Pinus massoniana alcohol dehydrogenase gene, product and application thereof, and pinus massoniana alcohol dehydrogenase and application thereof

Abstract

The invention provides a masson pine alcohol dehydrogenase gene, a product and application thereof, masson pine alcohol dehydrogenase and application thereof, and relates to the technical field of biology. The pinus massoniana alcohol dehydrogenase gene is cloned from pinus massoniana for the first time, the nucleotide sequence and the amino acid sequence of the pinus massoniana alcohol dehydrogenase gene are determined, the blank that in the prior art, terpene synthase in pinus massoniana and biosynthetic genes of the terpene synthase are unknown is filled up, and theoretical and basic support is provided for related research of pinus massoniana. Besides, the masson pine alcohol dehydrogenase gene is recombined to obtain a recombinant vector and an engineering bacterium, the masson pine alcohol dehydrogenase is successfully purified to obtain the masson pine alcohol dehydrogenase, the masson pine alcohol dehydrogenase can catalyze cis-3-hexenal to synthesize cis-3-hexenol in an in-vitro enzyme activity experiment, and the masson pine alcohol dehydrogenase has important significance on prevention of phytophagous diseases and insect pests.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a pine alcohol dehydrogenase gene, its products and applications, and the pine alcohol dehydrogenase and its applications. Background Technology

[0002] Masson pine ( Pinus massoniana The existing pine forest area is approximately 8.04 million hectares. 2 It ranks second among coniferous tree species, producing 500,000-700,000 tons of resin annually, accounting for 70% of the total resin production. It holds a fundamental and strategic position in the construction of the "four reservoirs" of forest resources. However, as one of the highly susceptible hosts of pine wilt disease, Masson pine severely hinders the development of the Masson pine industry.

[0003] Plant volatiles (HIPVs), such as monoterpenes, sesquiterpenes, and green leaf volatiles, released when plants are harmed by herbivores are crucial to plant defense mechanisms. Cis-3-hexenol is an important green leaf volatile, and its dual nature—attracting, repelling, and toxic—directly influences the physiology and behavior of herbivores. However, the biosynthetic gene for cis-3-hexenol synthase in *Pinus massoniana* has not yet been isolated and identified.

[0004] In view of this, the present invention is hereby proposed. Summary of the Invention

[0005] The primary objective of this invention is to provide a pine alcohol dehydrogenase gene to fill the gap in existing technology regarding the unknown terpene synthases and their biosynthetic genes in pine.

[0006] A second objective of this invention is to provide a recombinant carrier.

[0007] A third objective of this invention is to provide a cell.

[0008] A fourth objective of this invention is to provide the above-mentioned pine alcohol dehydrogenase gene, or vector, or cell for use in the preparation of pine alcohol dehydrogenase or the production of cis-3-hexenol.

[0009] The fifth objective of this invention is to provide a primer pair for amplifying the above-mentioned pine alcohol dehydrogenase gene, which can rapidly and accurately obtain the pine alcohol dehydrogenase gene.

[0010] The sixth objective of this invention is to provide a pine alcohol dehydrogenase to fill the gap in the prior art that lacks a pine alcohol dehydrogenase.

[0011] The seventh objective of this invention is to provide the application of the above-mentioned pine alcohol dehydrogenase in catalyzing the production of cis-3-hexenal from cis-3-hexenol.

[0012] The eighth objective of this invention is to provide a method for producing cis-3-hexenol, thereby filling the gap in the prior art where there is no method for the industrial production of cis-3-hexenol using biotechnology.

[0013] To achieve the above objectives, the following technical solution is adopted: In a first aspect, the present invention provides a pine alcohol dehydrogenase gene, the nucleic acid sequence of which is shown in SEQ ID NO.1 or SEQ ID NO.2.

[0014] Secondly, the present invention provides a recombinant vector comprising the aforementioned pine alcohol dehydrogenase gene and vector.

[0015] As a further technical solution, the vector includes an expression vector or a cloning vector; The expression vector includes pET-32a; The cloning vector includes pCone007.

[0016] Thirdly, the present invention provides a cell that carries the aforementioned pine alcohol dehydrogenase gene or contains the aforementioned vector.

[0017] As a further technical solution, the cells include Escherichia coli DH5α or Escherichia coli BL21.

[0018] Fourthly, the present invention provides the application of the above-mentioned pine alcohol dehydrogenase gene, vector, or cell in the preparation of pine alcohol dehydrogenase or the production of cis-3-hexenol.

[0019] Fifthly, the present invention provides primer pairs for the amplification of the above-mentioned pine alcohol dehydrogenase gene, wherein the sequences of the primer pairs are shown in SEQ ID NO.5 and SEQ ID NO.6, or the sequences of the primer pairs are shown in SEQ ID NO.7 and SEQ ID NO.8.

[0020] In a sixth aspect, the present invention provides a pine alcohol dehydrogenase, the amino acid sequence of which is shown in SEQ ID NO.3 or SEQ ID NO.4.

[0021] In a seventh aspect, the present invention provides the application of the above-mentioned pine alcohol dehydrogenase in catalyzing the production of cis-3-hexenal from cis-3-hexenol.

[0022] Eighthly, the present invention provides a method for producing cis-3-hexenol, wherein the pine alcohol dehydrogenase described above is used to catalyze cis-3-hexenal to obtain cis-3-hexenol.

[0023] Compared with the prior art, the present invention has the following beneficial effects: This invention marks the first time that the pine alcohol dehydrogenase gene has been cloned from *Pinus massoniana*, and its nucleotide and amino acid sequences have been determined. This fills the gap in existing technologies regarding the unknown terpene synthases and their biosynthetic genes in *Pinus massoniana*, providing theoretical and fundamental support for related research. Furthermore, the inventors successfully purified the pine alcohol dehydrogenase by recombining the gene into a recombinant vector and engineered bacteria. In in vitro enzyme activity experiments, this enzyme catalyzes the synthesis of cis-3-hexenol from cis-3-hexenal, which is of significant importance for the defense against phytophagous pests and diseases. Attached Figure Description

[0024] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0025] Figure 1 The results of protein purification from Masson pine ADH11 and ADH24; Figure 2 The peak elution time of cis-3-hexenol from *Pinus massoniana*. Figure 3 The peaks are characteristic of the cis-3-hexenol ion in Pinus massoniana (the top image shows the cis-3-hexenol ion characteristic peaks matched to the NIST08 standard library; the bottom image shows the cis-3-hexenol ion characteristic peaks in Pinus massoniana). Detailed Implementation

[0026] The embodiments and examples of the present invention will be described in detail below. However, those skilled in the art will understand that the following embodiments and examples are for illustrative purposes only and should not be considered as limiting the scope of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention. Unless otherwise specified, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0027] In a first aspect, the present invention provides a pine alcohol dehydrogenase gene, the nucleic acid sequence of which is shown in SEQ ID NO.1 or SEQ ID NO.2.

[0028] The nucleic acid sequence of the pine alcohol dehydrogenase gene ADH11 is as follows:

[0029] The nucleic acid sequence of the pine alcohol dehydrogenase gene ADH24 is as follows:

[0030] This invention is the first to clone the pine alcohol dehydrogenase gene (also referred to as "ADH11 and ADH24" in this invention) from Masson pine, and determine its nucleotide and amino acid sequences, filling the gap in the prior art regarding the unknown terpene synthase and its biosynthetic genes in Masson pine, and providing theoretical and basic support for related research on Masson pine.

[0031] Secondly, the present invention provides a recombinant vector comprising the aforementioned pine alcohol dehydrogenase gene and vector.

[0032] This recombinant vector can be used for the amplification and delivery of the pine alcohol dehydrogenase gene.

[0033] In some alternative implementations, the vector includes an expression vector or a cloning vector; The expression vectors include, but are not limited to, pET-32a; The cloning vector includes, but is not limited to, pCone007.

[0034] The vector of the present invention may include an expression vector or a cloning vector. The pine alcohol dehydrogenase gene is recombined in the expression vector or cloning vector to construct a biological module, which can realize the simple and rapid use of the pine alcohol dehydrogenase gene and quickly obtain the target gene or target protein in large quantities.

[0035] Thirdly, the present invention provides a cell that carries the aforementioned pine alcohol dehydrogenase gene or contains the aforementioned vector.

[0036] By placing the Masson pine alcohol dehydrogenase gene into host cells, the target gene and enzyme can be obtained rapidly and in large quantities. The biological module composed of engineered bacteria avoids the cumbersome operation of PCR amplification from the Masson pine genome when using the target gene.

[0037] In some alternative embodiments, the cells include, but are not limited to, Escherichia coli DH5α or Escherichia coli BL21, or other cells well known to those skilled in the art.

[0038] Fourthly, the present invention provides the application of the above-mentioned pine alcohol dehydrogenase gene, vector, or cell in the preparation of pine alcohol dehydrogenase or the production of cis-3-hexenol.

[0039] The pine alcohol dehydrogenase gene, vector, or cell provided by this invention can be used to prepare pine alcohol dehydrogenase, and then the prepared pine alcohol dehydrogenase can be used to catalyze cis-3-hexenal to achieve the preparation of cis-3-hexenol.

[0040] Fifthly, the present invention provides primer pairs for the amplification of the above-mentioned pine alcohol dehydrogenase gene, the sequences of which are shown in SEQ ID NO.5 and SEQ ID NO.6: The forward primer is: 5'-ATGGCGATCTCTACTGCAGGCC-3' (SEQ ID NO.5); The reverse primer is: 3'-TTAATTACCAGTCATGTTAATC-5' (SEQ ID NO.6); Alternatively, the sequences of the primer pairs may be as shown in SEQ ID NO.7 and SEQ ID NO.8: The forward primer is: 5'-ATGGCGAGCTCTACTGCAGGCC-3' (SEQ ID NO.7) The reverse primer is: 3'-TTAATTACCAATCATGTTAATC-5' (SEQ ID NO.8).

[0041] The amplification primer pair provided by this invention can rapidly and accurately amplify the pine alcohol dehydrogenase gene from the pine genome, with good specificity and high efficiency.

[0042] Sixthly, the present invention provides a pine alcohol dehydrogenase, the amino acid sequence of which is shown in SEQ ID NO.3 or SEQ ID NO.4: The amino acid sequence of Pinus massoniana alcohol dehydrogenase ADH11 is as follows: MAISTAGQVIKCKAAVAWGAGEPLKIEEVEVAPPQAMEVRIKVHYTALCHTDLYFWEAKGQTPLFPRILGHEAAGVVESVGEGVTDLKEGDSVLPVFTGECGECRHCKSEDSNMCDLLRINTDRGVMINDGKSRFSINGKPIYHFLGTSTFSEYTVVHVGCVAKINPEAPLNKVCILSCGVSTGMGATLNVAK PKKGSSVAIFGLGGVGLAAAEGARIAGASRIIGIDLNSDRFEKAKLFGVTEFINPLDHQKPIQQVIAEKTDGGVDYSIECTGNVKAMIQAFESCHDGWGVAVLVGVPHSDAVFATSPVNFLNERTLKGTFFGNYKPRSDLPGLVELYLDKKLELEKFITHEVSFADINKAFDYMIKGESLRCVINMTGN (SEQ ID NO.3).

[0043] The amino acid sequence of pine alcohol dehydrogenase ADH24 is as follows: MASSTAGQVIKCKAAVAWGAGEPLKIEEVEVAPPQAMEVRIKVHYTALCHTDLYFWEAKGQTPLFPRILGHEAAGVVESVGEGVTDLKEGDSVLPVFTGECGECRHCKSEESNMCDLLRINTDRGVMVNDGKSRFSINGKPIYHFLGTSTFSEYTVVHVGCVAKINPEAPLNKVCILSCGVSTGMGATLNVAK PKKGSSVAIFGLGGVGLAAAEGARIAGASRIIGIDLNSDRFEKAKLFGVTEFINPLDHQKPIQQVISEKTDGGVDYSIECTGNVKAMIQAFESCHDGWGVAVLVGVPHSDAVFATSPVNFLNERTLKGTFFGNYKPRSDLPGLVELYLDKKLELEKFITHEVSFADINKAFDYMIKGESLRCVINMIGN (SEQ ID NO.4) In a seventh aspect, the present invention provides the application of the above-mentioned pine alcohol dehydrogenase in catalyzing the production of cis-3-hexenal from cis-3-hexenol.

[0044] The pine alcohol dehydrogenase of the present invention can catalyze the synthesis of cis-3-hexenol using cis-3-hexenal as a substrate. Cis-3-hexenol is of great significance for the defense against phytophagous diseases and pests.

[0045] Eighthly, the present invention provides a method for producing cis-3-hexenol, wherein the pine alcohol dehydrogenase described above is used to catalyze cis-3-hexenal to obtain cis-3-hexenol.

[0046] The production method provided by this invention is simple and convenient, and can be used for the mass production of cis-3-hexenol.

[0047] In some alternative implementations, the production method includes the following steps: A solution containing cis-3-hexenal was prepared, and then pine alcohol dehydrogenase was added. The mixture was reacted at 30°C for 1 hour to prepare cis-3-hexenol. The solution containing cis-3-hexenal contains: Tris-HCl, 50 mM, pH 7.5; DTT, 1 mM; NADPH, 0.1 mM; and cis-3-hexenal, 0.1 mM.

[0048] The present invention will be further illustrated below with specific embodiments. However, it should be understood that these embodiments are merely for the purpose of more detailed illustration and should not be construed as limiting the present invention in any way.

[0049] Example 1: Cloning the pine alcohol dehydrogenase gene, constructing a cloning vector and transforming it into prokaryotic cells. RNA was extracted from the stem tissue of *Pinus massoniana*, and cDNA was synthesized using reverse transcriptase M-MLV. The following primers were used as a template for amplification of this cDNA: The forward primer is: 5'-ATGGCGATCTCTACTGCAGGCC-3' (SEQ ID NO.5); The reverse primer is: 3'-TTAATTACCAGTCATGTTAATC-5' (SEQ ID NO.6); The forward primer is: 5'-ATGGCGAGCTCTACTGCAGGCC-3' (SEQ ID NO.7) The reverse primer is: 3'-TTAATTACCAATCATGTTAATC-5' (SEQ ID NO.8).

[0050] PCR amplification was performed using Phanta Max Super-Fidelity DNA polymerase from Vazyme.

[0051] PCR conditions were: 95℃ for 3 min; 95℃ for 15 sec; 55℃ for 15 sec; 72℃ for 2 min; 35 cycles; extension at 72℃ for 5 min.

[0052] The PCR products were detected by 1% agarose gel electrophoresis. The fragment size of the target gene, the pine alcohol dehydrogenase gene, was approximately 1149 bp, which was in line with expectations.

[0053] The target gene fragment was recovered using an agarose gel electrophoresis recovery kit, the target fragment was TA cloned, ligated into the pClone007 vector, and then transformed into Escherichia coli DH5α clone strain.

[0054] The transformation conditions were as follows: 5 μl of ligation product was added to 50 μl of competent cells, gently mixed, and incubated on ice for 25 min. The cells were then subjected to heat shock in a 42°C water bath for 45 s, followed by an immediate ice bath and incubation for 2 min. 500 μl of antibiotic-free LB medium was added, mixed, and the cells were incubated at 37°C and 200 rpm for 1 h. The bacterial culture was centrifuged at 3000 rpm for 1 min, 400 μl of supernatant was discarded, the bacterial culture was resuspended, and spread onto solid LB plates containing antibiotics (Amp). The cells were then incubated upside down at 37°C for 12-16 h.

[0055] Positive clones were screened using colony PCR. The screening method was as follows: a single colony was randomly picked from the transformation plate and cultured in liquid culture medium in a 1.5 ml centrifuge tube. Each tube was numbered, and 1 μl of each tube was used as a template for PCR detection. The remaining culture was stored at 4°C. Positive colonies were stored on plates or in glycerol tubes for later use.

[0056] Sequencing revealed the successful cloning of two pine alcohol dehydrogenase genes, with nucleotide sequences shown in SEQ ID NO.1 and SEQ ID NO.2 (named ADH11 and ADH24, respectively). Each gene contains 1149 bases and encodes 382 amino acids. A recombinant vector containing the pine alcohol dehydrogenase gene inserted into the pClone007 cloning vector was also obtained, along with a positive-positive engineered bacterium successfully transformed into prokaryotic Escherichia coli DH5α.

[0057] Example 2: Construction of an expression vector for the alcohol dehydrogenase gene and its transformation into prokaryotic cells The target fragment cloned in Example 1 was transformed into the pET32a linearized vector through homologous recombination.

[0058] The ligation reaction conditions were as follows: After mixing the reaction system, incubate at 37°C for 30 min, then at 20°C for 1 h. Add 5 μl of the reaction solution to 50 μl of E. coli DH5α competent cells, mix well, and incubate on ice for 30 min. Gently remove the cells, heat shock at 42°C for 60 s, immediately incubate on ice for 2 min, add 500 μl of LB medium, and incubate at 37°C for 1 h. Spread 100 μl of the bacterial culture onto LB plates containing Kan resistance and incubate overnight.

[0059] Positive colonies selected from antibiotic (Kan) screening were used to extract plasmids. The prokaryotic expression vector was transformed into *Escherichia coli* strain BL21 and cultured at 37°C and 200 rpm until OD500 was reached. 600 The concentration was 0.6. 0.1 mM IPTG was added to the culture medium in the test tubes, and then the tubes were incubated at 15℃ and 37℃ to induce expression, resulting in a large number of bacterial cultures expressing pine alcohol dehydrogenase.

[0060] Example 3: Prokaryotic expression, protein purification, and protein detection of Pinus massoniana alcohol dehydrogenase Select single colonies containing recombinant plasmids from Example 2 and incubate them in 3 mL LB liquid medium (ampicillin resistant) at 37°C overnight, then preserve them at -20°C.

[0061] Select single colonies containing the recombinant plasmid and add them to 3 mL of LB liquid medium (ampicillin resistant), then incubate at 37°C with shaking until OD reaches 1.5. 600Approximately 0.6 μg / mL was used as a control group. The remaining bacterial culture was incubated with IPTG inducer (final concentration 1 mM) at 37°C with shaking for 3 h. 0.15 mL of each culture was centrifuged at 12000×g for 2 min. The bacterial pellet was resuspended in 40 μL of 1× loading buffer for lysis, and analyzed by SDS-PAGE. Results are as follows: Figure 1 As shown, lane 1: P-broken precipitate; lane 2: Pre-column effluent; lane 3: Aft effluent protein; lane 4: 5% washed protein; lane 5: 30% eluted protein; lane 6: 30% eluted protein; lane 7: marker (kDd); lane 8: 100% eluted protein; lane 9: 100% eluted protein; lane 10: 30% eluted protein; lane 11: 30% eluted protein; lane 12: 5% washed protein; lane 13: Aft effluent protein; lane 14: Pre-column effluent; lane 15: P-broken precipitate.

[0062] Take 100 μL of the bacterial culture stored at -20℃ and inoculate it into 100 mL of LB liquid medium (ampicillin resistant) and culture overnight with shaking; take 100 mL of the bacterial culture and inoculate it into 2000 mL of LB liquid medium, and culture at 37℃ until OD reaches the target value. 600 Approximately 0.6, lower the culture temperature to 30℃; add IPTG inducer to a final concentration of 0.5 mM, and continue to culture at 30℃ with shaking for 3 h; centrifuge at 8000 rpm for 3 min to collect the cells, resuspend in 50 mL of pre-cooled NTA-0 buffer, and incubate on ice for 30 min.

[0063] The bacterial cells were disrupted by ultrasonication with the following parameters: power 200 W, operation time 3 s, pause time 4 s, 99 cycles; centrifuged at 16000 rpm at 4℃ for 50 min, and the supernatant and precipitate were collected; a small amount of supernatant and precipitate were taken for SDS-PAGE analysis, and the remaining supernatant and precipitate were stored at 4℃ for later use.

[0064] The supernatant protein solution was filtered through a 0.22 µm filter and set aside. A Ni-NTA column was prepared. The supernatant protein solution was loaded onto the column at a flow rate of 1 ml / min. The column was washed with NTA-0 buffer (pH 8.0) until the eluent was free of protein (G250 detection solution did not change color). Elution was performed with 20 mM, 60 mM, 200 mM, and 500 mM imidazole, and the eluent was collected in fractions until the G250 detection solution did not change color. The column stock was washed with 3 column volumes of deionized water, and the column was sealed with 20% ethanol. The collected eluent was analyzed by SDS-PAGE electrophoresis, and the pine alcohol dehydrogenase was detected by Western spectroscopy. The target protein was 58 kDa, the tag was 15 kDa, and the fusion protein was approximately 73 kDa.

[0065] Example 4 Biochemical function of Pinus massoniana alcohol dehydrogenase Using cis-3-hexenal as a substrate, the enzymatic reaction system was as follows: Tris-HCl, 50 mM, pH 7.5; DTT, 1 mM; NADPH, 0.1 mM; cis-3-hexenal, 0.1 mM; 50 ng of purified pine alcohol dehydrogenase was added and the reaction was carried out at 30℃ for 1 h.

[0066] After the reaction, volatile substances were extracted by headspace extraction using a SPME fiber PDMS 100μm filter for 30 min at 40℃ for 30 min. The catalytic products were then detected by GC-MS (Ag11entGC-MS789OB-5977A).

[0067] Detection methods GC-MS conditions: Column: DB-5MS (60m × 0.25mm ID × 0.25μm film thickness). Temperature program parameters: Initial temperature 50℃, hold for 2 min, increase to 80℃ at 3℃ / min, hold for 2 min, increase to 180℃ at 5℃ / min, hold for 1 min, increase to 230℃ at 10℃ / min, hold for 5 min, and finally increase to 250℃ at 20℃ / min, hold for 3 min. Injector temperature: 220℃. Splitless pulse injection, 1μL injection, carrier gas: high-purity helium (99.999%), column flow rate: 1.5mL / min. GC-MS interface temperature: 250℃. Ion source temperature: 230℃. Ionization mode: EI. Electron energy: 70eV. Scan mass range: 50-500m / z.

[0068] Qualitative analysis: Each component was searched and matched using the NIST08 standard spectral library, fragments were compared, and qualitative analysis was performed in conjunction with relevant literature reports and the relative retention times of each component.

[0069] The detection results of cis-3-hexenol in the catalyst products of ADH11 and ADH24 are as follows: Figure 2 As shown, the elution time of cis-3-hexenol was 10.848 min.

[0070] Figure 3 The results show the detection of the characteristic peak of cis-3-hexenol ion in Pinus massoniana; and the comparison results of the characteristic peak of cis-3-hexenol ion with the NIST08 standard spectral library.

[0071] The results showed that the pine alcohol dehydrogenases (ADH11 and ADH24) in this embodiment successfully catalyzed the synthesis of cis-3-hexenol using cis-3-hexenal as a substrate.

[0072] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A gene for a taxol dehydrogenase, characterized in that, The nucleic acid sequence of the pinometreol dehydrogenase gene is shown as SEQ ID NO. 1 or SEQ ID NO.

2.

2. A recombinant vector, characterized in that, The recombinant vector comprises the pinometreol dehydrogenase gene of claim 1 and a vector.

3. The recombinant vector of claim 2, wherein, The vector comprises an expression vector or a cloning vector; The expression vector comprises pET-32a; The cloning vector comprises pCone007.

4. A cell, characterized in that, The cell carries the pinometreol dehydrogenase gene of claim 1 or contains the vector of claim 2 or 3.

5. The cell of claim 4, wherein, The cell comprises Escherichia coli DH5α or Escherichia coli BL21.

6. Use of the pinometreol dehydrogenase gene of claim 1 or the vector of claim 2 or 3 or the cell of claim 4 or 5 in the preparation of pinometreol dehydrogenase or the production of cis-3-hexenol.

7. A primer pair for amplifying the gene of the pullulanase of claim 1, characterized by, The sequences of the primer pair are shown as SEQ ID NO. 5 and SEQ ID NO. 6 or the sequences of the primer pair are shown as SEQ ID NO. 7 and SEQ ID NO.

8.

8. A taxifolin dehydrogenase, characterized in that, The amino acid sequence of the pinometreol dehydrogenase is shown as SEQ ID NO. 3 or SEQ ID NO.

4.

9. Use of the pinometreol dehydrogenase of claim 8 in catalyzing cis-3-hexenal to generate cis-3-hexenol.

10. A method for producing cis-3-hexenol, characterized by, Cis-3-hexenol is obtained by using the pinometreol dehydrogenase of claim 8 to catalyze cis-3-hexenal.

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