Proteins and methods for synthesizing insect pheromone precursor compounds

Abstract

The present application provides a protein and a method for synthesizing an insect sex pheromone precursor compound. The present application finds that co-expressing a protein with a hexadecanoic fatty acid specific thioesterase in plants, which amino acid sequence is shown in SEQ ID No. 2, can synthesize 10-hexadecenoic acid. The process of synthesizing 10-hexadecenoic acid by plants is green and pollution-free, has low environmental pollution degree due to simple and convenient purification, and has high purity of extraction, which changes the path of relying on chemical synthesis, solves the disadvantages of complex chemical synthesis, large amount of by-products, environmental pollution, high cost and low purity of final product, etc.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of enzymology, in particular to a protein and a method for synthesizing a precursor compound of a moth sex pheromone. BACKGROUND

[0002] As an important chemical signal substance in the process of mating behavior, insect sex pheromones are mainly synthesized and released by female moths. Sex pheromones can not only attract male moths at a long distance, but also regulate the courtship behavior of male moths at a short distance. The pest behavior control agents developed based on insect sex pheromones can not only effectively monitor, disorient and trap target pests, but also eliminate negative impacts on non-target organisms and the environment.

[0003] However, at present, insect sex pheromones mainly rely on chemical synthesis, and the high cost of raw materials and harsh reaction conditions make the price of artificially synthesized sex pheromone products very expensive. In addition, the reaction technology of some catalysts is not mature enough, resulting in a large number of by-products, which leads to low product purity, not only greatly reduces the effect of pest control, but also secondarily pollutes the environment. Therefore, green and efficient synthesis of insect sex pheromones is one of the most effective strategies for preventing and controlling agricultural pests.

[0004] E / Z 10-hexadecenoic acid (E / Z 10-16:COOH) is a precursor of insect sex pheromone E / Z 10-hexadecenal (E / Z 10-16:Ald). After synthesizing E / Z 10-hexadecenoic acid (E / Z 10-16:COOH), only known reduction and oxidation reactions are needed to generate E / Z 10-hexadecenal. At present, E / Z 10-hexadecenoic acid mainly relies on chemical synthesis, resulting in the generation of by-products in the synthesis process, environmental pollution, high cost and low purity of the final product.

[0005] Therefore, it is urgent to develop a synthesis method of E / Z 10-hexadecenoic acid which can greatly solve the problems of environmental pollution, high cost and low purity in the chemical synthesis process. SUMMARY

[0006] One of the present application provides a CpunFADV protein, and the amino acid sequence thereof is shown as SEQ ID No. 2.

[0007] The second of the present application provides a nucleic acid encoding the CpunFADV protein as described in one of the present application.

[0008] In one specific embodiment, the base sequence of the nucleic acid is shown as SEQ ID No. 1.

[0009] The third invention provides the use of the CpunFADV protein according to the first invention or the nucleic acid according to the second invention in the biosynthesis of 10-hexadecenoic acid.

[0010] In one specific embodiment, the 10-hexadecenoic acid is cis-10-hexadecenoic acid and / or trans-10-hexadecenoic acid.

[0011] In one specific embodiment, the CpunFADV protein is co-expressed in plants with a hexadecanoic acid-specific thioesterase for the synthesis of 10-hexadecenoic acid; or the CpunFADV protein is co-expressed in plants with a hexadecanoic acid-specific thioesterase and a gene silencing repressor for the synthesis of 10-hexadecenoic acid; wherein the amino acid sequence of the hexadecanoic acid-specific thioesterase is registered at AGG79283.1, and the amino acid sequence of the gene silencing repressor is shown in SEQ ID No. 4.

[0012] In one specific embodiment, the accession number of the nucleic acid encoding the hexadecimal fatty acid-specific thioesterase is KC675176.1, and the nucleic acid encoding the gene silencing repressor is shown in SEQ ID No. 3.

[0013] In one specific embodiment, the plant is tobacco and / or flax.

[0014] The fourth invention provides a method for synthesizing 10-hexadecenoic acid, comprising expressing the CpunFADV protein according to one invention and a hexadecanoic acid-specific thioesterase in plants to synthesize the 10-hexadecenoic acid; or expressing the CpunFADV protein according to one invention, a hexadecanoic acid-specific thioesterase, and a gene silencing repressor in plants to synthesize the 10-hexadecenoic acid; wherein the amino acid sequence of the hexadecanoic acid-specific thioesterase is registered as AGG79283.1, and the amino acid sequence of the gene silencing repressor is shown in SEQ ID No. 4.

[0015] In one specific embodiment, the accession number of the nucleic acid encoding the hexadecimal fatty acid-specific thioesterase is KC675176.1, and the nucleic acid encoding the gene silencing repressor is shown in SEQ ID No. 3.

[0016] In one specific embodiment, the 10-hexadecenoic acid is cis-10-hexadecenoic acid and / or trans-10-hexadecenoic acid.

[0017] In one specific embodiment, the plant is tobacco and / or flax.

[0018] In one specific implementation, the promoter for initiating the transcription of the CpunFADV gene in *Capsella bursa-pastoris* is the pOleosin promoter.

[0019] In one specific implementation, the nucleic acid sequence of the pOleosin promoter is shown in SEQ ID No. 5.

[0020] In one specific embodiment, the 10-hexadecenoic acid is primarily found in tobacco leaves.

[0021] In one specific embodiment, the 10-hexadecenoic acid is mainly found in the seeds of *Capsella bursa-pastoris*.

[0022] The beneficial effects of this invention are as follows: This invention discovers that 10-hexadecenoic acid can be synthesized by co-expressing a protein with the amino acid sequence shown in SEQ ID No. 2, along with a hexadecanoic acid-specific thioesterase and a gene silencing repressor in plants. The process of synthesizing 10-hexadecenoic acid using plants is green and pollution-free. Due to the simplicity and convenience of purification, the degree of environmental pollution is low, and the extracted purity is high. This changes the reliance on chemical synthesis and solves the drawbacks of chemical synthesis, such as complexity, large amounts of byproducts, environmental pollution, high cost, and low purity of the final product. Attached Figure Description

[0023] Figure 1 The GC-MS chromatographic analysis of total fatty acids after methyl esterification in tobacco leaves co-transformed with CpunFADV, TE16 and P19 is shown.

[0024] Figure 2 The GC-MS mass spectra of monounsaturated hexadecenoic acid methyl ester in tobacco leaves co-transformed with CpunFADV, TE16 and P19 are shown.

[0025] Figure 3 The GC-MS chromatographic analysis of total fatty acids after methyl esterification in tobacco leaves co-transformed into SlitDes5, TE16 and P19 is shown.

[0026] Figure 4 The GC-MS mass spectra of monounsaturated hexadecenoic acid methyl esters in tobacco leaves co-transformed into SlitDes5, TE16, and P19 are shown.

[0027] Figure 5 The GC-MS chromatographic analysis of total fatty acids after methyl esterification in tobacco leaves transferred to TE16 and P19 is shown.

[0028] Figure 6The GC-MS chromatogram and mass spectrum of monounsaturated hexadecenoic acid methyl ester in tobacco leaves co-transformed with CpunFADV, TE16 and P19 after treatment with dimethyl disulfide are shown.

[0029] Figure 7 The GC-MS chromatograms and mass spectra of monounsaturated hexadecenoic acid methyl esters co-transformed into SlitDes5, TE16 and P19 in tobacco leaves after treatment with dimethyl disulfide are shown.

[0030] Figure 8 GC-MS chromatographic analysis of total fatty acid methyl esterification in T3 generation flaxseeds stably expressing CpunFADV is shown.

[0031] Figure 9 The GC-MS chromatographic analysis of total fatty acids in wild-type flaxseeds after methyl esterification is shown. Detailed Implementation

[0032] The present invention will be further described below with reference to the embodiments. However, the embodiments of the present invention are merely illustrative examples and should not be construed as limiting the present invention under any circumstances.

[0033] Example 1: Construction of GV3101 / pXZP393a-35s:CpunFADV engineered bacteria

[0034] From peach borer ( Conogethes punctiferalis The coding region nucleic acid sequence of the CpunFADV gene is shown in SEQ ID No. 1, and the amino acid sequence of the protein encoded by the CpunFADV gene is shown in SEQ ID No. 2.

[0035] The attL1 sequence was added to the 5' end and the attL2 sequence to the 3' end of the CpunFADV gene, and the complete sequence was synthesized by Zhejiang Youkang Biotechnology Co., Ltd. The synthesized nucleic acid was cloned into the pXZP393a plant expression vector (Xue-Rong Zhou, Pushkar Shrestha, Fang Yin, James R. Petrie, Surinder P. Singh) using an LR reaction kit (Gateway LR reaction, Invitrogen). AtDGAT2 is a functional acyl-CoA:diacylglycerolacyltransferase and displays different acyl-CoA substrate preferences than AtDGAT1. (2013.) This led to the cloning of the CpunFADV gene downstream of the 35S promoter in pXZP393a. The ligation product was transformed into Trans1-T1 Escherichia coli and cultured on a medium containing 75 mg / L spectinomycin antibiotic. Single clones were picked for colony PCR and sequencing identification. Positive transformants Trans1-T1 / pXZP393a-35s:CpunFADV were selected. The culture was expanded and the plasmid was extracted to obtain the positive plasmid pXZP393a-35s:CpunFADV.

[0036] Electroporation transformation of Agrobacterium: 1 μg of pXZP393a-35s:CpunFADV expression vector was added to 50 μL of Agrobacterium competent cells GV3101 (Biomed) for electroporation transformation. Then, 800 μL of LB liquid was added and mixed thoroughly. The mixture was transferred to a 2 mL centrifuge tube and incubated at 28°C and 220 rpm for 1.5 h. The bacterial culture was centrifuged at 12000 rpm for 1 minute, and the supernatant was discarded. 100 μL of LB liquid was added to fully resuspend the culture, and it was evenly spread on LB agar plates (containing 20 mg / L rifampin and 75 mg / L spectinomycin). The plates were incubated upside down at 28°C for 3 to 4 days. Single colonies were picked, and positive clones were identified by PCR and sequencing to obtain the positive strain GV3101 / pXZP393a-35s:CpunFADV.

[0037] Example 2: Construction of engineered bacteria GV3101 / pXZP393a-35s:SlitDes5

[0038] From the sea gray-winged moth ( Spodoptera litura The nucleotide accession number for the SlitDes5 gene is XM_022972138.1, and the amino acid accession number is XP_022827905.1.

[0039] The SlitDes5 protein is the protein with the highest known sequence identity to the CpunFADV protein. Its gene sequence shares 62.103% identity with the CpunFADV gene sequence; its amino acid sequence shares 54.819% identity with the CpunFADV amino acid sequence.

[0040] The CpunFADV gene in Example 1 was replaced with the SlitDes5 gene, and everything else remained the same as in Example 1. Specifically, the SlitDes5 gene was ligated into the pXZP393a plant expression vector, and positive transformants Trans1-T1 / pXZP393a-35s:SlitDes5 were screened out. The transformants were then expanded and cultured, and plasmids were extracted to obtain the positive plasmid pXZP393a-35s:SlitDes5. pXZP393a-35s:SlitDes5 was then electroporated into Agrobacterium competent cells GV3101, and positive strain GV3101 / pXZP393a-35s:SlitDes5 was obtained.

[0041] Example 3: Construction of engineered bacteria pXZP393a-35S:TE16 and pXZP393a-35S:P19

[0042] The accession number for the gene encoding a hexadecanoic acid-specific thioesterase (TE16) is KC675176.1, and the accession number for the amino acid of the hexadecanoic acid-specific thioesterase is AGG79283.1.

[0043] The nucleic acid sequence of the gene (P19) encoding the gene silencing repressor is shown in SEQ ID No. 3, and the amino acid sequence of the gene silencing repressor is shown in SEQ ID No. 4.

[0044] Referring to Example 1, TE16 and P19 were respectively ligated downstream of the 35S promoter in the pXZP393a plant expression vector to obtain pXZP393a-35S:TE16 and pXZP393a-35S:P19. These vectors were then electroporated into GV3101 to obtain GV3101 / pXZP393a-35S:TE16 and GV3101 / pXZP393a-35S:P19 positive engineered bacteria, respectively.

[0045] Example 4: Transient conversion of CpunFADV and SlitDes5 in tobacco

[0046] Activate GV3101 / pXZP393a-35s:CpunFADV in liquid LB at 28°C on a shaker at 230 rpm for 24 hours. Add 100 μL of the activated bacterial solution to 10 mL of liquid LB and incubate at 28°C on a shaker at 230 rpm for 16 hours until OD=1.0. Centrifuge at 6000 rpm for 5 minutes and discard the supernatant. Resuspend the bacterial cells in tobacco injection buffer (10 mmol / L MgCl2, 10 mmol / L 2-morpholine ethanesulfonic acid, 100 μmol / L acetylsylphenone, pH=5.6) to OD=1.0. The procedure for GV3101 / pXZP393a-35S:TE16 and GV3101 / pXZP393a-35S:P19 is the same. Then, mix the three bacterial solutions in equal volumes to obtain a mixed bacterial solution. Select healthy and uniformly growing tobacco plants, inject a mixed bacterial solution into the underside of the leaves, wipe off the excess solution after injection, and culture in an artificial climate chamber at 26 degrees Celsius and 16L:8D light conditions for 96 hours to obtain CpunFADV transient conversion tobacco.

[0047] The transient conversion of tobacco by SlitDes5 into *Nicotiana benthamiana* differed from the previous method in that equal volumes of three bacterial suspensions—GV3101 / pXZP393a-35s:SlitDes5, GV3101 / pXZP393a-35S:TE16, and GV3101 / pXZP 393a-35S:P19—were mixed and injected onto the underside of *Nicotiana benthamiana* leaves. The mixture was then incubated for 96 hours in an artificial climate chamber at 26°C and 16L:8D light conditions to obtain the transient conversion of tobacco by SlitDes5.

[0048] Equal volumes of two bacterial suspensions, GV3101 / pXZP393a-35S:TE16 and GV3101 / pXZP393a-35S:P19, were mixed to prepare a mixed bacterial suspension, which was then injected onto the underside of *Nicotiana benthamiana* leaves. The mixture was then cultured for 96 hours in an artificial climate chamber at 26°C and 16L:8D light conditions to obtain a negative control of transiently transformed tobacco.

[0049] Because GC-MS has a low response value to hexadecenoic acid or octadecenoic acid and can remain in the column and acidify the column, the sample needs to be esterified before testing.

[0050] 0.7 g of injected leaves were weighed and placed in a 4 mL glass vial for esterification: 1 mL of methanol (containing 2% sulfuric acid) was added, and the mixture was incubated at 90°C for 1 hour; after cooling to room temperature, 1 mL of distilled water and 1 mL of heptane were added, and the mixture was vortexed for 15 seconds; after standing at room temperature for 5 minutes, the upper heptane layer was transferred to a 2 mL sample vial to obtain the extracted total fatty acid methyl ester solution. The fatty acid methyl esters in each 1 μL total fatty acid methyl ester solution were determined using GC-MS (Agilent). The chromatogram results for the CpunFADV transient conversion of tobacco are shown below. Figure 1 The mass spectrum of monounsaturated hexadecenoic acid methyl ester is shown below. Figure 2 The chromatogram results of the transient conversion of tobacco by SlitDes5 are shown in [reference needed]. Figure 3 The mass spectrum of monounsaturated hexadecenoic acid methyl ester is shown below. Figure 4 The chromatogram results of the negative control transient conversion of tobacco are shown in [reference needed]. Figure 5 .

[0051] Will Figure 1 and Figure 2 Based on the analysis, it can be seen that Figure 1 The chromatographic peak pointed to by the middle arrow is the chromatographic peak of monounsaturated hexadecenoic acid methyl ester; Figure 3 and Figure 4 Based on the analysis, it can be seen that Figure 3 The chromatographic peak pointed to by the middle arrow is the chromatographic peak of monounsaturated hexadecenoic acid methyl ester; although Figure 1 and Figure 3 The middle arrows both point to monounsaturated hexadecenoic acid methyl esters, but their retention times are different; therefore, they are monounsaturated hexadecenoic acid methyl esters with different double bond positions. Figure 5 At the positions of the two monounsaturated hexadecenoic acid methyl esters ( Figure 1 and Figure 3 No chromatographic peaks were observed in any of the samples. This indicates that expression of the CpunFADV, TE16, and P19 genes in tobacco can enable tobacco to synthesize a monounsaturated hexadecenoic acid; expression of the SlitDes5, TE16, and P19 genes in tobacco can enable tobacco to synthesize another monounsaturated hexadecenoic acid.

[0052] Example 5: Dimethyl disulfide treatment of monounsaturated hexadecenoic acid methyl ester

[0053] Take 50 μL of the total fatty acid methyl ester solution obtained from the CpunFADV transient conversion of tobacco in Example 4, add 50 μL of dimethyl disulfide (DMDS) and 5 μL of iodine solution; react overnight at 40°C; add 200 μL of heptane and 50 μL of 5% Na2SO3 aqueous solution, let stand for 5 minutes, pipette the supernatant into a 2 mL sample vial, concentrate to 50 μL under nitrogen blowing, and use GC-MS to detect the double bond positions of monounsaturated fatty acid methyl esters. The GC-MS chromatogram and mass spectrum results corresponding to the peaks of monounsaturated hexadecenoic acid are shown below. Figure 6 The characteristic ion fragment molecular masses corresponding to the mass spectra are 131 and 231, respectively. Therefore, it can be concluded that the newly synthesized substances in tobacco leaves through the transient conversion of CpunFADV are cis-10-hexadecenoic acid and trans-10-hexadecenoic acid.

[0054] The same procedure was performed on the total fatty acid methyl esters of SlitDes5 transiently converted tobacco using DMDS for GC-MS detection of the double bond positions of monounsaturated fatty acid methyl esters. The GC-MS chromatograms and mass spectra of the peaks corresponding to monounsaturated hexadecenoic acid are shown below. Figure 7 The characteristic ion fragment molecular masses corresponding to these values ​​in the mass spectra are 117.05 and 245.15, respectively. Therefore, it can be concluded that the newly synthesized substance in tobacco leaves through the transient conversion of SlitDes5 is cis-11-hexadecenoic acid.

[0055] Example 6: Construction of engineered bacteria GV3101 / pBinGlyBar:Glycinin:TE16+pOleosin:CpunFADV

[0056] Add attL1 to the 5' end and attR5 to the 3' end of the TE16 gene to obtain attL1-TE16-attR5.

[0057] The pOleosin promoter is derived from flaxseed ( Camelina sativa Its nucleotide sequence is shown in SEQ ID No. 5.

[0058] The pOleosin promoter and the CpunFADV gene are linked in the order from 5' to 3' (written as pOleosin:CpunFADV). The attL5 sequence is added to the 5' end of the pOleosin promoter, and the attL2 sequence is added to the 3' end of the CpunFADV gene, resulting in attL5-pOleosin:CpunFADV-attL2.

[0059] The two fragments, attL1-TE16-attR5 and attL5-pOleosin:CpunFADV-attL2, were synthesized by Zhejiang Youkang Biotechnology Co., Ltd.

[0060] Using the Gateway LR reaction kit (Invitrogen), attL1-TE16-attR5 and attL5-pOleosin:CpunFADV-attL2 were cloned into the pBinGlyBar vector (guyen HT, Silva JE, Podicheti R, Macrander J, Yang W, Nazarenus TJ, Nam JW, Jaworski JG, Lu C, Scheffler BE, Mockaitis K, Cahoon EB. Camelina seed transcriptome: a tool for meal and oil improvement and translational research. 2013.). The TE16 gene was cloned downstream of the Glycinin promoter in the pBinGlyBar vector. The ligation product was transferred to Trans1-T1 E. coli and cultured on a medium containing 50 mg / L kanamycin. Single colonies were picked for colony PCR and sequencing identification. Positive transformants Trans1-T1 / pBinGlyBar:Glycinin:TE16+ pOleosin:CpunFADV were selected. The plasmid was extracted by amplification culture to obtain the positive plasmid pBinGlyBar:Glycinin:TE16+pOleosin:CpunFADV.

[0061] pBinGlyBar:Glycinin:TE16+pOleosin:CpunFADV was electroporated into Agrobacterium competent cells GV3101, and screened with 20 mg / L rifampin and 50 mg / L kanamycin to obtain positive strain GV3101 / pBinGlyBar:Glycinin:TE16+ pOleosin:CpunFADV.

[0062] Example 7: Stable synthesis of 10-hexadecenoic acid (E / Z10-16:COOH) using linseed.

[0063] In this embodiment, *Elaeagnus pungens* (…) Camelina sativa The only difference between the operation of esterifying total fatty acids in seeds and Example 4 is that the tobacco leaves in Example 4 are replaced with flaxseeds.

[0064] The procedure for determining total fatty acid methyl esters in this embodiment is the same as that for determining total fatty acid methyl esters in Example 4.

[0065] 2 mL of GV3101 / pBinGlyBar:Glycinin:TE16+ pOleosin:CpunFADV was activated on a shaker at 230 rpm for 24 hours at 28°C. The activated bacteria were then transferred to 50 mL of liquid LB and cultured overnight, followed by a complete transfer to 1 L LB and cultured for 24 h. The cells were centrifuged at 6000 rpm for 30 min, and the supernatant was discarded. The cells were resuspended in buffer (1.3 g Murashige and Skoog vitamin solution, 30 g sucrose, 300 μL of surfactant Silwet L-77, and 600 μL of acetylsyleugenone dissolved in 600 mL of distilled water). Flaxseed plants that had grown for approximately 5 weeks were transformed using the flower immersion method. The basta-resistant gene carried by the pBinGlyBar vector was used as a selection marker, and seeds from basta-resistant plants, i.e., the T1 generation seeds, were harvested.

[0066] All T1 seeds were sown in the soil. Four to five days after emergence, a 0.01 wt% glufosinate solution was sprayed. Surviving plants were considered positive T1 generation plants. T2 seeds were harvested from each T1 generation plant. Twenty seeds were randomly selected from each T1 generation plant for total fatty acid analysis. The plants were then ranked according to 10-hexadecenoic acid (10-HCA) yield, and the top five plants were selected for further propagation. Each T1 generation plant produced more than 25 T2 seeds for T3 seed production. Twenty T3 seeds were then selected from each T2 generation plant for total fatty acid analysis, using wild-type flaxseed as a control. The results showed that both cis-10-hexadecenoic acid and trans-10-hexadecenoic acid could be extracted from the T3 seeds harvested from each T2 generation plant. The GC-MS chromatogram of the T3 seeds is shown below. Figure 8 As shown in the figure, the chromatogram of wild-type flaxseed seeds analyzed by GC-MS is as follows. Figure 9 As shown. The T3 seeds from the plant with the highest 10-hexadecenoic acid yield were further propagated to produce T4 seeds, which were identified as homozygous.

[0067] While the present invention has been described with reference to specific embodiments, those skilled in the art will understand that various changes can be made without departing from the true spirit and scope of the invention. Furthermore, numerous modifications can be made to the subject, spirit, and scope of the invention to suit specific situations, materials, material compositions, and methods. All such modifications are included within the scope of the claims of the present invention.

Claims

1. A CpunFADV protein, the amino acid sequence of which is shown in SEQ ID No.

2.

2. The nucleic acid encoding the CpunFADV protein as described in claim 1.

3. The nucleic acid according to claim 2, characterized in that, The base sequence of the nucleic acid is shown in SEQ ID No.

1.

4. The application of the CpunFADV protein according to claim 1 in the biosynthesis of 10-hexadecenoic acid; wherein, The CpunFADV protein was co-expressed in *Capsella linteus* with a hexadecanoic acid-specific thioesterase for the synthesis of 10-hexadecenoic acid; or the CpunFADV protein was co-expressed in tobacco with a hexadecanoic acid-specific thioesterase and a gene silencing repressor for the synthesis of 10-hexadecenoic acid; wherein... The amino acid sequence of the hexadecimal fatty acid-specific thioesterase is accessed under accession number AGG79283.1, and the amino acid sequence of the gene silencing repressor is shown in SEQ ID No.

4.

5. The application of the nucleic acid according to claim 2 or 3 in the biosynthesis of 10-hexadecenoic acid; wherein, The nucleic acid described in claim 2 or 3, together with a nucleic acid encoding a hexadecanoic acid-specific thioesterase, is expressed in *Capsella linteus* for the synthesis of 10-hexadecenoic acid; or the nucleic acid described in claim 2 or 3, together with a nucleic acid encoding a hexadecanoic acid-specific thioesterase and a nucleic acid encoding a gene silencing repressor, is expressed in tobacco for the synthesis of 10-hexadecenoic acid; wherein... The amino acid sequence of the hexadecimal fatty acid-specific thioesterase is accessed under accession number AGG79283.1, and the amino acid sequence of the gene silencing repressor is shown in SEQ ID No.

4.

6. The application according to claim 4 or 5, characterized in that, The 10-hexadecenoic acid is cis-10-hexadecenoic acid and / or trans-10-hexadecenoic acid.

7. The application according to claim 5, characterized in that, The accession number for the nucleic acid encoding the hexadecanoic acid-specific thioesterase is KC675176.1, and the nucleic acid encoding the gene silencing repressor is shown in SEQ ID No.

3.

8. A method for synthesizing 10-hexadecenoic acid, comprising expressing the CpunFADV protein according to claim 1 in *Capsella bursa-pastoris* to synthesize the 10-hexadecenoic acid; or expressing the CpunFADV protein according to claim 1 in tobacco in combination with a hexadecenoic acid-specific thioesterase and a gene silencing repressor; wherein, The amino acid sequence of the hexadecimal fatty acid-specific thioesterase is accessed under accession number AGG79283.1, and the amino acid sequence of the gene silencing repressor is shown in SEQ ID No.

4.

9. The method according to claim 8, characterized in that, The 10-hexadecenoic acid is cis-10-hexadecenoic acid and / or trans-10-hexadecenoic acid.

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