Method for synthesizing sex pheromone intermediate of moth insects

Abstract

The invention relates to the technical field of synthesizing a sex pheromone component precursor substance by a biological method, further relates to a method for synthesizing a moth insect sex pheromone intermediate, and particularly relates to a recombinant plasmid containing cnaphalocrocis medinalis desaturase and elongase, a recombinant cell, an expression system and a construction method and application thereof. By adopting the method provided by the invention, the sex pheromone component precursor substance can be obtained through a non-chemical synthesis method for the first time, and when the sex pheromone component precursor substance is synthesized, the method is high in specificity, free of impurities or almost free of byproducts, and environment-friendly.

Description

Technical Field

[0001] This application relates to the technical field of precursor substances for the biological synthesis of sex pheromone components, and further to methods for synthesizing intermediates of sex pheromones in moths, specifically including recombinant plasmids, recombinant cells, expression systems, construction methods, and applications of rice leaf roller desaturase and elongation enzyme. Background Technology

[0002] Z13-18:Ald is a chemical substance, specifically an aldehyde compound with the chemical name (Z)-9-Octadecenal. This substance is commonly used in agriculture as a sex pheromone for moths such as the rice leaf folder and rice stem borer, enabling the large-scale trapping of these species to control rice pests. Currently, Z13-18:Ald mainly relies on chemical catalytic industrial synthesis. Chemical synthesis involves multiple complex catalytic reactions, making it difficult and generating numerous byproducts. These byproducts may affect the activity and purity of the sex pheromone. Furthermore, the rice leaf folder's sex pheromone is an unsaturated fatty aldehyde with cis-trans isomers, making it difficult to control the isomer ratio during chemical synthesis, which can also lead to impurities and low biological activity. Moreover, the organic solvents and other potentially toxic chemical reagents used in the production process may pose a health risk, and the production process may also generate toxic byproducts, causing secondary pollution to the environment.

[0003] Current insect sex pheromone synthesis technologies do not yet involve fatty acid carbon chain elongation reactions. Taking the synthesis of Z11-16:Acid as an example, 16:Acid is first used as a precursor, and an unsaturated double bond is introduced at the 11th carbon position to generate Z11-16:Acid. In the common fatty acid synthesis metabolic pathway in organisms, the reaction begins with acetyl-CoA as the starting two-carbon unit. In each round of the reaction, one malonyl-CoA is incorporated, and after seven cycles of dehydrogenation, hydration, re-dehydrogenation, and thiolysis, palmitoyl-ACP is generated. Since β-ketoacyl-ACP synthase can only accept acyl groups with a maximum of 14 carbon atoms, fatty acid synthases can only synthesize up to hexadecanoic acid. The synthesis of longer-chain fatty acids requires the catalysis of ultra-long-chain fatty acid elongation enzymes.

[0004] The synthesis of Z13-18:Ald, the main sex pheromone component of rice leaf roller, depends on carbon chain elongation. During the synthesis process, the precursor Z11-16:Acid is further elongated into Z13-18:Acid, and then Z13-18:Ald is generated through a redox reaction.

[0005] This technology solves the problem that carbon chains cannot be elongated by elongation enzymes during the biosynthesis of sex pheromones. Summary of the Invention

[0006] This application provides a method for synthesizing intermediates of sex pheromones in moths, specifically including recombinant plasmids, recombinant cells, expression systems, their construction methods, and applications containing rice leaf roller desaturase and elongation enzymes. This application screens desaturases and elongation enzymes capable of successfully synthesizing sex pheromone precursors in a tobacco expression system; these methods exhibit high specificity, produce few byproducts, and solve the problem of not obtaining elongation products in existing biosynthetic methods; the method is green, environmentally friendly, and energy-saving; the concept and method for synthesizing sex pheromone precursors can also be further applied to the synthesis of more fatty acid derivatives catalyzed by fatty acid elongation enzymes, thus possessing great application potential.

[0007] This application involves the following:

[0008] In a first aspect, this application provides a desaturase, the amino acid sequence of which is shown in any one or more of SEQ ID NO. 62-70; or has at least 80% sequence identity with the amino acid sequences shown in any one or more of SEQ ID NO. 62-70.

[0009] Optionally, the amino acid sequence of the desaturase is as shown in SEQ ID NO. 65; or has at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO. 65. For example, it has 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO. 65.

[0010] Optionally, the nucleotide sequence encoding the open reading frame of the desaturase is as shown in any one or more of SEQ ID NO. 33-41; or has at least 80% sequence identity with any one or more of the nucleotide sequences shown in SEQ ID NO. 33-41.

[0011] Optionally, the nucleotide sequence encoding the open reading frame of the desaturase is as shown in SEQ ID NO. 36; or has at least 80% sequence identity with the nucleotide sequence shown in SEQ ID NO. 36. For example, it has 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the nucleotide sequence shown in SEQ ID NO. 36.

[0012] Secondly, this application provides an elongation enzyme whose amino acid sequence is shown in any one or more of SEQ ID NO. 55-61; or has at least 80% sequence identity with the amino acid sequences shown in any one or more of SEQ ID NO. 55-61.

[0013] Optionally, the amino acid sequence of the elongation enzyme is as shown in any one or more of SEQ ID NO. 55-58, 61; or has at least 80% sequence identity with the amino acid sequences shown in any one or more of SEQ ID NO. 55-58, 61. For example, it has 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequences shown in any one or more of SEQ ID NO. 55-58, 61.

[0014] Optionally, the nucleotide sequence encoding the open reading frame of the elongase is as shown in any one or more of SEQ ID NO. 42-48; or has at least 80% sequence identity with any one or more of the nucleotide sequences shown in SEQ ID NO. 42-48. For example, it has 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with any one or more of the nucleotide sequences shown in SEQ ID NO. 42-48.

[0015] Optionally, the nucleotide sequence encoding the open reading frame of the elongase is as shown in any one or more of SEQ ID NO. 42-45, 48; or has at least 80% sequence identity with any one or more of the nucleotide sequences shown in SEQ ID NO. 42-45, 48. For example, it has 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with any one or more of the nucleotide sequences shown in SEQ ID NO. 42-45, 48.

[0016] The aforementioned amino acid sequences with sequence identity can refer to enzyme mutants obtained by conservatively substituting amino acids into an enzyme containing a specific amino acid sequence (e.g., SEQ ID NO. 36), which are also within the scope of protection of this application. Conservative substitutions can be substitutions between amino acids of the same class: for example, substitutions between polar amino acids, such as glycine, serine, threonine, tyrosine, cysteine, asparagine, and glutamine; substitutions between nonpolar amino acids, such as alanine, valine, tryptophan, leucine, proline, and methionine; and substitutions between acidic amino acids, basic amino acids, and hydrophobic amino acids, etc. It can also be any enzyme mutant obtained by functionally mutating an enzyme with a specific amino acid sequence (e.g., SEQ ID NO. 36), where the "functional mutation" does not change or substantially does not change the catalytic activity of the desaturated enzyme; "substantially does not change the catalytic activity of the desaturated enzyme" means that the catalytic activity of the enzyme mutant, compared with that before mutation, has an activity change of up to 10%, up to 5%, up to 4%, up to 3%, up to 2%, or up to 1%; specifically, for example, an activity change of 1%, 2%, 3%, 4%, 5%, 7%, 9%, or 10%. This activity change can be an increase or a decrease in activity.

[0017] Thirdly, this application provides a recombinant plasmid comprising:

[0018] (1) Plasmid vectors; and

[0019] (2) The nucleotide sequence encoding the above-mentioned desaturase; and

[0020] (3) The nucleotides encoding the elongation enzyme mentioned above;

[0021] The nucleotide sequence encoding the desaturase is recombined on the plasmid vector;

[0022] The nucleotide sequence encoding the elongase is recombined on the plasmid vector.

[0023] Optionally, the nucleotide sequence encoding the desaturase comprises or is a nucleotide sequence encoding an open reading frame of the desaturase.

[0024] Optionally, the nucleotide sequence encoding the elongase comprises or is a nucleotide sequence encoding the open reading frame of the elongase.

[0025] Optionally, the plasmid vector is selected from pYEX-CHT-U-DEST and pXZP393.

[0026] Fourthly, this application provides recombinant cells, said recombinant cells comprising:

[0027] (1) Competent cells; and

[0028] (2) The nucleotide sequence encoding the above-mentioned desaturase and the nucleotide sequence encoding the above-mentioned elongation enzyme;

[0029] And / or, the recombinant cells contain the recombinant plasmids described above.

[0030] Optionally, the competent cells are Agrobacterium competent cells.

[0031] Fifthly, this application provides a tobacco expression system for synthesizing precursor substances of sex pheromones, the tobacco expression system comprising a nucleotide sequence encoding the aforementioned desaturase and a nucleotide sequence encoding the aforementioned elongation enzyme;

[0032] Or the aforementioned recombinant plasmids;

[0033] Or the aforementioned recombinant cells.

[0034] Optionally, the precursor of the sex pheromone component is a long-chain unsaturated fatty acid; further, it is (Z)-11-hexadecenoic acid and / or (Z)-13-octadecenoic acid.

[0035] Sixthly, this application provides an attB primer set, wherein the attB primer set is selected from any one or more pairs of the following group of primer pairs:

[0036] As shown in SEQ ID NO:1-2, the attB primer pair;

[0037] As shown in SEQ ID NO:3-4, the attB primer pair;

[0038] As shown in SEQ ID NO:5-6, the attB primer pair;

[0039] The attB primer pair shown in SEQ ID NO:7-8;

[0040] As shown in SEQ ID NO:9-10, the attB primer pair;

[0041] The attB primer pair shown in SEQ ID NO:11-12;

[0042] The attB primer pair shown in SEQ ID NO:13-14;

[0043] The attB primer pair shown in SEQ ID NO:15-16;

[0044] The attB primer pair shown in SEQ ID NO:17-18;

[0045] The attB primer pair shown in SEQ ID NO:19-20;

[0046] The attB primer pairs shown in SEQ ID NO:21-22;

[0047] The attB primer pair shown in SEQ ID NO:23-24;

[0048] The attB primer pair shown in SEQ ID NO:25-26;

[0049] The attB primer pair shown in SEQ ID NO:27-28;

[0050] The attB primer pair shown in SEQ ID NO:29-30;

[0051] The attB primer pairs are shown in SEQ ID NO:31-32.

[0052] Sixthly, this application provides a method for constructing recombinant plasmids for synthesizing precursor substances of sex pheromones, the method comprising the following steps:

[0053] Construction of the entry vector: The cDNA of the sex pheromone gland is mixed with the above attB primer set, and after amplification, the amplification product is mixed with the initial vector and BP cloning enzyme and reacted to obtain the entry clone;

[0054] Construction of recombinant plasmid: The initiation vector, plasmid vector, and LR cloning enzyme are mixed and reacted to obtain the recombinant plasmid.

[0055] Optionally, the initial vector is the pDONR221 plasmid.

[0056] Optionally, the plasmid vector is pYEX-CHT-U-DEST or pXZP393.

[0057] Seventhly, this application provides a method for constructing recombinant cells, the method comprising the following steps:

[0058] The recombinant plasmid described above, and / or the recombinant plasmid constructed using the above method, is mixed with competent cells and introduced into the competent cells.

[0059] Eighthly, this application provides a method for constructing a tobacco expression system for synthetic sex pheromone component precursors, the method comprising the following steps:

[0060] The recombinant cells described above, and / or the recombinant cells constructed using the methods described above, are injected into tobacco tissue to obtain the tobacco expression system for synthesizing precursor substances of sex pheromones.

[0061] Optionally, the tobacco tissue is a tobacco leaf.

[0062] Ninthly, this application provides a method for synthesizing precursor substances of sex pheromones, the method comprising the following steps:

[0063] This enables the expression of the tobacco expression system described above, and / or the tobacco expression system constructed using the methods described above, to synthesize pheromone precursor substances, namely long-chain unsaturated fatty acids, through tobacco tissue.

[0064] Invention Effects

[0065] 1. This application is the first to screen suitable desaturases and elongases, enabling the successful co-expression of the open reading frame nucleic acid sequences encoding them in a tobacco system, so as to successfully prepare the precursor substances of sex pheromones, namely long-chain unsaturated fatty acids, such as (Z)-11-hexadecenoic acid and / or (Z)-13-octadecenoic acid, by biological methods; the desaturases and elongases obtained by this special screening solve the problem that the carbon chain cannot be elongated by elongases when synthesizing sex pheromones by biological methods.

[0066] 2. Based on the coding sequences of the desaturase and elongation enzyme obtained by this special screening, this application also proposes a biosynthesis method for sex pheromone component precursors. This method has the following advantages: (1) The synthesis of the sex pheromone component precursor does not depend on chemical synthesis, thus avoiding the use of organic reagents, especially toxic reagents, in the process of chemical synthesis, resulting in less environmental pollution and greater environmental friendliness; (2) The synthesis of the sex pheromone component precursor is completed under enzyme catalysis, which has stronger specificity, and the product is unique, free of impurities or almost free of impurities, especially without the production of corresponding isomers, and also avoids the production of toxic byproducts; (3) This method synthesizes sex pheromone component precursors in a tobacco expression system. Unlike the microbial synthesis method, the method of this application does not require sugar, glycerol, etc. as carbon sources, but directly uses carbon dioxide in the air as a carbon source, which is more energy-efficient and environmentally friendly.

[0067] 3. The method of this application can further utilize the obtained sex pheromone precursor material to synthesize more fatty acid derivatives catalyzed by fatty acid elongation enzymes. For example, common 18-carbon fatty acid derivatives can be derived from 16-carbon fatty acids and their derivatives through carbon chain elongation. Further examples include the synthesis of stearic acid, oleic acid, linoleic acid, linolenic acid, arachidonic acid, etc. Therefore, the discovery of this application has a significant impact on the synthesis of long-chain fatty acids and their derivatives. Attached Figure Description

[0068] Figure 1Four candidate desaturase genes, Cmed070400, Cmed116530, Cmed116590, and Cmed116600, were heterologously expressed in Nicotiana benthamiana. Compounds present in the original tobacco leaves are indicated in black, while substances produced by the introduced desaturases are shown in red.

[0069] Figure 2 In Nicotiana benthamiana, Cmed070400 and five candidate elongation enzyme genes, Cmed092430, Cmed092440, Cmed092450, Cmed153350, and Cmed063180, were co-expressed. Compounds originally present in tobacco leaves are indicated in black; introduced desaturases and substances produced by elongation enzymes are shown in red.

[0070] Figure 3 Cmed070400 and the elongase candidate gene Cmed092440 were co-expressed in Nicotiana benthamiana. Detailed Implementation

[0071] It should be noted that certain terms are used in the specification and claims to refer to specific components. Those skilled in the art will understand that different terms may be used to refer to the same component. This specification and claims do not distinguish components based on differences in terminology, but rather on differences in function. Specific embodiments of this application will now be described in more detail with reference to the accompanying drawings. Although specific embodiments of this application are shown in the drawings, it should be understood that this application can be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided to enable a more thorough understanding of this application and to fully convey the scope of this application to those skilled in the art.

[0072] Reagents and materials

[0073] Rice leaf roller larvae were collected and cultured in the laboratory until they emerged (photoperiod 14L:10D). Before emergence, males and females were cultured separately, and after emergence, they were fed 10% honey water every day.

[0074] The main reagent kits used are as follows: the reverse transcription kit was HiScriptⅢRT SuperMix for qPCR, purchased from Nanjing Novizan Biotechnology Co., Ltd.; the PCR high-fidelity enzyme KOD One... TMPCR Master Mix-Blue- was purchased from Takara Bio Inc.; the GeneJET Gel Extraction Kit was purchased from Thermo Fisher Scientific; and the Gateway BP Clonase II Enzyme Mix and Gateway LR Clonase II Enzyme Mix were purchased from Thermo Fisher Scientific.

[0075] The activity of calling behavior in the rice leaf roller is roughly synchronized with the titer changes of (cis)-13-octadecenal (Z13-18:Ald), the main sex pheromone component in its pheromone gland (PG). In the laboratory, this calling behavior was observed to occur primarily during the dark period 2-4 days after adult emergence, specifically 4-6 hours after entering the dark period (photoperiod 16L:8D). Therefore, unmated adults 2-4 days after emergence were selected for tissue extraction. Within 4-6 hours of entering the dark period, both male and female adults were dissected, and the head, thorax, abdomen, legs, wings, antennae, and sex pheromone glands or taste brushes were collected.

[0076] RNA was extracted from various tissues using the Trizol method. The extracted RNA was then sent to Beijing Novogene Biotechnology Co., Ltd. for reference transcriptome sequencing using the Novaseq-PE150 sequencing platform.

[0077] The protein-coding sequences of the rice stem borer desaturase CsupYPAQ (GenBank accession number: MN453822) and the rice stem borer ultra-long chain fatty acid elongation enzyme Csup16941 were used as homology alignment sequences. The protein-coding sequences of the rice leaf folder reference genome (GenBank: GCA_014851415.1) were used as a local database. Candidate genes for desaturases and elongations were screened from the rice leaf folder genome using BLAST (E-value = 0.05), resulting in nine candidate desaturase genes (Cmed009890, C...). The nine candidate desaturases (Cmed022650, Cmed054120, Cmed070400, Cmed077910, Cmed116530, Cmed116590, Cmed116600, Cmed138850) and seven candidate elongases (Cmed092430, Cmed092440, Cmed092450, Cmed153350, Cmed153360, Cmed153370, Cmed063180) are listed in Table 1. The open reading frames (ORF) of these nine candidate desaturases and seven candidate elongases are shown in Table 2.

[0078] Table 1

[0079]

[0080]

[0081]

[0082]

[0083]

[0084]

[0085] Table 2

[0086]

[0087]

[0088]

[0089]

[0090] RNA extracted from various tissues during the peak synthesis period of the rice leaf folder sex pheromone was used as cDNA. Using PG cDNA as a template, candidate genes were screened and amplified by PCR. Gene-specific primers containing attB1 and attB2 recombination sites were used for gateway cloning. PCR products were separated by agarose gel electrophoresis, and the DNA was purified by gel recovery. Subsequently, the open reading frames (ORFs) of the candidate genes were cloned into the pDONR221 vector using BP recombinase to construct entry clones. After sequencing verification, LR reaction was used to construct yeast (pYEX-CHT-U-DEST) and tobacco expression vectors (pXZP393). The function of each gene was analyzed in the yeast eukaryotic expression system and the Agrobacterium-mediated tobacco expression system, respectively. Fatty acids were extracted and fatty acid methyl esters were prepared. Gas chromatography-mass spectrometry was used to detect the presence of the target products: Z11-16:Me and Z13-18:Me.

[0091] The following will describe in detail the steps of reverse transcription, polymerase chain reaction, agarose gel DNA recovery, and BP reaction.

[0092] Example

[0093] Example 1. Cloning of candidate genes for rice leaf roller desaturase and elongation enzyme

[0094] 1. Tissue extraction

[0095] Unmated adult rice leaf rollers, 2-4 days after emergence, were selected for tissue extraction. Within 4-6 hours of entering the dark period, both female and male adults were dissected, and heads (30), thoraxes (3), abdomen (1, after removing the sex pheromone glands), legs (90), wings (15 pairs), antennae (30 pairs), and sex pheromone glands (female, 30) or taste brushes (male, 30) were collected. The dissection method for the female sex pheromone glands and male taste brushes was as follows: the abdomen was gently squeezed to extend the gonads or taste brushes, which were then cut off with dissecting scissors and frozen in liquid nitrogen for later use.

[0096] 2. RNA extraction

[0097] (1) Use a tissue homogenizer to fully break the cells and add Trizol to 1 mL. Let stand on ice for 5 min.

[0098] (2) Centrifuge at 4℃ and 12000rpm for 10min, and transfer 800μL of supernatant into a new 1.5mL centrifuge tube.

[0099] (3) Add 200 μL of pre-cooled chloroform, shake well to mix, and let stand on ice for 5 min.

[0100] (4) Centrifuge at 4℃ and 12000rpm for 15min, and aspirate 700μL of supernatant into a new centrifuge tube.

[0101] (5) Add 700 μL of pre-cooled isopropanol, shake thoroughly to mix, and let stand at -80℃ for 30 min.

[0102] (6) Centrifuge at 4℃ and 12000rpm for 15min and discard the supernatant.

[0103] (7) Add 1 mL of 75% ethanol solution, centrifuge at 4℃ and 12000 rpm for 5 min, and discard the supernatant.

[0104] (8) Repeat (7) and dry the remaining ethanol in a fume hood.

[0105] (9) Dissolve in 20 μL of RNase-free water and measure RNA concentration using a NanoDrop micro spectrophotometer.

[0106] 3. Reverse transcription

[0107] (1) Prepare the reverse transcription system: 4 μL 4×gDNA wiper Mix, 500 ng RNA, and RNase-free water to make up to 16 μL.

[0108] (2) After reacting at 42℃ for 2 min, add 4 μL of 5×HiScriptⅢqPCR Super Mix.

[0109] (3) React at 37℃ for 15 min, then deactivate at 85℃ for 5 s.

[0110] 4. Polymerase chain reaction

[0111] (1) Using PG cDNA (cDNA of sex pheromone glands) as a template, the selected gene fragments were specifically amplified. The reaction system was prepared according to the components in Table 3, and amplification was performed on a PCR instrument according to the procedure in Table 4. The specific Primers in Table 3 are shown in Table 5.

[0112] Table 3 KOD One PCR Reaction System

[0113]

[0114] Table 4 KOD One PCR Reaction Procedure

[0115]

[0116] Table 5 Primer Information

[0117]

[0118]

[0119]

[0120] Note in Table 5 that attB1_Cmed92430 For and attB2_Cmed092430Rev are primers for the elongase candidate gene Cmed092430; attB1_Cmed92440 For and attB2_Cmed092440Rev are primers for the elongase candidate gene Cmed092440; attB1_Cmed92450 For and attB2_Cmed092450Rev are primers for the elongase candidate gene Cmed092450; attB1_Cmed153350For and attB2_Cmed0153350Rev are primers for the elongase candidate gene Cmed153350; attB1_Cmed153360For and attB2_Cmed0153360Rev are primers for the elongase candidate gene Cmed153360; attB1_Cmed153370For and attB2_Cmed092450Rev are primers for the elongase candidate gene Cmed153360; ed0153370Rev is a primer targeting the elongase candidate gene Cmed153370; attB1_Cmed063180For and attB2_Cmed063180Rev are primers targeting the elongase candidate gene Cmed063180; attB1_Cmed009890For and attB2_Cmed009890Rev are primers targeting the desaturase candidate gene Cmed009890; attB1_Cmed022650For and attB2_Cmed022650Rev are primers targeting the desaturase candidate gene Cmed009890. Primers for the desaturase candidate gene Cmed022650; attB1_Cmed054120For and attB2_Cmed054120Rev are primers for the desaturase candidate gene Cmed054120; attB1_Cmed070400For and attB2_Cmed070400Rev are primers for the desaturase candidate gene Cmed070400; attB1_Cmed077910For and attB2_Cmed077910Rev are primers for the desaturase candidate gene Cm Primers for ed077910; attB1_Cmed116530For and attB2_Cmed116530Rev are primers for the desaturase candidate gene Cmed116530; attB1_Cmed116590For and attB2_Cmed116590Rev are primers for the desaturase candidate gene Cmed116590; attB1_Cmed116600For and attB2_Cmed116600Rev are primers for the desaturase candidate gene Cmed116600.attB1_Cmed138850For and attB2_Cmed138850Rev are primers targeting the desaturase candidate gene Cmed138850.

[0121] (2) Prepare agarose gel in advance. Dissolve 0.8% agarose in 1×TAE, heat in microwave until the agarose is completely dissolved, cool slightly and add GelBlue nucleic acid dye at a ratio of 1:1000. Pour into a clean gel holder with comb teeth inserted and let stand until solidification.

[0122] (3) The PCR products were subjected to agarose gel electrophoresis. After loading the samples, electrophoresis was performed at 130V for 20 min. The gel was then imaged under ultraviolet light, and the images were saved after taking pictures.

[0123] 5. Agarose gel DNA recovery

[0124] (1) Cut off the gel electrophoresis band, put it into a 1.5mL EP tube, add 600μL Buffer GDP, and incubate in a 55℃ water bath for 10min.

[0125] (2) Let the solution stand at room temperature, transfer it into the adsorption column, insert the adsorption column into the collection tube, and centrifuge at 12000 rpm for 30s.

[0126] (3) Transfer the filtrate into the adsorption column and centrifuge at 12,000 rpm for 30 seconds.

[0127] (4) Discard the waste liquid, add 700μL Buffer GW, and centrifuge at 12000rpm for 30s.

[0128] (5) Repeat (4), discard the filtrate, centrifuge at 12000 rpm for 2 min, and let stand for 2 min with the lid open.

[0129] (6) Place the adsorption column in a clean 1.5 mL centrifuge tube, add 30 μL of deionized water, and centrifuge at 12000 rpm for 1 min.

[0130] (7) Transfer the filtrate into the adsorption column and centrifuge at 12,000 rpm for 30 seconds.

[0131] (8) The concentration of DNA was measured using a NanoDrop micro-spectrophotometer.

[0132] 6. BP reaction

[0133] (1) Prepare the reaction system: 100 ng of the gel recovery product obtained in step 5 above, 100 ng of pDONR221 plasmid (commonly available), and 1 μL of BP clonase.

[0134] (2) React at 37℃ for 1 hour.

[0135] 7. Transformation

[0136] (1) Take 50 μL of competent cells (HB101), add BP reaction product, let stand on ice for 20 min, heat shock at 42℃ for 90 s, and let stand on ice for 5 min.

[0137] (2) Add 400 μL of LB liquid medium and incubate at 37℃ and 180 rpm for 50-60 min.

[0138] (3) Collect bacteria by centrifugation at 4000 rpm for 2 min, and take about 100 μL of supernatant to resuspend the bacterial culture and spread it on kanamycin sulfate-resistant LB solid medium. Invert the plate and incubate at 37°C for 14 to 16 h.

[0139] 8. Colony PCR

[0140] (1) Pick a few colonies on the plate, poke the bottom of the PCR tube a few times, and then incubate in 400 μL LB (Kan+) liquid medium at 37℃ and 24 rpm for 6-8 h.

[0141] (2) Prepare the reaction system according to the components in Table 6. After preparation, perform amplification on the PCR instrument according to the procedure in Table 7. The primers for PCR amplification are shown in Table 5, and the primers for colony PCR are shown in Table 8.

[0142] (3) The PCR products were identified by agarose gel electrophoresis. The corresponding bacterial cultures were inoculated and cultured overnight before being sent to Sanger for sequencing.

[0143] Table 6 2×Rapid Taq Master Mix enzyme PCR reaction system

[0144]

[0145] Table 7 2×Rapid Taq Master Mix Enzyme PCR Reaction Procedure

[0146]

[0147] Table 8

[0148]

[0149] 9. Plasmid extraction

[0150] (1) Extract plasmids from the bacterial culture with correct sequencing. Take 1.5 mL of bacterial culture, centrifuge at 12000 rpm for 2 min to collect bacteria, and discard the upper culture medium.

[0151] (2) Add 100 μL SolutionⅠ, mix well by blowing, add 200 μL SolutionⅡ, gently shake a few times, add 150 μL SolutionⅢ, gently shake to mix, and centrifuge at 12000 rpm for 15 min.

[0152] (3) Transfer about 500 μL of the supernatant to a new centrifuge tube, add 1 mL of anhydrous ethanol, invert several times, and centrifuge at 12000 rpm for 2 min.

[0153] (4) Discard the supernatant, add 500 μL of 75% ethanol solution, and centrifuge at 12000 rpm for 5 min.

[0154] (5) Discard the supernatant, open the centrifuge tube cap and let it stand for 5 minutes to allow the ethanol to evaporate. Add 100 μL TE to dissolve it. The resulting product is the entry clone. Measure the DNA concentration using a NanoDrop micro spectrophotometer.

[0155] 10. LR reaction

[0156] (1) The reaction systems were configured according to Table 9 and Table 10 respectively to construct yeast (pYEX-CHT-U-DEST) and tobacco expression vector (pXZP393).

[0157] (2) React at 37℃ for 1.5h.

[0158] Table 9 LR Reaction System - Construction of Yeast Expression Vector (pYEX-CHT-U-DEST)

[0159]

[0160] Table 10 LR reaction system - Construction of tobacco expression vector (pXZP393)

[0161]

[0162] 11. Transformation

[0163] The transformation steps are the same as above. The pYEX-CHT-U-DEST vector is coated with ampicillin-resistant LB solid medium, and the pXZP393 is coated with spectinomycin hydrochloride LB solid medium.

[0164] 12. Colony PCR

[0165] The colony PCR procedure is the same as above. Plasmids are extracted from the bacterial cultures that have been correctly sequenced to obtain yeast and tobacco expression vectors for candidate genes.

[0166] Example 2. Agrobacterium-mediated transient expression in tobacco.

[0167] 1. Agrobacterium-mediated transformation

[0168] (1) Take 50 μL of competent cells (GV3101) and add 100 ng of plasmid. Place on ice for 5 min, place on liquid nitrogen for 5 min, incubate in water at 37℃ for 5 min, and place on ice for 5 min.

[0169] (2) Add 700 μL of LB liquid medium and incubate at 30℃ and 220 rpm for 2-3 hours.

[0170] (3) Centrifuge at 6000 rpm for 1 min to collect bacteria, and take 100 μL of supernatant. Resuspend the bacteria by blowing and spreading it on Rif+Gen+Spc LB solid medium. Incubate at 30℃ upside down for 2-3 days.

[0171] 2. Injection of tobacco leaves

[0172] (1) Positive clones were identified by colony PCR. The corresponding colonies were inoculated into 2 mL of LB liquid medium and cultured at 30℃ and 300 rpm for 36 h.

[0173] (2) Transfer 2 mL of bacterial culture to an Erlenmeyer flask, add culture medium to 50 mL, and continue to incubate at 30℃ and 300 rpm for 36 h.

[0174] (3) Add 50 μL of 100 μM acetylsalicylic acid and incubate at 30℃ and 300 rpm for 2-3 h.

[0175] (4) Transfer the bacterial culture to a 50 mL centrifuge tube, centrifuge at 4200 g for 5 min, discard the supernatant, and resuspend in 10 mL of infiltration buffer.

[0176] (5) Measure the OD600 value of each culture tube and prepare 20 mL of Agrobacterium injection solution with different combinations, wherein the OD600 of each culture is 0.2.

[0177] (6) Take a piece of Nicotiana benthamiana that is about one month old and in its vigorous growth period. Use a 1 mL disposable syringe to draw up the bacterial solution, remove the needle, press your finger against the front of the leaf, and let the Agrobacterium solution penetrate from the back of the leaf. Blot up the excess bacterial solution on the leaf surface, make a mark, and make three biological replicates for each combination. Collect the samples after culturing for 3-4 days.

[0178] Example 3. Extraction of fatty acids from tobacco leaves and preparation of fatty acid methyl esters

[0179] (1) Take about 300 mg of each leaf randomly and put it into a 4 mL glass tube. Add 2 mL of 2% sulfuric acid methanol solution and 50 μL of internal reference 19:Me (1 mM, dissolved in heptane). Place it in a 90℃ oven and react for 1 h.

[0180] (2) Add 1 mL of water and 1 mL of heptane, and shake to mix thoroughly.

[0181] (3) Centrifuge at 2000 rpm for 2 min, and take 650 μL of the upper heptane phase into a GC vial.

[0182] (4) Take another GC vial, pipette 4 μL of fatty acid methyl ester sample into 196 μL of heptane, i.e., dilute to 1 / 50 of the original concentration, for GC / MS analysis.

[0183] Example 4. Transcriptome sequencing

[0184] After filtering the raw data, checking the sequencing error rate, and checking the GC content distribution, clean reads were obtained for subsequent analysis. The data summary is shown in Table 11 below.

[0185] Table 11 Summary of Sample Sequencing Data Quality

[0186]

[0187] The specific information for each sample in Table 11 is shown in Table 12.

[0188] Specific information for each sample in Tables 12 and 11

[0189]

[0190]

[0191] Example 5. Agrobacterium-mediated transient expression in tobacco.

[0192] 1. Functional analysis of candidate genes for desaturases

[0193] The following candidate genes for desaturases were successfully cloned and constructed into plant expression vectors: Cmed070400, Cmed116530, Cmed116590, and Cmed116600. The combinations for tobacco injection are as follows: ① P19 + TE16; ② P19 + TE16 + Cmed070400; ③ P19 + TE16 + Cmed116530; ④ P19 + TE16 + Cmed116590; ⑤ P19 + TE16 + Cmed116600.

[0194] The P19 protein is used to suppress post-transcriptional gene silencing, and TE16 catalyzes the hydrolysis of the thioester bond between 16:ACP, thereby releasing free hexadecanoic acid, which provides a substrate for desaturase. Figure 1 GC / MS analysis showed that Z11-16:Me was detected only in tobacco leaves expressing Cmed070400, and that Cmed070400 can act on 16:Acid to produce Z11-16:Acid.

[0195] 2. Functional analysis of candidate genes for elongation enzymes

[0196] The following candidate genes for elongation enzymes were successfully cloned and constructed into plant expression vectors: Cmed092430, Cmed092440, Cmed092450, Cmed153350, and Cmed063180. The combinations for tobacco injection are as follows: ① P19 + TE16 + Cmed070400; ② P19 + TE16 + Cmed070400 + Cmed092430; ③ P19 + TE16 + Cmed070400 + Cmed092440; ④ P19 + TE16 + Cmed070400 + Cmed092450; ⑤ P19 + TE16 + Cmed070400 + Cmed153350; ⑥ P19 + TE16 + Cmed070400 + Cmed063180.

[0197] Figure 2 GC / MS analysis showed that Z13-18:Me could be detected in tobacco leaves expressing several candidate genes for desaturase.

[0198] The compounds are shown in Table 13.

[0199] Table 13 Specific information for each compound

[0200]

[0201]

[0202] Figure 3 GC / MS analysis showed that Z13-18:Me was detected in tobacco leaves expressing the Cmed070400 desaturase candidate gene and the Cmed092440 elongase candidate gene.

[0203] The description in this application is given for illustrative purposes and is not intended to be exhaustive or to limit the disclosure to the forms disclosed. Many modifications and variations will be apparent to those skilled in the art. The embodiments were chosen and described to better illustrate the principles and practical application of this disclosure and to enable those skilled in the art to understand this disclosure and design various embodiments with various modifications suitable for a particular purpose.

Claims

1. A desaturase, characterized in that, the amino acid sequence of the desaturase is set forth in any one or more of SEQ ID NOs. 62-70; or has at least 80% sequence identity to the amino acid sequence set forth in any one or more of SEQ ID NOs. 62-70; Preferably, the nucleotide sequence of the open reading frame encoding the desaturase is set forth in any one or more of SEQ ID NOs. 33-41; or has at least 80% sequence identity to the nucleotide sequence set forth in any one or more of SEQ ID NOs. 33-41.

2. An elongase characterized in that, the amino acid sequence of the elongase is set forth in any one or more of SEQ ID NOs. 55-61; or has at least 80% sequence identity to the amino acid sequence set forth in any one or more of SEQ ID NOs. 55-61; Preferably, the nucleotide sequence of the open reading frame encoding the elongase is set forth in any one or more of SEQ ID NOs. 42-48; or has at least 80% sequence identity to the nucleotide sequence set forth in any one or more of SEQ ID NOs. 42-48.

3. A recombinant plasmid characterized in that, the recombinant plasmid comprises: (1) a plasmid vector; and (2) a nucleotide sequence encoding the desaturase of claim 1; and (3) a nucleotide sequence encoding the elongase of claim 2; wherein the nucleotide sequence encoding the desaturase is recombined on the plasmid vector; wherein the nucleotide sequence encoding the elongase is recombined on the plasmid vector.

4. A recombinant cell, characterized in that, the recombinant cell comprises: (1) a competent cell; and (2) a nucleotide sequence encoding the desaturase of claim 1, and a nucleotide sequence encoding the elongase of claim 2; and / or, the recombinant cell comprises the recombinant plasmid of claim 3.

5. A tobacco expression system for the synthesis of a precursor substance of a semiochemical component, characterized in that the tobacco expression system comprises a nucleotide sequence encoding the desaturase of claim 1 and a nucleotide sequence encoding the elongase of claim 2; or the recombinant plasmid of claim 3; or the recombinant cell of claim 4.

6. An attB primer set, characterized in that, the attB primer set is selected from any one or more combinations of the following primer pairs: the attB primer pair set forth in SEQ ID NOs: 1-2; the attB primer pair set forth in SEQ ID NOs: 3-4; the attB primer pair set forth in SEQ ID NOs: 5-6; the attB primer pair set forth in SEQ ID NOs: 7-8; the attB primer pair set forth in SEQ ID NOs: 9-10; the attB primer pair set forth in SEQ ID NOs: 11-12; the attB primer pair set forth in SEQ ID NOs: 13-14; the attB primer pair set forth in SEQ ID NOs: 15-16; the attB primer pair set forth in SEQ ID NOs: 17-18; the attB primer pair set forth in SEQ ID NOs: 19-20; the attB primer pair set forth in SEQ ID NOs: 21-22; the attB primer pair set forth in SEQ ID NOs: 23-24; an attB primer pair as shown in SEQ ID NO: 25-26; an attB primer pair as shown in SEQ ID NO: 27-28; an attB primer pair as shown in SEQ ID NO: 29-30; an attB primer pair as shown in SEQ ID NO: 31-32.

7. A method of constructing a recombinant plasmid for the synthesis of a precursor substance of a semiochemical component, characterized in that, The method comprises the following steps: constructing a gateway vector: mixing the cDNA of the sex pheromone gland and the attB primer set of claim 6, amplifying, mixing the amplification product and the original vector, and BP clonase, and reacting to obtain a gateway clone; constructing a recombinant plasmid: mixing the gateway vector and the plasmid vector, and LR clonase, and reacting to obtain a recombinant plasmid.

8. A method of constructing a recombinant cell, characterized in that, The method comprises the following steps: mixing the recombinant plasmid of claim 3, and / or the recombinant plasmid constructed by the method of claim 7, with competent cells and into the competent cells.

9. A method of constructing a tobacco expression system for the synthesis of a precursor substance of a semiochemical component, characterized in that, The method comprises the following steps: injecting the recombinant cell of claim 4, and / or the recombinant cell constructed by the method of claim 8, into tobacco tissue to obtain the tobacco expression system for synthesizing sex pheromone component precursor substances.

10. A method of synthesizing a pheromone component precursor, characterized in that, The method comprises the following steps: allowing the tobacco expression system of claim 5, and / or the tobacco expression system constructed by the method of claim 9, to express to synthesize sex pheromone component precursor substances, i.e. long-chain unsaturated fatty acids, through tobacco tissue.

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