CN116622751A - Curcuma rhizome citronellol synthetase gene HcTPS38 and application thereof - Google Patents
Curcuma rhizome citronellol synthetase gene HcTPS38 and application thereof Download PDFInfo
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- CN116622751A CN116622751A CN202310615103.8A CN202310615103A CN116622751A CN 116622751 A CN116622751 A CN 116622751A CN 202310615103 A CN202310615103 A CN 202310615103A CN 116622751 A CN116622751 A CN 116622751A
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- hctps38
- citronellol
- gene
- synthase
- gingerol
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- QMVPMAAFGQKVCJ-UHFFFAOYSA-N citronellol Chemical compound OCCC(C)CCC=C(C)C QMVPMAAFGQKVCJ-UHFFFAOYSA-N 0.000 title claims abstract description 144
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- 235000000484 citronellol Nutrition 0.000 title claims abstract description 72
- QMVPMAAFGQKVCJ-SNVBAGLBSA-N (R)-(+)-citronellol Natural products OCC[C@H](C)CCC=C(C)C QMVPMAAFGQKVCJ-SNVBAGLBSA-N 0.000 title claims abstract description 71
- 235000014375 Curcuma Nutrition 0.000 title description 6
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/88—Lyases (4.)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- 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
- C12N15/8243—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 involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
-
- 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
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
- C12P7/22—Preparation of oxygen-containing organic compounds containing a hydroxy group aromatic
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention discloses a citronellol synthase gene HcTPS38 and application thereof, wherein the full-length cDNA sequence of the HcTPS38 gene is shown as SEQ ID NO. 1, the coding sequence is shown as SEQ ID NO. 2, and the coded amino acid sequence is shown as SEQ ID NO. 3. The research of the invention shows that the HcTPS38 gene is highly expressed in the rhizome of the stigmariurus petalitis, and the expression quantity of the HcTPS38 gene in the rhizome is consistent with that of citronellol; exogenous recombinant protein of HcTPS38 is prepared, monoterpene citronellol can be generated after a substrate is catalyzed, and the exogenous recombinant protein can be used for preparing citronellol; in addition, hcTPS38 is connected with a plant transformation vector and then is introduced into ginger flowers or other plant cells, so that a transgenic plant expressing the HcTPS38 gene can be obtained, and the method is beneficial to culturing citronellol plants or constructing transgenic materials containing monoterpene citronellol synthase genes.
Description
Technical Field
The invention relates to the technical field of plant genetic engineering, in particular to a citronellol synthase gene HcTPS38 of a ginger flower and application thereof.
Background
The gingerol is a perennial herb of the genus gingerol of the family Zingiberaceae, native to india and southwest China, and now distributed throughout tropical regions. The gingerol plant not only has extremely high ornamental value, but also is an important aromatic medicinal plant. The flowers, rhizomes and leaves of the gingers contain rich essential oil, and researches show that the gingers essential oil has various biological activities of antioxidation, anti-tumor, anti-inflammation, antibiosis and the like, wherein terpene compounds are main active ingredients. Terpenoids are a generic name for compounds composed of several Isoprene (C5) structural units, and can be classified into monoterpenes (monoterenes, C10), sesquiterpenes (sesterpenes, C15), diterpenes (diterpenes, C20) and the like, depending on the number of structural units. Wherein the oxygen-containing derivatives (alcohols, aldehydes, ketones) of monoterpenes have strong fragrance and biological activity, and are important raw materials in the industries of medicine, food and cosmetics. Terpene synthases are terminal key enzymes for the biosynthesis of terpenes, which directly determine the type, quantity and yield of terpenes. 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.
Terpene synthases are key enzymes for terpene biosynthesis, and thus have also been the most studied and deepest enzymes in terpene biosynthesis, since two sesquiterpene synthase genes have been cloned in tobacco in 1992 (Facchini and Chappell, 1992), more than 200 monoterpene and sesquiterpene synthase genes have been cloned in more than 40 plants by scientists (Degenhardt et al, 2009), related to crops (Chen et al, 1995;Kollner et al, 2008, uan et al, 2008; chen et al, 2014), needle plants (Bohlmann et al, 1999;Martin et al, 2004), medicinal plants (Deguerry et al, 2006), spice plants and ornamental plants and arabidopsis thaliana (Chen et al, 2003).
At present, domestic researches on gingerol terpene synthases mainly concentrate on gingerol sesquiterpene synthase genes, for example, chinese patent CN109797161A discloses a gingerol sesquiterpene synthase gene HcTPS12 and application thereof, but reports on gingerol synthase genes are fresh. Citronellol is an important monoterpene compound and is also an important broad-spectrum perfume raw material, and is widely used as a flavoring agent in industry, natural citronellol exists in more than 70 kinds of essential oil, has sweet floral odor and is commonly used in essence with rose fragrance and citrus fragrance. Citronellol also has a potent antimicrobial effect, and studies have demonstrated that citronellol has a significant bacteriostatic effect on streptococcus mutans without compromising the viability of fibroblasts (rib eiro et al, 2018), and in most cases, citronellol imparts antimicrobial activity by disrupting cell walls and membranes, resulting in cell lysis, is a very potent antimicrobial compound (Lopez-Romero et al, 2015). The existing research reports that the TwMTS gene in the tripterygium wilfordii can catalyze GPP to produce beta-citronellol in vitro, but the TwMTS gene has a few related reports in other plants.
Therefore, cloning of the citronellol synthase gene is a precondition for research of the formation mechanism of the citronellol component of the curcuma, lays a theoretical foundation for comprehensively explaining the formation and regulation mechanism of the citronellol component of the curcuma and improving the content of the citronellol component of the curcuma through a genetic engineering means, and provides a novel bioengineering method for preparation of the citronellol.
Disclosure of Invention
The invention aims to overcome the defects of researches on the monoterpene synthase genes of the ginger flowers and provide a single-function enzyme gene HcTPS38 for controlling the monoterpene component citronellol in the ginger flowers.
The first object of the invention is to provide a citronellol synthase gene HcTPS38 from rhizoma Zingiberis recens.
A second object of the present invention is to provide a citronellol synthase HcTPS38 from ginger flowers.
The third object of the invention is to provide the application of the gingerol synthase gene HcTPS38 and/or gingerol synthase HcTPS38.
It is a fourth object of the present invention to provide a recombinant vector and recombinant bacterium comprising the recombinant vector and a cell line comprising the recombinant bacterium.
It is a fifth object of the present invention to provide a process for preparing citronellol.
It is a sixth object of the present invention to provide a method for constructing a transgenic material containing a monoterpene citronellol synthase gene.
The above object of the present invention is achieved by the following technical scheme:
cloning a citronellol synthase gene HcTPS38 with the length of 2146bp in the rhizome of the gingers, wherein the full-length cDNA sequence of the HcTPS38 gene is shown as SEQ ID NO. 1, the coding region (CDS) is 1752bp, and the nucleotide sequence is shown as SEQ ID NO. 2; the predicted code 584 amino acid with the amino acid sequence shown in SEQ ID NO. 3, the predicted protein molecular weight of 67.61kDa, isoelectric point (pI) of 5.33 and the gene sequence comprising the DDXXD conserved sequence.
The research of the invention shows that the expression level of the gingerol gene HcTPS38 in the rhizome of the gingerol is higher, and the expression level of the gingerol in the rhizome is positively correlated, and the enzyme generated by encoding the HcTPS38 gene is a functional enzyme gene capable of catalyzing GPP to generate monoterpene citronellol, so that the HcTPS38 gene can endow the rhizome of the gingerol with the citronellol component.
Thus, the present invention provides the use of the curcuma species citronellol synthase gene HcTPS38 or the curcuma species citronellol synthase HcTPS38 in the preparation of citronellol, in the breeding of citronellol plants, or in the construction of transgenic materials containing monoterpene citronellol synthase genes.
The invention provides a recombinant vector, which comprises a citronellol synthase gene HcTPS38 of gingerol.
The invention provides a recombinant bacterium comprising the recombinant vector.
The invention provides a cell line comprising the recombinant bacterium.
The invention obtains high-purity in-vitro recombinant protein through in-vitro induction by connecting the full-length cDNA sequence of the citronellol synthase gene HcTPS38 of the gingerol to a prokaryotic expression vector. In vitro enzyme activity experiments were performed on recombinant proteins in vitro by administering a reaction substrate to geranyl pyrophosphate (GPP). The result shows that the HcTPS38 protein catalyzes GPP to generate monoterpene citronellol, which indicates that the HcTPS38 protein is monoterpene synthetase with a catalytic function, and can be used for preparing citronellol by microbial metabolism engineering through a curcuma citronellol synthetase gene HcTPS38, so that the method can be used for preparing plant essential oil or medicines.
According to the sequence information of the HcTPS38 gene provided by the invention, the person skilled in the art can easily obtain the gene equivalent to HcTPS38 by: (1) obtained by database retrieval; (2) Screening a genomic library or a cDNA library of the ginger flower or other plants by taking the HcTPS38 gene fragment as a probe; (3) Designing an oligonucleotide primer according to the HcTPS38 gene sequence information, and obtaining the oligonucleotide primer from the genome, mRNA and cDNA of the ginger flower or other plants by using a PCR amplification method; (4) The gene is obtained by modifying a gene engineering method on the basis of an HcTPS38 gene sequence; (5) obtaining the gene by chemical synthesis.
The invention provides a method for preparing citronellol, which takes geranyl pyrophosphate GPP as a substrate and adopts a gingerol synthase HcTPS38 to catalyze and prepare the citronellol.
The invention also provides a method for constructing the transgenic material containing the monoterpene citronellol synthase gene, which constructs the gingerol synthase gene HcTPS38 on a plant expression vector, and obtains the transgenic material containing the monoterpene citronellol synthase gene by exogenous transformation of the plant material.
The invention has the following beneficial effects:
the invention provides a novel citronellol synthase gene HcTPS38, which can catalyze GPP to form monoterpene compound citronellol, and plays an important role in improving the content and resistance of plant terpenoid components; the citronellol synthase gene HcTPS38 can be used for preparing citronellol and further preparing essential oil, essence and medicines. The HcTPS38 gene segment is constructed on a plant expression vector, and other plant materials can be transformed exogenously, so that a transgenic material containing monoterpene citronellol synthase genes is obtained, and an effective method is provided for cultivating medicinal plants. The invention clones citronellol synthase genes, and solves the problem that genes cannot be transferred among plant species in traditional breeding.
In addition, the present invention can further provide or use a transgenic plant and corresponding seed having a medicinal value obtained using the above DNA fragment, and a plant transformed with the gene of the present invention or a recombinant based on the gene or a seed obtained from such a plant. The genes of the invention can be transferred into other plants by means of sexual crossing.
Drawings
FIG. 1 shows the cloning of the HcTPS38 gene and the analysis of the homology of the amino acid sequence of HcTPS38 (A: agarose gel electrophoresis of HcTPS38 clone; B: analysis of the homology of the amino acid sequence of HcTPS 38).
FIG. 2 shows the expression specificity of HcTPS38 gene of the present invention in different ginger flower variety rootstocks (A: different ginger flower varieties; B: hcTPS38 gene expression in 4 ginger flower rootstocks; citronellol content).
FIG. 3 shows prokaryotic expression of HcTPS38 gene and in-vitro enzyme catalysis reaction of recombinant protein (A: hcTPS38 recombinant protein SDS-PAGE gel electrophoresis, M is Marker, C is precipitate after breaking recombinant protein bacteria, S is supernatant after breaking recombinant protein bacteria, E is recombinant protein eluent, B: in-vitro catalysis GPP to produce citronellol).
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 sample ginger flower rootstocks adopted in the following examples are respectively taken from ginger flowers of Emei, red ginger flowers, ginger flowers of 'Mingyu' and ginger flowers of round-petal, and the 4 ginger flowers are planted in a ginger flower germplasm resource garden of a flower research center of agricultural university in south China.
EXAMPLE 1 obtaining of full-length cDNA of HcTPS38 Gene
Extracting total RNA of the rhizome of the gingers: the rhizome of the gingers 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 the rhizome of the gingers according to the specification of Trizol (TaKaRa) by adopting a Trizol method. 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.
The total RNA of the rhizome 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 gingerol transcriptome database, designing a primer, and an upstream primer F1:5'-ATGTCTCTTTTCCTTGCTCCAC-3' (SEQ ID NO: 4). Downstream primer R1:5'-AATGATAGGGTTGATCAACAGCGA-3' (SEQ ID NO: 5). And is synthesized by Shanghai bioengineering company.
PCR amplification was performed using the synthesized cDNA as a template using Phanta high fidelity enzyme, and the specific method was performed according to the instructions. Procedure for PCR amplification: 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.
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 of ampicillin was coated with 30. Mu.L of X-gal (20 mg/mL) and 30. Mu.L of IPTG (20 mg/mL), then with an appropriate amount of a conversion solution, and after the conversion solution was completely absorbed, the plate was inverted and incubated overnight at 37℃in an incubator, and the results were observed after about 16 hours.
White colonies were then screened by X-gal/IPTG blue-white spots and the recombinant plasmid was initially identified and the plates were stored 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 vector universal primers M13-47 and M13-48 according to specific methods.
The agarose gel electrophoresis result of the cloned gene is shown in figure 1A, the result shows that a single band which accords with the estimated size is obtained, the bacterial liquid containing the target fragment is selected for DNA sequence determination after the pMD19-T carrier is connected, and sequencing work is completed by Shanghai Biotechnology Co. Comparing the obtained sequence with original sequence information of genome, the comparison result shows that the invention clones a gene with the total length of 2146bp from the rhizome of the ginger flower, and the nucleotide sequence of the gene is shown as SEQ ID NO. 1; the total 1752bp of the gene coding region (CDS) has the nucleotide sequence shown in SEQ ID NO. 2; 584 amino acids are deduced according to cDNA sequence, the amino acid sequence is shown as SEQ ID NO. 3, the molecular weight of the deduced protein is 67.61kDa, and the isoelectric point (pI) is 5.33. The amino acid sequence information alignment and homology analysis were performed, as shown in FIG. 1B, and the Hc38 gene sequence contained a DDXXD conserved sequence, while the alignment and homology analysis were performed at NCBI, preliminarily identified as a TPS gene family member, designated as HcTPS38 gene.
EXAMPLE 2 analysis of expression of HcTPS38 Gene
The method comprises the steps of selecting different varieties of ginger flowers (ginger flowers, safflower flowers, ginger flowers of 'open moon' (ginger flowers of round-petal ginger flowers) (the phenotype of the different varieties of ginger flowers is shown in figure 2A)) to extract RNA, wherein the RNA extraction adopts a Trizol method (TaKaRa), the fluorescence quantitative PCR adopts a Hieff qPCR SYBR Green Master Mix (low Rox) method, and 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'-ACCGTCATACTTTCCTTTCCG-3' (SEQ ID NO: 6). P2:5'-ATTGCTGGTCGGCTGTCAT-3' (SEQ ID NO: 7). The reference gene GAPDH is designed according to the design principle of Real-time PCR Primer by using Primer premier 5.0, GAPDH-P1:5'-TAACATCATTCCCAGCAGCACT-3' (SEQ ID NO: 8), GAPDH-P2:5'-GAGCCTGACAGTGAGATCCAC-3' (SEQ ID NO: 9).
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 in ddH 2 O is 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 HcTExpression of the PS38 gene in different samples.
The results of the gene expression analysis are shown in FIG. 2B, from which it can be seen that: the HcTPS38 gene is highly expressed in the rhizome of the curcuma longa, the expression level of red Jiang Huazhong is low, the content of citronellol in the rhizome of the curcuma longa is highest, no expression is shown in the curcuma longa, the relative expression of the HcTPS38 gene in the rhizome of the curcuma longa is consistent with the expression level of citronellol in the rhizome, the result shows that the HcTPS38 gene is a related gene involved in regulating and controlling the synthesis of citronellol which is a monoterpene substance of the curcuma longa, and the HcTPS38 gene is attributed to terpene synthases (TPS) and identified as follows: the citronellol synthase HcTPS38 gene of the gingerol.
EXAMPLE 3 prokaryotic expression of the HcTPS38 Gene
(1) And (3) constructing a carrier: according to the coding region of the HcTPS38 gene obtained (SEQ ID NO: 2), a homologous recombination primer F:5'-gccatggctgatatcggatccATGTCTCTTTTCCTTGC TCCAC-3' (shown as SEQ ID NO: 10) comprising BamH I and Hind III cleavage sites was used; r:5'-gcaagcttgtcgacggagctcAATGATAGG GTTGATCAACAGCGA-3' (SEQ ID NO: 11), PCR amplification was performed. The pET-30a 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 Kan) liquid medium, cultured overnight at 37℃and 180 rpm. Transfer 100. Mu.L seed solution to fresh 100mL LB (containing 100 mg/LKan) liquid medium, culture at 37℃and 180rpm to OD 600 The value was 0.4-0.6, 10. Mu.L of IPTG (1M) was added, and induction was performed at 16℃for 20 hours. Another control group was also taken without IPTG induction. The thalli are collected by centrifugation,cells were suspended in 5mL lysis buffer (50 mM phosphate buffer pH 8.0), cooled, and sonicated on ice. 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. 50. Mu.L of each of the supernatant and the pellet was stored at-20℃and analyzed by SDS-PAGE. 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 the pre-chilled column, mixed well 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 20. Mu.L each was analyzed by SDS-PAGE. 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 catalytic function identification: 20. Mu.L of 30mM HEPES (pH 7.5), 50mM DTT,25mM MgCl 2 20. Mu.L each, 20. Mu.L of protein extract, 1. Mu.L of GPP, 20. Mu.L of pure glycerol, and ddH were added 2 O99 mu L, sealing in sample bottle, reacting at 28deg.C for 1 hr, inserting 75 μm polydimethyl oxolane (PMDS) extraction fiber head into glass bottle, performing solid-phase microextraction for 1 hr, and placing the extraction fiber head into gas phase after reactionThe analysis is carried out in a spectrum-mass spectrometer, and the gas chromatography conditions are as follows: 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 of the HcTPS38 gene is shown in FIG. 3A, which shows that the HcTPS38 recombinant protein can be expressed in supernatant after being induced by IPTG, and the size of the protein is about 67kDa and is close to the expected size; the in vitro enzyme activity identification result is shown in figure 3B, which shows that when GPP is used as a substrate, the in vitro enzyme catalysis reaction product of pET-30a-HcTPS38 is identified as monoterpene substance citronellol through mass spectrum. The enzyme coded and generated by the HcTPS38 gene is a functional enzyme gene which can catalyze GPP to generate monoterpene citronellol.
In conclusion, the invention provides a novel citronellol synthase gene HcTPS38, which can catalyze GPP to form monoterpene compound citronellol, and plays an important role in improving the content and resistance of plant terpenoid components; the citronellol synthase gene HcTPS38 can be used for preparing citronellol and further preparing essential oil, essence and medicines. The gene fragment of the invention is constructed on a plant expression vector, and other plant materials can be transformed exogenously, so that a transgenic material containing monoterpene citronellol synthase genes is obtained, and an effective method is provided for cultivating medicinal plants. Cloning of citronellol synthase genes is a precondition to overcome the problem of inability to transfer genes between plant species in traditional breeding. In addition, the present invention can further provide or use a transgenic plant and corresponding seed having a medicinal value obtained using the above DNA fragment, and a plant transformed with the gene of the present invention or a recombinant based on the gene or a seed obtained from such a plant. The genes of the invention can be transferred into other plants by means of sexual crossing.
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 citronellol synthase gene HcTPS38 is characterized in that the full-length cDNA sequence of the gene is shown as SEQ ID NO. 1, and the gene coding sequence is shown as SEQ ID NO. 2.
2. The citronellol synthase HcTPS38 is characterized in that the amino acid sequence of the synthase is shown as SEQ ID NO. 3.
3. Use of the gingerol synthase gene HcTPS38 of claim 1 or the gingerol synthase HcTPS38 of claim 2 for the preparation of citronellol.
4. Use of the gingerol synthase gene HcTPS38 of claim 1 or the gingerol synthase HcTPS38 of claim 2 in the breeding of citronellol plants.
5. Use of the gingerol synthase gene HcTPS38 of claim 1 or the gingerol synthase HcTPS38 of claim 2 for constructing transgenic material containing monoterpene citronellol synthase gene.
6. A recombinant vector comprising the gingerol synthase gene HcTPS38 according to claim 1.
7. A recombinant bacterium comprising the recombinant vector of claim 6.
8. A cell line comprising the recombinant bacterium of claim 7.
9. A method for preparing citronellol, characterized in that geranyl pyrophosphate GPP is used as a substrate, and the citronellol is prepared by catalysis with the curcuma species citronellol synthase HcTPS38 according to claim 2.
10. A method for constructing a transgenic material containing a monoterpene citronellol synthase gene, which is characterized in that the gingerol synthase gene HcTPS38 according to claim 1 is constructed on a plant expression vector, and the plant material is transformed by exogenous transformation, so as to obtain the transgenic material containing the monoterpene citronellol synthase gene.
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