Protein for synthesizing octadecene compound and method for synthesizing octadecene compound

Abstract

The invention provides a protein for synthesizing an octadecene compound and a method for synthesizing the octadecene compound. Relates to the field of enzymology. The amino acid sequence of the protein has more than 95% of consistency with the sequence as shown in SEQ ID No.3. The protein, hexadecanoic acid specific thioesterase, Z11 desaturase, gene silencing suppressor and vegetable fat synthesis related transcription factors are jointly expressed in plants and can be used for synthesizing (Z)-13-octadecenoic acid.

Description

Technical Field

[0001] The present invention relates to the field of enzymology, and in particular to a protein for synthesizing octadecene compounds and a method for synthesizing octadecene compounds. Background Art

[0002] Sex pheromones are important chemical signal substances in the process of mating, which are mainly synthesized and released by female moths. Sex pheromones can attract male moths from a distance and regulate their courtship behavior at close range. In recent years, pheromones have emerged as a new force in pest control, especially sex pheromones, which have been used in the control of many moth pests.

[0003] However, insect pheromones are currently mainly produced by chemical synthesis. The high cost of raw materials and harsh reaction conditions make the price of artificially synthesized pheromones very expensive. In addition, the technology of some catalyst reactions is not mature enough, and a large number of by-products are produced, resulting in insufficient purity of the product, which not only greatly reduces the effect of pest control, but also pollutes the environment in disguised form. Therefore, green and efficient synthesis of insect pheromones is one of the most effective strategies for controlling agricultural pests.

[0004] (Z)-13-octadecenoic acid is a precursor of three sex pheromones: (Z)-13-octadecenoic acetate, (Z)-13-octadecenoic aldehyde and (Z)-13-octadecenoic alcohol. After the synthesis of (Z)-13-octadecenoic acid, the three sex pheromones can be generated by simply undergoing known reduction or esterification reactions. Currently, (Z)-13-octadecenoic acid mainly relies on chemical synthesis, which leads to the generation of by-products during the synthesis process, environmental pollution, high costs and low purity of the final product.

[0005] Therefore, there is an urgent need to develop a method for synthesizing (Z)-13-octadecenoic acid that can greatly solve the problems of environmental pollution, high cost and low purity during chemical synthesis. Summary of the invention

[0006] One of the present inventions provides a protein whose amino acid sequence has more than 95% identity with the sequence shown in SEQ ID No. 3.

[0007] In a specific embodiment, the amino acid sequence of the protein is shown as SEQ ID No. 3.

[0008] The second aspect of the present invention provides a nucleic acid encoding the protein as described in the first aspect of the present invention.

[0009] In a specific embodiment, the base sequence of the nucleic acid is shown as SEQ ID No. 2.

[0010] The third aspect of the present invention provides the use of the protein according to the first aspect of the present invention or the nucleic acid according to the second aspect of the present invention in the biosynthesis of (Z)-13-octadecenoic acid.

[0011] In a specific embodiment, the protein is co-expressed in plants with a hexadecene fatty acid-specific thioesterase, a Z11 desaturase, a gene silencing suppressor, and a transcription factor related to plant oil synthesis for the synthesis of (Z)-13-octadecenoic acid.

[0012] In a specific embodiment, the accession number of the amino acid sequence of the hexadecane fatty acid-specific thioesterase is AGG79283.1, the accession number of the amino acid sequence of the Z11 desaturase is QGA89354.1, the amino acid sequence of the gene silencing suppressor is shown in SEQ ID No. 5, and the accession number of the amino acid sequence of the plant oil synthesis-related transcription factor is NP_191000.3.

[0013] In a specific embodiment, the accession number of the base sequence of the nucleic acid encoding the hexadecanoic acid-specific thioesterase is KC675176.1, the accession number of the base sequence of the nucleic acid encoding the Z11 desaturase is MN453822.1, the base sequence of the nucleic acid encoding the gene silencing suppressor is shown in SEQ ID No. 4, and the accession number of the base sequence of the nucleic acid encoding the plant oil synthesis-related transcription factor is NM_115292.4.

[0014] In a specific embodiment, the (Z)-13-octadecenoic acid is subjected to an esterification reaction to generate (Z)-13-octadecenoic acid acetate.

[0015] In a specific embodiment, the (Z)-13-octadecenoic acid is subjected to a reduction reaction to generate (Z)-13-octadecenal.

[0016] In a specific embodiment, the (Z)-13-octadecenoic acid is subjected to a reduction reaction to generate (Z)-13-octadecenol.

[0017] The fourth aspect of the present invention provides a method for synthesizing (Z)-13-octadecenoic acid, which comprises expressing the protein according to one of the present invention together with a hexadecanoic acid-specific thioesterase, a Z11 desaturase, a gene silencing suppressor and a transcription factor related to plant oil synthesis in tobacco to synthesize the (Z)-13-octadecenoic acid.

[0018] In a specific embodiment, the (Z)-13-octadecenoic acid is synthesized by transferring into the tobacco a nucleic acid encoding a protein as described in one of the present invention, a nucleic acid encoding a hexadecanoic acid-specific thioesterase, a nucleic acid encoding a Z11 desaturase, a nucleic acid encoding a gene silencing suppressor, and a nucleic acid encoding a transcription factor related to plant oil synthesis, so that they are expressed together in the tobacco.

[0019] In a specific embodiment, the base sequence of the nucleic acid encoding the protein as described in one of the present invention is shown as SEQ ID No. 2.

[0020] In a specific embodiment, the accession number of the base sequence of the nucleic acid encoding the hexadecanoic acid-specific thioesterase is KC675176.1, the accession number of the base sequence of the nucleic acid encoding the Z11 desaturase is MN453822.1, the base sequence of the nucleic acid encoding the gene silencing suppressor is shown in SEQ ID No. 4, and the accession number of the base sequence of the nucleic acid encoding the plant oil synthesis-related transcription factor is NM_115292.4.

[0021] Beneficial effects of the present invention: The present invention finds that the protein having an amino acid sequence as shown in SEQ ID No. 3, a hexadecanoic acid-specific thioesterase, a Z11 desaturase, a gene silencing suppressor and a transcription factor related to plant oil synthesis can be expressed in plants to synthesize (Z)-13-octadecenoic acid. The process of synthesizing (Z)-13-octadecenoic acid using plants is green and pollution-free. Since the purification is simple and convenient, the degree of environmental pollution is low, and the extraction purity is high (Hong-Lei Wang, Bao-Jian Ding, Jian-Qing Dai, Tara J. Nazarenus, Rafael Borges, Agenor Mafra-Neto, Edgar B. Cahoon, Per Hofvander, Sten Stymne & Christer Löfstedt. Insect pest management with sex pheromone precursors from engineered oilseed plants. 2022.), which changes the path that relies on chemical synthesis and solves the disadvantages of complex chemical synthesis, large amount of by-products, environmental pollution, high cost and low purity of the final product. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1The figure shows the expression analysis of Csa7610, TE16, YPAQ, P19 and WRI1 in tobacco leaves into which Csa7610, TE16, YPAQ, P19 and WRI1 were co-transformed.

[0023] Figure 2 The graph shows the expression analysis of eGFP in eGFP-transfected tobacco leaves.

[0024] Figure 3 Shown is the GC-MS chromatogram analysis of total fatty acid methyl esters in tobacco leaves co-transformed with Csa7610, TE16, YPAQ, P19, and WRI1.

[0025] Figure 4 Shown is a GC-MS chromatogram analysis of total fatty acid methyl esters in eGFP-transfected tobacco leaves.

[0026] Figure 5 Shown is the GC-MS mass spectrum of methyl octadecenoate after treatment with dimethyl disulfide. DETAILED DESCRIPTION

[0027] The present invention will be further described below in conjunction with examples, but the examples of the present invention are only exemplary descriptions, and the implementation methods do not constitute limitations of the present invention under any circumstances. Example 1: Construction of engineered bacteria such as GV3101 / pXZP393c-Csa7610::Csa7610

[0028] From camelina Camelina sativa ) Csa7610 The nucleic acid sequence of the gene promoter is shown in SEQ ID No. 1. Csa7610 The coding region nucleic acid sequence of the gene is shown in SEQ ID No. 2. Csa7610 The amino acid sequence of the gene-encoded protein is shown in SEQ ID No. 3.

[0029] Will Csa7610 Gene promoter and Csa7610 The genes were linked in 5' to 3' order (written as Csa7610::Csa7610) and based on the LR reaction, Csa7610 Add attL1 to the 5' end of the gene promoter, and Csa7610The attR1 sequence was added to the 3' end of the gene, and then the whole sequence was synthesized by Zhejiang Youkang Biotechnology Co., Ltd. The synthesized nucleic acid was connected to the pXZP393c plant expression vector (Bao-Jian Ding, PerHofvander, Hong-Lei Wang, Timothy P. Durrett, Sten Stymne & ChristerLofstedt. A plant factory for moth pheromone production. 2014.) using the LR kit (invitrogene), and the ligation product was transferred to Trans1-T1 Escherichia coli, and the transformation product was cultured under 75 mg / L spectinomycin antibiotics, and then the bacterial liquid PCR and sequencing were performed to obtain the positive transformant Trans1-T1 / pXZP393c-Csa7610::Csa7610, among which the positive plasmid was pXZP393c- Csa7610::Csa7610.

[0030] Electroporation of Agrobacterium: Agrobacterium GV3101 was cultured in LB liquid medium to prepare GV3101 competent cells. 10 μL of pXZP393c- Csa7610::Csa7610 expression vector was added to 50 μL of Agrobacterium competent cell solution for electroporation, then 800 μL of liquid LB was added to mix, transferred to a 1.5 ml centrifuge tube, and cultured at 28 degrees Celsius and 220 rpm for 1.5 hours; the bacterial solution was centrifuged at 12000 rpm for 1 minute, and the supernatant was discarded; 100 μL of liquid LB was added to fully suspend, and evenly coated on LB plate culture medium (containing 50 mg / L rifampicin and 75 mg / L spectinomycin), and cultured inverted at 28 degrees Celsius for 2 days. Single clone colonies were picked and positive clones were identified by agarose after PCR to obtain GV3101 / pXZP393c-Csa7610::Csa7610.

[0031] The accession number of the gene encoding the hexadecane fatty acid-specific thioesterase (TE16) is KC675176.1, and the accession number of the hexadecane fatty acid-specific thioesterase amino acid is AGG79283.1.

[0032] The accession number of the gene encoding the Z11 desaturase (YPAQ) is MN453822.1, and the accession number of the Z11 desaturase amino acid is QGA89354.1.

[0033] The nucleic acid sequence of the gene (P19) encoding the gene silencing suppressor is shown in SEQ ID No. 4, and the amino acid sequence of the gene silencing suppressor is shown in SEQ ID No. 5.

[0034] The nucleic acid accession number of plant oil synthesis related transcription factor (WRI1) is NM_115292.4, and the amino acid accession number of plant oil synthesis related transcription factor is NP_191000.3.

[0035] The nucleic acid sequence of the gene encoding eGFP is shown in SEQ ID No. 6, and the amino acid sequence of the eGFP protein is shown in SEQ ID No. 7.

[0036] Referring to Csa7610, TE16, YPAQ, P19, WRI1, and eGFP were connected to the downstream of the 35S promoter in the pXZP393a vector (Bao-Jian Ding, Per Hofvander, Hong-Lei Wang, Timothy P. Durrett, Sten Stymne & ChristerLofstedt. A plant factory for moth pheromone production. 2014.) to obtain pXZP393a-35S::TE16, pXZP393a-35S::YPAQ, pXZP393a-35S::P19, pXZP393a-35S::WRI1, and pXZP393a-35S::eGFP. The above vectors were transformed into GV3101 by electroporation to obtain GV3101 / pXZP393a-35S::TE16 positive engineering bacteria, GV3101 / pXZP393a-35S::YPAQ positive engineering bacteria, GV3101 / pXZP393a-35S::P19 positive engineering bacteria, GV3101 / pXZP393a-35S::WRI1 positive engineering bacteria and GV3101 / pXZP393a-35S::eGFP positive engineering bacteria. Example 2: Transient transformation of Csa7610 in tobacco

[0037] The study “Release of moth pheromone compounds from Nicotiana benthamiana upon transient expression of heterologous biosynthetic genes (Yi Han Xia, Bao JianDing, Shuang Lin Dong, Hong Lei Wang, Per Hofvander & Christer Löfstedt; BMC Biology; 2022)” showed that (Z)-13-octadecenoic acid is not produced in wild-type tobacco.

[0038] Activate GV3101 / pXZP393c-Csa7610::Csa7610 in liquid LB at 28 degrees Celsius on a shaker at 230 rpm for 24 hours. Take 20 microliters of the activated bacterial solution and add it to 10 ml of liquid LB. Incubate it at 28 degrees Celsius on a shaker at 230 rpm for 16 hours until OD = 1.0. Centrifuge at 4000 rpm for 10 minutes and remove the supernatant. Resuspend the cells in tobacco injection buffer (10 mmol / L MgCl2, 10 mmol / L monohydrate morpholineethanesulfonic acid, 100 micromol / L acetosyringone, pH = 5.6) to OD = 1.0. Let it stand in the dark at room temperature for 1 to 2 hours. The operation of GV3101 / pXZP393a-35S::TE16, GV3101 / pXZP393a-35S::YPAQ, GV3101 / pXZP393a-35S::P19, GV3101 / pXZP393a-35S::WRI1 is the same as above, and it is left to stand for 1 to 2 hours in a dark room. Then, the five bacterial solutions are mixed in equal amounts to prepare a mixed bacterial solution. The mixed bacterial solution is injected from the back of the well-growing and consistent Nicotiana benthamiana leaves, and the excess bacterial solution is wiped off after injection. It is placed in a dark environment of an artificial climate chamber for 12 hours, and then transferred to normal light conditions and cultured for 72 hours to obtain Csa7610 instantaneous transformation tobacco.

[0039] Based on the same operation as above, only GV3101 / pXZP393a-35S::eGFP was transferred into Nicotiana benthamiana leaves and cultured for 72 hours to obtain eGFP transiently transformed tobacco.

[0040] Since octadecenoic acid is not volatile, it cannot be directly detected by GC-MS. In order to determine whether octadecenoic acid is synthesized in plants, an esterification reaction is required first.

[0041] The injected leaves of the Csa7610 transiently transformed tobacco and the eGFP transiently transformed tobacco cultured for 72 hours were cut and divided into two parts, one for determining the expression level of the gene and the other for determining the synthesized target compound.

[0042] For the determination of gene expression levels, wild-type tobacco (WT) without any transformation was used as a blank control. The leaves were placed in liquid nitrogen and ground with a mortar. RNA from tobacco leaves was extracted using a plant total RNA extraction kit (Nanjing Novozyme). RNA was then reverse transcribed into cDNA using an RNA reverse transcription kit (Nanjing Novozyme), and tobacco's own housekeeping gene Actin was used as an internal reference (A Abdul Kader Jailani, Vikas Solanki, Anirban Roy, T Sivasudha, Bikash Mandal. A CGMMV genome-replicon vector with partial sequences of coatprotein gene efficiently expresses GFP in Nicotiana benthamiana. 2017), and the expression levels of the injected genes were detected using real-time fluorescence quantitative PCR. Among them, the base sequence of the internal reference upstream primer AF is shown in SEQ ID No. 8, and the base sequence of the internal reference downstream primer AR is shown in SEQ ID No. 9; the base sequence of the Csa7610 gene upstream primer Csa7610F is shown in SEQ ID No. 10, and the base sequence of the Csa7610 gene downstream primer Csa7610R is shown in SEQ ID No. 11; the base sequence of the TE16 gene upstream primer TE16F is shown in SEQ ID No. 12, and the base sequence of the TE16 gene downstream primer TE16R is shown in SEQ ID No. 13; the base sequence of the YPAQ gene upstream primer YPAQF is shown in SEQ ID No. 14, and the base sequence of the YPAQ gene downstream primer YPAQR is shown in SEQ ID No. 15; the base sequence of the P19 gene upstream primer P19F is shown in SEQ ID No. 16, and the base sequence of the P19 gene downstream primer P19R is shown in SEQ ID The base sequence of the upstream primer WRI1F of the WRI1 gene is shown in SEQ ID No. 18, and the base sequence of the downstream primer WRI1R of the WRI1 gene is shown in SEQ ID No. 19; the base sequence of the upstream primer eGFPF of the eGFP gene is shown in SEQ ID No. 20, and the base sequence of the downstream primer eGFPR of the eGFP gene is shown in SEQ ID No. 21. The expression levels of the five genes in Csa7610 transiently transformed tobacco are shown in Figure 1 The expression level of eGFP gene in eGFP transiently transformed tobacco is shown in Figure 2 . Figure 1The results showed that five genes, Csa7610, TE16, YPAQ, P19 and WRI1, were successfully expressed in Csa7610 transiently transformed tobacco; Figure 2 The results showed that the eGFP gene was successfully expressed in eGFP transiently transformed tobacco.

[0043] For the determination of the synthetic target compound, the leaf veins were removed, weighed, and placed in a 4 ml glass bottle for esterification reaction: 1 ml of methanol (containing 2% sulfuric acid and 10 μg of (Z)-10-heptadecenoic acid methyl ester as an internal standard, wherein no (Z)-10-heptadecenoic acid or (Z)-10-heptadecenoic acid methyl ester was produced in the tobacco leaves), 90 degrees Celsius, 1 hour; 1 ml of water (containing 0.075 mol / L acetic acid) was added, vortexed for 15 seconds; 1 ml of heptane was added, vortexed for 15 seconds; let stand at room temperature for 5 minutes, and the supernatant was taken to a new injection bottle to obtain the extracted total fatty acid methyl ester solution. GC-MS (Shimadzu) was used to determine the fatty acid methyl esters in 1 μl of each total fatty acid methyl ester solution. Among them, the chromatogram results of Csa7610 instantaneous transformation of tobacco are shown in Figure 3 The chromatogram results of eGFP transient transformation of tobacco are shown in Figure 4 .

[0044] Will Figure 3 and Figure 4 By comparison, Figure 3 Chromatographic peaks appeared at the position of methyl octadecenoate, while Figure 4 There are no chromatographic peaks at the corresponding positions, indicating that after the Csa7610, TE16, YPAQ, P19 and WRI1 genes are expressed in tobacco, tobacco can synthesize octadecenoic acid. Example 3: Dimethyl disulfide treatment of methyl octadecenoate

[0045] Take 50 μl of the total fatty acid methyl ester solution obtained by the Csa7610 instantaneous tobacco transformation operation in Example 2, add 50 μl of dimethyl disulfide (DMDS) and 5 μl of iodine solution; place in the dark and react at 40°C overnight; add 200 μl of heptane and 50 μl of 5% Na2SO3 aqueous solution, let it stand for 5 min, use a pipette to draw the lower layer of liquid into a new glass injection bottle, concentrate it to 50 μl with nitrogen blowing, and use GC-MS to perform mass spectrometry on each fatty acid methyl ester, among which the mass spectrum result of the peak corresponding to octadecenoic acid methyl ester is shown in Figure 5 .according to Figure 5 The molecular weights of the characteristic ion fragments in the expression of methyl octadecenoate (117.05 and 273.20) indicate that methyl octadecenoate is methyl (Z)-13-octadecenoate. Therefore, the newly synthesized substance in the leaves of tobacco transiently transformed with Csa7610 is (Z)-13-octadecenoic acid.

[0046] Although the present invention has been described with reference to specific embodiments, it will be appreciated by those skilled in the art that various changes may be made without departing from the true spirit and scope of the present invention. In addition, the subject matter, spirit and scope of the present invention may be varied to accommodate specific situations, materials, material combinations and methods. All of these changes are included within the scope of the claims of the present invention.

Claims

1. A protein whose amino acid sequence has more than 95% identity with the sequence shown in SEQ ID No.

3.

2. The protein according to claim 1, characterized in that The amino acid sequence of the protein is shown in SEQ ID No.

3.

3. A nucleic acid encoding the protein according to claim 1 or 2.

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

2.

5. Use of the protein according to claim 1 or 2 or the nucleic acid according to claim 3 or 4 in the biosynthesis of (Z)-13-octadecenoic acid.

6. The use according to claim 5, characterized in that: The protein is co-expressed in plants with hexadecanoic acid-specific thioesterase, Z11 desaturase, gene silencing suppressor and plant oil synthesis-related transcription factors for the synthesis of (Z)-13-octadecenoic acid.

7. The use according to claim 6, characterized in that The accession number of the amino acid sequence of the hexadecane fatty acid-specific thioesterase is AGG79283.1, the accession number of the amino acid sequence of the Z11 desaturase is QGA89354.1, the amino acid sequence of the gene silencing suppressor is shown in SEQ ID No. 5, and the accession number of the amino acid sequence of the plant oil synthesis-related transcription factor is NP_191000.

3.

8. The use according to claim 6, characterized in that The accession number of the base sequence of the nucleic acid encoding the hexadecane fatty acid-specific thioesterase is KC675176.1, the accession number of the base sequence of the nucleic acid encoding the Z11 desaturase is MN453822.1, the base sequence of the nucleic acid encoding the gene silencing suppressor is shown in SEQ ID No. 4, and the accession number of the base sequence of the nucleic acid encoding the plant oil synthesis-related transcription factor is NM_115292.

4.

9. The use according to claim 5, characterized in that The (Z)-13-octadecenoic acid is subjected to an esterification reaction to generate (Z)-13-octadecenoic acid acetate; and / or The (Z)-13-octadecenoic acid is subjected to a reduction reaction to generate (Z)-13-octadecenoic aldehyde; and / or The (Z)-13-octadecenoic acid is subjected to a reduction reaction to generate (Z)-13-octadecenol.

10. A method for synthesizing (Z)-13-octadecenoic acid, comprising expressing the protein according to claim 1 or 2 together with a hexadecanoic acid-specific thioesterase, a Z11 desaturase, a gene silencing suppressor and a transcription factor related to plant oil synthesis in tobacco to synthesize the (Z)-13-octadecenoic acid.

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