A gene for the biosynthesis of (Z)-13-octadecenoic acid and its application

Abstract

This invention relates to the field of genetic engineering technology, and more particularly to a gene for the biosynthesis of (Z)-13-octadecenoic acid and its application. A gene for the biosynthesis of (Z)-13-octadecenoic acid is provided, the amino acid sequence of which is shown in SEQ ID NO: 1. This invention discovers that expressing a protein with the amino acid sequence shown in SEQ ID NO: 1 in plants in conjunction with a hexadecimal fatty acid-specific thioesterase and a Z11 desaturase can synthesize (Z)-13-octadecenoic acid. The process of synthesizing (Z)-13-octadecenoic acid using plants is green and pollution-free, purification is simple and convenient, environmental pollution is low, and the extraction purity is high.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and in particular to a gene for the biosynthesis of (Z)-13-octadecenoic acid and its application. Background Technology

[0002] Currently, insect sex pheromones are mainly synthesized chemically. The high cost of raw materials and demanding reaction conditions make artificially synthesized sex pheromone products very expensive. Furthermore, some catalytic reaction technologies are not yet mature enough, producing numerous byproducts and resulting in insufficient product purity. This not only significantly reduces their effectiveness in controlling pests but also indirectly causes secondary environmental pollution. Therefore, green and efficient synthesis of insect sex pheromones is one of the most effective strategies for controlling agricultural pests.

[0003] (Z)-13-Octadecenoic acid is a precursor to three sex pheromones: (Z)-13-octadecenoic acid acetate, (Z)-13-octadecenoic acid aldehyde, and (Z)-13-octadecenoic acid alcohol. These three sex pheromones can be generated from (Z)-13-octadecenoic acid through known reduction, esterification, and oxidation reactions. Currently, the synthesis of (Z)-13-octadecenoic acid, (Z)-13-octadecenoic acid acetate, (Z)-13-octadecenoic acid aldehyde, and (Z)-13-octadecenoic acid alcohol mainly relies on chemical synthesis, which leads to drawbacks such as the generation of byproducts, environmental pollution, high costs, and low purity of the final products.

[0004] 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 in the chemical synthesis process. Summary of the Invention

[0005] The purpose of this invention is to provide a gene for the biosynthesis of (Z)-13-octadecenoic acid and its application.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] The present invention provides a gene for the biosynthesis of (Z)13 octadecenoic acid, the amino acid sequence encoded by the gene being shown in SEQ ID NO: 1.

[0008] Preferably, the nucleotide sequence encoded by the gene is shown in SEQ ID NO: 2.

[0009] The present invention also provides an expression vector, comprising an initial vector and the gene described herein.

[0010] Preferably, the initial carrier is pXZP393a.

[0011] The present invention also provides a host, which is transformed or transfected with the expression vector described above; the host is a microorganism.

[0012] The present invention also provides the use of the gene, the expression vector, or the host in the biosynthesis of (Z)-13-octadecenoic acid.

[0013] Preferably, the gene is co-expressed in plants with a hexadecanoic acid-specific thioesterase and a Z11 desaturase to synthesize (Z)-13-octadecenoic acid.

[0014] Preferably, the accession number for the amino acid sequence of the hexadecimal fatty acid-specific thioesterase is AGG79283.1, and the accession number for the amino acid sequence of the Z11 desaturase is QGA89354.1; the accession number for the nucleotide sequence of the nucleic acid encoding the hexadecimal fatty acid-specific thioesterase is KC675176.1, and the accession number for the nucleotide sequence of the nucleic acid encoding the Z11 desaturase is MN453822.1.

[0015] The present invention also provides a method for synthesizing (Z)-13-octadecenoic acid by co-expressing the gene with a hexadecanoic acid-specific thioesterase and a Z11 desaturase to synthesize (Z)-13-octadecenoic acid.

[0016] Preferably, the plant is tobacco, Arabidopsis thaliana, or rice.

[0017] Among them, SEQ ID NO: 1

[0018] CmedELVL1 amino acid sequence

[0019] MSHDEERQRGLLKRAKMSYLEKVDKYLDSLKVGKSALVDSWFMMSSPVPILCVVIAYLVVIRVIPVLMKNRPPLQMNKLITYYNASQVFMATMICTKALRLNLFRDGIMYAGCRFPSNTQNPMLLDLGWWYFFAKFTELLDTVFFVLRKKNSQVSFLHVYHHVIMAVYSWSYLKFAAGGEGAILALLNSIVHVVMYSYYLLSGMGPRFQKYLWWKKYVTKMQLIQFVLMLVYCAWTHFSPRCQYASGFTYFICANVMIFLFLFLNFYTKSYKGKKNVEKEIKEEYHKAKNGIKTNGVKTNGVTQNSEKENGLHRNDSKNAEVDDVPYEVNNVCGDYEKDAPYVENWPLMTSPGPILAILLSYLLVVLKLGPWIMQKRAPMTLNNLLIVYNAVQVLVSAYLFNVGYQILRSNGLVHTTCLLNNEHVMSAITTGIYSYLLAKISELLDTVFFILRKKHNQLTFLHVYHHTSMVLFTWISLKYEPTHSVVFLGTINSFIHIMMYAYYGLAAFPALTKYLWWKKYITSMQLIQFVLIILHSLFDYVYGACPPSYLLLSLIVCNAAFFVFLFGDFYIKSYTKEAPSKTVKQKVK

[0020] SEQ ID NO: 2

[0021] Nucleotide sequence of CmedELVL1

[0022]

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] This invention discovers that (Z)-13-octadecenoic acid can be synthesized by co-expressing a protein with the amino acid sequence shown in SEQ ID NO: 1, along with a hexadecanoic acid-specific thioesterase and a Z11 desaturase in plants. The process of synthesizing (Z)-13-octadecenoic acid from plants is green and pollution-free. Due to its simple and convenient purification, it has a low degree of environmental pollution and yields high purity. 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

[0025] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0026] Figure 1 The chromatogram results are for the transient conversion of tobacco by CmedELVL1.

[0027] Figure 2 The chromatogram results are for the transient conversion of tobacco by eGFP.

[0028] Figure 3 This is the mass spectrum of the peak corresponding to methyl (Z)-13-octadecenoate after treatment with dimethyl disulfide.

[0029] Figure 4 The spectrum of the CmedELVL1-Puc57 vector. Detailed Implementation

[0030] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0031] Example 1: Construction of engineered bacteria GV3101 / pXZP393c-35S::CmedELVL1.

[0032] Derived from the rice leaf roller (Cnaphalocrocis medinalis) CmedELVL1 The nucleic acid sequence of the coding region of the gene is shown in SEQ ID NO: 2. CmedELVL1 The amino acid sequence of the gene-encoded protein is shown in SEQ ID NO: 1.

[0033] Will CmedELVL1 The gene was ligated downstream of the 35S promoter in the pXZP393a vector (Bao-Jian Ding, Per Hofvander, Hong-Lei Wang, Timothy P. Durrett, Sten Stymne & Christer Lofstedt. A plant factory for moth pheromone production. 2014.) in 5' to 3' order (written as 35s::CmedELVL1). Based on the LR response, in CmedELVL1 Add attL1 to the 5' end of the gene, and in CmedELVL1 The attR1 sequence was added to the 3' end of the gene, and the full sequence was synthesized by Zhejiang Youkang Biotechnology Co., Ltd. The vector map is shown below. Figure 4 As shown.

[0034] The synthesized nucleic acid was ligated into the pXZP393a plant expression vector using an LR kit (invitrogene). The ligation product was then transformed into Trans1-T1 Escherichia coli and cultured on a medium containing 75 mg / L spectinomycin antibiotic. After bacterial PCR and sequencing identification, the positive transformant Trans1-T1 / pXZP393a-35s::CmedELVL1 was obtained, in which the positive plasmid was pXZP393a-35s::CmedELVL1.

[0035] Electroporation transformation of Agrobacterium: Agrobacterium GV3101 was cultured in LB liquid medium to prepare GV3101 competent cells. 10 μL of pXZP393a-35s::CmedELVL1 expression vector was added to 50 μL of Agrobacterium competent cell culture for electroporation transformation. Then, 800 μL of LB liquid was added and mixed well. The mixture was transferred to a 1.5 mL centrifuge tube and incubated at 28°C and 220 rpm for 1.5 h. The 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 cells, and the mixture was evenly spread on LB agar plates (containing 50 mg / L rifampin and 75 mg / L spectinomycin). The plates were incubated upside down at 28°C for 2 days. Single colonies were picked and identified by PCR and agarose gel chromatography to obtain positive clones, yielding GV3101 / pXZP393a-35s::CmedELVL1.

[0036] The accession number for the gene (TE16) encoding a 16-carbon fatty acid-specific thioesterase is KC675176.1, and the accession number for its amino acid is AGG79283.1.

[0037] The accession number for the gene encoding the Z11 desaturase (YPAQ) is MN453822.1, and the accession number for its amino acid is QGA89354.1.

[0038] The nucleic acid sequence of the gene encoding eGFP is shown in SEQ ID NO: 3, and the amino acid sequence of the eGFP protein is shown in SEQ ID NO: 4.

[0039] SEQ ID No. 3

[0040] eGFP gene coding region nucleic acid sequence

[0041] ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACC CTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTG GTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCC GACCACTACCAGCAGAACACCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAA

[0042] SEQ ID No. 4

[0043] The amino acid sequence of the protein encoded by the eGFP gene

[0044] MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDT LVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK

[0045] Referring to Example 1, TE16, YPAQ, and eGFP were ligated downstream of the 35S promoter in the pXZP393a vector (Bao-Jian Ding, Per Hofvander, Hong-Lei Wang, Timothy P. Durrett, Sten Stymne & Christer Lofstedt. A plant factory for moth pheromone production. 2014. DOI:10.1038 / ncomms4353), resulting in pXZP393a-35S::TE16, pXZP393a-35S::YPAQ, and pXZP393a-35S::eGFP. The above vectors were electroporated into GV3101 to obtain GV3101 / pXZP393a-35S::TE16 positive engineered bacteria, GV3101 / pXZP393a-35S::YPAQ positive engineered bacteria and GV3101 / pXZP393a-35S::eGFP positive engineered bacteria, respectively.

[0046] Example 2: Transient conversion of CmedELVL1 in tobacco.

[0047] 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)” indicates that (Z)-13-octadecenoic acid is not produced in wild-type tobacco.

[0048] Activate GV3101 / pXZP393a-35S::CmedELVL1 in liquid LB at 28°C on a shaker at 230 rpm for 24 hours. Add 20 μL of the activated bacterial culture 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 4000 rpm for 10 minutes and discard the supernatant. Resuspend the cells in tobacco injection buffer (10 mmol / L MgCl2, 10 mmol / L morpholinoethanesulfonic acid monohydrate, 100 μmol / L acetylsylphenone, pH=5.6) to OD=1.0. Incubate in the dark at room temperature for 1 to 2 hours. The procedure for GV3101 / pXZP393a-35S::TE16 and GV3101 / pXZP393a-35S::YPAQ is the same as above, incubating in the dark at room temperature for 2 hours. Then, equal amounts of the three bacterial solutions were mixed to prepare a mixed bacterial solution. The mixed bacterial solution was injected into the back of well-grown and uniformly grown tobacco leaves. After injection, excess bacterial solution was wiped off, and the leaves were placed in a dark environment in an artificial climate chamber for 12 hours. Then, they were transferred to normal light conditions and cultured for 72 hours to obtain CmedELVL1 transiently converted tobacco.

[0049] Following the same procedure described above, GV3101 / pXZP393a-35S::eGFP was transferred into tobacco leaves. After culturing for 72 hours, transient eGFP conversion in tobacco was obtained.

[0050] Since (Z)-13-octadecenoic acid is not volatile, it cannot be directly detected by GC-MS. Therefore, esterification is required to determine whether (Z)-13-octadecenoic acid is synthesized in plants.

[0051] Leaves were cut from tobacco plants cultured for 72 hours using CmedELVL1 transient transformation and eGFP transient transformation after injection, and the synthesized target compounds were determined.

[0052] For the determination of the target compounds synthesized, the leaf veins were removed, the samples were weighed, and placed in a 4 mL glass bottle for esterification: 1 mL of methanol (containing 2% sulfuric acid and 10 μg of (Z)-10-heptadecenoic acid methyl ester as an internal standard, wherein (Z)-10-heptadecenoic acid or (Z)-10-heptadecenoic acid methyl ester is not produced in tobacco leaves) was added, and the reaction was carried out at 90°C for 1 hour; 1 mL of water (containing 0.075 mol / L acetic acid) was added, and the mixture was vortexed for 15 seconds; 1 mL of heptane was added, and the mixture was vortexed for 15 seconds; the mixture was allowed to stand at room temperature for 5 minutes, and the supernatant was transferred to a new 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 (Shimadzu). The chromatogram results of the transient conversion of tobacco by CmedELVL1 are shown below. Figure 1 The chromatogram results of transient conversion of tobacco with eGFP are shown below. Figure 2 .

[0053] Will Figure 1 and Figure 2 The comparison shows that, Figure 1 A chromatographic peak appeared at the position of (Z)-13-octadecenoic acid methyl ester, while Figure 2 The absence of chromatographic peaks at the corresponding positions indicates that the expression of CmedELVL1, TE16, and YPAQ genes in tobacco can enable the synthesis of octadecenoic acid.

[0054] Example 3: Dimethyl disulfide treatment of (Z)-13-octadecenoic acid methyl ester.

[0055] Take 50 μL of the total fatty acid methyl ester solution obtained from the CmedELVL1 transient conversion of tobacco in Example 2, add 50 μL of dimethyl disulfide (DMDS) and 5 μL of iodine solution; place in the dark and react overnight at 40°C; add 200 μL of heptane and 50 μL of 5% Na2SO3 aqueous solution, let stand for 5 min, pipette the lower layer liquid into a new glass sample vial, concentrate to 50 μL by nitrogen blowing, and use GC-MS to perform mass spectrometry on each fatty acid methyl ester. The mass spectrum of the peak corresponding to (Z)-13-octadecenoic acid methyl ester is shown in […]. Figure 3 .according to Figure 3 Based on the molecular weights (117.05 and 273.20) of the characteristic ion fragments, methyl octadecenoate is (Z)-13-octadecenoate. Therefore, the newly synthesized substance in the leaves of tobacco transiently converted by CmedELVL1 is (Z)-13-octadecenoic acid.

[0056] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A gene for biosynthesis of (Z)-13-octadecenoic acid, characterized by, The amino acid sequence encoded by the gene is shown as SEQ ID NO:

1.

2. The gene of claim 1, wherein The nucleotide sequence encoded by the gene is shown as SEQ ID NO:

2.

3. An expression vector, characterized by, The initial vector and the gene according to claim 1 or 2.

4. The expression vector of claim 3, wherein, The initial vector is pXZP393a.

5. A host, characterized in that, The host is a microorganism.

6. Use of the genes according to claim 1 or 2 for the biosynthesis of (Z)-13-octadecenoic acid, characterized in that, Co-expressing the gene according to claim 1 or 2 with a hexadecanoate-specific thioesterase and a Z11 desaturase in tobacco to synthesize (Z)-13-octadecenoic acid; the accession number of the amino acid sequence of the hexadecanoate-specific thioesterase is AGG79283.1, and the accession number of the amino acid sequence of the Z11 desaturase is QGA89354.1; the accession number of the nucleotide sequence of the nucleic acid encoding the hexadecanoate-specific thioesterase is KC675176.1, and the accession number of the nucleotide sequence of the nucleic acid encoding the Z11 desaturase is MN453822.

1.

7. A method of synthesizing (Z)-13-octadecenoic acid, characterized by, Co-expressing the gene according to claim 1 or 2 with a hexadecanoate-specific thioesterase and a Z11 desaturase in tobacco to synthesize (Z)-13-octadecenoic acid; the accession number of the amino acid sequence of the hexadecanoate-specific thioesterase is AGG79283.1, and the accession number of the amino acid sequence of the Z11 desaturase is QGA89354.1; the accession number of the nucleotide sequence of the nucleic acid encoding the hexadecanoate-specific thioesterase is KC675176.1, and the accession number of the nucleotide sequence of the nucleic acid encoding the Z11 desaturase is MN453822.1.

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