CN109797161B - Ginger flower sesquiterpene synthase gene HcTPS12 and application thereof - Google Patents

Abstract

The invention discloses a ginger flower sesquiterpene synthase gene HcTPS12 and application thereof. The full-length cDNA sequence of the HcTPS12 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 HcTPS12 gene has high expression level in ginger flower leaf tissue, hardly expresses in organs such as rhizome and flower, and the expression level is regulated and controlled by leaf development. The exogenous recombinant protein of HcTPS12 can catalyze a substrate to generate sesquiterpene medicinal ingredient bisabolene, and can be used for preparing bisabolene and further preparing essential oil, essence and medicines; connecting HcTPS12 with a plant transformation vector, and then introducing into ginger flower or other plant cells to obtain a transgenic plant expressing the gene; and specific molecular markers are generated according to the gene sequence information, are used for identifying sesquiterpene bisabolene synthetase genes of the ginger flowers or other plants, and are used for molecular marker-assisted selective breeding, so that the breeding selection efficiency is improved, and the method has a wide application prospect.

Description

Ginger flower sesquiterpene synthase gene HcTPS12 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 HcTPS12 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. The sesquiterpene is an important medicinal component and an important essence component. Sesquiterpene paclitaxel has been reported to be useful in the treatment of tumors (wuna et al, 2016), and sesquiterpene artemisinin is a specific drug for the treatment of malaria (Mann, et al, 2000). The sesquiterpene bisabolene has the effects of anti-itching, anti-inflammation, anti-cancer and the like, and is an important edible essence (Pompe and picnic, and the like, 2018).

Terpene synthases (TPS) are the terminal key enzymes in the biosynthesis of terpenoids, which directly determine the type, quantity and yield of terpenoid 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.

At present, the domestic research on the terpene synthase of the zingiber officinale flower is mainly focused on a monoterpene synthase gene, but the sesquiterpene synthase gene of the zingiber officinale flower is 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, and 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 by a genetic engineering means.

Bisabolene is a sesquiterpene natural active compound derived from plants, is widely distributed in nature, and mainly exists in natural plant essential oil. Can be divided into 3 isomers according to the difference of double bond positions: α -bisabolene, β -bisabolene, and γ -bisabolene; the bisabolene has the smell similar to fruit fragrance and balm, wherein the beta-bisabolene has the smell similar to sesame oil and can be used as edible essence and daily chemical essence; bisabolene is also a novel biofuel with great potential; the beta-bisabolene and the gamma-bisabolene also have the functions of anti-itching and anti-inflammation and anticancer activity. In addition, bisabolene is also a precursor substance for synthesizing various high-value-added industrial products, such as biofuel, bioplastic, cosmetics, health products, medicines and the like.

At present, the industrial production of the bisabolene is mainly realized by a plant extraction method, but the extraction of the bisabolene from plant tissues has the defects of low product content, difficult separation and purification and the like. The rapid development of microbial metabolic engineering has provided a more potential biosynthetic route to the production of these plant natural products. The microbial cell factory which is constructed and produced by using the microbial metabolic engineering technology and produces the valuable plant natural products has the unique advantages of green and clean property, sustainable development, good economic benefit and the like.

Disclosure of Invention

The invention aims to solve the technical problems of overcoming the defects and shortcomings in the aspects of researches on terpene synthase genes of ginger flowers and preparation of bisabolene and providing a monofunctional enzyme gene HcTPS12 for controlling the bisabolene serving as a sesquiterpene component in ginger flowers.

The first purpose of the invention is to provide a ginger flower sesquiterpene synthetase gene HcTPS 12.

The second purpose of the invention is to provide a zingiber officinale roscoe sesquiterpene synthetase HcTPS 12.

The third purpose of the invention is to provide the application of the zingiber officinale roscoe sesquiterpene synthetase gene HcTPS12 and/or the zingiber officinale roscoe sesquiterpene synthetase HcTPS12 in preparation of bisabolene.

The above object of the present invention is achieved by the following technical solutions:

a zingiber officinale roscoe sesquiterpene synthase gene HcTPS12 has a full-length cDNA sequence shown in SEQ ID NO. 1, and can endow the zingiber officinale roscoe sesquiterpene bisabolene components with the effect of improving the specificity of the zingiber officinale roscoe sesquiterpene synthase gene. The ginger flower gene HcTPS12 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 HcTPS12 gene is 1823bp, and the nucleotide sequence is shown as SEQ ID NO 1; the total 1653bp of gene coding region (CDS), its nucleotide sequence is shown as SEQ ID NO: 2; the presumed 550 amino acids are coded, the amino acid sequence is shown in SEQ ID NO. 3, and the presumed protein molecular weight is 64.2 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 HcTPS12, the amino acid sequence of which is shown in SEQ ID NO. 3.

Based on the sequence information of the HcTPS12 gene provided by the present invention, a person skilled in the art can easily obtain a gene equivalent to HcTPS12 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 HcTPS12 gene fragment as a probe to obtain the HcTPS12 gene fragment; (3) designing oligonucleotide primers according to the HcTPS12 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 HcTPS12 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 HcTPS12, wherein the nucleotide sequence of the primer pair is shown in SEQ ID NO. 4-5.

Meanwhile, the invention also provides a recombinant vector containing the ginger flower sesquiterpene synthetase gene HcTPS 12.

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 HcTPS12 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 results show that the HcTPS12 protein catalyzes FPP to generate the sesquiterpene component bisabolene, which accounts for 100 percent of the total product of the reaction. The bisabolone is one of the components of the essential oil of the ginger flower. The HcTPS12 protein is shown to be sesquiterpene synthetase with a single catalytic function, and meanwhile, the bisabolene can be prepared by utilizing the sesquiterpene synthetase gene HcTPS12 from the ginger flower through microbial metabolic engineering, so that plant essential oil or medicine can be prepared.

Therefore, the application of the zingiberene sesquiterpene synthetase gene HcTPS12 and/or the zingiberene sesquiterpene synthetase HcTPS12 in preparing bisabolene or preparing spices, essential oil or medicines is also within the protection scope of the invention.

Specifically, the application is to produce bisabolene by using farnesyl pyrophosphate (FPP) as a substrate and catalyzing by using a zingiberene sesquiterpene synthetase HcTPS 12.

The zingiber officinale roscoe sesquiterpene synthase gene HcTPS12 provided by the invention has important application value. One application is that the HcTPS12 gene sequence is connected to any plant transformation vector, and HcTPS12 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 the regulatory sequence thereof can be modified appropriately, and other promoters can be used for replacing the original promoter of the gene before the transcription initiation codon, so that the capability of the plant for producing the sesquiterpene bisabolene and enhancing the resistance is widened and enhanced.

The sesquiterpene synthetase gene HcTPS12 provided by the invention can be further applied to generating 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) and CAP (cut amplified fragment polymorphism). The markers can be used for identifying sesquiterpene bisabolene synthetase genes of the ginger flowers or other plants, and are used for molecular marker-assisted selection breeding, so that the selection efficiency of breeding is improved.

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

the invention provides a new sesquiterpene synthase gene HcTPS12, which can catalyze FPP to form a sesquiterpene compound bisabolene and plays an important role in improving the content and resistance of plant terpenoid components; the hemiterpene synthase gene HcTPS12 can be used for preparing bisabolene and further preparing essential oil, essence and medicines. The gene fragment is constructed on a plant expression vector, other plant materials can be exogenously transformed, so that a transgenic material containing the sesquiterpene bisabolene 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 HcTPS12 gene in different tissues of Zingiber officinale Roscoe; pe is petal; ri is rhizome; le, leaf.

FIG. 2 shows the expression of the HcTPS12 gene in different developmental stages of the ginger flower leaf organ.

FIG. 3 shows the in vitro enzyme-catalyzed reaction of the recombinant protein HcTPS12 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 HcTPS12 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'-GTCATGGAGCTTGCTGGTACT-3' (shown in SEQ ID NO: 4). The downstream primer R1: 5'-GGCTTCAACAATATTAACAAGACC-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 HcTPS12 gene is shown as SEQ ID NO. 1, and the full length of the cDNA is 1823 bp; the coding region (CDS) of the gene is shown as SEQ ID NO. 2, and 1653bp in total; the amino acid sequence is presumed to be shown in SEQ ID NO 3.

Example 2 expression analysis of HcTPS12 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'-TGGAAGGCGTGGTTGTTGAT-3' (shown in SEQ ID NO: 6). P2: 5'-AGACCGTGATTTCTGATTTGT-3' (shown in SEQ ID NO: 7). The internal reference gene RPS uses Primer premier 5.0 to design a Primer according to the design principle of Real-time PCR Primer, and the 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^－△△CtData analysis was performed by the method (Livak et al, 2001) and the amount of the HcTPS12 of the ginger flowers in different samples was calculatedThe expression of (1).

2. Results

The results of the gene expression analysis are shown in FIGS. 1 and 2. As can be seen from fig. 1: HcTPS12 has high expression level in leaf tissue, and hardly expresses in nutritive organs such as rhizome and leaf. FIG. 2 shows that: the expression level of the gene is regulated and controlled by the development of leaves, the expression level of the gene in old leaves is higher, and the expression level is consistent with the change trend of the content of sesquiterpene bisabolene in the ginger flower essential oil, which indicates that HcTPS12 is a related gene participating in regulating and controlling the synthesis of terpene substances in the ginger flower.

Example 3 prokaryotic expression of HcTPS12 Gene

S1, vector construction: based on the coding region of the HcTPS12 gene obtained, 5 '-GATCTG was digested with a specific primer F: 5' -GATCTG containing KpnI and EcoRI cleavage sitesGGTACCATGGAGCTTGCTGGTACTCC-3' (shown in SEQ ID NO: 10); r: 5' -GAGCTCGAATTCAATAGGGATAGGATGAACAA-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 performed overnight at 16 ℃ and the ligation product was transformed into E.coli (E.coli) DH 5. 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 subjected to ultrasonic disruption on ice. 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 and analyzed by SDS-PAGE. 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, 100. mu.L pH 7.5,5mMDTT,20mM MgCl₂Each 100 μ L, and protein extract 100 μ L, 2 μ L GPP or FPP, plus ddH₂And 3, adding O588 mu L of the mixture into a sample bottle with the final volume of 1mL, sealing the sample bottle, carrying out water bath at 30 ℃ for 1h, inserting a 75 mu m Polydimethylsiloxane (PMDS) extraction fiber head into a glass bottle, carrying out headspace solid phase microextraction for 1h, and after the reaction is finished, putting the extraction fiber head into a gas chromatography-mass spectrometer for analysis, 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 mass range 35-335 amu, and analyzing the collected mass spectrum image by using a WILLEY/MAINLIB library.

The result of prokaryotic expression of the HcTPS12 gene is shown in FIG. 3. FIG. 3 shows: when FPP is taken as a substrate, the product of the in vitro enzyme catalysis reaction of pET-30a-HcTPS12 is identified as the sesquiterpenoid bisabolene by mass spectrum. The HcTPS12 gene is used to encode and produce enzyme which is a monofunctional enzyme gene capable of catalyzing FPP to produce the hemiterpene bisabolene.

Sequence listing

<120> ginger flower sesquiterpene synthase gene HcTPS12 and application thereof

<141> 2019-02-28

<160> 11

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1823

<212> DNA

<213> ginger flower (Hedychium coronarium Koen)

<400> 1

caagtcatgg agcttgctgg tactccatca gtcgaggttt tggaaggcgt ggttgttgat 60

cgtcagttgg caggcttcga tcccagctct tggggtgaat actttattac aaatcagaaa 120

tcacggtctg aggcatggat gaacgaaaga gctgaagagc tcaagaatga agtaaggagc 180

atgttccaaa acgtgactgg cgtcctacaa accatgaatc taattgatac aattcaactc 240

ctcggacttg attaccattt catggaggaa atagacagag ctttagatca tctcaaggat 300

gttgacatga gcaaaaacgg gctctatgag gttgctcttc attttcgact gcttagacaa 360

aaaggagtca acatttcttc agatgtattt aaaaaataca tggataaaga gggaaaattt 420

atagaagaac taaaagatga tgctaagggg ctcctgagct tatataatgc ggcttacctc 480

ggaaccaaaa aagagactat actcgacgaa gccatttctt ttactagaga taaccttaca 540

tctttgttaa aagatctaaa tcctacattt gcaaagttag tgtctctcac tctaaagaca 600

cctattccac gaagcatgaa acgacttttc acaagatgct acatctctat ttaccaagat 660

gaaccgactc gaaatgaaac aatatttgag cttgcaaaat tggacttcaa catactacaa 720

tgtctccacc aggaggagct caagaaacta agcatgtggt ggaagcagtt gaatttagac 780

atcatgcatc taaattttgt tcgagatcga gtggtggaat gtttctgttg gtcgatggag 840

atacgccatg aacccagttg ttctcgtgct cgactgatag cgtctaagct acttatgttc 900

attactgtct tggatgactt ctatgatagc tacagcacat tagaagagag tcgactactt 960

acagatgcaa tcgaaaggtg gagtcctgat gcagtagatc aactaccaga atacctgagg 1020

gagttctttc tcaaaatgtt gaacattttt caagaatttg aagatgaact tgcaccggaa 1080

gagaagtttc gaatattgta cttcaaggaa gaatggaaaa ctcaagctca aagttacttc 1140

aaggaatgcc aatggaggga tgacaattat gtgcccaagt tagaagagca catgcgtgtt 1200

tcaatcataa gtgtgggatt tgtcttgttt tattgcggat ttttgagtgg catggaggag 1260

acagtggcca caaaggatgc atttgaatgg ttcgcaagct tccccaagat catagaagct 1320

tgtgcaacaa ttcttcgtat cactaatgac ataacttcaa aggagcgaga acaaaagagg 1380

gcacatgttg cctcgacggt agattgctat atgaaggaat atggaacatc aaaagatgtt 1440

gcatgcgaga agctcctagg ctttgttgaa gatgcatgga agactatcaa cgaggagctc 1500

cttactgcaa ctggattgtc gagggaagta attgaactat cactccactg tgcgcaaact 1560

acagaatttg tatacaagga tgtcgacgca ttcacagaac ctaatacctc gatgaaggaa 1620

agcatctttt tcctacttgt tcatcctatc cctatttgat gacagtagtg ctacaatcat 1680

gtactatttg gtatctcata tggttgtgtg cttaagttat tattatagaa ataaaagggg 1740

ggagaaagag agaaataact tggtcttgtt aatattgttg aagccaaata agcttatatg 1800

tacaagttgt ttaccagttg tac 1823

<210> 2

<211> 1653

<212> DNA

<213> ginger flower (Hedychium coronarium Koen)

<400> 2

atggagcttg ctggtactcc atcagtcgag gttttggaag gcgtggttgt tgatcgtcag 60

ttggcaggct tcgatcccag ctcttggggt gaatacttta ttacaaatca gaaatcacgg 120

tctgaggcat ggatgaacga aagagctgaa gagctcaaga atgaagtaag gagcatgttc 180

caaaacgtga ctggcgtcct acaaaccatg aatctaattg atacaattca actcctcgga 240

cttgattacc atttcatgga ggaaatagac agagctttag atcatctcaa ggatgttgac 300

atgagcaaaa acgggctcta tgaggttgct cttcattttc gactgcttag acaaaaagga 360

gtcaacattt cttcagatgt atttaaaaaa tacatggata aagagggaaa atttatagaa 420

gaactaaaag atgatgctaa ggggctcctg agcttatata atgcggctta cctcggaacc 480

aaaaaagaga ctatactcga cgaagccatt tcttttacta gagataacct tacatctttg 540

ttaaaagatc taaatcctac atttgcaaag ttagtgtctc tcactctaaa gacacctatt 600

ccacgaagca tgaaacgact tttcacaaga tgctacatct ctatttacca agatgaaccg 660

actcgaaatg aaacaatatt tgagcttgca aaattggact tcaacatact acaatgtctc 720

caccaggagg agctcaagaa actaagcatg tggtggaagc agttgaattt agacatcatg 780

catctaaatt ttgttcgaga tcgagtggtg gaatgtttct gttggtcgat ggagatacgc 840

catgaaccca gttgttctcg tgctcgactg atagcgtcta agctacttat gttcattact 900

gtcttggatg acttctatga tagctacagc acattagaag agagtcgact acttacagat 960

gcaatcgaaa ggtggagtcc tgatgcagta gatcaactac cagaatacct gagggagttc 1020

tttctcaaaa tgttgaacat ttttcaagaa tttgaagatg aacttgcacc ggaagagaag 1080

tttcgaatat tgtacttcaa ggaagaatgg aaaactcaag ctcaaagtta cttcaaggaa 1140

tgccaatgga gggatgacaa ttatgtgccc aagttagaag agcacatgcg tgtttcaatc 1200

ataagtgtgg gatttgtctt gttttattgc ggatttttga gtggcatgga ggagacagtg 1260

gccacaaagg atgcatttga atggttcgca agcttcccca agatcataga agcttgtgca 1320

acaattcttc gtatcactaa tgacataact tcaaaggagc gagaacaaaa gagggcacat 1380

gttgcctcga cggtagattg ctatatgaag gaatatggaa catcaaaaga tgttgcatgc 1440

gagaagctcc taggctttgt tgaagatgca tggaagacta tcaacgagga gctccttact 1500

gcaactggat tgtcgaggga agtaattgaa ctatcactcc actgtgcgca aactacagaa 1560

tttgtataca aggatgtcga cgcattcaca gaacctaata cctcgatgaa ggaaagcatc 1620

tttttcctac ttgttcatcc tatccctatt tga 1653

<210> 3

<211> 550

<212> PRT

<213> ginger flower (Hedychium coronarium Koen)

<400> 3

Met Glu Leu Ala Gly Thr Pro Ser Val Glu Val Leu Glu Gly Val Val

1 5 10 15

Val Asp Arg Gln Leu Ala Gly Phe Asp Pro Ser Ser Trp Gly Glu Tyr

20 25 30

Phe Ile Thr Asn Gln Lys Ser Arg Ser Glu Ala Trp Met Asn Glu Arg

35 40 45

Ala Glu Glu Leu Lys Asn Glu Val Arg Ser Met Phe Gln Asn Val Thr

50 55 60

Gly Val Leu Gln Thr Met Asn Leu Ile Asp Thr Ile Gln Leu Leu Gly

65 70 75 80

Leu Asp Tyr His Phe Met Glu Glu Ile Asp Arg Ala Leu Asp His Leu

85 90 95

Lys Asp Val Asp Met Ser Lys Asn Gly Leu Tyr Glu Val Ala Leu His

100 105 110

Phe Arg Leu Leu Arg Gln Lys Gly Val Asn Ile Ser Ser Asp Val Phe

115 120 125

Lys Lys Tyr Met Asp Lys Glu Gly Lys Phe Ile Glu Glu Leu Lys Asp

130 135 140

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

145 150 155 160

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

165 170 175

Leu Thr Ser Leu Leu Lys Asp Leu Asn Pro Thr Phe Ala Lys Leu Val

180 185 190

Ser Leu Thr Leu Lys Thr Pro Ile Pro Arg Ser Met Lys Arg Leu Phe

195 200 205

Thr Arg Cys Tyr Ile Ser Ile Tyr Gln Asp Glu Pro Thr Arg Asn Glu

210 215 220

Thr Ile Phe Glu Leu Ala Lys Leu Asp Phe Asn Ile Leu Gln Cys Leu

225 230 235 240

His Gln Glu Glu Leu Lys Lys Leu Ser Met Trp Trp Lys Gln Leu Asn

245 250 255

Leu Asp Ile Met His Leu Asn Phe Val Arg Asp Arg Val Val Glu Cys

260 265 270

Phe Cys Trp Ser Met Glu Ile Arg His Glu Pro Ser Cys Ser Arg Ala

275 280 285

Arg Leu Ile Ala Ser Lys Leu Leu Met Phe Ile Thr Val Leu Asp Asp

290 295 300

Phe Tyr Asp Ser Tyr Ser Thr Leu Glu Glu Ser Arg Leu Leu Thr Asp

305 310 315 320

Ala Ile Glu Arg Trp Ser Pro Asp Ala Val Asp Gln Leu Pro Glu Tyr

325 330 335

Leu Arg Glu Phe Phe Leu Lys Met Leu Asn Ile Phe Gln Glu Phe Glu

340 345 350

Asp Glu Leu Ala Pro Glu Glu Lys Phe Arg Ile Leu Tyr Phe Lys Glu

355 360 365

Glu Trp Lys Thr Gln Ala Gln Ser Tyr Phe Lys Glu Cys Gln Trp Arg

370 375 380

Asp Asp Asn Tyr Val Pro Lys Leu Glu Glu His Met Arg Val Ser Ile

385 390 395 400

Ile Ser Val Gly Phe Val Leu Phe Tyr Cys Gly Phe Leu Ser Gly Met

405 410 415

Glu Glu Thr Val Ala Thr Lys Asp Ala Phe Glu Trp Phe Ala Ser Phe

420 425 430

Pro Lys Ile Ile Glu Ala Cys Ala Thr Ile Leu Arg Ile Thr Asn Asp

435 440 445

Ile Thr Ser Lys Glu Arg Glu Gln Lys Arg Ala His Val Ala Ser Thr

450 455 460

Val Asp Cys Tyr Met Lys Glu Tyr Gly Thr Ser Lys Asp Val Ala Cys

465 470 475 480

Glu Lys Leu Leu Gly Phe Val Glu Asp Ala Trp Lys Thr Ile Asn Glu

485 490 495

Glu Leu Leu Thr Ala Thr Gly Leu Ser Arg Glu Val Ile Glu Leu Ser

500 505 510

Leu His Cys Ala Gln Thr Thr Glu Phe Val Tyr Lys Asp Val Asp Ala

515 520 525

Phe Thr Glu Pro Asn Thr Ser Met Lys Glu Ser Ile Phe Phe Leu Leu

530 535 540

Val His Pro Ile Pro Ile

545 550

<210> 4

<211> 21

<212> DNA

<213> ginger flower (Hedychium coronarium Koen)

<400> 4

gtcatggagc ttgctggtac t 21

<210> 5

<211> 24

<212> DNA

<213> ginger flower (Hedychium coronarium Koen)

<400> 5

ggcttcaaca atattaacaa gacc 24

<210> 6

<211> 20

<212> DNA

<213> ginger flower (Hedychium coronarium Koen)

<400> 6

tggaaggcgt ggttgttgat 20

<210> 7

<211> 21

<212> DNA

<213> ginger flower (Hedychium coronarium Koen)

<400> 7

agaccgtgat ttctgatttg t 21

<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 ccatggagct tgctggtact cc 32

<210> 11

<211> 32

<212> DNA

<213> ginger flower (Hedychium coronarium Koen)

<400> 11

gagctcgaat tcaataggga taggatgaac aa 32

Claims (6)

1. Ginger flower sesquiterpene synthase geneHcTPS12It 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 HcTPS12 is characterized in that an amino acid sequence of the protein is shown in SEQ ID NO. 3.

3. A recombinant vector characterized in thatComprising the zingiber officinale roscoe sesquiterpene synthase gene of claim 1HcTPS12。

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 1HcTPS12Or the use of the zingiberene sesquiterpene synthase HcTPS12 of claim 2 in the preparation of bisabolene.

Images