CN109722443B - Ginger flower sesquiterpene synthase gene HcTPS14 and application thereof - Google Patents

Abstract

The invention discloses a ginger flower sesquiterpene synthase gene HcTPS14 and application thereof. The full-length cDNA sequence of the HcTPS14 gene is shown as SEQ ID NO 1; the coding sequence is shown as SEQ ID NO. 2; the coded amino acid sequence is shown in SEQ ID NO. 3. The HcTPS14 gene has certain expression in leaf tissue and rhizome, almost no expression in flower organ, and its expression is controlled by leaf development. Catalyzing the exogenous recombinant protein of HcTPS14 to generate sesquiterpene medicinal component alpha-caryophyllene, which can be used for preparing alpha-caryophyllene and further preparing essential oil, essence and medicine; connecting HcTPS14 with a plant transformation vector, and then introducing into ginger flower or other plant cells to obtain a transgenic plant expressing the gene; specific molecular markers can be generated according to the gene sequence information, and the molecular markers are used for identifying sesquiterpene alpha-caryophyllene synthetase genes of the zingiber officinale or other plants and used for molecular marker-assisted selective breeding, so that the breeding selection efficiency is improved, and the molecular markers have a wide application prospect.

Description

Ginger flower sesquiterpene synthase gene HcTPS14 and application thereof

Technical Field

The invention relates to the technical field of genetic engineering, and particularly relates to a zingiber officinale roscoe sesquiterpene synthetase gene HcTPS14 and application thereof.

Background

The zingiber officinale roscoe is a perennial herb of the genus zingiberaceae, and is a traditional medicinal plant in indian areas (Ray, et al, 2016). A large amount of essential oil can be extracted from rhizome, petal and leaf of Zingiber officinale Roscoe, and research shows that the Zingiber officinale Roscoe essential oil has antibacterial, antioxidant and antiinflammatory medicinal properties, and can be widely used in skin care products, essence and perfume industries, and contains terpene compounds (Santos, et al., 2010; Dixit,2018) as main component. Terpenoids are a general term for a class of compounds consisting of several Isoprene (Isoprene, C5) structural units, and are classified into monoterpenes (C10), sesquiterpenes (Sesquiterpene, C15), and diterpenes (Diterpene, C20), etc. (Martin et al, 2015), depending on the number of structural units. Sesquiterpenes are important medicinal ingredients, and are reported to be used for treating tumors (Wuna et al, 2016), sesquiterpene artemisinin is a specific drug for treating malaria (Mann, et al, 2000), sesquiterpene alpha-caryophyllene has local anesthetic, anti-inflammatory, analgesic and antidepressant effects, is an important raw material of various drugs, and is an important spice (Liuxiayu et al, 2012). .

Terpene synthases are the terminal key enzymes in terpenoid biosynthesis, which directly determine the type, quantity and yield of terpene products. TPS is classified into monoterpene synthase (MonoTPS), sesquiterpene synthase (SesquiTPS) and diterpene synthase (DiTPS) which catalyze the formation of the corresponding monoterpene, sesquiterpene and diterpene products by the substrates GPP, FPP and GGPP, respectively, depending on the products which TPS catalyzes.

Terpene synthases are key enzymes of terpenoid biosynthesis and have therefore become the most studied and most intensive enzymes in terpene biosynthesis, since the cloning of two sesquiterpene synthase genes in tobacco in 1992 (Facchini and Chappell,1992), scientists have cloned over 200 monoterpene and sesquiterpene synthase genes in over 40 plants (Degenhardt et al, 2009), related to crop plants (Pen et al, 1995; Kollner et al, 2008; Yuan et al, 2008; Chen et al, 2014), conifer plants (Bohlmann et al, 1999; Martin et al, 2004), medicinal plants (Deguerrer et al, 2006), spice and ornamental plants, and Arabidopsis thaliana (Chen et al, 2003), among others.

Patent CN201810701370.6 discloses a Machilus sesquiterpene synthetase SgSTPS1, wherein the Machilus sesquiterpene synthetase SgSTPS1 protein can generate active sesquiterpene synthetase after prokaryotic expression, and a substrate FPP is catalyzed to generate beta-caryophyllene, isocaryophyllene and alpha-caryophyllene. However, at present, the research on the terpene synthases of the ginger flowers in China is mainly focused on monoterpene synthase genes, and the sesquiterpene synthase genes of the ginger flowers are rarely reported. The cloning of the sesquiterpene synthase gene is a precondition for researching the formation mechanism of the sesquiterpene component in the zingiber officinale roscoe, lays a theoretical foundation for comprehensively explaining the formation and regulation mechanism of the sesquiterpene component in the zingiber officinale roscoe and improving the content of the sesquiterpene component in the zingiber officinale roscoe through a genetic engineering means, and provides a new bioengineering method for preparing caryophyllene.

Disclosure of Invention

The invention aims to solve the technical problem of overcoming the defects and shortcomings of the study on the sesquiterpene synthase gene of the ginger flower and provides a monofunctional enzyme gene HcTPS14 for controlling the sesquiterpene component alpha-caryophyllene in the ginger flower.

The first purpose of the invention is to provide a ginger flower sesquiterpene synthetase gene HcTPS 14.

The second purpose of the invention is to provide a zingiber officinale roscoe sesquiterpene synthetase HcTPS 14.

The third purpose of the invention is to provide the application of the zingiber officinale roscoe sesquiterpene synthetase gene HcTPS14 and/or the zingiber officinale roscoe sesquiterpene synthetase HcTPS14 in preparation of alpha-caryophyllene.

The invention realizes the above purpose by the following technical scheme:

a zingiber officinale roscoe sesquiterpene synthase gene HcTPS14 has a full-length cDNA sequence shown in SEQ ID NO. 1, and can endow a zingiber officinale roscoe leaf sesquiterpene alpha-caryophyllene component with the full-length cDNA sequence. The ginger flower gene HcTPS14 shown in the invention has higher expression level in leaf tissue, and the expression is related to the development process of leaves. The full length of the cDNA of the HcTPS14 gene is 1852bp, and the nucleotide sequence is shown as SEQ ID NO 1; 1515bp gene coding region (CDS), the nucleotide sequence is shown in SEQ ID NO: 2; the presumed code 504 amino acids, the amino acid sequence is shown in SEQ ID NO. 3, the presumed molecular weight of the protein is 58.7 kDa. The gene sequence contains a DDXXD conserved sequence, and gene phylogenetic tree analysis shows that the gene sequence belongs to the Tps-a subfamily of a plant terpene synthase gene family.

Therefore, the invention also claims a zingiber officinale roscoe sesquiterpene synthetase HcTPS14, the amino acid sequence of which is shown in SEQ ID NO. 3.

Based on the sequence information of the HcTPS14 gene provided by the present invention, a person skilled in the art can easily obtain a gene equivalent to HcTPS14 by the following method: (1) obtaining through database retrieval; (2) screening a genomic library or a cDNA library of the ginger flower or other plants by using the HcTPS14 gene fragment as a probe to obtain the HcTPS14 gene fragment; (3) designing oligonucleotide primers according to the HcTPS14 gene sequence information, and obtaining from the genome, mRNA and cDNA of the ginger flower or other plants by using a PCR amplification method; (4) obtained by modifying a gene engineering method on the basis of the HcTPS14 gene sequence; (5) the gene is obtained by a chemical synthesis method.

The invention also provides a primer pair for amplifying the ginger flower sesquiterpene synthetase gene HcTPS14, wherein the primer sequence is shown in SEQ ID NO. 4-5.

Meanwhile, the invention also provides a recombinant vector containing the ginger flower sesquiterpene synthetase gene HcTPS 14.

A recombinant bacterium comprising the recombinant vector.

A cell line comprising the recombinant bacterium.

The invention obtains high-purity in vitro recombinant protein by connecting the full-length cDNA sequence of the sesquiterpene synthase gene HcTPS14 from the zingiber officinale roscoe to a prokaryotic expression vector and carrying out in vitro induction. The in vitro enzyme activity experiment is carried out on the in vitro recombinant protein by giving a reaction substrate FPP. The result shows that the HcTPS14 protein catalyzes FPP to generate a sesquiterpenoid substance alpha-caryophyllene, which accounts for 100 percent of the total product of the reaction. The alpha-caryophyllene is one of the components of the ginger flower essential oil. The HcTPS14 protein is shown to be sesquiterpene synthetase with a single catalytic function, and meanwhile, the zingiber officinale sesquiterpene synthetase gene HcTPS14 can be used for preparing alpha-caryophyllene through microbial metabolic engineering so as to prepare plant essential oil or medicines.

Therefore, the application of the zingiberene sesquiterpene synthetase gene HcTPS14 and/or the zingiberene sesquiterpene synthetase HcTPS14 in preparation of alpha-caryophyllene or in preparation of spices, essential oil or medicines is also within the protection scope of the invention.

Specifically, the application is to produce alpha-caryophyllene by using farnesyl pyrophosphate (FPP) as a substrate and catalyzing by using a zingiber officinale sesquiterpene synthetase HcTPS 14.

The zingiber officinale roscoe sesquiterpene synthase gene HcTPS14 provided by the invention has important application value. One application is that the HcTPS14 gene sequence is connected to any plant transformation vector, and HcTPS14 gene is introduced into ginger flower or other plant cells by any transformation method, so that transgenic plants expressing the gene can be obtained and can be applied to production. When the gene is constructed in 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 promoter of the gene before a transcription initiation codon of the gene, so that the capability of the plant for producing the sesquiterpene alpha-caryophyllene and enhancing the resistance is widened and enhanced.

Another application of the sesquiterpene synthetase gene HcTPS14 provided by the invention is to generate specific molecular markers according to the gene sequence information, including but not limited to SNP (single nucleotide polymorphism), SSR (simple sequence repeat polymorphism), RFLP (restriction endonuclease length polymorphism), CAP (cut amplified fragment polymorphism). The markers can be used for identifying sesquiterpene alpha-caryophyllene synthetase genes of the ginger flowers or other plants, and are used for molecular marker-assisted selection breeding, so that the breeding selection efficiency is improved.

Compared with the prior art, the invention has the following beneficial effects:

the invention provides a novel sesquiterpene synthase gene HcTPS14, which can catalyze FPP to form a sesquiterpene compound alpha-caryophyllene and plays an important role in improving the content and resistance of plant terpenoid components; the sesquiterpene synthetase gene HcTPS14 can be used for preparing alpha-caryophyllene and further preparing essential oil, essence and medicines. The gene fragment is constructed on a plant expression vector, and other plant materials can be exogenously transformed, so that a transgenic material containing the sesquiterpene alpha-caryophyllene synthetase gene is obtained, and an effective method is provided for cultivating medicinal plants. The cloning of sesquiterpene synthetase gene is a precondition for overcoming the problem that the gene can not be transferred between plant species in the traditional breeding. In addition, the present invention can further provide or use transgenic plants and corresponding seeds having medicinal value obtained by using the above DNA fragments, and plants transformed with the gene of the present invention or recombinants based on the gene or seeds obtained from such plants. The gene of the invention can be transferred to other plants by sexual crossing.

Drawings

FIG. 1 shows the expression specificity of HcTPS14 gene in different tissues of Zingiber officinale Roscoe; pe is petal; ri is rhizome; le, leaf.

FIG. 2 shows the expression of the HcTPS14 gene in different developmental stages of the ginger flower leaf organ.

FIG. 3 shows the in vitro enzyme-catalyzed reaction of the recombinant protein HcTPS14 of the present invention with FPP as the substrate.

Detailed Description

The present invention will be described in further detail with reference to the following drawings and specific examples. Unless otherwise indicated, the reagents and methods employed in the examples are those conventionally used in the art.

Example 1 obtaining of full Length of HcTPS14 Gene cDNA

S1, extracting the RNA of the ginger flower leaves: the ginger flower leaves stored in an ultra-low temperature refrigerator are used as a material for extracting RNA. Soaking a gun head and an Eppendorf tube with 0.1% DEPC at 37 ℃ overnight, sterilizing at 121 ℃ for 25min, wrapping with aluminum foil in a glass ware and a mortar, performing dry heat treatment at 180 ℃ for 3h, and cooling for later use. The total RNA of the ginger leaves is extracted by a Trizol method according to the instructions 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 microspectrophotometric method. Storing at-80 deg.C for use.

S2, using the total RNA of the ginger flower leaves as a template, and synthesizing First strand cDNA by using a raw M-MuLV First cDNA Synthesis Kit. According to related annotation gene sequences of a ginger flower transcriptome database, primers are designed, and an upstream primer F1: 5'-TCCGAAACTCCTTTCACTCA-3' (shown in SEQ ID NO: 4). The downstream primer R1: 5'-TGGTTGCCTCTAACGATTTT-3' (shown in SEQ ID NO: 5). And is synthesized by Shanghai bioengineering company. The cDNA synthesized above was used as a template for PCR Amplification reaction using TaKaRa PCR Amplification Kit, and the specific procedure was as described in the specification. The PCR procedure was: pre-denaturation at 94 ℃ for 4 min; denaturation at 94 ℃ for 30s, renaturation at 56 ℃ for 30s, extension at 72 ℃ for 2min, 35 cycles; then extended for 10min at 72 ℃. Storing at-20 deg.C for use. After the PCR reaction is finished, the band of the target fragment is detected in the PCR product by 1.0% agarose gel electrophoresis. After the PCR amplification product is detected by 1% Agarose Gel electrophoresis, a Gel block containing the target fragment is cut out under an ultraviolet lamp by a scalpel, and is recovered by a DNA Gel recovery Kit (Agarose Gel DNA Purification Kit, TaKaRa), and the recovery method basically refers to the Kit specification. And then, carrying out 1% agarose electrophoresis detection on the recovered product to see the recovery effect and approximate concentration of the recovered product so as to ensure the subsequent test. According to the size and effective concentration of the recovered target fragment, connecting a proper amount of recovered and purified product with a cloning vector, wherein the vector is TaKaRa pMD19-T vector, and the molar ratio of the target DNA to the cloning vector is controlled to be 3: about 1, the specific operation is performed according to the specification. Connecting at 16 ℃ for 3-6 h at constant temperature, wherein the connection time depends on the length of the target fragment. Taking out the competent cell DH5 alpha (TaKaRa) from a refrigerator at minus 80 ℃ in advance, placing the cell in an ice box for natural melting, adding 10 mu L of the whole connecting solution into a centrifuge tube of the competent cell, carrying out ice bath for 30min, carrying out water bath heat shock at 42 ℃ for 50s, rapidly placing the cell on ice for 2-5 min, adding 890 mu L of SOC liquid culture medium pre-warmed at 37 ℃ to complement to 1mL, mixing uniformly, and carrying out shake culture at 37 ℃ and 180rpm for 1 h. 30 mu L of X-gal (20mg/mL) and 30 mu L of IPTG (20mg/mL) are coated on the surface of an LB solid medium plate containing 100 mu g/mL of ampicillin, then a proper amount of transformation liquid is coated, the transformation liquid is placed in an incubator at 37 ℃ for overnight culture after being completely absorbed, the result is observed after about 16 hours, white colonies are screened by X-gal/IPTG blue white spots, the recombinant plasmid is preliminarily identified, and the plate is stored at 4 ℃. After primary screening by blue-white spots, 6 plaques are usually selected and shaken to extract plasmids for further identification. White single colonies were picked from LB plate medium with sterilized toothpicks and inoculated into LB liquid medium containing 100. mu.g/mL ampicillin, cultured overnight with shaking at 240rpm on a temperature-controlled shaking water bath shaker at 37 ℃ and plasmid DNA minipump kit (Shanghai Boya Biol Ltd.) to extract plasmids by the method described above. The upgraded particles were checked by 1.0% agarose gel electrophoresis, the plasmid sizes were compared and the significantly lagging plasmid was analyzed by double restriction digestion (EcoRI/Hind III, TaKaRa). After digestion at 37 ℃ for 1h, the digestion product was examined by electrophoresis on a 1.0% agarose gel. And (4) randomly selecting recombinant plasmids containing target fragments after enzyme digestion identification for DNA sequence determination. Sequencing work was carried out by Shanghai Biotechnology Ltd using an American ABI377 sequencer. The obtained sequence is compared with the original sequence information of the transcriptome, the comparison and the homology analysis are carried out at NCBI, the obtained gene sequence is determined to be the complete full-length sequence of the TPS family, and the protein sequence is deduced according to the cDNA sequence.

The result shows that the full-length cDNA sequence of the HcTPS14 gene is shown as SEQ ID NO. 1, and the full-length cDNA is 1852 bp; the gene coding region (CDS) is shown as SEQ ID NO. 2 and is 1515bp in total; the amino acid sequence is presumed to be shown in SEQ ID NO 3.

Example 2 expression analysis of HcTPS14 Gene

1. Selecting different tissue parts and leaves of the ginger flower at different development periods to extract RNA, wherein the RNA extraction adopts a Trizol method (TaKaRa), a SYBR green (TaRaKa) method is adopted for fluorescent quantitative PCR, and the specific principle of a dye method is shown in an instruction book. Designing real-time fluorescent quantitative PCR primers by using Primer Premier 5.0 software, respectively designing primers by using Primer Premier 5.0 according to the fluorescent quantitative PCR Primer design principle, detecting whether the primers have mismatching or Primer dimer and amplification efficiency thereof by fluorescent quantitative PCR, and selecting a pair of optimal primers from the primers, wherein the Primer is P1: 5'-ATCCAACAGCAGTAGCTCGAC-3' (shown in SEQ ID NO: 6). P2: 5'-GGTTCAACCAGCACCAAAGA-3' (shown in SEQ ID NO: 7). The reference gene RPS uses Primer according to the design principle of Real-time PCR Primerpremier 5.0 design primer, RPS-P1: 5'-TTAGTAGCATCGGCTGCAATAAG-3' (shown in SEQ ID NO: 8), RPS-P2: 5'-CTCAACCGTCTTCCCAAAAGAG-3' (shown in SEQ ID NO: 9). And (3) detecting by Real-time PCR, and making a standard curve to detect whether the amplification efficiency (E) is screened within the range of 90-110%. And (3) performing fluorescent quantitative PCR reaction on an ABI fluorescent quantitative PCR instrument by taking the cDNA of each sample as a template. 3 replicates per sample in ddH₂O is a negative control. The reaction system is SYBR Premix ExTaq (TaKaRa)10.0 μ L, upstream primer (10 μ M)0.4 μ L, downstream primer (10 μ M)0.4 μ L, cDNA 2.0 μ L, ddH₂O7.2. mu.L. The reaction program is 94 ℃ for 30 s; 15s at 94 ℃; 30s at 55 ℃; 72 ℃ for 30 min; 40 cycles, 94 ℃, 15 s; 72 ℃ for 30 s; melting curve analysis at 0.4 ℃/s. After completion of the reaction, the amplification curve and the melting curve were confirmed, and 2 was used^－△△CtThe method (Livak et al, 2001) performed data analysis to calculate the expression of the flowers of Zingiber officinale HcTPS14 in different samples.

2. Results

The results of the gene expression analysis are shown in FIGS. 1 and 2. As can be seen from fig. 1: HcTPS14 has some expression in both leaf tissue and rhizome, and hardly expressed in flower organs. FIG. 2 shows that: the expression level of the gene is regulated and controlled by the development of leaves, the expression level in old leaves is higher, and the expression level is consistent with the change trend of the content of sesquiterpene alpha-caryophyllene in the ginger flower essential oil, which indicates that HcTPS14 is a related gene participating in regulating and controlling the synthesis of terpene substances in ginger flowers.

Example 3 prokaryotic expression of HcTPS14 Gene

S1, vector construction: based on the coding region of the HcTPS14 gene obtained, 5 '-GATCTG was digested with a specific primer F: 5' -GATCTG containing KpnI and EcoRI cleavage sitesGGTACCATGATAAAGAGAGTGGAAGA-3' (shown in SEQ ID NO: 10); r: 5' -GAGCTCGAATTCTATAGGAATGGGTTCAACCAGCA-3' (shown in SEQ ID NO: 11). PCR amplification was performed. The PCR product is recovered by a Takara recovery kit, the recovered product is directly subjected to double enzyme digestion by KpnI and EcoRI restriction enzymes, and the target fragment is recovered by 1% agarose gel. The pET-30a prokaryotic expression vector is subjected to double digestion with KpnI and EcoRI restriction enzymes to recover a large fragment in 1% agarose gel. Ligation was carried out overnight at 16 ℃ andtransforming Escherichia coli (E.coli) DH5 alpha competent cells; and (3) carrying out enzyme digestion and sequencing identification on the extracted plasmid to obtain a recombinant prokaryotic expression vector.

S2, recombinant protein expression: BL21(DE3) competent cells were transformed with the identified recombinant plasmid DNA, and a single colony was picked and inoculated into 5mL of LB medium (containing 25mg/L Kan, 34mg/L Chl) and cultured overnight with shaking at 37 ℃. Transferring 100 mu L of seed solution into a fresh 100ml LB culture medium (containing 25mg/L Kan, 34mg/L Chl), culturing at 37 ℃ and 180rpm for 4-6 h, measuring OD value to 0.4-0.6, and inducing with a certain amount of IPTG (0.1-0.2 mM) at 14-18 ℃ for 14-16 h. At the same time, another control group was taken in which no IPTG induction was added. The cells were collected by centrifugation, suspended in 5ml lysis buffer (50mM phosphate buffer pH 8.0), cooled and placed on ice for cell disruption by sonication. Centrifuging at 12000rpm at 4 ℃ for 10min, transferring the supernatant into a new centrifuge tube, washing the precipitate once with double distilled water, and suspending the precipitate with 5mL lysis buffer. The supernatant and the precipitate were collected at 50. mu.L each, and stored at-20 ℃ for SDS-PAGE analysis. 12.5% SDS polyacrylamide gel was prepared and loaded sequentially. The gel was electrophoresed with 60V and 120V, respectively. And after the electrophoresis is finished, dyeing the gel with Coomassie brilliant blue for 30min, decoloring the gel with decoloring solution for 1-8 h, and observing and recording the test result.

S3, purifying the recombinant protein: a single colony is selected and inoculated in 5mL of liquid LB culture medium, cultured at 37 ℃ and 180rpm overnight, and then all the colonies are transferred to 500mL of fresh liquid LB culture collection, cultured at 37 ℃ and 180rpm for 4h, induced by IPTG (0.1-0.2 mM) at 18 ℃ for 16h, and then the thalli are collected. 200. mu.L of the cells were put into a centrifuge tube, stored at 4 ℃ and analyzed by SDS-PAGE electrophoresis. 5mL of lysed buffer was used to suspend the cells and transferred to a centrifuge tube, which was placed on ice to keep the cells cool all the time, and the cells were disrupted by ultrasound. Centrifuging at 4 ℃ for 20-30 min at 10,000g, and collecting supernatant. 20 μ L of the supernatant was stored at-20 ℃ and analyzed by SDS-PAGE. Adding 1-2 mL of Ni-NTA resin (nickel-nitrilotriacetic acid resin filler) into 5mL of cell lysate, mixing uniformly, and combining on a shaker at low speed at 4 ℃ for 60 min. The well-bound cell lysate and resin mixture was loaded onto a chromatography column, the bottom cap was removed and the effluent fraction (F) was collected, 20. mu.L of the effluent was taken, stored at-20 ℃ and analyzed by SDS-PAGE. The column was eluted twice with 4ml of wash buffer, and the eluates (wash fraction, W) were collected from each fraction, and 20. mu.L of each fraction was stored in a-20 ℃ freezer for SDS-PAGE analysis. The column was washed four times with 0.5mL elution buffer, and the eluates (E) from each fraction were collected sequentially using separate collection tubes, labeled E1, E2, E3, and E4, and 20. mu.L of each fraction was analyzed by SDS-PAGE. According to the SDS-PAGE detection result, the fractions containing the target protein eluate were pooled in a centrifuge tube, transferred to a dialysis bag by a pipette, and dialyzed overnight at 4 ℃. Collecting dialyzed solution, adding glycerol to make total content of glycerol in protein solution 20%, subpackaging at 200 μ L/tube, collecting trace amount, performing SDS-PAGE protein concentration detection, and storing the rest in-80 deg.C ultra-low temperature refrigerator for use.

S4, enzyme catalysis function identification: 30mM HEPES,100ul pH 7.5,5mM DTT,20mM MgCl₂Each 100ul, and protein extract 100ul, 2ul GPP or FPP, plus ddH₂And O588 mu L, the final volume is 1ml, the mixture is added into a sample bottle and sealed, water bath at 30 ℃ is carried out for 1h, a 75 mu m Polydimethylsiloxane (PMDS) extraction fiber head is inserted into a glass bottle, headspace solid phase microextraction is carried out for 1h, and the extraction fiber head is put into a gas chromatography-mass spectrometer for analysis after the reaction is finished, wherein the gas chromatography conditions are as follows: the chromatographic column is HP-1NNOWAX column (30m × 0.25 mm); the carrier gas is high-purity helium, and the split ratio is 20: 1, the column front pressure is 50Pa, and the flow rate is 1 mL/min; sampling time is 2 min; temperature programming: the column was started at 45 ℃ for 2min, ramped at 5 ℃/min to 80 ℃ for 1min, and ramped at 10 ℃/min to 250 ℃ for 5 min. The mass spectrum conditions are as follows: the interface temperature of GC-MS is 220 ℃, electron bombardment sources EI and 350V; the ion source temperature is 170 ℃; electron energy 70 eV; scanning the mass range from 35 to 335aum, and analyzing the collected mass spectrum by using a WILLEY/MAINLIB library. The result of prokaryotic expression of the HcTPS14 gene is shown in FIG. 3. FIG. 3 shows: when FPP is used as a substrate, a product of in vitro enzyme catalysis reaction of pET-30a-HcTPS14 is identified as a sesquiterpene substance alpha-caryophyllene by mass spectrometry. The enzyme generated by the coding of the HcTPS14 gene is a monofunctional enzyme gene which can catalyze FPP to generate a hemiterpene substance alpha-caryophyllene.

Sequence listing

<120> ginger flower sesquiterpene synthase gene HcTPS14 and application thereof

<141> 2019-02-28

<160> 11

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1852

<212> DNA

<213> ginger flower (Hedychium coronarium Koen)

<400> 1

gaagcaattg ctaaattttg ttggtgatga tgaagtgata gttcgtaagt ctacacaata 60

tcatccaagt gtttggggtg attttttcat ccgaaactcc tttcactcac aaacacagga 120

gtcatttcaa aggatgataa agagagtgga agaattaaag gtgcgagtaa agacaatgtt 180

caaggacact agtgacattc tgcaacttat gaatttgatt gattcaattc aacttcttag 240

actggactat cattttgagg atgaaatagc tggcgcatta agattgatct ttgaggttga 300

cgataagata tatgggctct atgaaacttc tcttagattt cgattgctta ggcagcatgg 360

atataatatt tctgcagata cttttaacaa gttcaaagat gacaatggaa gctttatatc 420

taccttgaat ggagatgcaa agggactact aagcttatat aatgcgtctt atcttgcaac 480

acatggagag actatacttg atgatgccaa gtatttttca aagtctcaac tagtatcctt 540

attgagtgaa cttgaacaac ctttagagac acaagtatct cttttccttg aggttccact 600

atgtcgaaga attaaaagtc tcttggcgag aatatatata cctatttatc aaaaggatgc 660

aacgcgagat gatgtcatat tagaacttgc aaaattggat tttaatctac tgcaatctct 720

ttatcaagag gaattgaaga aagtttcgat atggtggaat aatttagcac ttgctaaatc 780

actaaagttt gctcgtgatc gaattgtgga aggttattat tgggttcttg gtatgtatta 840

tgagcctcaa tattctcgtg cacgagtgat gtgcacaaaa gtattttgtc ttctatcagt 900

tatggatgat acctatgata actatagcac attggaagag agcaaactat taactgaagc 960

aatcaagagg tggaattgtc aagctgctga ttctttacca gaatgcataa atttttttta 1020

tctcaagcta ttaaagagtc tcaaagaatt tgaagcagag ttggaactta atgagaagta 1080

tcgtgtggaa taccttaaaa atgaatttaa agttgtagcc atagcatatt ttgaagaatc 1140

taagtggggt gcggagcgat atgttccaat acttgatgaa cacttgcatg tttcattgat 1200

ctcctccgca tgttctttgg ttatttgttc catgtatttg ggcatgggag aagtggcaac 1260

aaaagaggtt ttcgagtggt attctagttt tcccaagccc gtggaagctt gctctgtaat 1320

tggtagactc ctcaatgata taagatcaca tgagacagag caagagagag accatgttgc 1380

ctctacagtg gaaagttaca tgaaagagca tggcacagat gtaaaagttg catgcaagaa 1440

gctacgagag atagtggaaa aagcatggaa ggatctaaat aaggaacgtc tcaatccaac 1500

agcagtagct cgacctataa ttgaaagaat actcagcttt tcaatatcaa tggaagacgt 1560

ctataggtac accgatgagt acactaattc tgataataaa atgaaagata atatctcttt 1620

ggtgctggtt gaacccattc ctatataaga gtgtgacttc ttcaataaat caatgggaag 1680

agcatgttga aaatcgttag aggcaaccat tacagtgtga ggatgttcaa tacattatca 1740

atgtttgaat aatgtaaact catatttgga tgtttttatg gaaaagttat aattgatatg 1800

tagttatata ttatgtaact tgcttaataa aatttgcacg tttgcttgga ag 1852

<210> 2

<211> 1515

<212> DNA

<213> ginger flower (Hedychium coronarium Koen)

<400> 2

atgataaaga gagtggaaga attaaaggtg cgagtaaaga caatgttcaa ggacactagt 60

gacattctgc aacttatgaa tttgattgat tcaattcaac ttcttagact ggactatcat 120

tttgaggatg aaatagctgg cgcattaaga ttgatctttg aggttgacga taagatatat 180

gggctctatg aaacttctct tagatttcga ttgcttaggc agcatggata taatatttct 240

gcagatactt ttaacaagtt caaagatgac aatggaagct ttatatctac cttgaatgga 300

gatgcaaagg gactactaag cttatataat gcgtcttatc ttgcaacaca tggagagact 360

atacttgatg atgccaagta tttttcaaag tctcaactag tatccttatt gagtgaactt 420

gaacaacctt tagagacaca agtatctctt ttccttgagg ttccactatg tcgaagaatt 480

aaaagtctct tggcgagaat atatatacct atttatcaaa aggatgcaac gcgagatgat 540

gtcatattag aacttgcaaa attggatttt aatctactgc aatctcttta tcaagaggaa 600

ttgaagaaag tttcgatatg gtggaataat ttagcacttg ctaaatcact aaagtttgct 660

cgtgatcgaa ttgtggaagg ttattattgg gttcttggta tgtattatga gcctcaatat 720

tctcgtgcac gagtgatgtg cacaaaagta ttttgtcttc tatcagttat ggatgatacc 780

tatgataact atagcacatt ggaagagagc aaactattaa ctgaagcaat caagaggtgg 840

aattgtcaag ctgctgattc tttaccagaa tgcataaatt ttttttatct caagctatta 900

aagagtctca aagaatttga agcagagttg gaacttaatg agaagtatcg tgtggaatac 960

cttaaaaatg aatttaaagt tgtagccata gcatattttg aagaatctaa gtggggtgcg 1020

gagcgatatg ttccaatact tgatgaacac ttgcatgttt cattgatctc ctccgcatgt 1080

tctttggtta tttgttccat gtatttgggc atgggagaag tggcaacaaa agaggttttc 1140

gagtggtatt ctagttttcc caagcccgtg gaagcttgct ctgtaattgg tagactcctc 1200

aatgatataa gatcacatga gacagagcaa gagagagacc atgttgcctc tacagtggaa 1260

agttacatga aagagcatgg cacagatgta aaagttgcat gcaagaagct acgagagata 1320

gtggaaaaag catggaagga tctaaataag gaacgtctca atccaacagc agtagctcga 1380

cctataattg aaagaatact cagcttttca atatcaatgg aagacgtcta taggtacacc 1440

gatgagtaca ctaattctga taataaaatg aaagataata tctctttggt gctggttgaa 1500

cccattccta tataa 1515

<210> 3

<211> 504

<212> PRT

<213> ginger flower (Hedychium coronarium Koen)

<400> 3

Met Ile Lys Arg Val Glu Glu Leu Lys Val Arg Val Lys Thr Met Phe

1 5 10 15

Lys Asp Thr Ser Asp Ile Leu Gln Leu Met Asn Leu Ile Asp Ser Ile

20 25 30

Gln Leu Leu Arg Leu Asp Tyr His Phe Glu Asp Glu Ile Ala Gly Ala

35 40 45

Leu Arg Leu Ile Phe Glu Val Asp Asp Lys Ile Tyr Gly Leu Tyr Glu

50 55 60

Thr Ser Leu Arg Phe Arg Leu Leu Arg Gln His Gly Tyr Asn Ile Ser

65 70 75 80

Ala Asp Thr Phe Asn Lys Phe Lys Asp Asp Asn Gly Ser Phe Ile Ser

85 90 95

Thr Leu Asn Gly Asp Ala Lys Gly Leu Leu Ser Leu Tyr Asn Ala Ser

100 105 110

Tyr Leu Ala Thr His Gly Glu Thr Ile Leu Asp Asp Ala Lys Tyr Phe

115 120 125

Ser Lys Ser Gln Leu Val Ser Leu Leu Ser Glu Leu Glu Gln Pro Leu

130 135 140

Glu Thr Gln Val Ser Leu Phe Leu Glu Val Pro Leu Cys Arg Arg Ile

145 150 155 160

Lys Ser Leu Leu Ala Arg Ile Tyr Ile Pro Ile Tyr Gln Lys Asp Ala

165 170 175

Thr Arg Asp Asp Val Ile Leu Glu Leu Ala Lys Leu Asp Phe Asn Leu

180 185 190

Leu Gln Ser Leu Tyr Gln Glu Glu Leu Lys Lys Val Ser Ile Trp Trp

195 200 205

Asn Asn Leu Ala Leu Ala Lys Ser Leu Lys Phe Ala Arg Asp Arg Ile

210 215 220

Val Glu Gly Tyr Tyr Trp Val Leu Gly Met Tyr Tyr Glu Pro Gln Tyr

225 230 235 240

Ser Arg Ala Arg Val Met Cys Thr Lys Val Phe Cys Leu Leu Ser Val

245 250 255

Met Asp Asp Thr Tyr Asp Asn Tyr Ser Thr Leu Glu Glu Ser Lys Leu

260 265 270

Leu Thr Glu Ala Ile Lys Arg Trp Asn Cys Gln Ala Ala Asp Ser Leu

275 280 285

Pro Glu Cys Ile Asn Phe Phe Tyr Leu Lys Leu Leu Lys Ser Leu Lys

290 295 300

Glu Phe Glu Ala Glu Leu Glu Leu Asn Glu Lys Tyr Arg Val Glu Tyr

305 310 315 320

Leu Lys Asn Glu Phe Lys Val Val Ala Ile Ala Tyr Phe Glu Glu Ser

325 330 335

Lys Trp Gly Ala Glu Arg Tyr Val Pro Ile Leu Asp Glu His Leu His

340 345 350

Val Ser Leu Ile Ser Ser Ala Cys Ser Leu Val Ile Cys Ser Met Tyr

355 360 365

Leu Gly Met Gly Glu Val Ala Thr Lys Glu Val Phe Glu Trp Tyr Ser

370 375 380

Ser Phe Pro Lys Pro Val Glu Ala Cys Ser Val Ile Gly Arg Leu Leu

385 390 395 400

Asn Asp Ile Arg Ser His Glu Thr Glu Gln Glu Arg Asp His Val Ala

405 410 415

Ser Thr Val Glu Ser Tyr Met Lys Glu His Gly Thr Asp Val Lys Val

420 425 430

Ala Cys Lys Lys Leu Arg Glu Ile Val Glu Lys Ala Trp Lys Asp Leu

435 440 445

Asn Lys Glu Arg Leu Asn Pro Thr Ala Val Ala Arg Pro Ile Ile Glu

450 455 460

Arg Ile Leu Ser Phe Ser Ile Ser Met Glu Asp Val Tyr Arg Tyr Thr

465 470 475 480

Asp Glu Tyr Thr Asn Ser Asp Asn Lys Met Lys Asp Asn Ile Ser Leu

485 490 495

Val Leu Val Glu Pro Ile Pro Ile

500

<210> 4

<211> 20

<212> DNA

<213> ginger flower (Hedychium coronarium Koen)

<400> 4

tccgaaactc ctttcactca 20

<210> 5

<211> 20

<212> DNA

<213> ginger flower (Hedychium coronarium Koen)

<400> 5

tggttgcctc taacgatttt 20

<210> 6

<211> 21

<212> DNA

<213> ginger flower (Hedychium coronarium Koen)

<400> 6

atccaacagc agtagctcga c 21

<210> 7

<211> 20

<212> DNA

<213> ginger flower (Hedychium coronarium Koen)

<400> 7

ggttcaacca gcaccaaaga 20

<210> 8

<211> 23

<212> DNA

<213> ginger flower (Hedychium coronarium Koen)

<400> 8

ttagtagcat cggctgcaat aag 23

<210> 9

<211> 22

<212> DNA

<213> ginger flower (Hedychium coronarium Koen)

<400> 9

ctcaaccgtc ttcccaaaag ag 22

<210> 10

<211> 32

<212> DNA

<213> ginger flower (Hedychium coronarium Koen)

<400> 10

gatctgggta ccatgataaa gagagtggaa ga 32

<210> 11

<211> 35

<212> DNA

<213> ginger flower (Hedychium coronarium Koen)

<400> 11

gagctcgaat tctataggaa tgggttcaac cagca 35

Claims (6)

1. Ginger flower sesquiterpene synthase geneHcTPS14It 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. A zingiber officinale roscoe sesquiterpene synthetase HcTPS14 is characterized in that an amino acid sequence of the protein is shown in SEQ ID NO. 3.

3. A recombinant vector comprising the zingiberene synthase gene of claim 1HcTPS14。

4. A recombinant bacterium comprising the recombinant vector of claim 3.

5. A cell line comprising the recombinant bacterium of claim 4.

6. The zingiberene sesquiterpene synthase gene of claim 1HcTPS14Or the use of the zingiberene sesquiterpene synthase HcTPS14 of claim 2 in the preparation of α -caryophyllene.

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