CN111378675A

CN111378675A - Biosynthesis gene of eremophilane sesquiterpene in catharanthus roseus and application

Info

Publication number: CN111378675A
Application number: CN201811651116.6A
Authority: CN
Inventors: 于荣敏; 梁锦才; 訾佳辰; 朱建华
Original assignee: Individual
Current assignee: Individual
Priority date: 2018-12-31
Filing date: 2018-12-31
Publication date: 2020-07-07

Abstract

The invention discloses an eremophilane sesquiterpene 5-epi-jinkoh-eremol and debneyol biosynthetic gene pair in Catharanthus roseus (Catharanthus roseus) and application thereof. The entire gene pair contains 2 genes: a gene encoding terpene synthase CrTPS3 and a gene encoding cytochrome P450 enzyme CrCPY. The biosynthesis of 5-epi-jinkoh-eremol and debneyol can be blocked or enhanced by genetic manipulation of the above biosynthetic genes. The gene and the protein thereof provided by the invention can be used for gene engineering, protein expression, enzyme catalytic reaction and the like of 5-epi-jinkoh-eremol and debneyol compounds, and can also be used for searching and discovering compounds or genes and proteins which can be used for medicine, industry or agriculture.

Description

Biosynthesis gene of eremophilane sesquiterpene in catharanthus roseus and application

Technical Field

The invention belongs to the technical field of plant genetic engineering and medicine, and particularly relates to a biosynthesis method and application of eremophilane sesquiterpene 5-epi-jinkoh-eremol and Debneyol with an antibacterial effect.

Background

Plant mycoses are diseases caused by phytopathogenic fungi. Accounts for about 70-80% of plant diseases, and is one of the main causes of yield reduction of economic plants. Several or even dozens of fungal diseases can be found in one crop, and common diseases include downy mildew, powdery mildew, white rust, black powder, rust powder, tobacco mold, black nevus, mildew, mushroom, cotton floc, granules, ropes, clay grains, small black spots and the like. The crop is infected with diseases to cause yield reduction and quality deterioration, and some pathogenic fungi (such as fusarium and aspergillus flavus) also generate various mycotoxins harmful to human and livestock in the process of infecting plants, so that the food safety is influenced. Many fungal diseases do not manifest signs at all when environmental conditions are inappropriate. The infection cycle types of fungal diseases are the most, many germs can form special tissues or spores to live through the winter, the damage cycle is long, and huge economic losses are caused. At present, methods for coping with plant fungal diseases are limited, and besides selecting good disease-resistant varieties and strengthening cultivation management, the method mainly depends on pesticide application for prevention and control. The existing plant fungi control drugs are mostly of microbial sources and have the defects of being environment-friendly, easy to resist drugs, easy to remain and the like. Therefore, the search for new and highly effective plant-derived antifungal agents is urgent.

Terpenoids in plants play an important role in their own chemical defense system and thus often have strong antifungal and antibacterial effects. Eremophilane (eremophilane) sesquiterpenes exist in various plants, and various eremophilane sesquiterpene derivatives are found to have antitumor effect. In addition, the eremophilane sesquiterpene also has strong antifungal effect. Wherein the eremophilane sesquiterpene debneyol found in the tobacco infected by the virus has better inhibiting effect on fungi such as Cladosporium cuprinum, Colletotrichum lindleyanum and the like, and the eremophilane sesquiterpene debneyol and the analogues thereof have the effect of resisting plant fungal diseases [ Phytochemistry,24,2191-219(1985) ]. At present, eremophilane sesquiterpenes are mainly obtained by a method of extracting from plants or chemically synthesizing, and have the defects of high energy consumption, environmental friendliness and the like. The eremophilane sesquiterpene is produced by excavating plant gene resources and utilizing a biosynthesis method, so that the new plant-derived eremophilane sesquiterpene with plant fungal disease resistance can be found in depth, and the method has practical significance for the development and utilization of the sesquiterpene.

Disclosure of Invention

The invention aims to overcome the defects of a plant fungal disease antibacterial agent from microorganisms, and determines the eremophilane sesquiterpene 5-epi-jinkoh-eremol and debneyol biosynthesis related gene pair CrTPS3-CrCYP by performing genome data analysis and biochemical function verification on a medicinal plant catharanthus roseus with strong anti-germ capacity. The antibacterial effect of 5-epi-jinkoh-eremol and debneyol in phytochemical defense is determined.

The invention provides a nucleotide sequence for coding terpene synthase CrTPS3, wherein the nucleotide sequence is positioned at the 1st to 1686 th base in SEQ ID NO.1, and the coded amino acid sequence is SEQ ID NO. 2.

The invention also provides a nucleotide sequence for coding the cytochrome P450 enzyme CrCYP, wherein the nucleotide sequence is positioned at the 1687-3201 base in SEQ ID NO.1, and the coded amino acid sequence is SEQ ID NO. 3.

The invention also provides a recombinant expression plasmid containing the CrTPS3 and CrCYP encoding genes, and a recombinant microorganism escherichia coli C41(DE3) and saccharomyces cerevisiae BY4741 containing the recombinant plasmid.

The invention discloses a biosynthesis way capable of efficiently producing 5-epi-jinkoh-eremol and Debneyol in Escherichia coli C41(DE3) and Saccharomyces cerevisiae BY4741, and the biosynthesis way can be used for producing 5-epi-jinkoh-eremol of more than or equal to 100mg/L and Debneyol of more than or equal to 3mg/L BY fermentation under laboratory conditions.

The invention also discloses the antibacterial action of eremophilane sesquiterpene 5-epi-jinkoh-eremol and debneyol on 3 common plant pathogenic fungi and 2 plant pathogenic bacteria.

Starting from cloning plant biosynthesis gene pairs, the biosynthesis is researched by adopting a method combining microbiology, molecular biology, biochemistry and organic chemistry, and the biosynthesis pathway of eremophilane sesquiterpenes 5-epi-jinkoh-eremol and debneyol is disclosed through the research on the biosynthesis mechanism. On the basis, the principle of metabolic engineering is applied, and the heterologous expression quantity of the 5-epi-jinkoh-eremol and debneyol is improved by reasonably optimizing a biosynthesis pathway by combining biology.

The application of eremophilane sesquiterpene 5-epi-jinkoh-eremol and debneyol biosynthetic gene pairs of the invention includes (but is not limited to):

(1) comprising the nucleotide sequence provided by the present invention or at least part of the nucleotide sequence may be modified or mutated. These include insertions, substitutions or deletions, polymerase chain reaction, error-mediated polymerase chain reaction, site-specific mutations, reconnection of different sequences, directed evolution of different parts of a sequence or homologous sequences from other sources, or mutagenesis by ultraviolet light or chemical reagents, and the like.

(2) Cloned genes comprising the nucleotide sequences provided by the invention or at least part of the nucleotide sequences can be expressed in an exogenous host by means of a suitable expression system to obtain the corresponding enzymes or other higher biological activity or yield. Such foreign hosts include Streptomyces, Pseudomonas, Escherichia, Bacillus, yeast, plants, and animals.

(3) The amino acid sequences provided by the invention can be used for separating the required protein and can be used for preparing antibodies.

(4) Polypeptides comprising the amino acid sequences or at least partial sequences provided herein may have biological activity, even new biological activity, after removal or substitution of certain amino acids, or increased yield or optimized protein kinetics or other properties sought to be achieved.

(5) Genes comprising the nucleotide sequences provided by the invention or at least part of the nucleotide sequences can be expressed in heterologous hosts and their function in the metabolic chain of the host is understood by DNA chip technology.

(6) The gene comprising the nucleotide sequence or at least part of the nucleotide sequence provided by the present invention can be used for constructing a recombinant plasmid through genetic recombination to obtain a novel biosynthetic pathway, and can also be used for obtaining a novel biosynthetic pathway through insertion, replacement, deletion or inactivation.

(7) The protein encoded by the nucleotide sequence provided by the invention can catalyze farnesyl pyrophosphate to synthesize 5-epi-jinkoh-eremol and debneyol, and can be recombined by a biosynthesis pathway or a partial biosynthesis pathway with other natural products to obtain the novel eremophane sesquiterpene compounds.

Therefore, a recombinant vector, an expression cassette, a transgenic cell line or a recombinant bacterium containing the above gene pair is also within the scope of the present invention.

The application of the protein, the gene cluster, the recombinant vector, the expression cassette, the transgenic cell line or the recombinant bacteria in the synthesis of 5-epi-jinkoh-eremol and debneyol is also within the protection scope of the invention.

Another objective of the invention is to provide a method for synthesizing 5-epi-jinkoh-eremol and debneyol, which is to ferment the recombinant bacteria and collect fermentation products to obtain the eremophilane sesquiterpene 5-epi-jinkoh-eremol and debneyol compounds.

In conclusion, the information of all genes and proteins related to 5-epi-jinkoh-eremol and debneyol biosynthesis provided by the invention can help people to understand the biosynthesis mechanism of eremophilane sesquiterpene compounds, and provides materials and knowledge for further genetic modification. The gene and the protein thereof provided by the invention can also be used for searching and discovering compounds or genes and proteins which can be used for medicine, industry or agriculture.

Description of the drawings

FIG. 1 biosynthetic pathway of 5-epi-jinkoh-eremol and Debneyol in Catharanthus roseus;

FIG. 2 shows the GC-MS detection result of the catalytic generation of 5-epi-jinkoh-eremol in Saccharomyces cerevisiae by CrTPS 3;

FIG. 3 GC-MS detection of the catalytic formation of 5-epi-jinkoh-eremol in E.coli by CrTPS 3;

FIG. 4 shows the GC-MS detection result of Debneyol catalyzed by CrCYP in Saccharomyces cerevisiae;

FIG. 5 shows the GC-MS detection result of Debneyol catalyzed by CrCYP in E.coli;

FIG. 6. optimization results of 5-epi-jinkoh-eremol biosynthetic pathway in Saccharomyces cerevisiae;

FIG. 7.5-zone of inhibition of epi-jinkoh-eremol and Debneyol against Rhizoctonia solani;

FIG. 8.5-epi-jinkoh-eremol and Debneyol quantification of zone of inhibition against Rhizoctonia solani;

FIG. 9.5-zone of inhibition of epi-jinkoh-eremol and Debneyol against early blight of tomato;

FIG. 10.5-epi-jinkoh-eremol and Debneyol quantification results of inhibition zones for early blight of tomato;

FIG. 11.5 zone of inhibition of F.oxysporum by epi-jinkoh-eremol and Debneyol;

FIG. 12.5-epi-jinkoh-eremol and Debneyol quantification results for inhibition zones of Fusarium oxysporum.

Fifth, detailed description of the invention

The invention is further illustrated by the following specific examples in conjunction with the accompanying drawings. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, molecular cloning is generally performed according to conventional conditions such as Sambrook et al: the conditions described in the laboratory Manual (New York: Cold spring harbor laboratory Press,1989), or according to the manufacturer's recommendations. Percentages and parts are by weight unless otherwise indicated.

Example 1

Catharanthus roseus RNA extraction and CrTPS3-CrCYP gene cloning

cDNA sequence of vinca sesquiterpene 5-epi-jinkoh-eremol and Debneyol synthetase gene in vinca genomeThe biosynthetic pathway is shown in FIG. 1. Using Trizol (Invitrogen) separately according to the kit instructions^TM) And HiScript II 1st Strand cDNA Synthesis Kit (Novozam Vazyme) for the extraction of total RNA from Catharanthus roseus leaves and reverse transcription to synthesize cDNA. The full-length coding sequences of the 5-epi-jinkoh-eremol synthetase gene CrTPS3 and the Debneyol synthetase gene CrTPS3 were amplified using two pairs of primers (CrTPS 3-F/R; CrCYP-F/R), and the amplified products were purified by electrophoresis and ligated with a blunt-end cloning vector pEASY-blunt (all-type gold) for DNA sequencing.

Example 2

Construction of Saccharomyces cerevisiae expression vector

For improving the content of farnesyl pyrophosphate serving as a precursor for sesquiterpene synthesis in saccharomyces cerevisiae, farnesyl pyrophosphate synthetase gene ERG20 and truncated hydroxymethyl glutaryl coenzyme A reductase gene tHMGp are respectively constructed on a pESC-LEU vector by a homologous recombination method to obtain two plasmids of pESC-LEU-ERG20 and pESC-LEU-tHMGp; for the biosynthesis of 5-epi-jinkoh-eremol, the full-length sequence of CrTPS3 is respectively constructed on pESC-LEU-ERG20 and pESC-LEU-tHMGp vectors by a homologous recombination method after AACA is added at the 5' end, so as to obtain two plasmids of pESC-LEU-ERG20-CrTPS3 and pESC-LEU-tHMGp-CrTPS 3; for the biosynthesis of Debneyol, the full-length sequence of CrCYP is added with AACA at the 5' end and then constructed on a pESC-URA vector by a homologous recombination method to obtain a pESC-URA-CrCYP plasmid; because the oxidation function of CrCYP needs to participate in NADPH dependent cytochrome P450 reductase, AtR1 of Arabidopsis thaliana is inserted into pESC-URA-CrCYP vector by using a homologous recombination method to obtain pESC-URA-CrCYP-AtR1 plasmid, and AtR1 of Arabidopsis thaliana is inserted into pESC-URA vector by using the homologous recombination method to obtain pESC-URA-AtR1 plasmid in order to provide negative control in biochemical function verification.

Example 3

Construction of E.coli expression vectors

In order to optimize the content of farnesyl pyrophosphate in Escherichia coli, ERG20 was inserted into pETDuet-1 vector by homologous recombination to obtain pETDuet-1-ERG20 plasmid; for the biosynthesis of 5-epi-jinkoh-eremol, the full-length sequence of CrTPS3 is inserted into a pETDuet-1-ERG20 vector by a homologous recombination method to obtain a pETDuet-1-ERG20-CrTPS3 plasmid; for the biosynthesis of Debneyol, the full-length sequence of the CrCYP is firstly truncated by 108bp at the 5' end and connected with a section of peptization peptide MAKKTSSKGKLPPGPS, and then the CrCYP is inserted into a pACYCDuet-1 vector by a homologous recombination method to obtain a pACYCDuet-1-CrCYP plasmid; similarly, the plasmid pACYCDuet-1-AtR1 and pACYCDuet-1-CrCYP-AtR1 are obtained by inserting AtR1 into the vectors pACYCDut-1 and pACYCDut-1-CrCYP by homologous recombination.

Example 4

Biosynthesis of the antibacterial sesquiterpenes Debneyol and 5-epi-jinkoh-eremol in Saccharomyces cerevisiae

For the strain for biosynthesis of 5-epi-jinkoh-eremol, only pESC-LEU-tHMGp-CrTPS3 plasmid needs to be transformed into Saccharomyces cerevisiae BY 4741; for the strain for biosynthesis of Debneyol, simultaneous transformation of pESC-LEU-ERG20-CrTPS3 plasmid and pESC-URA-CrCYP-AtR1 plasmid into BY4741 was required. The transformed strain was inoculated into 5ml SD medium, shake-cultured overnight at 30 ℃ and 220RPM, and then inoculated into 50ml SG (synthetic Galactose minimum Dropout Medium) medium at a ratio of 1:50 the next day, and shake-cultured at 30 ℃ and 220RPM for 5 days. Extracting the 5-epi-jinkoh-eremol fermentation liquor by using normal hexane to obtain a 5-epi-jinkoh-eremol crude extract, and extracting the Debneyol fermentation liquor by using ethyl acetate to obtain a Debneyol crude extract. Plasmid transformation of all saccharomyces cerevisiae strains in this experiment was based on the electroporation transformation method: adding 40ul of saccharomyces cerevisiae shock competence and 300ng of plasmid DNA into an EP tube, gently mixing, transferring into a precooled 2mm specification shock cup, quickly placing into a BIO-RAD MicroPulser shock tank, selecting a Sc2 mode for shock transformation, immediately resuspending the competence cells in the shock cup with 1M sorbitol after the shock is finished, transferring into the EP tube, incubating at 30 ℃ for 1 hour, coating in SD (synthetic Dextrose Minimal DropouthMedium) culture with corresponding auxotrophy, and carrying out inverted culture at 30 ℃ for 48-72 hours.

Example 5

Biosynthesis of antibacterial sesquiterpenes in E.coli

For the biosynthesis of 5-epi-jinkoh-eremol, the pIRS plasmid (Cyr, A.; Wilderman, P.R.; Determan, M.; Peters, R.J.J.J.am.chem.Soc.2007, 129,6684.) and pETDuet-1-ERG20-CrTPS3 plasmid were transformed simultaneously, for the biosynthesis of Debneol, the pIRS plasmid, pETDuet-1-ERG20-Cr 3 and CYpACDuet-1-CrCYP-AtR 1 plasmid were transformed simultaneously, positive clones were picked directly into 5ml TB medium containing the corresponding antibiotic at the time of biosynthesis of 5-epi-jinkoh-eremol, activated at 37 ℃ at 220RPM for 4 hours, then inoculated into 50ml TB medium containing the corresponding antibiotic at a ratio of 1:50, the strain was cultured at 37 ℃ at 0.600 ℃ for 0.6.7.7 ℃ in a gentle stroke of 5-100 min, the strain was inoculated into a gentle tapping of the strain, and the E.C.E.E.B.E.coli was extracted immediately after incubation with the heat-DNA at 37 ℃ for 5-10 min, and the strain was added to 5-100 min, and the E.7 min under a gentle tapping of the expression of the strain, and the strain was added to the E.E.C.16-E.E.E.E.C.E.coli strain was added to induce the expression of the strain, and the strain was added to the strain was expressed in a gentle strain under the conditions of the heat-strain under the heat-strain, and the conditions of the strain, the strain was added to induce the strain, the strain was added to the strain, the strain was added to the strain, the strain was added to the strain, the strain was added to the strain, the strain was added to.

Example 6

Detection, separation and purification of antibacterial sesquiterpene

The detection of 5-epi-jinkoh-eremol and Debneyol is based on gas mass spectrometry: dissolving and diluting a sample to be detected by using n-hexane, and carrying out gas phase mass spectrometry according to the following conditions, wherein the sample injection amount is 1ul, the sample injection is not carried out in a split-flow manner, the sample injection inlet temperature is 250 ℃, the oven temperature is kept at 50 ℃ for 3 minutes, then the rate of 20 ℃ per minute is increased to 70 ℃ for 1 minute, and then the rate of 15 ℃ per minute is increased to 300 ℃ for 3 minutes. Electron Ionization ion source, energy intensity 70 eV. Ultrasonically extracting the fermentation liquor for 20 minutes by using n-hexane or ethyl acetate with the same volume, carrying out rotary evaporation drying on an organic layer under the reduced pressure condition, redissolving the organic layer in 5ml of n-hexane, then passing through a 200-mesh 300-mesh silica gel column according to the proportion of 1:40, eluting the silica gel column by using n-hexane with the volume of 6 times of the column volume after loading, and then carrying out 40: 1 elution 6 column volumes, followed by n-hexane acetone 30: 1 eluted 3 column volumes followed by n-hexane acetone 20:1 eluting 3 column volumes followed by 10:1 eluting 3 column volumes. Fractions containing the desired product were combined, concentrated and further purified by HPLC using C18 reverse phase column chromatography with methanol in water as the mobile phase, followed by 70% methanol for 30 min and then 100% methanol for 10 min. Fractions containing the target product were analyzed by gas mass spectrometry, combined, blown dry with nitrogen, and dissolved in deuterated chloroform for NMR analysis. As shown in FIGS. 2 to 6, 5-epi-jinkoh-eremol and Debneyol compounds can be heterologously produced with high efficiency BY expressing a gene cluster comprising CrTPS3 and CrCYP genes in Saccharomyces cerevisiae BY4741 and Escherichia coli E.coli C41(DE 3).

Example 7

Inhibition of phytopathogenic fungi by 5-epi-jinkoh-eremol and Debneyol

Respectively dissolving 5-epi-jinkoh-eremol and Debneyol in DMSO (dimethylsulfoxide), preparing a potato glucose agar (PDA) culture medium, respectively adding the culture medium to a final concentration of 50mg/l when the culture medium is cooled to 50 ℃, adding DMSO (dimethyl sulfoxide) with the same volume in a blank control group, respectively inoculating rhizoctonia solani, early blight of tomato and fusarium oxysporum after preparing a PDA culture dish, culturing for 72 hours at 28 ℃, and measuring the size of a colony. 5-epi-jinkoh-eremol and Debneyol are respectively prepared into 1.5mg/ml to-be-detected solution by DMSO. Selecting pseudomonas syringae/xanthomonas campestris, carrying out monoclonal inoculation on the pseudomonas syringae/xanthomonas campestris, inoculating the pseudomonas syringae/xanthomonas campestris into 3ml KB/NYG liquid culture medium containing 50mg/L Rif, culturing the liquid culture medium at 30 ℃ and 220RPM until OD600 is 1.0-1.5, preparing 50ml of agar KB/NYG culture medium with 1.5 percent, adding 50mg/L Rif and 1ml of pseudomonas syringae/xanthomonas campestris bacterial liquid when the culture medium is cooled to 50 ℃, shaking up gently to prepare a solid culture plate, punching a puncher with the diameter of 0.5mm after bacterial plaque is solidified, adding 20ul of solution to be tested into the hole, culturing the liquid culture plate at 30 ℃ for 48 hours, and measuring the size of a colony. As shown in fig. 7-12. The 5-epi-jinkoh-eremol and debneyol compounds have remarkable antibacterial effects on 3 plant fungal diseases of rhizoctonia solani, early blight of tomato and fusarium oxysporum, wherein the debneyol has the best antibacterial effect on the early blight of tomato; 5-epi-jinkoh-eremol has the best antibacterial effect on rhizoctonia solani.

Sequence listing

<110> Yirongming

<120> eremophilane sesquiterpene biosynthesis gene in catharanthus roseus and application

<130>2018.12.27

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<213>Catharanthus roseus

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atgggcaggg aagtagtgat aatgatggct tctttcgtcg acaatggggt tcttcgtcca 60

ataaaaaaat tccctcctag catatggggt gataccttca attctttcga gtgggatcat 120

gaggcatcag aaaaacttgg caaggaaatg aaaatattgg aaaaagatgc gaggaacatg 180

ttacaagctg atacaagaaa tgagacagta aaggacataa ttactttgat caatactttt 240

gagcggctcg ggctttcgta taagtttgag aaagagatag aagatcatct ggaacgactt 300

gtccattctt ttgattatga tggaaatcaa catgatttgc tcacggtttc tctcctgttt 360

agaattctca ggcaacacgg atatgaaatc tcctcaggta tcttcaaaaa attcatggac 420

aagaatgggg aatttaaaga agagatcatt ggcaatgatg tgaaaggtgt attaagcctg 480

tatgaagcat cttacgtgag ggggcatgga gaagatattc ttgaaaaagc attggttttt 540

acaaaaggcc atctcatgag aattcttcct gaaataatag aatctcctct tggaaaacaa 600

gtaatatatg ctcttgaaca accactccat agaggtgttc caagacttga ggctcgtcat 660

tacatctcca tttatgagga agaccaagaa actaaaaatg aagcacttct tagactcgca 720

aaattggatt tcaatctatt gcaaatgtta cacaagaaag agatatgtga gattacaagg 780

tggtggaaga aatcggactt tatgggaaaa cttccttatc taagagatag ggtggtggaa 840

tgctattttt gggcaatagg aatatacttt gaacctaagt attctcttgc tcgtataatg 900

gccagcaaag ttgtggccat gacttcaatc atggatgaca cctatgactc ttatggcata 960

attgaagaac ttgaagtttt cacttctgct gtcgaaaggt ggagcattga agaaattgat 1020

agactcccaa gttacatgaa gatagcctac atggcacttc tcaacctgta cgaagaattt 1080

gatgaaaaac taaaagaaca gggacgatcc tttgcagttc aatattccaa agaaagaatg 1140

aagcagctaa taagaagcta tgacaaagaa gctaagtggt tttatgaaag atcagatgat 1200

gtgcctagtt ttgatgaata catggaaaat gcaatatcaa ctagcactta cctcgtactc 1260

atgccgtcgt tgttgttggg gatggaatct gcaagcaggg aagtgtttga ttgggtcatg 1320

aacaatccta gtatagttgt ggctagtgcc aaagttggtc gatgcacaga cgacgtcgct 1380

acttattcgg ttgagaaagc aaggggtcaa ccagcctgcg gaatcgaatg gtacatgaaa 1440

gaacatggtg tctctaaaga agaaactttc aaaaaatttc atgaaattgt tgaagattct 1500

tggaaagata ttaataagga acttgtccga tcatcctcga ttccaatgga tattctagtg 1560

agggctttaa accaagcaag agtgatcgac gtggtttata agcatgatca agatggttat 1620

actcaccctg aaaaggttct caaaccccat atcaaggcct tacttgttga tcctataagc 1680

atttgaatgg gctttcagat tcctttaaac ttcattgcct tctttgtatt cctattgttg 1740

tcttctattt tattagtgaa acagaggaat agaaaatcat taggaaagaa aaaactgccc 1800

ccaggacccc ggaagttacc attgattgga aacctacaca atttgatagg tggacttcct 1860

catcatattt tcagagattt atctcgaaaa tatggacctc ttattcacct acaattgggt 1920

caagtaggta ccattttgat atcttcacca cgtttagcaa aagaggtgat gaaaactcat 1980

gatcttacct ttgcaacaag gccggacaat cttgccggag atgtcatgtt ctatggtagc 2040

acagatattg tatttgccaa atacggcgag tactggagac aaatgcgtaa aatcagtgtc 2100

ttagaactct tcagcgcgaa aaatgtccgg tcatttggtt ctataagaat ggatgaatca 2160

ttacttatga ttgcgtctat acgagaatca gttggtaaag cggttaatct aagcacaaaa 2220

cttgcaaact atacaagttc tgtggtttgc agggcagcat ttggtaggtt atgtcctgac 2280

caacatgagt ttattgagtt agttgatgaa gcatctgttt tagcagcagg ctttgatatt 2340

ggtgaccttt ttccatcatt aaagtttatt cagtttttga ctggattaaa gcctaaatta 2400

atgaaggttc ataataaggt tgacaagatt ttggaccatg taattaatga gcatagaaaa 2460

aacatgggaa ggagaaatgg tgagtttggt gaagaagact taactgattc acttctaaga 2520

attcaacaaa gtggtggtga ccttcaattt cccatctccg acaacaatat taaggcaatc 2580

ttgtttgatg tgtttggtgc gggaacagaa acttcatcca caataacaga atgggccttg 2640

tcagaattaa ttaaaaatcc agatatgatg aacaaggcac aaactgaaat aaggcaagcc 2700

ttcaagggaa agaaaaggcc gattgaagag gctgatcttc aaggcctaag ttatctcaag 2760

tgtgtaatta aagaaacact gaggctatat cctgcagcgc ctttattggt tcctcgtgaa 2820

tgcagagagg actgtgaatt ggatggatat tttataccaa agaaatcaag ggtaattgtt 2880

aatgcttggg caattggaag agatcctgag tattggccta atgcaaacag ttttattccc 2940

gaaagatttg agaattcttc aactgatttc accggtaatc actttgaatt aataccattt 3000

gggtcaggaa ggaggagttg tcctggaatg ctgtttggta tagctaatat tgagcttcct 3060

ttagctcttc ttttatacca cttcaactgg agtctcccag atggccttac ttccgaaact 3120

ttggacatgt ctgagacttg gggaataaca actccaagga aatatgatct tcacctaatc 3180

cctacatctt attatcctta a 3201

<210>2

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<212>PRT

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Met Gly Arg Glu Val Val Ile Met Met Ala Ser Phe Val Asp Asn Gly

1 5 10 15

Val Leu Arg Pro Ile Lys Lys Phe Pro Pro Ser Ile Trp Gly Asp Thr

20 25 30

Phe Asn Ser Phe Glu Trp Asp His Glu Ala Ser Glu Lys Leu Gly Lys

35 40 45

Glu Met Lys Ile Leu Glu Lys Asp Ala Arg Asn Met Leu Gln Ala Asp

50 55 60

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

65 70 75 80

Glu Arg Leu Gly Leu Ser Tyr Lys Phe Glu Lys Glu Ile Glu Asp His

85 90 95

Leu Glu Arg Leu Val His Ser Phe Asp Tyr Asp Gly Asn Gln His Asp

100 105 110

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

115 120 125

Glu Ile Ser Ser Gly Ile Phe Lys Lys Phe Met Asp Lys Asn Gly Glu

130 135 140

Phe Lys Glu Glu Ile Ile Gly Asn Asp Val Lys Gly Val Leu Ser Leu

145 150 155 160

Tyr Glu Ala Ser Tyr Val Arg Gly His Gly Glu Asp Ile Leu Glu Lys

165 170 175

Ala Leu Val Phe Thr Lys Gly His Leu Met Arg Ile Leu Pro Glu Ile

180 185 190

Ile Glu Ser Pro Leu Gly Lys Gln Val Ile Tyr Ala Leu Glu Gln Pro

195 200 205

Leu His Arg Gly Val ProArg Leu Glu Ala Arg His Tyr Ile Ser Ile

210 215 220

Tyr Glu Glu Asp Gln Glu Thr Lys Asn Glu Ala Leu Leu Arg Leu Ala

225 230 235 240

Lys Leu Asp Phe Asn Leu Leu Gln Met Leu His Lys Lys Glu Ile Cys

245 250 255

Glu Ile Thr Arg Trp Trp Lys Lys Ser Asp Phe Met Gly Lys Leu Pro

260 265 270

Tyr Leu Arg Asp Arg Val Val Glu Cys Tyr Phe Trp Ala Ile Gly Ile

275 280 285

Tyr Phe Glu Pro Lys Tyr Ser Leu Ala Arg Ile Met Ala Ser Lys Val

290 295 300

Val Ala Met Thr Ser Ile Met Asp Asp Thr Tyr Asp Ser Tyr Gly Ile

305 310 315 320

Ile Glu Glu Leu Glu Val Phe Thr Ser Ala Val Glu Arg Trp Ser Ile

325 330 335

Glu Glu Ile Asp Arg Leu Pro Ser Tyr Met Lys Ile Ala Tyr Met Ala

340 345 350

Leu Leu Asn Leu Tyr Glu Glu Phe Asp Glu Lys Leu Lys Glu Gln Gly

355 360 365

Arg Ser Phe Ala Val Gln Tyr SerLys Glu Arg Met Lys Gln Leu Ile

370 375 380

Arg Ser Tyr Asp Lys Glu Ala Lys Trp Phe Tyr Glu Arg Ser Asp Asp

385 390 395 400

Val Pro Ser Phe Asp Glu Tyr Met Glu Asn Ala Ile Ser Thr Ser Thr

405 410 415

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

420 425 430

Arg Glu Val Phe Asp Trp Val Met Asn Asn Pro Ser Ile Val Val Ala

435 440 445

Ser Ala Lys Val Gly Arg Cys Thr Asp Asp Val Ala Thr Tyr Ser Val

450 455 460

Glu Lys Ala Arg Gly Gln Pro Ala Cys Gly Ile Glu Trp Tyr Met Lys

465 470 475 480

Glu His Gly Val Ser Lys Glu Glu Thr Phe Lys Lys Phe His Glu Ile

485 490 495

Val Glu Asp Ser Trp Lys Asp Ile Asn Lys Glu Leu Val Arg Ser Ser

500 505 510

Ser Ile Pro Met Asp Ile Leu Val Arg Ala Leu Asn Gln Ala Arg Val

515 520 525

Ile Asp Val Val Tyr Lys His Asp Gln AspGly Tyr Thr His Pro Glu

530 535 540

Lys Val Leu Lys Pro His Ile Lys Ala Leu Leu Val Asp Pro Ile Ser

545 550 555 560

Ile

<210>3

<211>504

<212>PRT

<213>Catharanthus roseus

<400>3

Met Gly Phe Gln Ile Pro Leu Asn Phe Ile Ala Phe Phe Val Phe Leu

1 5 10 15

Leu Leu Ser Ser Ile Leu Leu Val Lys Gln Arg Asn Arg Lys Ser Leu

20 25 30

Gly Lys Lys Lys Leu Pro Pro Gly Pro Arg Lys Leu Pro Leu Ile Gly

35 40 45

Asn Leu His Asn Leu Ile Gly Gly Leu Pro His His Ile Phe Arg Asp

50 55 60

Leu Ser Arg Lys Tyr Gly Pro Leu Ile His Leu Gln Leu Gly Gln Val

65 70 75 80

Gly Thr Ile Leu Ile Ser Ser Pro Arg Leu Ala Lys Glu Val Met Lys

85 90 95

Thr His Asp Leu Thr Phe Ala Thr Arg Pro Asp Asn Leu Ala Gly Asp

100 105 110

Val Met Phe Tyr Gly Ser Thr Asp Ile Val Phe Ala Lys Tyr Gly Glu

115 120 125

Tyr Trp Arg Gln Met Arg Lys Ile Ser Val Leu Glu Leu Phe Ser Ala

130 135 140

Lys Asn Val Arg Ser Phe Gly Ser Ile Arg Met Asp Glu Ser Leu Leu

145 150 155 160

Met Ile Ala Ser Ile Arg Glu Ser Val Gly Lys Ala Val Asn Leu Ser

165 170 175

Thr Lys Leu Ala Asn Tyr Thr Ser Ser Val Val Cys Arg Ala Ala Phe

180 185 190

Gly Arg Leu Cys Pro Asp Gln His Glu Phe Ile Glu Leu Val Asp Glu

195 200 205

Ala Ser Val Leu Ala Ala Gly Phe Asp Ile Gly Asp Leu Phe Pro Ser

210 215 220

Leu Lys Phe Ile Gln Phe Leu Thr Gly Leu Lys Pro Lys Leu Met Lys

225 230 235 240

Val His Asn Lys Val Asp Lys Ile Leu Asp His Val Ile Asn Glu His

245 250 255

Arg Lys Asn Met Gly Arg Arg Asn Gly Glu Phe Gly Glu Glu Asp Leu

260 265 270

Thr Asp Ser Leu Leu Arg Ile Gln Gln Ser Gly Gly Asp Leu Gln Phe

275 280 285

Pro Ile Ser Asp Asn Asn Ile Lys Ala Ile Leu Phe Asp Val Phe Gly

290 295 300

Ala Gly Thr Glu Thr Ser Ser Thr Ile Thr Glu Trp Ala Leu Ser Glu

305 310 315 320

Leu Ile Lys Asn Pro Asp Met Met Asn Lys Ala Gln Thr Glu Ile Arg

325 330 335

Gln Ala Phe Lys Gly Lys Lys Arg Pro Ile Glu Glu Ala Asp Leu Gln

340 345 350

Gly Leu Ser Tyr Leu Lys Cys Val Ile Lys Glu Thr Leu Arg Leu Tyr

355 360 365

Pro Ala Ala Pro Leu Leu Val Pro Arg Glu Cys Arg Glu Asp Cys Glu

370 375 380

Leu Asp Gly Tyr Phe Ile Pro Lys Lys Ser Arg Val Ile Val Asn Ala

385 390 395 400

Trp Ala Ile Gly Arg Asp Pro Glu Tyr Trp Pro Asn Ala Asn Ser Phe

405 410 415

Ile Pro Glu Arg Phe Glu Asn Ser Ser Thr Asp Phe Thr Gly Asn His

420425 430

Phe Glu Leu Ile Pro Phe Gly Ser Gly Arg Arg Ser Cys Pro Gly Met

435 440 445

Leu Phe Gly Ile Ala Asn Ile Glu Leu Pro Leu Ala Leu Leu Leu Tyr

450 455 460

His Phe Asn Trp Ser Leu Pro Asp Gly Leu Thr Ser Glu Thr Leu Asp

465 470 475 480

Met Ser Glu Thr Trp Gly Ile Thr Thr Pro Arg Lys Tyr Asp Leu His

485 490 495

Leu Ile Pro Thr Ser Tyr Tyr Pro

500

Claims

1. A biosynthetic gene pair derived from eremophilane sesquiterpenes 5-epi-jinkoh-eremol and debneyol in Catharanthus roseus (Catharanthus roseus), characterized in that the nucleotide sequence of the gene cluster is shown as 1-3201 in SEQ ID NO. 1; the genes for coding eremophilane sesquiterpene 5-epi-jinkoh-eremol and debneyol biosynthesis related genes included in the gene pair are specifically as follows:

1) a nucleotide sequence for coding terpene synthase CrTPS3 is located at 1-1686 base of SEQ ID NO.1, and the coded amino acid sequence is SEQ ID NO. 2.

2) A nucleotide sequence for coding a cytochrome P450 enzyme CrCYP, wherein the nucleotide sequence is positioned at 1687-3201 base in SEQ ID NO.1, and the coded amino acid sequence is SEQ ID NO. 3.

2. A recombinant vector, expression cassette, transgenic cell line or recombinant bacterium comprising the biosynthetic gene pair of eremophilane sesquiterpenes 5-epi-jinkoh-eremol and debneyol of claim 1.

3. Use of the recombinant vector, expression cassette, transgenic cell line or recombinant bacterium of claim 1 for the synthesis of 5-epi-jinkoh-eremol and debneyol.

4. A method for synthesizing 5-epi-jinkoh-eremol and debneyol, which is the recombinant bacterium in claim 3, and collecting and extracting fermentation products to obtain the 5-epi-jinkoh-eremol and debneyol compounds.