CN118064464A - Multi-monoterpene product synthetase gene, product and application - Google Patents

Multi-monoterpene product synthetase gene, product and application Download PDF

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CN118064464A
CN118064464A CN202410167614.2A CN202410167614A CN118064464A CN 118064464 A CN118064464 A CN 118064464A CN 202410167614 A CN202410167614 A CN 202410167614A CN 118064464 A CN118064464 A CN 118064464A
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alpha
synthetase
monoterpene
polyterpene
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邓小敏
石彬
廖宏泽
晁金泉
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Rubber Research Institute Chinese Academy Tropical Agricultural Sciences
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Abstract

The invention discloses a multi-monoterpene product synthetase gene derived from a rubber tree, a product and application thereof, and belongs to the technical field of biology. The invention clones the synthetase genes of the polyterpene products (alpha-pinene, beta-myrcene, D-limonene, gamma-terpinene, terpinolene and alpha-terpineol) from the rubber tree for the first time, determines the nucleotide sequence of the synthetase genes to be shown as SEQ ID NO.1 and the amino acid sequence to be shown as SEQ ID NO.2, fills the blank that terpene synthetases and biosynthesis genes thereof in the rubber tree are unknown in the prior art, and provides theoretical and basic support for related researches of the rubber tree. The expression product of the multi-monoterpene product synthetase has obvious biological activity for inhibiting fusarium oxysporum which is pathogenic bacteria of banana vascular wilt, has obvious faint scent, and can be used for development of pesticides for preventing and controlling pathogenic bacteria and research and development of products such as essence, perfume and essential oil preparations.

Description

Multi-monoterpene product synthetase gene, product and application
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a polyterpene product synthetase gene, a product and application thereof.
Background
The monoterpene compounds are natural compounds containing 2 isoprene units, are widely existing in plant specialized secretion tissues, are important components in plant aroma volatile matters, have better physiological activity, are important raw material sources in pesticide and essence and spice industries, and therefore have very important application values.
In plants, in addition to monoterpene synthetases whose main product is relatively single, there are reports of terpene synthetases capable of producing multiple products. These multiproduct terpene synthases give the source plant more options, possibly with multiple physiological functions than a single product. Although a multi-monoterpene product synthetase is reported, a monoterpene synthetase has not been reported so far, and the monoterpene synthetase can simultaneously synthesize characteristic monoterpenes such as alpha-pinene (alpha-pinene), beta-pinene (beta-pinene), beta-myrcene (beta-myrcene), D-limonene (D-limonene), gamma-terpinene (gamma-terpinene), terpinolene (terpinolene), alpha-terpineol (alpha-terpineol) and the like. Each of these seven monoterpene compounds is an important and highly valuable compound alone. For example, alpha-pinene and beta-pinene can be used for expelling parasites, diminishing inflammation, and synthesizing artificial spice verbenone as a precursor. Meanwhile, the beta-myrcene can be used for preparing cologne and deodorant. However, limonene has been developed as an anti-oral cancer synergistic composition (CN 201710122379) and has been registered in 2016 as a tomato bemisia tabaci control agent, and thus has a very important value. In addition, the gamma-terpinene has strong oxidation resistance, can be used for preparing an oral antioxidant for scavenging free radicals, and can be used as a potential high-density fuel. The alpha-terpineol can be used for edible essence such as lemon, sweet orange, peach and the like, and main agent and flavoring agent of the syringa oblata essence, and can be used as soap essence due to alkali resistance. However, no report on the product containing the compounds which can be synthesized naturally or prepared artificially is available at present.
In view of this, the present invention has been made.
Disclosure of Invention
The invention aims at providing a multi-monoterpene product synthetase gene, a product and application aiming at the defects of the prior art. The invention utilizes the efficient microorganism synthesis chassis to screen and identify the genes of the polyterpene synthetase derived from the rubber tree, and successfully realizes the biosynthesis of polyterpene products including alpha-pinene, beta-myrcene, D-limonene, gamma-terpinene, terpinolene and alpha-terpinol by utilizing the genes.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
The first object of the present invention is to provide a polyterpene product synthetase gene, the nucleotide sequence of which is shown in SEQ ID NO. 1.
A second object of the present invention is to provide an amplification primer pair for the above-mentioned polyterpene product synthase gene, which is: the nucleotide sequence of the forward primer is shown as SEQ ID NO.3, and the nucleotide sequence of the reverse primer is shown as SEQ ID NO. 4.
A third object of the present invention is to provide a polyterpene product synthetase having an amino acid sequence as shown in SEQ ID NO. 2.
Further, the substrate of the polyterpene product synthetase comprises geranyl pyrophosphate.
A fourth object of the present invention is to provide the above-mentioned multiple monoterpene product synthetase gene or the above-mentioned multiple monoterpene product synthetase, and the use thereof in preparing monoterpene compounds, wherein the monoterpene compounds comprise at least one of alpha-pinene, beta-myrcene, D-limonene, gamma-terpinene, terpinolene and alpha-terpinol.
A fifth object of the present invention is to provide a recombinant expression vector comprising the above gene as shown in SEQ ID NO. 1.
Further, the recombinant expression vector comprises prokaryotic expression vectors such as escherichia coli and the like, lower eukaryotic expression vectors such as yeast and the like and plant expression vectors
It is a sixth object of the present invention to provide a recombinant expression strain comprising the recombinant expression vector described above.
A seventh object of the present invention is to provide a method for producing a monoterpene compound, culturing the recombinant expression strain of claim 7, obtaining the monoterpene compound from the culture, and separating and purifying to obtain at least one of α -pinene, β -myrcene, D-limonene, γ -terpinene, terpinolene and α -terpinol.
An eighth object of the present invention is to provide a method for producing monoterpene compounds, culturing the recombinant expression strain, obtaining monoterpene compounds from the culture, and separating and purifying to obtain at least one of α -pinene, β -myrcene, D-limonene, γ -terpinene, terpinolene and α -terpinol.
Further, the product comprises fusarium oxysporum inhibitors of banana vascular wilt pathogens.
A ninth object of the present invention is to provide a plant cell or a transgenic plant into which the polyterpene product synthetase gene according to claim 1 is introduced.
The term "expression" as used in the present invention includes any step involving the production of a polypeptide, including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.
The term "recombinant vector" as used in the present invention means a linear or circular DNA molecule comprising a polynucleotide encoding a polypeptide operably linked to control sequences for its expression.
The term "host cell" as used in the present invention means any cell type that is readily transformed, transfected, transduced, or the like with a nucleic acid construct or expression vector comprising a polynucleotide of the present invention. The term "host cell" encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication.
Compared with the prior art, the technical scheme provided by the invention has the beneficial effects that:
(1) The invention clones the synthetase genes of the polyterpene products (alpha-pinene, beta-myrcene, D-limonene, gamma-terpinene, terpinolene and alpha-terpinol) from the rubber tree for the first time, determines the nucleotide sequence and the amino acid sequence thereof, and provides theoretical and basic support for the related research of the rubber tree.
(2) The invention prepares recombinant vector and engineering bacteria by recombining the synthetase genes of the synthetic polymonoterpene products (alpha-pinene, beta-myrcene, D-limonene, gamma-terpinene, terpinolene and alpha-terpinol) from rubber tree sources, and successfully constructs the recombinant vector and engineering bacteria into production strains, thereby laying an important foundation for the application of the polymonoterpene products (alpha-pinene, beta-myrcene, D-limonene, gamma-terpinolene, terpinolene and alpha-terpinol).
(3) The invention proves that the metabolite of the polyterpene synthetase has obvious growth inhibition effect on fusarium oxysporum through growth inhibition experiments on fusarium oxysporum (Fusarium oxysporum f.sp.), which provides a new thought and raw material source for developing green control of banana wilt.
(4) The invention researches the synthetase with special functions and the coding genes thereof in the rubber tree by carrying out gene excavation and functional characterization in the rubber tree, has important value for expanding research and application of other small molecular compounds except natural rubber synthesis of the rubber tree, and the novel rubber tree polymonoterpene synthetase gene identified by the invention provides important gene synthesis elements and technical basis for producing polymonoterpene products including alpha-pinene, beta-myrcene, D-limonene, gamma-terpinene, terpinolene and alpha-terpinol by adopting biotechnology.
Drawings
FIG. 1 is a GC analysis of YHbMTS strain products, wherein peak 1-peak 7 represents the synthetic product of HbMTS1 polyterpene synthase and peak 8-peak 14 represents the α -pinene, β -myrcene, D-limonene, γ -terpinene, terpinolene, and α -terpinol standard, respectively;
FIG. 2 is a GC-corresponding MS diagram of YHbMTS strain products, wherein peak 1-peak 7 represents the synthetic product of HbMTS polyterpene synthetase, and peak 8-peak 14 represents alpha-pinene, beta-myrcene, D-limonene, gamma-terpinene, terpinolene, and alpha-terpinol standard, respectively;
FIG. 3 shows the results of inhibition of Fusarium oxysporum (sp.) growth in treated group (A) and control group (B) containing HbMTS fermentation products.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the following detailed description of the specific embodiments of the present invention will be given with reference to the accompanying drawings. The examples are not to be construed as limiting the specific techniques or conditions described in the literature in this field or as per the specifications of the product. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
The nucleotide sequence of the multi-monoterpene product synthetase gene which is derived from the rubber tree and comprises alpha-pinene, beta-myrcene, D-limonene, gamma-terpinene, terpinolene and alpha-terpinol is shown as SEQ ID NO. 1; the amino acid sequence of the rubber tree polyterpene product synthetase is shown as SEQ ID NO. 2.
The invention clones and obtains the multi-monoterpene product synthetase gene including alpha-pinene, beta-myrcene, D-limonene, gamma-terpinene, terpinolene and alpha-terpineol from rubber tree for the first time, can be used for efficiently synthesizing special multi-monoterpene products, determines the nucleotide sequence and amino acid sequence thereof, fills the blank of synthesizing the multi-monoterpene products including alpha-pinene, beta-myrcene, D-limonene, gamma-terpinene, terpinolene and alpha-terpineol and biosynthesis thereof in the rubber tree in the prior art, and provides theoretical and technical support for related researches of rubber tree monoterpenes and biosynthesis thereof.
In some embodiments, the amplification primer pairs for the rubber tree polyterpene product synthetase gene are as follows:
The forward primer P1 is:
5’- GGCCCGGGCGTCGACATGGCCCTTCAATTGTTTG-3’（SEQ ID NO.3）；
The reverse primer P2 is:
5’-CGGATCTTAGCTAGCTTAAATGGTTACAGATAGGCAG-3’（SEQ ID NO.4）。
The amplification primer pair provided by the invention can be used for rapidly, efficiently and specifically amplifying the rubber tree polyterpene product synthetase gene from the rubber tree genome.
The rubber tree-derived polymonoterpene product synthetase of the invention can take geranyl pyrophosphate as a substrate to catalyze and synthesize polymonoterpene products comprising alpha-pinene, beta-myrcene, D-limonene, gamma-terpinene, terpinolene and alpha-terpinol, and the product has faint scent and can be applied to the essence and perfume industry.
The invention also provides a recombinant vector containing the multi-monoterpene product synthetase gene derived from the rubber tree, the multi-monoterpene product synthetase gene of the rubber tree is recombined in an expression vector, a biosynthesis module is constructed, and the simple and rapid utilization of the multi-monoterpene product synthetase gene of the rubber tree can be realized, so that a large amount of target genes or target proteins can be obtained. In the present invention, the expression vector is preferably a pESC yeast expression vector.
The invention also provides engineering bacteria containing the rubber tree-derived multi-monoterpene product synthetase gene, wherein the engineering bacteria comprise the rubber tree-derived multi-monoterpene product synthetase gene and host cells.
The multi-monoterpene product synthetase genes which are derived from rubber trees and comprise alpha-pinene, beta-myrcene, D-limonene, gamma-terpinene, terpinolene and alpha-terpinol are introduced into host cells, a large amount of target genes and target protease can be obtained rapidly, and a biosynthesis module formed by engineering bacteria avoids the complicated operation of PCR amplification from rubber tree genome when the target genes are used. The host cell is preferably a yeast JCR27 strain.
The present invention also provides a plant cell or transgenic plant into which the above-described rubber tree-derived polyterpene product synthase gene is introduced. As a technical method well known to those skilled in the art, a plant expression vector is constructed by using the rubber tree gene, and a plant cell and callus are introduced by agrobacterium, protoplast, gene gun and the like or a transgenic plant is regenerated to synthesize and produce a polymonoterpene product including alpha-pinene, beta-myrcene, D-limonene, gamma-terpinolene, terpinolene and alpha-terpinol.
The invention also provides a production method of the polyterpene product synthetase derived from the rubber tree, which comprises the steps of culturing the engineering bacteria to obtain a culture, and separating from the culture to obtain the polyterpene product synthetase comprising alpha-pinene, beta-myrcene, D-limonene, gamma-terpinene, terpinolene and alpha-terpinol.
Alpha-pinene (CAS: 7785-70-8), beta-pinene (CAS: 18172-67-3), beta-myrcene (CAS: 123-35-3), D-limonene (CAS: 5989-27-5), gamma-terpinene (CAS: 99-85-4), terpinolene (CAS: 586-62-9).
The invention also provides application of the polyterpene product synthetase in preparing monoterpene compounds, wherein the monoterpene compounds comprise at least one of alpha-pinene, beta-myrcene, D-limonene, gamma-terpinene, terpinol and alpha-terpinol. When the polyterpene synthetase is applied to preparing one of alpha-pinene, beta-myrcene, D-limonene, gamma-terpinene, terpinolene and alpha-terpineol, the target substance is separated after catalyzing a substrate GPP (geranyl pyrophosphate) by using the polyterpene synthetase; or fermenting and culturing the recombinant strain constructed by the method, and separating out the target. When using a polymonoterpene product synthase for the simultaneous production of several of these substances, for example, alpha-pinene and beta-myrcene are produced; preparing gamma-terpinene and D-limonene; or preparing a mixture of beta-myrcene, terpinolene and alpha-terpineol, catalyzing a substrate by using the polymonoterpene synthase, and separating out target substances or removing impurities. Since the polymonoterpene synthases are one type of controlled multiproducts, terpene-based compounds that may be synthesized include alpha-pinene, beta-myrcene, D-limonene, gamma-terpinene, terpinolene, and alpha-terpinol, in some alternative embodiments, the polymonoterpene synthases may be used to synthesize a mixture of alpha-pinene, beta-myrcene, D-limonene, gamma-terpinolene, and alpha-terpinol.
The invention also provides application of the polyterpene synthetase in preparing a product taking the monoterpene compound as an active substance, wherein the monoterpene compound comprises at least one of alpha-pinene, beta-myrcene, D-limonene, gamma-terpinene, terpinolene and alpha-terpinol. The product using the monoterpene compounds as active substances can be selected from one of alpha-pinene, beta-myrcene, D-limonene, gamma-terpinene, terpinolene and alpha-terpinol as active ingredients, and can also be selected from several of alpha-pinene, beta-myrcene, D-limonene, gamma-terpinolene, terpinolene and alpha-terpinol as active ingredients, for example, alpha-pinene and beta-myrcene; gamma-terpinene and alpha-terpineol are used as active ingredients; since the polymonoterpene synthases are a type of synthase that controls multiple products, terpenes that can be synthesized include alpha-pinene, beta-myrcene, D-limonene, gamma-terpinene, terpinolene, and alpha-terpinol, in some alternative embodiments, the products have a mixture of alpha-pinene, beta-myrcene, D-limonene, gamma-terpinolene, and alpha-terpinol as the active ingredient.
The invention is further illustrated by the following specific examples, but it should be understood that these examples are for the purpose of illustration only and are not to be construed as limiting the invention in any way.
The biological reagent information used in the invention is as follows:
2x PrimerSTAR Max Permix DNA polymerase from Bao Ri doctor Material technology (Beijing) Co., ltd; restriction enzymes were purchased from new intel biotechnology (beijing) limited; the total plant RNA extraction kit is purchased from Tiangen Biochemical technology (Beijing) limited company; pTOPO-Blunt Simple vector was purchased from Beijing Edley Biotechnology Co., ltd; yeast expression plasmid pESC was purchased from Novagen company; the first strand cDNA reverse transcription kit, the DNA gel recovery kit, the plasmid extraction kit and the homologous recombination kit are all purchased from Nanjinopran biotechnology Co., ltd; the primer pair P1/P2 for cloning specific genes was synthesized by the company Shanghai Co., ltd.
Rubber tree cDNA: total RNA is extracted from the rubber tree latex of conventional rubber tapping and reverse transcribed to synthesize first strand cDNA, which is performed according to the reaction conditions suggested by the manufacturer, which are well known to those skilled in the art, and the present invention is not limited thereto.
YPDHG medium: the medium composition was 1% yeast extract, 2% tryptone, 1% glucose and 1% galactose.
Yeast strain JCR27, strain reference "Systematic identification ofOcimum sanctumsesquiterpenoid synthases and (-)-eremophilene overproduction in engineered yeast" Metab Eng. 2022,69:122-133. doi: 10.1016/j.ymben.2021.11.005.
The product detection method comprises the following steps: GC-MS detection was performed using Thermo TRACE GC Ultra system and TSQ 9000 system. The GC detection procedure was set as follows: the initial oven temperature was 50 ℃ for 1 minute; then raising the temperature to 280 ℃ at a speed of 15 ℃/min and keeping the temperature for 1 min; then, the temperature was raised to 300℃at a rate of 20℃per minute, and the mixture was kept for 2 minutes. The volatile samples were injected at 240℃and the MS transfer temperature was maintained at 270 ℃.
Example 1
(1) Gene cloning
The present inventors have found a rubber tree monoterpene synthase gene by analyzing the genomic sequence of rubber tree. The nucleotide sequence is shown as SEQ ID NO. 1.
The method comprises the steps of collecting a rubber gum emulsion tissue sample, extracting total RNA by using a plant total RNA extraction kit of Tiangen biochemical technology (Beijing) limited company according to a kit using instruction, and synthesizing a cDNA template by using a first-strand cDNA reverse transcription kit of Nanjinozan biological technology limited company. The HbMTS gene was cloned from the above cDNA template using a conventional PCR method using primer pairs P1 (SEQ ID NO. 3) and P2 (SEQ ID NO. 4). PCR reaction system: 2x PrimerSTAR Max Permix 10. Mu.L each of forward and reverse primers (10. Mu.M), 0.5. Mu.L of cDNA template, and ddH 2 O8. Mu.L. The PCR reaction procedure was: 94℃for 3 minutes, 98℃for 10 seconds, 54℃for 20 seedlings, 72℃for 2 minutes, 30 cycles, 72℃for 5 minutes. And (3) cutting out a gene fragment with the correct size after running DNA agarose electrophoresis, connecting pTOPO-Blunt Simple cloning vector after recovering by using a Norvezan DNA gel recovery kit, transforming DH5 alpha escherichia coli competence, coating on an ampicillin resistance plate for screening, and sending to a biological sequencing company for sequencing confirmation after the PCR verification of correctness.
(2) Expression vector construction
The HbMTS gene fragment containing the homology arm and the pESC yeast linearization vector after enzyme digestion react for 30 minutes at 37 ℃ through a Norvezan homologous recombination kit, TOP10 bacteria competence is transformed at low temperature, then ampicillin resistance plates are coated for screening, and yeast expression vector pHbMTS containing HbMTS gene is obtained after PCR re-verification.
(3) Construction and function evaluation of engineering bacteria
① Yeast competent preparation and transformation
The JCR27 strain cultured overnight is transferred into 50mL fresh YPD culture medium, shake-cultured at 30 ℃ until OD 600 is about 0.6-0.8, collected after centrifugation for 5 minutes at 500 and g, resuspended in 10-20 mL LiAC solution, collected after centrifugation for 5 minutes at 500 and g, added with 1mL LiAC to be resuspended to obtain the competence. Transferring the recombinant expression vector pHbMTS to JCR27 yeast competence through LiAC/PEG solution, standing at 30 ℃ for 30 minutes, carrying out heat shock at 42 ℃ for about 10 minutes, centrifuging at 500 g for 5 minutes, coating on a corresponding screening plate, standing at 30 ℃ for 3d, carrying out colony PCR verification by using a primer pair P1/P2, and naming positive bacteria as YHbMTS1 respectively.
② Functional evaluation
Inoculating YHbMTS-10 mL SD engineering bacteria into a defect culture medium for culturing overnight, transferring to a YPDHG culture medium, adding 0.5-1% isopropyl myristate IMP, culturing at 30 ℃ for 3d, centrifuging at 4000 rpm for 5-10min, collecting IMP, and preparing a test sample for GC-MS detection. As shown in fig. 1, the result of the detection of YZHbMTS a fermentation sample is that the sample is matched with the retention time and mass spectrum peak of the existing standard substance one by one, at least 7 product peaks are identified, and are respectively alpha-pinene (RT 1=4.74 min, beta-pinene (RT 2=5.46 min), beta-myrcene (RT 3=5.71 min), D-limonene (RT 4=6.40 min), gamma-terpinene (RT 5=6.93 min), terpinolene (RT 6=7.49 min), and alpha-terpinol (RT 7=9.92 min), thus HbMTS is a multiple monoterpene product synthetase, which can synthesize multiple monoterpene products including alpha-pinene, beta-myrcene, D-limonene, gamma-terpinene, terpinene and alpha-terpinol by using GPP, wherein alpha-pinene accounts for about 33%, D-limonene accounts for about 21%, gamma-terpinene accounts for about 23%, which is an important byproduct of the new application, and the new product can not be synthesized by using the new product.
(4) Antibacterial Activity test
And adding the YHbMTS engineering bacteria fermentation product with the working concentration of 100 ppm into a 5ml PDA culture medium to prepare the PDA plate containing the fermentation filtrate. The center of the mixing plate is inoculated with a cake of fusarium oxysporum to be tested having a diameter of 5mm. The PDA is used as a reference, the culture dish is sealed by a sealing film, and then is placed in a 28 ℃ incubator for culturing about 5 d in a reverse manner, and the growth condition of pathogenic bacteria is observed and recorded. The results are shown in figure 2, and the growth diameter of Fusarium oxysporum (sp.) in the treated group containing 100 ppm active polymonoterpene products is significantly smaller than that of the control group. The bacteriostasis experiment obviously shows that the fermentation product containing HbMTS functional enzyme derived from rubber tree can inhibit the growth of Fusarium oxysporum (sp.) of banana Fusarium wilt pathogenic bacteria, and the active product can be used for developing and utilizing biochemical control agents of the pathogenic bacteria.
The embodiments described above and features of the embodiments herein may be combined with each other without conflict.
The foregoing description of the preferred embodiments of the invention is not intended to limit the invention to the precise form disclosed, and any such modifications, equivalents, and alternatives falling within the spirit and scope of the invention are intended to be included within the scope of the invention.

Claims (10)

1. A polyterpene product synthetase gene is characterized in that the nucleotide sequence of the polyterpene synthetase gene is shown as SEQ ID NO. 1.
2. The amplification primer pair of the polyterpene product synthetase gene according to claim 1, wherein the amplification primer pair is: the nucleotide sequence of the forward primer is shown as SEQ ID NO.3, and the nucleotide sequence of the reverse primer is shown as SEQ ID NO. 4.
3. The multi-monoterpene product synthetase is characterized in that the amino acid sequence of the multi-monoterpene product synthetase is shown as SEQ ID NO. 2.
4. The use of the polyterpene product synthetase gene according to claim 1 or the polyterpene product synthetase according to claim 3 for preparing monoterpene compounds, wherein the monoterpene compounds comprise at least one of alpha-pinene, beta-myrcene, D-limonene, gamma-terpinene, terpinolene and alpha-terpinol.
5. A recombinant expression vector comprising the gene of claim 1.
6. A recombinant expression strain comprising the recombinant expression vector of claim 5.
7. A method for producing monoterpene compounds, characterized in that the recombinant expression strain of claim 6 is cultivated, the monoterpene compounds are obtained from the culture, and at least one of alpha-pinene, beta-myrcene, D-limonene, gamma-terpinene, terpinolene and alpha-terpinol is obtained through separation and purification.
8. Use of the polyterpene product synthetase gene as claimed in claim 1 or the polyterpene product synthetase as claimed in claim 3 for preparing a product with monoterpene compounds as active substances, wherein the monoterpene compounds comprise at least one of alpha-pinene, beta-myrcene, D-limonene, gamma-terpinene, terpinolene and alpha-terpinol.
9. The use according to claim 8, wherein the product comprises a fusarium oxysporum inhibitor of banana vascular wilt pathogen.
10. A plant cell or transgenic plant into which the polyterpene product synthetase gene of claim 1 has been introduced.
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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101896607A (en) * 2007-09-11 2010-11-24 蓝宝石能源公司 Produce molecule by photosynthetic organism
JP2018164464A (en) * 2018-08-02 2018-10-25 株式会社カロテノイド生産技術研究所 Terpene synthase gene, acetoacetic ester hydrolase gene, and manufacturing method of terpene
CN110951788A (en) * 2019-12-09 2020-04-03 中国林业科学研究院亚热带林业研究所 Application of masson pine α -pinene synthetase in preparation of terpene compounds and products containing terpene compounds
CN114350713A (en) * 2021-12-30 2022-04-15 中国林业科学研究院亚热带林业研究所 Application of camptothecine synthetase in preparation of terpene compounds and products containing terpene compounds
CN115725560A (en) * 2022-10-28 2023-03-03 广东省林业科学研究院 Pinus massoniana multifunctional terpene synthetase mutant and application thereof in sesquiterpene product production

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101896607A (en) * 2007-09-11 2010-11-24 蓝宝石能源公司 Produce molecule by photosynthetic organism
JP2018164464A (en) * 2018-08-02 2018-10-25 株式会社カロテノイド生産技術研究所 Terpene synthase gene, acetoacetic ester hydrolase gene, and manufacturing method of terpene
CN110951788A (en) * 2019-12-09 2020-04-03 中国林业科学研究院亚热带林业研究所 Application of masson pine α -pinene synthetase in preparation of terpene compounds and products containing terpene compounds
CN114350713A (en) * 2021-12-30 2022-04-15 中国林业科学研究院亚热带林业研究所 Application of camptothecine synthetase in preparation of terpene compounds and products containing terpene compounds
CN115725560A (en) * 2022-10-28 2023-03-03 广东省林业科学研究院 Pinus massoniana multifunctional terpene synthetase mutant and application thereof in sesquiterpene product production

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
GENBANK: "terpene synthase 10-like[Hevea brasiliensis]", pages 1, Retrieved from the Internet <URL:https://www.ncbi.nlm.nih.gov/protein/XP_021684617.1?report=genpept> *

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