Genes for producing (z)-9-tetradecenoic acid from brassica napus and use thereof
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- XIANGHU LABORATORY
- Filing Date
- 2026-04-15
- Publication Date
- 2026-06-23
Smart Images
Figure CN122256387A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of genetic engineering technology, and in particular to a gene for producing (Z)-9-tetradecenoic acid from rapeseed and its application. Background Technology
[0002] Insect pheromones are informational compounds released by insects that effectively regulate their behavior. Pheromone-based pest behavior control technologies and products are crucial for effective pest monitoring and behavioral influence, enabling green pest control and playing a vital role in ensuring the quality and safety of agricultural products. Compared to traditional pesticides, using pheromones to control pests is an environmentally friendly and long-term effective method. Pheromone pheromones can specifically affect the behavior of target pests while having virtually no toxicity to humans and animals, and minimal impact on beneficial insects and pollinators. More importantly, target pests do not develop resistance to pheromones in the short term.
[0003] Currently, all insect pheromones and related products on the market are obtained through chemical synthesis. However, chemical synthesis severely pollutes the environment and harms human health, and the high cost of raw materials makes it prohibitive for many. This is a key factor restricting the widespread application of insect pheromone products. Therefore, developing green and low-cost pheromone synthesis technologies is crucial for the development of the insect pheromone industry.
[0004] Currently, researchers have developed several alternative methods for heterologous biosynthesis to replace traditional production methods. For example, by genetically engineering Saccharomyces cerevisiae, it has been made to synthesize specific moth sex pheromone precursors (Yuguo Jiang, et al. “Denovo biosynthesis of sex pheromone components of Helicoverpa armigera through an artificial pathway in yeast.” Green Chemistry.). Ding et al. successfully synthesized two insect sex pheromone precursors in tobacco leaves by transiently transfecting tobacco and introducing multiple genes involved in the biosynthesis steps of sex pheromone (BaoJian Ding, et al. “A plant factory for moth pheromone production.” Nature Communications.). Wang et al. used flaxseed seeds and transgenic technology to produce bollworm sex pheromone precursors, with the target compound accounting for about 20% of the total fatty acids in the seeds (HongLei Wang, et al. “Insect pest management with sex pheromone precursors from engineered oil seed plants.” Nature Sustainability). However, there are currently no reports on the biosynthesis of insect sex pheromone precursors in rapeseed seeds. Compared to flaxseed (approximately 1995 kg / ha with an oil content of 40%), rapeseed has a higher yield per acre (approximately 3000 kg / ha with an oil content of 49%). Therefore, utilizing rapeseed to synthesize insect sex pheromone precursors has a broader prospect.
[0005] (Z)-9-Hexadecenoic acid is a precursor to three sex pheromones: (Z)-9-tetradecenoic acid acetate, (Z)-9-tetradecenoic aldehyde, and (Z)-9-tetradecenoic alcohol. These three sex pheromones can be generated from (Z)-9-tetradecenoic acid through known reduction or esterification reactions. Currently, more than a dozen known moth pests use these three compounds as sex pheromones, such as the fall armyworm, the armyworm, and the cutworm (Database of Pheromones and Semiochemicals o http: / / www.pherobase.net4).
[0006] In plant systems, by coordinating the expression of exogenous desaturase genes and related enzymes such as endogenous acyltransferases, it is hoped that this precursor substance can be efficiently accumulated during rapeseed seed oil synthesis. Rapeseed, as a globally important oilseed crop, not only boasts high seed oil content and suitable oil composition but also possesses a mature genetic transformation and large-scale cultivation system, providing an ideal platform for the large-scale, low-cost production of insect sex pheromone precursors. Utilizing rapeseed for the synthesis of such high-value-added compounds can not only reduce the cost of pheromone synthesis in field pest management but also promote the practice of green pest control strategies, achieving both ecological and economic benefits. Summary of the Invention
[0007] The purpose of this invention is to provide a gene for producing (Z)-9-tetradecenoic acid from rapeseed and its application.
[0008] To achieve the above-mentioned objectives, the present invention provides the following technical solution: The present invention provides a gene combination comprising a gene encoding a 14-carbon fatty acid-specific thioesterase and at least one gene encoding a Z9 desaturase.
[0009] Preferably, the nucleotide sequence of the gene encoding a 14-carbon fatty acid-specific thioesterase is as shown in SEQ ID NO:1, or a variant thereof encoding the same function.
[0010] Preferably, the gene encoding the Z9 desaturase is selected from any one or more of the following: Genes with nucleotide sequences as shown in SEQ ID NO:2; Genes with nucleotide sequences as shown in SEQ ID NO:3; Genes with nucleotide sequences as shown in SEQ ID NO:4; Or a variant of it that encodes the same Z9 desaturase function.
[0011] The present invention provides a recombinant vector comprising the aforementioned gene combination.
[0012] Preferably, the recombinant vector is a plant expression vector.
[0013] Preferably, the plant expression vector is pBinGlyBar.
[0014] The present invention provides a method for synthesizing (Z)-9-tetradecenoic acid in rapeseed, comprising co-expressing the aforementioned gene combination in rapeseed.
[0015] This invention provides the application of the gene combination or the recombinant vector described herein in the production of (Z)-9-tetradecenoic acid or in the preparation of insect sex pheromones.
[0016] Compared with the prior art, the present invention has the following beneficial effects: This invention represents a technological breakthrough in the heterologous synthesis of (Z)-9-tetradecenoic acid, a key precursor to insect sex pheromones, from rapeseed seeds, and successfully established a rapeseed bioreactor platform for the efficient synthesis of this compound.
[0017] Compared to traditional chemical synthesis methods, this invention uses rapeseed seeds as the production carrier, eliminating the need for precious metal catalysts. The reaction conditions are mild and energy consumption is low, significantly reducing environmental pollution and production costs. Furthermore, rapeseed has higher yield per unit area and oil content than flaxseed, allowing for higher yields of the target product within the same planting area. This demonstrates good potential for large-scale production and a cost-competitive advantage, providing a feasible path for the green and low-cost commercial supply of insect pheromones.
[0018] The (Z)-9-tetradecenoic acid produced by this invention can serve as a universal precursor to various moth sex pheromones and can be further developed into a behavior regulation product with high specificity and environmental compatibility. The widespread application of this technology helps reduce the use of chemical pesticides, delays the development of pesticide resistance in pests, protects the ecological environment and biodiversity, and aligns with the strategic needs of green and high-quality agricultural development, thus yielding significant social, ecological, and economic benefits. Attached Figure Description
[0019] 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.
[0020] Figure 1 The image shows the GC-MS chromatogram of total fatty acid methyl esters in the seeds of Bna-Z9-14 transgenic rapeseed.
[0021] Figure 2 This is a GC-MS chromatogram of total fatty acid methyl esters in rapeseed seeds from the control group.
[0022] Figure 3 GC-MS chromatogram of (Z)-9-tetradecenoic acid methyl ester standard.
[0023] Figure 4 A vector map for inserting T-DNA fragments into plants. Detailed Implementation
[0024] 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.
[0025] The sequences used in the following examples: SEQ ID NO: 1 SEQ ID NO:2 SEQ ID NO:3 SEQ ID NO:4 SEQ ID NO:5 MVAAAASAAFFSVATPRTNISPSSLSVPFKPKSNHNGGFQVKANASAHPKANGSAVSLKSGSLETQEDKTSSSSPPPRTFINQLPVWSMLLSAVTTVFGVAEKQWPMLDRKSKRPDMLVEPLGVDRIVYDGVSFRQSFSIRSYEIGADRTASIETLMNMFQETSLNHCKIIGLLNDGFGRTPEMCKRDLIWVVTKMQIEVNRYPTWGDTIEVNTWVSASGKHGMGRDWLISDCHTGEILIRATSVWAMMNQKTRRLSKIPYEVRQEIEPQFVDSAPVIVDDRKFHKLDLKTGDSICNGLTPRWTDLDVNQHVNNVKYIGWILQSVPTEVFETQELCGLTLEYRRECGRDSVLESVTAMDPSKEGDRSLYQHLLRLEDGADIVKGRTEWRPKNAGAKGAILTGKTSNGNSIS SEQ ID NO:6 MAPYSRIYHQDKSSRETGVLFEDDAQTVDSDLTTDRFQLKRAEKRRLPLVWRNIILFALVHLAALYGLHSIFTRAKLATTLFAAGLYIIGMLGVTAGAHRLWAHRTYKAKWPLRLLLVIFNTIAFQDAVYHWARDHRVHHKYSETDADPHNATRGFFFSHVGWLLCKKHPDIKEKGRGLDLSDLRADPILMFQRKHYYILMPLACFVLPTVIPMVYWNETLASSWFVATMFRWCFQLNMTWLVNSAAHKFGNRPYDKTMNPTQNAFVSAFTFGEGWHNYHHAFPWDYKTAEWGCYSLNITTAFIDLFAKIGWAYDLKTVAPDVIQRRVLRTGDGSHELWGWGDKDLTAEDARNVLLVDKSR SEQ ID NO:7 MAPYSRIYHQDKSSRETGVLFEDDAQTVDSDLTTDRFQLKRAEKRRLPLVWRNIILFALVHLAALYGLHSIFTRAKLATTLFAAGLYIIGMLGVTAGAHRLWAHRTYKAKWPLRLLLVIFNTIAFQDAVYHWARDHRVHHKYSETDADPHNATRGFFFSHVGWLLCKKHPDIKEKGRGLDLSDLRADPILMFQRKHYYILMPLACFVLPTVIPMVYWNETLASSWFVATMFRWCFQLNMTWLVNSAAHKFGNRPYDKTMNPTQNAFVSAFTFGEGWHNYHHAFPWDYKTAEWGCYSLNITTAFIDLFAKIGWAYDLKTVAPDVIQRRVLRTGDGSHELWGWGDKDLTAEDARNVLLVDKSR SEQ ID NO: 8 MAPYSRIYHQDKSSRETGVLFEDDAQTVDSDLTTDRFQLKRAEKRRLPLVWRNIILFALVHLAALYGLHSIFTRAKLATTLFAAGLYIIGMLGVTAGAHRLWAHRTYKAKWPLRLLLVIFNTIAFQDAVYHWARDHRVHHKYSETDADPHNATRGFFFSHVGWLLCKKHPDIKEKGRGLDLSDLRADPILMFQRKHYYILMPLACFVLPTVIPMVYWNETLASSWFVATMFRWCFQLNMTWLVNSAAHKFGNRPYDKTMNPTQNAFVSAFTFGEGWHNYHHAFPWDYKTAEWGCYSLNITTAFIDLFAKIGWAYDLKTVAPDVIQRRVLRTGDGSHELWGWGDKDLTAEDARNVLLVDKSR
[0026] Example 1: Construction of GV3101 / pBinGlyBar-Z9-14 engineering bacteria
[0027] The full sequences of TE14, DME_Ath, DME_Bna, and DME_Nta were synthesized by Zhejiang Youkang Biotechnology Co., Ltd., and homologous arm sequences were added to both ends of each gene according to the LR kit (invitrogene) instructions. Based on the LR reaction, the above four genes were ligated into the pBinGlyBar plant expression vector (BaoJian Ding, et al. “A plant factory for moth pheromone production.” Nature Communications.). The ligation product was transformed into Trans1-T1 Escherichia coli and cultured under 75 mg / L spectinomycin antibiotic. Then, the transformation product was identified by bacterial PCR and sequencing, and the positive transformant Trans1-T1 / pBinGlyBar-Z9-14 was obtained, of which the positive plasmid was pBinGlyBar-Z9-14.
[0028] Electroporation transformation of Agrobacterium: Agrobacterium GV3101 was cultured in LB liquid medium to prepare GV3101 competent cells. 10 μL of pBinGlyBar-Z9-14 expression vector was added to 50 μL of Agrobacterium competent cell culture for electroporation transformation. Then, 800 μL of LB liquid medium 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 medium 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 electrophoresis to obtain positive clones, yielding GV3101 / pBinGlyBar-Z9-14.
[0029] Gene encoding 14-carbon fatty acid thioesterase ( TE14 The sequence of ) is SEQ ID NO: 1, and the sequence of the amino acid of the fourteen-carbon fatty acid thioesterase is SEQ ID NO: 5.
[0030] The gene encoding the first Z9 desaturase ( DME_Ath The nucleotide sequence of ) is SEQ ID NO: 2, and the amino acid sequence of the first Z9 desaturase is SEQ ID NO: 6.
[0031] The gene encoding the second Z9 desaturase ( DME_Bna The nucleotide sequence of the first Z9 desaturase is SEQ ID NO: 3, and the amino acid sequence of the second Z9 desaturase is SEQ ID NO: 7.
[0032] The gene encoding the third Z9 desaturase ( DME_NtaThe nucleotide sequence of the third Z9 desaturase is SEQ ID NO: 4, and the amino acid sequence of the third Z9 desaturase is SEQ ID NO: 8.
[0033] Example 2: Genetic transformation of pBinGlyBar-Z9-14 in rapeseed
[0034] Select plump and healthy rapeseed seeds (Zhongshuang 11), disinfect them with 75% alcohol for 30 seconds, then rinse once with sterile water, followed by soaking in 0.15% HgCl2 for 10 minutes, rinsing 2-3 times with sterile water, and finally soaking in sterile water for 30 minutes. Sow the disinfected rapeseed seeds on MO medium. Culture conditions: 23℃, dark culture for 7 days. When the hypocotyl of the sterile rapeseed seedlings reaches the mouth of the bottle, remove the sterile seedlings with forceps, remove the cotyledon petioles and cotyledon tips, and cut the hypocotyl into 1-2 cm segments as explants, placing them on pre-culture medium. Incubate in the dark at 23℃ for 3 days.
[0035] Two days before transformation, Agrobacterium was streaked onto a petri dish using a fine inoculation loop. The corresponding strain was then inoculated into an Agrobacterium suspension (containing MS liquid medium and acetylsuccine), and the Erlenmeyer flask was shaken on a shaker for 30 minutes. The cultured bacterial solution was transferred to a centrifuge tube, and the Agrobacterium concentration (OD) was measured. 600 (value), OD 600 The value was 0.1~0.2. Rapeseed explants, pre-cultured for 3 days, were placed in Agrobacterium suspension for 10 minutes for infection. The infected explants were then dried on filter paper and cultured on M1 medium for 2 days. Next, the explants were transferred to M2 medium for callus induction, allowing callus tissue to grow. The culture was carried out at 23℃ under light for 7 days. Selected callus tissues were transferred to M3 medium containing resistance for selection. The culture was carried out at 23℃ under light for 30~45 days. During differentiation, if seedlings appeared on the callus, they were transferred to M4 medium containing the corresponding resistance to root. This yielded rapeseed seedlings containing pBinGlyBar-Z9-14 (T0 generation), which are Bna-Z9-14 transgenic rapeseed. T0 generation rapeseed seeds were sown in a greenhouse, and when they grew to 3~4 leaves, glufosinate was sprayed, and positive plants were selected. Propagate to the T3 generation using this method, and screen out positive homozygous plants for the detection of the target product.
[0036] Table 1 Culture medium formulation
[0037] Example 3: Detection of (Z)-9-tetradecenoic acid in Bna-Z9-14 transgenic rapeseed
[0038] Since (Z)-9-tetradecenoic acid cannot be directly measured, it needs to be esterified first. The leaf veins were removed, the sample was weighed, and placed in a 4ml glass bottle for esterification: 1ml of methanol (containing 2% sulfuric acid) was added, and the solution was incubated at 90℃ for 1 hour; 1ml of water (containing 0.075mol / L acetic acid) was added, and the solution was vortexed for 15 seconds; 1ml of heptane was added, and the solution was vortexed for 15 seconds; the solution 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 1µL total fatty acid methyl ester solution were determined using GC-MS (Agilent). The chromatogram results for Bna-Z9-14 transgenic rapeseed seeds are shown below. Figure 1 The chromatogram results of the control group rapeseed seeds are shown in the figure. Figure 2 The chromatographic results of the (Z)-9-tetradecenoic acid methyl ester standard are shown in [the table below]. Figure 3 .
[0039] Will Figure 1 , Figure 2 and Figure 3 The comparison shows that, Figure 1 and Figure 3 Chromatographic peaks appeared at the positions of methyl (Z)-9-tetradecenoate, with the same retention time. Figure 2 The absence of chromatographic peaks at the corresponding positions indicates that (Z)-9-tetradecenoic acid can be biosynthesized in Bna-Z9-14 transgenic rapeseed seeds.
[0040] 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 combination of genes, characterized in that, The gene combination includes a gene encoding a 14-carbon fatty acid-specific thioesterase and at least one gene encoding a Z9 desaturase.
2. The combination of claim 1, wherein, The nucleotide sequence of the gene encoding a 14-carbon fatty acid-specific thioesterase is shown in SEQ ID NO:1, or a variant thereof encoding the same function.
3. The combination of claims 1 or 2, wherein The gene encoding the Z9 desaturase is selected from any one or more of the following: Genes with nucleotide sequences as shown in SEQ ID NO:2; Genes with nucleotide sequences as shown in SEQ ID NO:3; Genes with nucleotide sequences as shown in SEQ ID NO:4; Or a variant of it that encodes the same Z9 desaturase function.
4. A recombinant vector, characterized in that, It comprises the gene combination as described in any one of claims 1 to 3.
5. The recombinant vector of claim 4, wherein, The recombinant vector is a plant expression vector.
6. The recombinant vector of claim 5, wherein, The plant expression vector is pBinGlyBar.
7. A method for synthesizing (Z)-9-tetradecenoic acid in rapeseed, characterized in that, This includes co-expressing the gene combination described in any one of claims 1 to 3 in rapeseed.
8. The use of the gene combination according to any one of claims 1 to 3 or the recombinant vector according to any one of claims 4 to 6 in the production of (Z)-9-tetradecenoic acid or the preparation of insect sex pheromones.