CN112391371A

CN112391371A - Tobacco monoterpene synthetase TPS2b, and coding gene and application thereof

Info

Publication number: CN112391371A
Application number: CN201910749192.9A
Authority: CN
Inventors: 张汝兵; 李依婷; 刘炜; 咸漠; 杨爱国
Original assignee: Qingdao Institute of Bioenergy and Bioprocess Technology of CAS; Tobacco Research Institute of CAAS
Current assignee: Qingdao Institute of Bioenergy and Bioprocess Technology of CAS; Tobacco Research Institute of CAAS
Priority date: 2019-08-14
Filing date: 2019-08-14
Publication date: 2021-02-23
Anticipated expiration: 2039-08-14
Also published as: CN112391371B

Abstract

The invention discloses a tobacco monoterpene synthetase TPS2b, and a coding gene and application thereof, and relates to the technical field of genetic engineering. The amino acid sequence of the tobacco monoterpene synthetase TPS2b is shown as SEQ ID NO.1, and the sequence of the gene is shown as SEQ ID NO. 2. The invention provides a novel monoterpene synthetase, namely tobacco monoterpene synthetase TPS2b and a coding gene thereof, the monoterpene synthetase TPS2b can generate an active monoterpene synthetase after prokaryotic expression, and catalyze geranyl pyrophosphate (GPP) to generate a mixed monoterpene compound of geraniol and nerol which is close to 1:1, and can be used for mass production of the monoterpene compound.

Description

Tobacco monoterpene synthetase TPS2b, and coding gene and application thereof

Technical Field

The invention relates to the technical field of genetic engineering, in particular to a tobacco monoterpene synthetase TPS2b, and a coding gene and application thereof.

Background

Monoterpenes (monoterpenes) are one of the terpenoids. Generally refers to terpenoids and their oxygen-containing and unsaturated derivatives polymerized from two molecules of isoprene. Most of them have strong fragrance and physiological activity, and are important raw materials for medicine, food and cosmetic industries.

Geraniol (also known as Geraniol, with the chemical name of trans-3, 7-methyl-2, 6-octadien-1-ol, Geraniol) and its isomer Nerol (cis-3, 7-dimethyl-2, 6-octadienol, Nerol) are both acyclic monoterpene alcohol compounds, which are one of the main components of essential oils such as rose oil, clove oil and citronella oil. Geraniol is the main component (about 40-60%) of rose oil, has the fragrance of rose, and is a rare spice. Nerol has pleasant fragrance of rose and orange flower, and has mild fragrance, and slight lemon-like fruity fragrance, which is softer and more beautiful than geraniol and is relatively cleaner. Geraniol and nerol can be used in essence and edible essence, are main agents of rose essence, and can be widely used in medicine, tobacco and food ingredients. In addition, the compound can also be used as a natural low-toxicity insect-resist agent, a novel chemical cancer-prevention preparation. At present, the annual demand of geraniol in China exceeds 1000 tons, the supply is short, and the gap reaches hundreds of tons. The domestic demand of nerol is not less than 500 tons, and the problem of insufficient production capacity also exists.

The industrial production of geraniol and nerol is mainly carried out by chemical synthesis method, using myrcene as raw material, reacting with hydrochloric acid under the action of catalyst to obtain first-grade chloride of myrcene, reacting with sodium acetate to obtain mixture of geranyl acetate and neryl acetate, hydrolyzing, distilling to obtain mixture of geraniol and nerol, and rectifying to obtain geraniol and nerol products. In addition, nerol and geraniol can be prepared by taking citral as a raw material, and the preparation method comprises a catalytic hydrogenation method, an aluminum alkoxide method and a sodium borohydride method. The sodium borohydride method is improved by adopting a phase transfer catalysis method in the Yi flood control, and water and benzene are used as mixed solvents to replace pure organic solvents, so that the reaction rate is improved. However, the chemical synthesis method has the problems of environmental pollution, harsh conditions and the like, and the chemically synthesized product is 'unnatural', is used as a food additive, has low consumer acceptance and influences the downstream application of the product.

Disclosure of Invention

In order to solve the problems, the invention provides a novel tobacco monoterpene synthetase TPS2b, and a coding gene and application thereof.

Firstly, the invention provides a tobacco monoterpene synthetase TPS2b, the amino acid sequence of which is shown in SEQ ID NO. 2.

Secondly, the invention provides a coding gene TPS2b of the tobacco monoterpene synthase TPS2b, the nucleotide sequence of which is shown in SEQ ID No. 1.

Thirdly, the invention also provides application of the coding gene TPS2b of the tobacco monoterpene synthase TPS2b in preparation of engineering bacteria for co-producing geraniol and nerol.

The engineering bacteria for co-producing geraniol and nerol also express geranyl pyrophosphate synthetase gene (GPPS2, GenBank No. AF513112.1).

The application specifically comprises the following steps:

(1) cloning the tobacco geraniol and nerol synthetase genes tps2 b;

(2) construction of expression vector: the nerol synthetase gene tps2b is connected between the BglII and XhoI double restriction enzyme sites of the pYJM26 plasmid to obtain recombinant plasmid pT2 b;

(3) and (3) recombinant bacterium transformation: and (3) transforming E.coli BL21(DE3) competent cells by the pYJM14 plasmid and the recombinant plasmid pT2b obtained in the step (2) to obtain the engineering bacteria for co-producing geraniol and nerol.

The step (1) is specifically as follows: extracting RNA of tobacco leaf tissue, converting the RNA into a cDNA template by adopting reverse transcriptase, taking the cDNA obtained by reverse transcription reaction as the template, and adopting a forward primer as follows: 5'-ATGGCCACCTCCATAAGACCTGCAA-3', the reverse primer is: 5'-TTATAGGGATGGATTGGGAGTCAAT-3', performing PCR amplification; the target gene tps2b with a fragment size of 1605bp was recovered by gel electrophoresis.

The step (2) is specifically as follows: the nerol synthetase gene tps2b was TA cloned and ligated to

-Blunt cloning vector to obtain recombinant plasmid pEASY-T2 b; taking a recombinant plasmid pEASY-T2b as a template, adopting a forward primer T2b-F (5'-GAAGATCTATGGCCACCTCCATAAGACCTGCAA-3') and a reverse primer T2b-R (5'-CCCTCGAGTTATAGGGATGGATTGGGAGTCAAT-3') to carry out PCR amplification, and recovering a target gene fragment with the fragment size of about 1610bp by gel electrophoresis; the obtained target gene fragment and pYJM26 plasmid are subjected to double enzyme digestion by BglII and XhoI respectively, exogenous gene fragments and vector fragments are obtained by recovery respectively, and the vector and the exogenous fragments are connected according to the molar ratio of 1:5 to obtain the recombinant plasmid pT2 b.

The plasmid pYJM26, namely pACY-mvaE-mvaS-GPPS2, carries acetyl coenzyme A acyltransferase gene/hydroxymethyl glutaryl coenzyme A reductase gene (mvaE, GenBank No. AAG02438) and 3-hydroxy-3-methylglutaryl coenzyme A synthase gene (mvaS, GenBank No. AAG02439) which are derived from enterococcus faecalis (enterococcus faecium); and pACYCDuet-1 derived from geranyl pyrophosphate synthetase gene (GPPS2, GenBank No. AF513112.1) of North American fir (Abies grandis).

The construction of pYJM26 is described in Yang J, et al, metabolism engineering of Escherichia coli for the biosynthesis of alpha-pinene Biotechnology for Biofuels,2013,6: 60.

The pYJM14 plasmid, pTrc-low plasmid, is pTrc His2B carrying mevalonate kinase gene (ERG12, GenBank No. NM-001182715.1), mevalonate-5-phosphate kinase gene (ERG8, GenBank No. NM-001182727.1), mevalonate-5-diphosphate decarboxylase gene (ERG19, GenBank No. X97557.1), isopentenyl pyrophosphate isomerase gene (IDI1, GenBank No. NM-001183931.1) derived from Saccharomyces cerevisiae.

The plasmid pYJM14 was constructed as described in Yang J, et al, metabolism engineering of Escherichia coli for the biosynthesis of alpha-pinene Biotechnology for Biofuels,2013,6: 60.

Advantageous effects

The invention provides a novel monoterpene synthetase, namely tobacco monoterpene synthetase TPS2b and a coding gene TPS2b thereof, TPS2b can catalyze geranyl pyrophosphate (GPP) to generate monoterpene compounds geraniol and nerol, a metabolic pathway for synthesizing geraniol and nerol by taking glucose as a raw material is constructed by successfully expressing the tobacco monoterpene synthetase TPS2b in escherichia coli and utilizing a genetic engineering means, and the metabolic pathway can be used for mass production of the geraniol and the nerol. The raw materials of the route are cheap and can be supplied continuously, and the route has the potential of large-scale development.

Drawings

FIG. 1 shows the PCR electrophoresis of the target gene tps2 b.

FIG. 2 is a GC chart of a mixed standard of geraniol and nerol.

FIG. 3 is a GC-MS molecular fragment mass spectrum of nerol standard.

FIG. 4 is a GC-MS molecular fragment mass spectrum of a geraniol standard.

FIG. 5 is a GC-MS diagram of nerol and geraniol synthesized by engineering bacteria.

FIG. 6 is a molecular fragment mass spectrum of nerol GC-MS synthesized by engineering bacteria.

FIG. 7 is a GC-MS molecular fragment mass spectrum of geraniol synthesized by engineering bacteria.

Detailed Description

The following examples are further illustrative of the present invention and are not intended to be limiting thereof.

The experimental methods not specifically mentioned in the following examples can be carried out according to conventional methods or according to the instructions of manufacturers of the products used; the materials, reagents and the like used are commercially available unless otherwise specified.

The detection method of nerol and geraniol comprises the following steps: taking 2ml of fermentation liquor, then centrifuging for 1min at 12000g, taking 1ml of supernatant, adding equal volume of ethyl acetate, uniformly mixing for 5min on a vortex oscillator, then standing for 10min, taking an upper layer organic phase, filtering, and placing in a liquid phase small bottle to be tested. Detection conditions are as follows: GC-MS instrument model: shimadzu TQ 8050; an ion source: EI; a detector: a four-stage lever; sample detection: taking the liquid in the liquid phase vial for detection. GC instrument model: shimadzu GC 2010 Pro; type of separation column: DB-5; sample introduction amount: 1 mul; a detector: a hydrogen flame detector (FID); column temperature: keeping the temperature at 80 ℃ for 2min, then heating to 160 ℃ at the speed of 30 ℃/min, then heating to 170 ℃ at the speed of 3 ℃/min, and finally heating to 300 ℃ at the speed of 50 ℃/min; and taking the upper organic phase for detection.

Plasmid: pYJM26, pACY-mvaE-mvaS-GPPS2, is a gene carrying acetyl-CoA acyltransferase gene/hydroxymethylglutaryl-CoA reductase gene (mvaE, GenBank No. AAG02438), 3-hydroxy-3-methylglutaryl-CoA synthase gene (mvaS, GenBank No. AAG02439) derived from Enterococcus faecalis (Enterococcus faecalis); and pACYCDuet-1 derived from geranyl pyrophosphate synthetase gene (GPPS2, GenBank No. AF513112.1) of North American fir (Abiesgrandis). pYJM26 is a plasmid which has been constructed and stored by Qingdao bioenergy and Process research institute of Chinese academy of sciences, and the specific construction method is as follows:

an HMG-CoA reductase gene (mvaE, GI: 9937382) and an HMG-CoA synthetase gene (mvaS, GI: 9937382) which are chemically synthesized and contain enzyme cutting sites Nco I and Pst I at two ends are connected to the first polyclonal expression site of the pACYDute-1 plasmid in a serial mode through restriction enzymes Nco I and Pst I to obtain a plasmid pACYC-mvaE-mvaS. The geranyl pyrophosphate synthetase gene (GPPS, GI: 22535958) chemically synthesized and containing restriction enzyme sites Bgl II and Xho I at both ends was ligated to the second polyclonal expression site of plasmid pACYC-mvaE-mvaS plasmid by using restriction enzymes Bgl II and Xho I to obtain plasmid pYJM 26. Specific construction methods can also be found in patent CN104120148A and in Yang J, et al, metabolic engineering for the biosynthesis of alpha-pinene, biotechnology for Biofuels,2013,6: 60.

pYJM14, pTrc-low, is pTrcHis2B carrying mevalonate kinase gene (ERG12, GenBank No. NM-001182715.1), mevalonate-5-phosphate kinase gene (ERG8, GenBank No. NM-001182727.1), mevalonate-5-diphosphate decarboxylase gene (ERG19, GenBank No. X97557.1), isopentenyl pyrophosphate isomerase gene (IDI1, GenBank No. NM-001183931.1) derived from Saccharomyces cerevisiae. The plasmid pYJM14 is a plasmid which has been constructed and stored by Qingdao bioenergy and Process research institute of Chinese academy of sciences, and the specific construction method is as follows:

amplifying partial fragments SFIBhB and SFB12hB of the plasmid pTrchis2B and a fragment ERG12(SF128h12 and SFB12h12) of 4 genes at the downstream of the MVA pathway of the saccharomyces cerevisiae by using commercial plasmids pTrchis2B (purchased from Invitrogen) and saccharomyces cerevisiae (S.cerevisiae) genomes as templates; ERG8(SF128h8, SF819h 8); ERG19(SF819h19, SF19 IhI); IDI (SF19IhI, SFIBhI), and then 6 SFs fragments SF128, SF819, SF19I, SFIB, SFB12, SF of the assembled plasmid were amplified by ordinary PCR or overlap PCR using these fragments or pTrchis2B backbone as template. 3 RBS sequences among 4 genes ERG12, ERG8, ERG19 and IDI are introduced during PCR amplification by designing primers so that the 4 genes are expressed by a single trc (trp-lac promoter) promoter. Table 1 shows the primers and templates for fragment amplification during the construction of pTrc-low plasmid, and Table 2 shows the sequence summary of the primers used.

TABLE 1 primers and templates for fragment amplification

Table1 PCR templates and primers for fragment construction

TABLE 2 summary of primer sequences constructed from pTrc-low

Table2 Primer sequences used to construct pTrc-low

The amplified 6 SFs fragments SF128, SF819, SF19I, SFIB, SFB12 and SFB were mixed in an EP tube of 1.5mL in an equimolar ratio, and the mixture was sealed, denatured by boiling water bath, and naturally cooled (annealed) at room temperature. Coli competent cells were transformed with a suitable volume of the ligation mixture, and after activation, 100. mu.L of the mixture was plated on LB plates (Amp-resistant) and cultured overnight at 37 ℃. 30 transformants are picked, the temperature is 37 ℃, the shaking culture is carried out at 180rpm for 8h, 2 mu L of bacterial liquid is taken as a template for colony PCR amplification identification, and positive clones are screened. After the amplification culture of the positive clone obtained by PCR, extracting plasmids, and respectively carrying out single enzyme digestion and double enzyme digestion identification by using different restriction enzymes so as to verify the correctness of the recombinant plasmid.

Specific construction methods can also be found in patent CN104120148A and in Yang J, et al, metabolism engineering of Escherichia coli for the biosynthesis of alpha-pinene Biotechnology for Biofuels,2013,6: 60.

EXAMPLE 1 cloning of tobacco monoterpene synthase Gene tps2b

Extracting RNA of tobacco leaf tissue, converting the RNA into a cDNA template by adopting reverse transcriptase, taking the cDNA obtained by reverse transcription reaction as the template, and adopting a forward primer as follows: 5'-ATGGCCACCTCCATAAGACCTGCAA-3', the reverse primer is: 5'-TTATAGGGATGGATTGGGAGTCAAT-3', PCR amplification using PrimeSTAR Max DNA polymerase from Takara; the PCR conditions were: 5min at 94 ℃; 30s at 94 ℃, 30s at 55 ℃, 30s at 72 ℃ and 35 cycles; extension at 72 ℃ for 5 min. The PCR product was detected by 1% agarose gel electrophoresis, and the results are shown in FIG. 1, where M in FIG. 1 is DNAmarker DL2000, and the size of the target gene tps2b fragment is 1605bp, which corresponds to the expected size.

Example 2 construction of engineering bacteria for coproduction of geraniol and nerol by using coding gene TPS2b of tobacco monoterpene synthetase TPS2b

(1) Cloning the tobacco geraniol and nerol synthetase genes tps2 b;

The method specifically comprises the following steps:

(1) cloning of the Nicotiana tabacum geraniol and nerol synthase genes tps2b

(2) Construction of expression vector: the nerol synthetase gene tps2b was ligated between the BglII and XhoI double cleavage sites of pYJM26 to obtain recombinant plasmid pT2b

Recovering target gene fragment by agarose gel electrophoresis gel recovery kit method, TA cloning the target fragment, and connecting to vector

Blunt cloning vector, which is then transformed into the E.coli DH 5. alpha. cloning strain under the following conditions: adding 5 mul of the ligation product into 100 mul of the competent cells, mixing the ligation product evenly and gently, and carrying out ice bath for 30 min; quickly putting into 42 deg.C water bath, thermally shocking for 90s, immediately putting on ice for 2-3 min; adding 800 μ L LB culture medium, culturing at 37 deg.C for 1 h; the bacterial liquid is centrifuged at 6000rpm for 1min, 700 mu L of supernatant is discarded, the bacterial strain is suspended and coated on an LB plate containing ampicillin (Amp, 100mg/L), and the culture is carried out for 12-16 h in an inverted dark mode at 37 ℃. Screening positive clones by colony PCR, selecting positive monoclonal colonies, extracting plasmid, and sequencing. Obtained by sequencing analysis and cloningThe nucleotide sequence of the tobacco monoterpene synthetase gene TPS2b is shown in SEQ ID No.1, the gene contains 1605 bases, the encoded protein is named as monoterpene synthetase TPS2b, 535 amino acids are total, and the specific amino acid sequence is shown in SEQ ID No. 2. And thereby inserting monoterpene synthase gene tps2b into

-recombinant plasmid of Blunt cloning vector, named pEASY-T2 b.

Carrying out PCR amplification by using a plasmid pEASY-T2b as a template and adopting a forward primer T2b-F (5'-GAAGATCTATGGCCACCTCCATAAGACCTGCAA-3') and a reverse primer T2cb-R (5'-CCCTCGAGTTATAGGGATGGATTGGGAGTCAAT-3'), wherein the PCR amplification conditions are as follows: pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 30s, annealing at 60 ℃ for 15s, and extension at 72 ℃ for 30s, repeating the above three steps of denaturation, annealing, and extension for 35 cycles, and then re-extension at 72 ℃ for 5 min. The desired gene fragment having a fragment size of about 1621bp was recovered using a gel recovery kit (purchased from Omega). Carrying out double enzyme digestion on the obtained gene fragment and a pYJM26 vector by BglII and XhoI respectively, connecting the vector and an exogenous fragment at the molar ratio of 1:5 at 16 ℃ for 4-6 h, converting a connecting product into E.coli DH5 alpha, coating an LB solid plate added with 34mg/L chloramphenicol (Cm), screening positive clones by PCR, extracting a recombinant plasmid pT2b (pACY-mvaE-mvaS-GPPS2-T2b) from the positive clones, and then carrying out restriction enzyme digestion and sequencing identification.

(3) And (3) recombinant bacterium transformation: transforming E.coli BL21(DE3) competent cells by pYJM14 plasmid and the recombinant plasmid pT2b obtained in the step (2) to obtain engineering bacteria for co-producing geraniol and nerol

E.coli BL21(DE3) competent cells were transformed by co-heat shock of pT2b and pYJM14, and plated on LB solid plates containing ampicillin and chloramphenicol antibiotics to obtain engineered E.coli BT2c containing pT2b and pYJM 4.

Example 3 fermentative production of nerol and geraniol

The white single clone obtained in example 1 was picked up in 10mL of LB liquid medium (containing Cm at 34. mu.g/mL and resistance at 100. mu.g/mLAmp), activated by shaking culture at 37 ℃ and used as a seed. When the concentration of seed bacteria is about 1.0, the bacteria after propagation culture are takenInoculating the solution into 100ml fermentation medium (pH7.0, yeast powder 5g/L, glucose 10g/L, K)₂HPO₄·3H₂9.8g/L of O, 2.1g/L of citric acid monohydrate and 0.3g/L of ferric ammonium citrate; MgSO (MgSO)₄·7H₂O0.49 g/L; and 100. mu.l of stock solution of trace elements ((NH) was added₄)₆Mo₇O₂₄·4H₂O 3.7g/L，ZnSO₄·7H₂O 2.9g/L，H₃BO₄24.7g/L，CuSO₄·5H₂O 2.5g/L，MnCl₂·4H₂O15.8 g/L); ampicillin 50. mu.g/mL; 34 mu g/mL of chloramphenicol), culturing at 37 ℃, adding IPTG with the final concentration of 0.5mM when the bacterial concentration reaches about 0.6, and continuing culturing at 30 ℃.

Product detection

After fermentation for 48h, the final fermentation liquid is taken to detect the yield, and GC-MS results (figure 5) show that 35.7mg/L of nerol and 31.3mg/L of geraniol are obtained.

Although the present invention has been described with reference to the above embodiments, it should be understood that the present invention is not limited to the above embodiments, and various changes and modifications can be made by those skilled in the art without departing from the spirit and scope of the present invention.

SEQUENCE LISTING

<110> Qingdao institute for bioenergy and Process of Chinese academy of sciences, institute for tobacco of Chinese academy of agricultural sciences

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gtgggagaca actatacgaa gcgattaaat aaactgaagg aggaaatgcg gaagaagttg 300

aagatgatgg acgatgttga tggagaagaa ttagacaagc tggagctgat tgacaactta 360

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caccaacaac atatattaca gaagaagggc agaaacataa tcgcgggaga atattcatta 480

ttatacgcta cagctttgaa atttaaactc ttgagagaac atgattttga tatttctcaa 540

gatatattga acagtttcaa gattgagaac ggtaatttca aggaaagtct tggtaaagac 600

ccaaaaggaa tgttgcaatt gtacgaagct tcgttttttg ctacagaaac agaaaacact 660

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cacggtgagg agaatataac agcaacatta gtccgccatg cattggaact cccttcgcat 780

tggatgatgt tgagattaga gacaagatgg ttcataaacg tttatgagaa aatgccaaac 840

gctaatcctc ttctgcttga gcttgccaag ttggacttca acattgttca agcaacacat 900

caagaagaat taagagatgt atcaaggtgg tggaagacca catgcctggc agagaagttg 960

ccattttcaa gggacagact agcggaggct ttcttttggg gagtagggat agtatttgag 1020

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1 MetAlaThrSerIleArgProAlaThrProSerLeuSerPhePheSerGlyArgLysAla

21 SerLeuValSerLysAlaLysAlaCysSerMetSerLysAlaAsnSerThrSerProLys

41 AlaGluProProLeuSerHisSerSerIleProThrAsnMetAsnThrHisArgArgSer

61 GlyAsnTyrLysProProThrTrpHisPheGluTyrIleGlnSerIleAsnAsnAspTyr

81 ValGlyAspAsnTyrThrLysArgLeuAsnLysLeuLysGluGluMetArgLysLysLeu

101 LysMetMetAspAspValAspGlyGluGluLeuAspLysLeuGluLeuIleAspAsnLeu

121 GlnArgLeuGlyLeuSerTyrHisPheLysGluGluIlePheGlnIleLeuArgSerIle

141 HisGlnGlnHisIleLeuGlnLysLysGlyArgAsnIleIleAlaGlyGluTyrSerLeu

161 LeuTyrAlaThrAlaLeuLysPheLysLeuLeuArgGluHisAspPheAspIleSerGln

181 AspIleLeuAsnSerPheLysIleGluAsnGlyAsnPheLysGluSerLeuGlyLysAsp

201 ProLysGlyMetLeuGlnLeuTyrGluAlaSerPhePheAlaThrGluThrGluAsnThr

221 LeuLysSerAlaThrArgValThrLysSerGluLeuLysAsnTyrLeuGluAsnTyrGlu

241 HisGlyGluGluAsnIleThrAlaThrLeuValArgHisAlaLeuGluLeuProSerHis

261 TrpMetMetLeuArgLeuGluThrArgTrpPheIleAsnValTyrGluLysMetProAsn

281 AlaAsnProLeuLeuLeuGluLeuAlaLysLeuAspPheAsnIleValGlnAlaThrHis

301 GlnGluGluLeuArgAspValSerArgTrpTrpLysThrThrCysLeuAlaGluLysLeu

321 ProPheSerArgAspArgLeuAlaGluAlaPhePheTrpGlyValGlyIleValPheGlu

341 ProGlnGlnGlyHisCysArgValMetLeuThrLysIleIleAlaPheValThrSerIle

361 AspAspIleTyrAspIleTyrGlyThrTyrProGluLeuLeuPheThrAspAlaValGlu

381 ArgTrpGluLeuThrAlaMetGluGlnLeuProGluTyrMetLysValMetTyrLeuAla

401 LeuPheAsnThrIleAsnGluMetAlaTyrGluValLeuLysGluGlnGlyIleAsnIle

421 LeuProTyrLeuSerLysSerAspGluLeuLysArgGlyAspValProLysSerIleGln

441 CysTyrIleAsnGluLysGlyValSerGluGluGluAlaArgGluArgIleGlnHisLeu

461 IleLysGluThrTrpGluMetMetAsnLysAlaGlnArgGluAsnLeuLeuPheSerArg

481 IlePheValGluIleAlaLysAsnIleAlaArgThrAlaGlnCysMetTyrLeuHisGly

501 AspGlyHisGlyIleGlnAsnAlaGluIleLysAsnSerIleSerLysIleLeuPheGlu

521 HisIleThrValProAsnProLeuThrProAsnProSerLeu***

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ccctcgagtt atagggatgg attgggagtc aat 33

Claims

1. A tobacco monoterpene synthase TPS2b, characterized in that: the amino acid sequence is shown in SEQ ID NO. 2.

2. A gene TPS2b encoding tobacco monoterpene synthase TPS2b according to claim 1, characterized in that: the nucleotide sequence is shown in SEQ ID NO. 1.

3. The use of the coding gene TPS2b of tobacco monoterpene synthase TPS2b according to claim 2 in the preparation of engineered bacteria that co-produce geraniol and nerol.

4. The use of the coding gene TPS2b of tobacco monoterpene synthase TPS2b according to claim 3 in the preparation of engineered bacteria for the co-production of geraniol and nerol, characterized in that: the engineering bacteria for co-producing geraniol and nerol also express geranyl pyrophosphate synthetase gene (GPPS2, GenBank No. AF513112.1).

5. The use of the coding gene TPS2b of tobacco monoterpene synthase TPS2b according to claim 3 in the preparation of engineered bacteria for the co-production of geraniol and nerol, characterized in that: the application specifically comprises the following steps:

(1) cloning the tobacco geraniol and nerol synthetase genes tps2 b;

6. The use of the coding gene TPS2b of tobacco monoterpene synthase TPS2b according to claim 5 in the preparation of engineered bacteria for the co-production of geraniol and nerol, characterized in that: the step (1) is specifically as follows: extracting RNA of tobacco leaf tissue, converting the RNA into a cDNA template by adopting reverse transcriptase, taking the cDNA obtained by reverse transcription reaction as the template, and adopting a forward primer as follows: 5'-ATGGCCACCTCCATAAGACCTGCAA-3', the reverse primer is: 5'-TTATAGGGATGGATTGGGAGTCAAT-3', performing PCR amplification; the target gene tps2b with a fragment size of 1605bp was recovered by gel electrophoresis.

7. The use of the coding gene TPS2b of tobacco monoterpene synthase TPS2b according to claim 5 in the preparation of engineered bacteria for the co-production of geraniol and nerol, characterized in that: the step (2) is specifically as follows: the nerol synthetase gene tps2b was TA cloned and ligated to

-Blunt cloning vector to obtain recombinant plasmid pEASY-T2 b; taking a recombinant plasmid pEASY-T2b as a template, adopting a forward primer T2b-F (5'-GAAGATCTATGGCCACCTCCATAAGACCTGCAA-3') and a reverse primer T2b-R (5'-CCCTCGAGTTATAGGGATGGATTGGGAGTCAAT-3') to carry out PCR amplification, and recovering a target gene fragment with the fragment size of 1621bp by gel electrophoresis; the obtained target gene fragment and pYJM26 plasmid are subjected to double enzyme digestion by BglII and XhoI respectively, exogenous gene fragments and vector fragments are obtained by recovery respectively, and the vector and the exogenous fragments are connected according to the molar ratio of 1:5 to obtain the recombinant plasmid pT2 b.

8. The use of the coding gene TPS2b of tobacco monoterpene synthase TPS2b according to claim 5 in the preparation of engineered bacteria for the co-production of geraniol and nerol, characterized in that: the pYJM26 plasmid carries acetyl-CoA acyltransferase gene/hydroxymethylglutaryl-CoA reductase gene (mvaE, GenBank No. AAG02438) derived from Enterococcus faecalis (Enterococcus faecalis), and 3-hydroxy-3-methylglutaryl-CoA synthase gene (mvaS, GenBank No. AAG02439); and geranyl pyrophosphate synthetase gene derived from Abies grandis (GPPS2, GenBank No. AF513112.1).

9. The use of the coding gene TPS2b of tobacco monoterpene synthase TPS2b according to claim 7 in the preparation of engineered bacteria for the co-production of geraniol and nerol, characterized in that: the pYJM14 plasmid carries a mevalonate kinase gene (ERG12, GenBank No. NM-001182715.1), a mevalonate-5-phosphate kinase gene (ERG8, GenBank No. NM-001182727.1), a mevalonate-5-diphosphate decarboxylase gene (ERG19, GenBank No. X97557.1), and an isopentenyl pyrophosphate isomerase gene (IDI1, GenBank No. NM-001183931.1) derived from Saccharomyces cerevisiae (Saccharomyces cerevisiae).