Gene for biosynthesis of (Z)-9-hexadecenoic acid and application thereof

Abstract

The invention relates to the technical field of enzymology, in particular to a gene for biosynthesis of (Z)-9-hexadecenoic acid and application of the gene. The invention discloses a gene for biosynthesis of (Z)-9-hexadecenoic acid. A nucleotide sequence coded by the gene is shown as SEQ ID NO: 1. The gene and hexadecanoic acid specific thioesterase are jointly expressed in plants, so that the biosynthesis yield of (Z)-9-hexadecenoic acid can be increased. The process of synthesizing the (Z)-9-hexadecenoic acid by using plants is green and pollution-free, and the purification is simple and convenient, so that the environmental pollution degree is low, and the extraction purity is high, thereby changing the path depending on chemical synthesis, and solving the defects of complex chemical synthesis, large by-product amount, environmental pollution, high cost, low final product purity and the like.

Description

Technical Field

[0001] This invention relates to the field of enzyme technology, and more particularly to a gene for the biosynthesis of (Z)-9-hexadecenoic acid and its application. Background Technology

[0002] In recent years, insect pheromones have emerged as a new force in pest control and have been applied to the control of various moth pests. Insect sex pheromones, as important chemical signaling substances during mating behavior, are mainly synthesized by female insects and released into the environment. Insect sex pheromones can attract males from a distance and also regulate their courtship behavior at close range.

[0003] However, 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 a large number of byproducts and resulting in insufficient product purity. This not only significantly reduces the effectiveness of the pheromones 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.

[0004] (Z)-9-hexadecenoic acid is a precursor to two sex pheromones: (Z)-9-hexadecenoic acid acetate and (Z)-9-hexadecenoic aldehyde. After the synthesis of (Z)-9-hexadecenoic acid, the corresponding pheromones can be generated through known reduction, esterification, or oxidation reactions. Currently, (Z)-9-hexadecenoic acid mainly relies on chemical synthesis, leading to drawbacks such as the generation of byproducts, 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)-9-hexadecenoic acid that can greatly solve the problems of environmental pollution, high cost and low purity in the chemical synthesis process. Summary of the Invention

[0006] The purpose of this invention is to provide a gene for the biosynthesis of (Z)-9-hexadecenoic acid and its application.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution: The present invention provides a gene for the biosynthesis of (Z)-9-hexadecenoic acid, the nucleotide sequence encoded by the gene being shown in SEQ ID NO: 1.

[0008] Preferably, the amino acid 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)-9-hexadecenoic acid.

[0013] Preferably, the gene is co-expressed in plants with a hexadecanoic acid-specific thioesterase to synthesize (Z)-9-hexadecenoic 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 nucleotide sequence is KC675176.1.

[0015] The present invention also provides a method for synthesizing (Z)-9-hexadecenoic acid, wherein the gene is co-expressed with a hexadecanoic acid-specific thioesterase in plants to synthesize (Z)-9-hexadecenoic acid.

[0016] Preferably, the plant is tobacco.

[0017] Compared with the prior art, the present invention has the following beneficial effects: This invention discovers that co-expressing a protein with the amino acid sequence shown in SEQ ID NO. 2 with a hexadecanoic acid-specific thioesterase in plants can increase the biosynthetic yield of (Z)-9-hexadecenoic acid. The process of synthesizing (Z)-9-hexadecenoic acid using 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

[0018] 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.

[0019] Figure 1 The expression levels of CmedFAD2 and TE16 are shown in tobacco leaves co-transfected with CmedFAD2 and TE16.

[0020] Figure 2The expression level of eGFP in tobacco leaves transformed with eGFP is shown.

[0021] Figure 3 The GC-MS chromatographic analysis of total fatty acid methyl esters in tobacco leaves co-transformed with CmedFAD2 and TE16 is shown.

[0022] Figure 4 The GC-MS chromatographic analysis of total fatty acid methyl esters in tobacco leaves transformed with eGFP is shown.

[0023] Figure 5 This study compares the in vitro conversion of hexadecenoic acid to methyl hexadecenoate in tobacco leaves co-transferred to CmedFAD2 and TE16 (the x-axis is described as CmedFAD2) with the in vitro conversion of hexadecenoic acid to methyl hexadecenoate in tobacco leaves transferred to eGFP (the x-axis is described as eGFP). This indicates a highly significant difference between the two at the p<0.01 level, and the statistical method used is the T-test.

[0024] Figure 6 The GC-MS mass spectrum of (Z)-9-hexadecenoic acid methyl ester after treatment with dimethyl disulfide is shown.

[0025] Figure 7 This is a vector map. Detailed Implementation

[0026] 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.

[0027] Example 1: Construction of engineered bacteria such as GV3101 / pXZP393a-35s::CmedFAD2.

[0028] Derived from rice leaf roller ( Cnaphalocrocis medinalis )of Cmed The coding region nucleic acid sequence of the FAD2 gene is shown in SEQ ID NO. 1. CmedFAD2 The amino acid sequence of the gene-encoded protein is shown in SEQ ID NO. 2.

[0029] SEQ ID NO. 1 CmedFAD2 gene coding region nucleic acid sequence

[0030] SEQ ID NO. 2 The amino acid sequence of the protein encoded by the CmedFAD2 gene MSSSLLLASTILTKEKQLDDDIPKITTRYVQGNNRTHEWQIVWRNVLAFVYLHASFLYGFYLLATVRVKLATWMFAIFFAVVSGLGVTAGAHRLWAHRAYKARWPLRVVLAVLQTMAFQNHIYEWVRDHRVHHKFTETDADPHNARRGFFFSHMGWLMVRKHKEVIEKGSTIDMSDLEKDPIVMFQK KTYLVVMPIICFIIPAWIPVHYWGEDPWNSWYIAAIFRYTASLHFTWLVNSAAHVWGNRPYDKYIRATDSKTVAICAFGEGWHNYHHVFPWDYKAAELGNYSTNMSTALIDIAAKMGLAYDMKTVSQEMIRNRVARTGDGSHPSSPQQAKQIDDDDHHHPENPVWGWDDKDLPEEDKQLAEIVHKKVE

[0031] Will Cmed The FAD2 gene was ligated downstream of the 35S promoter in the pXZP393a vector (Bao-Jian Ding, PerHofvander, Hong-Lei Wang, Timothy P. Durrett, Sten Stymne & Christer Lofstedt. A plant factory for moth pheromone production. 2014.) in a 5' to 3' sequence (written as 35s::CmedFAD2). Based on the LR response, in CmedFAD2 Add attL1 to the 5' end of the gene, and in CmedFAD2 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 synthesized nucleic acid was ligated into the pXZP393a plant expression vector using an LR kit (invitrogene). The ligation product was transformed into Trans1-T1 *E. coli* and cultured on a medium containing 75 mg / L spectinomycin antibiotic. Colony PCR and sequencing were then performed to identify the positive transformant Trans1-T1 / pXZP393a-35s::CmedFAD2, where the positive plasmid is pXZP393a-35s::CmedFAD2. Figure 7 ).

[0032] Electroporation transformation of Agrobacterium: Agrobacterium GV3101 was cultured in LB liquid medium to prepare GV3101 competent cells. 10 μL of pXZP393a-35s::CmedFAD2 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::CmedFAD2.

[0033] The accession number for the gene encoding a hexadecanoic acid-specific thioesterase (TE16) is KC675176.1, and the accession number for the amino acid sequence of the hexadecanoic acid-specific thioesterase is AGG79283.1. 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.

[0034] SEQ ID NO. 3 eGFP gene nucleic acid sequence ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAA

[0035] SEQ ID NO. 4 Amino acid sequence of the protein encoded by the eGFP gene MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDT LVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK

[0036] Reference Cmed The FAD2 gene was used to ligate TE16 and eGFP downstream of the 35S promoter in the pXZP393a vector, respectively, to obtain pXZP393a-35S::TE16 and pXZP393a-35S::eGFP. These vectors were then electroporated into GV3101 cells to obtain GV3101 / pXZP393a-35S::TE16 and GV3101 / pXZP393a-35S::eGFP positive engineered bacteria, respectively.

[0037] Example 2: Transient conversion of CmedFAD2 in tobacco.

[0038] 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; BMCBiology; 2022)” indicates that wild-type tobacco produces a small amount of (Z)-9-hexadecenoic acid.

[0039] Activate GV3101 / pXZP393a-35s::CmedFAD2 in liquid LB at 230 rpm for 24 h on a shaker at 28 °C. Add 20 μL of the activated bacterial solution to 10 mL of liquid LB and incubate at 230 rpm on a shaker at 28 °C for 16 h until OD = 1.0. Centrifuge at 4000 rpm for 10 min and discard the supernatant. Resuspend the bacterial cells in tobacco injection buffer (10 mmol / L MgCl2, 10 mmol / L morpholinoethanesulfonic acid monohydrate, 100 μmol / L acetylsyl syringone, pH = 5.6) to OD = 1.0. Incubate in the dark at room temperature for 1 to 2 h. Perform the same procedure for GV3101 / pXZP393a-35S::TE16, incubating in the dark at room temperature for 1 to 2 h. Then mix equal volumes of the three bacterial solutions to obtain a mixed bacterial solution. A mixed bacterial solution was injected into the underside of healthy and uniformly growing 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 CmedFAD2 transiently converted tobacco.

[0040] 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.

[0041] Since neither hexadecenoic acid nor octadecenoic acid is volatile, they cannot be directly detected by GC-MS. Therefore, esterification is necessary to determine whether hexadecenoic acid or octadecenoic acid is synthesized in plants.

[0042] Leaves were cut from the CmedFAD2 transiently transformed tobacco and eGFP transiently transformed tobacco cultured for 72 h after injection and divided into two parts. One part was used to determine the gene expression level, and the other part was used to determine the synthesized target compound.

[0043] To determine gene expression levels, untransformed wild-type tobacco (WT) was used as a blank control. Leaves were placed in liquid nitrogen and ground in a mortar. RNA was extracted from tobacco leaves using a plant total RNA extraction kit (Nanjing Novizan). The RNA was then reverse transcribed into cDNA using an RNA reverse transcription kit (Nanjing Novizan), with the tobacco's own housekeeping gene Actin as an internal control (A Abdul Kader Jailani, Vikas Solanki, Anirban Roy, T Sivasudha, Bikash Mandal. A CGMMV geNOme-replicon vector with partial sequences of coat protein gene efficiently expresses GFP in...). Nicotiana benthamiana (2017) The expression levels of the injected genes were detected by real-time quantitative PCR. The base sequences of the upstream primer AF (internal control) are shown in SEQ ID NO. 5, and the downstream primer AR (internal control) are shown in SEQ ID NO. 6; the base sequences of the upstream primer CmedFAD2F (CmedFAD2F) and the downstream primer CmedFAD2R (CmedFAD2R) are shown in SEQ ID NO. 8; the base sequences of the upstream primer TE16F (TE16F) and the downstream primer TE16R (TE16R) are shown in SEQ ID NO. 10; the base sequences of the upstream primer eGFPF (eGFPF) and the downstream primer eGFPR (eGFPR) are shown in SEQ ID NO. 12. The expression levels of the two genes in tobacco transiently transformed with CmedFAD2 are shown in […]. Figure 1 The expression level of the eGFP gene in transiently transformed tobacco is shown in [reference needed]. Figure 2 . Figure 1 The results showed that both CmedFAD2 and TE16 genes were successfully expressed in tobacco transiently transformed by CmedFAD2. Figure 2 The results showed that the eGFP gene was successfully expressed in tobacco transiently transformed with eGFP.

[0044] SEQ ID NO. 5

[0045] Internal reference upstream primer AF

[0046] GGTATTGTGTTGGACTCGGG

[0047] SEQ ID NO. 6

[0048] Internal reference downstream primer AR

[0049] GCTGTGGTAGTGGATGAGTAAC

[0050] SEQ ID NO. 7

[0051] CmedFAD2 gene upstream primer CmedFAD2F

[0052] TGGCCTTCCAGAACCACATC

[0053] SEQ ID NO. 8

[0054] CmedFAD2 gene downstream primer CmedFAD2R

[0055] GTCGATGGTGGACCCCTTTT

[0056] SEQ ID NO. 9

[0057] TE16 gene upstream primer TE16F

[0058] AATGCTGACTCCATCCGCAA

[0059] SEQ ID NO. 10

[0060] TE16 gene downstream primer TE16R

[0061] CCTCTCCAGAGGGATCCACA

[0062] SEQ ID NO. 11

[0063] eGFP gene upstream primer eGFPF

[0064] AGGACGACGGCAACTACAAG

[0065] SEQ ID NO. 12

[0066] eGFP gene downstream primer eGFPR

[0067] TTCTGCTTGTCGGCCATGAT

[0068] 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 mixture was incubated at 90 °C for 1 h; 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 CmedFAD2 are shown below. Figure 3 The chromatogram results of transient conversion of tobacco with eGFP are shown below. Figure 4 .

[0069] Will Figure 3 and Figure 4 The comparison shows that, Figure 3 Chromatographic peaks appeared at the positions of (Z)-9-hexadecenoic acid methyl ester, while Figure 4 The chromatographic peak area of ​​(Z)-9-hexadecenoic acid methyl ester at the corresponding position was significantly reduced, indicating that the expression of CmedFAD2 and TE16 genes in tobacco can increase the yield of (Z)-9-hexadecenoic acid.

[0070] Based on the content of the internal standard (Z)-10-heptadecenoate methyl ester and Figure 3 and Figure 4 The peak area of ​​(Z)-9-hexadecenoic acid methyl ester was used to calculate the content of (Z)-9-hexadecenoic acid methyl ester. Furthermore, the content of (Z)-9-hexadecenoic acid methyl ester produced by esterification of (Z)-9-hexadecenoic acid was calculated per gram of CmedFAD2 transiently converted tobacco leaf and per gram of eGFP transiently converted tobacco leaf. The bar chart results are shown below. Figure 5 .from Figure 5 The results showed that the content of (Z)-9-hexadecenoic acid in tobacco leaves transiently transformed with eGFP was 1.85 μg / g, while the content of (Z)-9-hexadecenoic acid in tobacco leaves transiently transformed with CmedFAD2 was approximately 7.40 μg / g. The content of (Z)-9-hexadecenoic acid in tobacco leaves transiently transformed with CmedFAD2 was significantly higher than that in tobacco leaves transiently transformed with eGFP, with the former being four times higher than the latter.

[0071] Example 3: Treatment of hexadecenoic acid and methyl octadecenoate with dimethyl disulfide.

[0072] Take 50 μL of the total fatty acid methyl ester solution obtained from the CmedFAD2 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)-9-hexadecenoic acid methyl ester is shown in […]. Figure 6 The characteristic ionic fragments of (Z)-9-hexadecenoic acid methyl ester have molecular weights of 145.10 and 217.10. In summary, the substance that increases the synthesis yield in tobacco leaves through CmedFAD2 transient conversion is (Z)-9-hexadecenoic acid.

[0073] 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 the biosynthesis of (Z)-9-hexadecenoic acid, characterized in that, The nucleotide sequence encoded by the gene is shown in SEQ ID NO:

1.

2. The gene according to claim 1, characterized in that, The amino acid sequence encoded by the gene is shown in SEQ ID NO:

2.

3. An expression carrier, characterized in that, Includes the initial vector and the gene as described in claim 1 or 2.

4. The expression vector according to claim 3, characterized in that, The initial carrier was pXZP393a.

5. A host, characterized in that, The transformation or transfection is performed using the expression vector as described in claim 3 or 4; the host is a microorganism.

6. The use of the gene of claim 1 or 2, the expression vector of claim 3 or 4, or the host of claim 5 in the biosynthesis of (Z)-9-hexadecenoic acid.

7. The application according to claim 6, characterized in that, The gene described in claim 1 or 2 is co-expressed in plants with a hexadecanoic acid-specific thioesterase to synthesize (Z)-9-hexadecenoic acid.

8. The application according to claim 7, characterized in that, The accession number for the amino acid sequence of the hexadecimal fatty acid-specific thioesterase is AGG79283.1, and the accession number for its nucleotide sequence is KC675176.

1.

9. A method for synthesizing (Z)-9-hexadecenoic acid, characterized in that, (Z)-9-hexadecenoic acid is synthesized by co-expressing the gene described in claim 1 or 2 with a hexadecanoic acid-specific thioesterase in plants.

10. The method according to claim 9, characterized in that, The plant in question is tobacco.

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