containing the nerolidol and linalool. The gene segment of the terpene synthase gene CaTPS2 is constructed on a plant expression vector, and other plant materials can be transformed through an external source, so that a transgenic material containing terpene floral genes is obtained, and an effective method is provided for cultivating fragrance and medicinal plants.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
Claims (10)
1. The ginger lotus terpene synthase gene CaTPS2 is characterized in that the coding sequence of the gene is shown as SEQ ID NO. 1.
2. The ginger lotus terpene synthase CaTPS2 is characterized in that the amino acid sequence of the synthase is shown as SEQ ID NO. 2.
3. Use of the gingerol terpene synthase gene calps 2 of claim 1 or the gingerol terpene synthase calps 2 of claim 2 for the preparation of nerol and/or linalool.
4. Use of the gingerol terpene synthase gene calps 2 of claim 1 or the gingerol terpene synthase calps 2 of claim 2 for cultivation of terpene floral plants.
5. A recombinant vector comprising the gingerol terpene synthase gene CaTPS2 according to claim 1.
6. A recombinant bacterium comprising the recombinant vector of claim 5.
7. A cell line comprising the recombinant bacterium of claim 6.
8. A method for cultivating a terpene floral plant, which is characterized in that the gingerol terpene synthase gene CaTPS2 according to claim 1 or the gingerol terpene synthase CaTPS2 according to claim 2 is linked to a plant transformation vector and then introduced into a plant to obtain a transgenic variety of CaTPS2.
9. The method according to claim 8, wherein the recombinant vector according to claim 6 or the recombinant bacterium according to claim 7 is transformed into a plant and a terpene floral plant is cultivated.
10. A method for preparing nerolidol and/or linalool, which is characterized in that farnesyl pyrophosphate FPP and geranyl pyrophosphate GPP are used as substrates, and nerolidol and/or linalool is produced by catalysis of the gingerol terpene synthase CaTPS2 according to claim 2.
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