N-methyltransferase for synthesizing horseradish peroxide and application thereof

Abstract

The application discloses an N-methyltransferase for synthesizing horverine and application, the N-methyltransferase capable of catalyzing tyramine to generate N-methyl tyramine and further catalyzing N-methyl tyramine to generate horverine is obtained by screening and identifying for the first time, and key enzyme resources for the scale production of horverine by microbial fermentation or plant metabolic engineering are provided.The N-methyltransferase can promote the accumulation of horverine in plants, and significantly enhances the anti-insect effects of plant on pests such as Spodoptera litura and Cnaphalocrocis medinalis, and provides an excellent anti-insect engineering gene source.

Description

(I) Technical Field

[0001] This invention belongs to the field of biotechnology and plant genetic engineering, specifically relating to an N-methyltransferase for synthesizing maltine, its encoding gene, and its application in catalyzing the production of maltine from tyramine, preparing plant insecticides, and cultivating insect-resistant transgenic crops. (II) Background Technology

[0002] Hordenine (N,N-dimethyltyramine) is a class of phenylethylamine alkaloids naturally found in barley and other grasses. Studies have shown that hordenine, as a plant secondary metabolite, possesses antifeedant activity against certain pests and has potential application value in the development of plant-derived pesticides. Current research suggests that the biosynthesis of hordenine may originate from the tyrosine pathway: tyrosine is first converted to tyramine by tyrosine decarboxylase (TyDC); subsequently, tyramine undergoes N-methylation under the action of tyramine N-methyltransferase, using S-adenosylmethionine (SAM) as a methyl donor, to generate N-methyltyramine; N-methyltyramine can further generate hordenine under the catalysis of the same type or specific N-methyltransferases.

[0003] However, to date, the acquisition of maltine mainly relies on all-chemical synthesis (cumbersome steps, use of hazardous reagents, and heavy environmental pollution) or direct extraction from plants (extremely low content and low efficiency). The key N-methyltransferases involved in the tyramine N-methylation reaction in barley during maltine biosynthesis and their encoding genes have not been clearly identified, resulting in a lack of functional enzyme resources available for the biosynthesis of maltine.

[0004] Therefore, screening and identifying N-methyltransferases and their encoding genes that can catalyze the production of N-methyltyramine and maltine from tyramine can solve the technical bottleneck of lacking core catalysts for the large-scale in vitro enzymatic production of maltine in industry. At the same time, it is of great significance for elucidating the biosynthetic pathway of maltine and developing plant-derived insect-resistant agents. (III) Summary of the Invention

[0005] This invention addresses the problems in existing technologies, such as the lack of a clearly defined key N-methyltransferase in the malt alkaloid biosynthesis pathway, the lack of functional enzyme resources for malt alkaloid biosynthesis, and the insufficient technology for the targeted accumulation of malt alkaloid in plants. It provides an N-methyltransferase, encoding gene, recombinant vector, engineered bacteria, and its application in catalyzing the production of malt alkaloid from tyramine, preparing plant insect repellents, and cultivating insect-resistant transgenic crops. This invention solves the technical bottleneck of lacking a core, highly efficient biocatalyst for large-scale green bio-manufacturing of malt alkaloid in industry, achieving efficient biosynthesis of malt alkaloid and improving plant insect resistance.

[0006] The technical solution adopted in this invention is: In a first aspect, the present invention provides an N-methyltransferase (HNMT) for the synthesis of maltine, wherein the N-methyltransferase has conserved catalytic active site residues and has the activities of catalyzing the formation of N-methyltyramine from tyramine and catalyzing the formation of maltine from N-methyltyramine; wherein the catalytic active site residues include tryptophan at position 255, leucine at position 257, and aspartic acid at position 262.

[0007] Furthermore, the N-methyltransferase has an amino acid sequence with 46.15% or more homology to the conserved sequence shown in SEQ ID NO.14, preferably with more than 60% homology, and more preferably as shown in SEQ ID NO.14.

[0008] Furthermore, the N-methyltransferase has an amino acid sequence with 42.24% or more homology to SEQ ID NO.1, preferably 75% or more homology, more preferably 80% or more homology, and more preferably 86% or more homology.

[0009] The N-methyltransferase of the present invention includes proteins with the same catalytic activity obtained by deleting, inserting, or substituting one or more amino acid residues in the amino acid sequence shown in SEQ ID NO.1.

[0010] Furthermore, the amino acid sequence of the N-methyltransferase is shown in one of SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, or SEQ ID NO.8, wherein the homology between SEQ ID NO.2-SEQ ID NO.8 and SEQ ID NO.1 is 90.34%, 73.86%, 86.86%, 73.07%, 73.64%, 79.55%, and 42.24%, respectively. The sequence homology was calculated using the BLASTP program.

[0011] In a second aspect, the present invention provides a nucleic acid molecule encoding the N-methyltransferase, wherein the nucleotide sequence of the nucleic acid molecule is shown in one of SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12 or SEQ ID NO. 13.

[0012] Thirdly, the present invention provides a recombinant expression vector comprising a nucleic acid molecule encoding the N-methyltransferase, wherein the recombinant expression vector is based on pET28a or pCAMBIA1305.

[0013] Fourthly, the present invention provides a recombinant genetically engineered bacterium containing the recombinant expression vector, wherein the recombinant genetically engineered bacterium uses Escherichia coli BL21(DE3) or Agrobacterium EHA105 as the host bacterium.

[0014] Fifthly, the present invention provides an application of the N-methyltransferase in the biosynthesis of malt alkaloids, wherein the application is carried out in the presence of S-adenosylmethionine, using tyramine as a substrate, under the action of the N-methyltransferase or an organism expressing the N-methyltransferase, to finally generate malt alkaloids.

[0015] Furthermore, the application method is as follows: tyramine, S-adenosylmethionine, vitamin C, MgCl2, and pure enzyme extracted from the induced culture of recombinant genetically engineered bacteria expressing N-methyltransferase after wet cell disruption are added to a buffer solution; the reaction is carried out at 20-40℃ for 20-40 min (preferably at 30℃ for 30 min) to obtain a reaction solution containing malt alkaloid; the buffer solution composition is: 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5% glycerol.

[0016] The concentrations of tyramine, S-adenosylmethionine, vitamin C, and MgCl2 added are all 1-5 mM (preferably 1 mM) based on the buffer volume, and the concentration of the pure enzyme added is 50-150 μg / mL (preferably 100 μg / mL) based on the protein content of the buffer.

[0017] Furthermore, the purified enzyme is prepared according to the following method: (1) The recombinant genetically engineered bacteria expressing N-methyltransferase were inoculated into LB liquid medium containing 50 μg / mL kanamycin and cultured overnight at 37°C with shaking at 200 rpm for activation; the overnight activated bacterial solution was added to LB liquid medium containing 50 μg / mL kanamycin at a volume concentration of 1% and cultured at 37°C with shaking at 200 rpm until OD 600When the value reached 0.6-0.8, 0.5 mM isopropyl-β-D-thiogalactoside (IPTG) was added to induce expression for 16 h at 16℃ and 120 rpm. After centrifugation, wet cells were collected. (2) The wet bacterial cells were resuspended in lysis buffer (20 mM Tris-HCl, pH 7.5, 300 mM NaCl, 10 mM imidazole, 10% glycerol, 1 mM DTT (dithiothreitol), 1× protease inhibitor), and sonicated for 15 min (5 s working time followed by 5 s intervals) under ice-water bath conditions at 200 W. After that, the cells were centrifuged at 12,000 rpm and 4 °C for 30 min and the supernatant was collected. The volume of the lysis buffer was 6 mL / g based on the weight of the wet bacterial cells. (3) Load the supernatant onto a Ni-NTA affinity chromatography gravity column packed with Ni-NTA packing material, and incubate at 4°C for 30 min by rotation. Wash with washing buffer (20 mM Tris-HCl pH 7.5, 300 mM NaCl, 20 mM imidazole, 10% glycerol) for 40 column volumes. Then elute the target protein with elution buffer (20 mM Tris-HCl pH 7.5, 300 mM NaCl, 250 mM imidazole, 10% glycerol) for 10 column volumes. Collect the eluent in a 10 kDa molecular weight cutoff dialysis bag. Add TEV protease at a TEV protease:target protein mass ratio of 1:10 to the dialysis buffer (dialysis buffer: 20 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol). After digesting the His tag with glycerol overnight at 4°C, the enzyme was re-loaded onto a new Ni-NTA column. The permeate was collected as the detagged target protein and concentrated using a 10 kDa ultrafiltration centrifuge tube to obtain the purified enzyme.

[0018] In a sixth aspect, the present invention provides the application of the N-methyltransferase in the preparation of plant insecticides, wherein the insecticides can promote the accumulation of maltose in plants.

[0019] Furthermore, the application involves transferring the N-methyltransferase into the plant genome using Agrobacterium infection to obtain transgenic plants with increased malt alkaloid content, thereby enhancing the insect resistance and antifeedant activity of the host plant.

[0020] Furthermore, the insecticides include those for the beet armyworm, diamondback moth, cotton bollworm, and rice leaf roller.

[0021] In a seventh aspect, the present invention provides the application of the N-methyltransferase in the cultivation of insect-resistant transgenic crops.

[0022] Furthermore, the application involves transferring the N-methyltransferase encoding gene into the crop genome to obtain transgenic crops with enhanced insect resistance.

[0023] Furthermore, the crops include rice and tomatoes.

[0024] Compared with the prior art, the present invention has the following beneficial effects: (1) This invention screened and identified for the first time N-methyltransferases that can catalyze the formation of N-methyltyramine from tyramine and further catalyze the formation of maltine from N-methyltyramine. These N-methyltransferases include HvHNMT1, HvHNMT2 and HvHNMT3 from barley, PmHNMT1 from millet, SCHNMT1 and SCHNMT2 from rye, AsHNMT1 from oats and TaHNMT1 from wheat. The key N-methylation steps in the biosynthesis pathway of maltine were elucidated and the core missing steps in the maltine synthesis pathway were completely elucidated. This provides key enzyme resources for the large-scale production of maltine using microbial fermentation or plant metabolic engineering.

[0025] (2) This invention provides N-methyltransferase protein and its encoding gene, recombinant vector and engineered bacteria, which can be used for in vitro enzymatic synthesis, heterologous biosynthesis and metabolic engineering of malt alkaloids, providing a new technical approach for the biomanufacturing of malt alkaloids.

[0026] (3) The N-methyltransferase of this invention can promote the accumulation of maltose in plants and significantly enhance the insect resistance of plants against pests such as the beet armyworm and rice leaf roller, which prevent feeding and inhibit growth. For example, the insect resistance performance of the HvHNMT1 and HvHNMT2 genes was verified by heterologous expression in tobacco (transient), Arabidopsis thaliana, rice, and tomato (stable transgenic). This invention provides an excellent source of insect-resistant engineered genes. (iv) Description of the attached drawings

[0027] Figure 1 This is a phylogenetic tree of methyltransferases highly expressed in barley roots, as described in Example 1.

[0028] Figure 2 The image shows the SDS-PAGE analysis of the purified HvHNMT protein in Example 2; lane 1 is HvHNMT1 protein; lane 2 is HvHNMT2 protein; lane 3 is HvHNMT3 protein; lane 4 is HvHNMT4 protein; lane 5 is HvHNMT5 protein; and lane 6 is HvHNMT6 protein.

[0029] Figure 3The image shows the UPLC-MS / MS detection of the in vitro catalytic reaction product of HvHNMT protein in Example 3; CK1 is the blank control reaction system without HvHNMT protein; CK2 is the blank control reaction system without HvHNMT protein.

[0030] Figure 4 The results show the maltine content in the leaves of *Tobacco Bunge* that transiently expressed HvHNMT1, HvHNMT2, and co-expressed HvHNMT1 and HvHNMT2 in Example 4.

[0031] Figure 5 The results of the resistance test of Spodoptera litura to tobacco leaves transiently expressing HvHNMT are shown below. A is a schematic diagram of the experiment on tobacco leaves consumed by Spodoptera litura larvae. B is the proportion of tobacco leaves transiently expressing HvHNMT1 that were consumed by Spodoptera litura at 6 h and 18 h. C is the proportion of tobacco leaves transiently expressing HvHNMT2 that were consumed by Spodoptera litura at 6 h and 18 h. D is the proportion of tobacco leaves transiently co-expressing HvHNMT1 / HvHNMT2 that were consumed by Spodoptera litura at 6 h and 18 h.

[0032] Figure 6 The results of maltol content detection in rice leaves were obtained by overexpressing HvHNMT1, HvHNMT2, and co-expressing HvHNMT1 and HvHNMT2.

[0033] Figure 7 The study investigated the overexpression of HvHNMT1, HvHNMT2, co-expression of HvHNMT1 and HvHNMT2, and the weight gain of rice after 14 days of feeding on rice seedlings without a growing crop.

[0034] Figure 8 Results of HvHNMT1 and HvHNMT2 co-expression and detection of maltine content in Arabidopsis thaliana leaves without a vector.

[0035] Figure 9 The weight gain of diamondback moth after feeding on overexpressed HvHNMT1 and HvHNMT2 co-expressed and unloaded Arabidopsis thaliana for 14 days.

[0036] Figure 10 The results were obtained for the overexpression of HvHNMT1 / HvHNMT2 and the detection of maltol content in unloaded tomato leaves.

[0037] Figure 11 The weight gain of cotton bollworms after feeding on HvHNMT1 / HvHNMT2 overexpressing tomatoes and unloaded tomatoes for 48 h.

[0038] Figure 12UPLC-MS / MS images of in vitro catalytic reaction products of wheat TaHNMT1, rye SCHNMT1, SCHNMT2, oat AsHNMT1 and millet PmHNMT1 proteins; CK is a blank control reaction system without HNMT protein.

[0039] Figure 13 This is a sequence alignment diagram of the major catalytic sites of amino acids in the proteins HvHNMT1, HvHNMT2, HvHNMT3 (barley), TaHNMT1 (wheat), SCHNMT1 (rye), SCHNMT2 (rye), AsHNMT1 (oat), and PmHNMT1 (millet). (V) Detailed Implementation Methods

[0040] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto: Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0041] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains. The experimental materials used in the embodiments of this invention are all conventional experimental materials in the art and are commercially available. Experimental methods not specifying detailed conditions were performed according to conventional experimental methods or the operating instructions recommended by the supplier.

[0042] Example 1: Screening and identification of HvHNMT candidate genes

[0043] S-adenosylmethionine (SAM) is the methyl donor in methylation reactions, and SAM-dependent methyltransferases typically possess a Rossmann-like domain that binds SAM. Transcriptome sequencing was performed on barley roots, shoots, and seeds. Comparative analysis of the transcriptome data from barley roots, shoots, and seeds was conducted. A phylogenetic tree was constructed using the Neighbor-Joining (NJ) method with MEGA7 software for cluster analysis. Figure 1Based on the phylogenetic tree results, six candidate methyltransferase genes with high expression levels in the roots were selected. These genes are identified in the GrainGenes database as HORVU1Hr1G003370.2 (denoted as HvHNMT1), HORVU1Hr1G011930.1 (denoted as HvHNMT2), HORVU4Hr1G001250.1 (denoted as HvHNMT3), HORVU3Hr1G116770.1 (denoted as HvHNMT4), HORVU7Hr1G018880.1 (denoted as HvHNMT5), and HORVU5Hr1G066150.1 (denoted as HvHNMT6). Domain analysis of the candidate proteins using the NCBI CDD and InterPro databases revealed that all of these candidate proteins possess SAM-dependent Rossmann-like methyltransferase domains.

[0044] Example 2: Prokaryotic expression and purification of candidate HvHNMT protein

[0045] 1. Constructing prokaryotic expression vectors

[0046] The nucleotide sequences encoding the genes HvHNMT1, HvHNMT2, HvHNMT3, HvHNMT4, HvHNMT5, and HvHNMT6 selected in Example 1 were optimized using E. coli preferred codons. The optimized nucleotide sequences were then artificially synthesized and constructed into the NcoI and XhoI restriction endonuclease sites of the pET28a vector. A His tag and a TEV protease cleavage site were fused to the N-terminus of the target protein to obtain the recombinant expression vectors pET28a-H8-TEV-HvHNMT1, pET28a-H8-TEV-HvHNMT2, pET28a-H8-TEV-HvHNMT3, pET28a-H8-TEV-HvHNMT4, pET28a-H8-TEV-HvHNMT5, and pET28a-H8-TEV-HvHNMT6, respectively.

[0047] 2. Construction of recombinant genetically engineered bacteria

[0048] The recombinant expression vector constructed in step 1 was transformed into *E. coli* BL21(DE3). After transformation, the cells were plated on LB solid medium containing 50 μg / mL kanamycin for resistance selection. The cells were incubated overnight at 37°C. Single colonies were picked and inoculated into LB liquid medium containing 50 μg / mL kanamycin, and incubated at 37°C for 12 h to obtain recombinant genetically engineered bacteria. E. coli BL21(DE3)-pET28a-H8-TEV-HvHNMT1, E. coliBL21(DE3)-pET28a- H8-TEV-HvHNMT2, E. coli BL21(DE3)-pET28a-H8-TEV-HvHNMT3, E. coli BL21(DE3)-pET28a-H8-TEV-HvHNMT4, E. coli BL21(DE3)-pET28a-H8-

[0049] TEV-HvHNMT5 and E. coli BL21(DE3)-pET28a-H8-TEV-HvHNMT6, bacterial culture was stored frozen at -80℃.

[0050] 3. Expression and purification of HvHNMT protein

[0051] After thawing the cryopreserved solution of the recombinant genetically engineered bacteria constructed in step 2, inoculate it into LB liquid medium containing 50 μg / mL kanamycin and activate it by shaking overnight at 37°C and 200 rpm. Take 10 mL of the overnight activated bacterial culture and add it to 1 L of LB liquid medium containing 50 μg / mL kanamycin, and incubate at 37°C and 200 rpm for 3.5 h, until the OD value is reached. 600 When the pH value reached 0.6–0.8, 0.5 mM isopropyl-β-D-thiogalactoside (IPTG) was added to induce the expression of the tagged protein. Expression was induced for 16 h at 16°C and 120 rpm, followed by centrifugation to collect the wet cells.

[0052] Five g of wet bacterial cells were resuspended in 30 mL of lysis buffer (20 mM Tris-HCl, pH 7.5, 300 mM NaCl, 10 mM imidazole, 10% glycerol, 1 mM DTT (dithiothreitol), 1× protease inhibitor), and sonicated for 15 min (5 s working time followed by 5 s intervals) at 200 W in an ice-water bath. The cells were then centrifuged at 12,000 rpm at 4 °C for 30 min. The supernatant was collected as the soluble protein fraction with a protein concentration of 3 mg / mL.

[0053] Load 5 mL of supernatant onto a Ni-NTA affinity chromatography gravity column containing 1 mL of Ni-NTA packing material. Incubate at 4°C for 30 min by rotation. Wash with washing buffer (20 mM Tris-HCl pH 7.5, 300 mM NaCl, 20 mM imidazole, 10% glycerol) for 40 column volumes. Elute the target protein with elution buffer (20 mM Tris-HCl pH 7.5, 300 mM NaCl, 250 mM imidazole, 10% glycerol) for 10 column volumes. Collect the eluent in a 10 kDa molecular weight cutoff dialysis bag. Add TEV protease at a TEV protease:target protein mass ratio of 1:10 to the dialysis buffer (dialysis buffer: 20 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol). After digestion of the His tag with glycerol at 4°C overnight, the protein was re-loaded onto a new Ni-NTA column, and the permeate was collected as the detagged target protein. The protein concentration was concentrated to 5 mg / mL using a 10 kDa ultrafiltration centrifuge tube to obtain purified HvHNMT1, HvHNMT2, HvHNMT3, HvHNMT4, HvHNMT5, and HvHNMT6 proteins. These proteins were then analyzed by SDS-PAGE gel electrophoresis. Figure 2 .

[0054] Example 3: In vitro enzymatic activity of HvHNMT protein

[0055] 1. In vitro reactions were carried out using tyramine or N-methyltyramine as substrates and SAM as a methyl donor, respectively.

[0056] The in vitro reaction system 1 consisted of: 100 μL buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5% glycerol), 1 mM tyramine, 1 mM SAM, 1 mM vitamin C, 1 mM MgCl2, and 10 μg of HvHNMT1-HvHNMT6 protein purified by the method in Example 2 (based on protein content); a buffer without HvHNMT protein was used as a blank control (denoted as CK1); the reaction was carried out at 30°C for 30 min, 100 μL of methanol was added to precipitate the protein, centrifuged at 12,000 rpm for 15 min, and filtered through a 0.22 μm needle filter. The filtrate (containing HNMT protein and tyramine) and tyramine standard were detected by UPLC-MS / MS (Waters UPLC-MS / MS system, Waters Corp., Milford, MA, USA), and the results are as follows. Figure 3 As shown.

[0057] The in vitro reaction system 2 consisted of: 100 μL buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5% glycerol), 1 mM N-methyltyramine, 1 mM SAM, 1 mM vitamin C, 1 mM MgCl2, and 10 μg of HvHNMT-HvHNMT6 protein purified by the method in Example 2 (based on protein content); a buffer without HvHNMT protein was used as a blank control (denoted as CK2); the reaction was carried out at 30°C for 30 min, 100 μL of methanol was added to precipitate the protein, centrifuged at 12,000 rpm for 15 min, and filtered through a 0.22 μm needle filter. The filtrate (containing HNMT protein and N-methyltyramine), N-methyltyramine standard, and maltol standard were detected by UPLC-MS / MS, respectively. The results are shown below. Figure 3 As shown.

[0058] UPLC-MS / MS detection conditions: The experimental instrument was an Agilent 6470 triple quadrupole LC-MS system; the column was a Zorbax SB C18 (3.5 μm, 2.1 × 150 mm); mobile phase A was ultrapure water containing 0.1% formic acid, and mobile phase B was acetonitrile; a gradient elution program was used: 0–2 min, 5% B; 2–6 min, 5%–70% B; 6–7 min, 70%–95% B; 7–8 min, 95% B. The flow rate was 0.3 mL / min. - ¹ The column temperature was maintained at 30 °C; MRM-positive ion scanning mode was used, and the injection volume was 10 μL each time.

[0059] Depend on Figure 3It is known that the gene expression product of HORVU1Hr1G003370.2 can catalyze the conversion of tyramine to N-methyltyramine, and further catalyze the conversion of N-methyltyramine to maltine. No N-methyltyramine or maltine was detected in the blank control CK1. Therefore, HORVU1Hr1G003370.2 is named HvHNMT1, and its encoded protein is named HvHNMT1 protein. The nucleotide sequence is shown in SEQ ID NO.9, and the amino acid sequence of the encoded protein is shown in SEQ ID NO.1. The gene expression product of HORVU1Hr1G011930.1 can also convert tyramine to N-methyltyramine and further catalyze the conversion of N-methyltyramine to maltine. No N-methyltyramine or maltine was detected in the blank control CK2. Therefore, HORVU1Hr1G011930.1 is named HvHNMT2, and its encoded protein is named HvHNMT2 protein, with the nucleotide sequence shown in SEQ ID NO.10 and the amino acid sequence shown in SEQ ID NO.2. The gene expression product of HORVU4Hr1G001250.1 can also convert tyramine to N-methyltyramine and further catalyze the formation of maltine from N-methyltyramine. N-methyltyramine and maltine were not detected in the blank control CK2. Therefore, HORVU4Hr1G001250.1 is named HvHNMT3, and its encoded protein is named HvHNMT3 protein, with the nucleotide sequence shown in SEQ ID NO.11 and the amino acid sequence shown in SEQ ID NO.3.

[0060] SEQ ID NO.1

[0061] MANEEALMFALQLASSAVLPMTLRTSIELGLLETLVGAGGKVLTPEEVAAKLPSKAEANPDAASMVDRLLRVLAAYKVVSCVVAESSDGSLSRRYGAEPVCKWLTPNEEGVSMAPFCLLAQDKLFMEAWCHMKDAVLEGGSAFTKAFGASWFDYAGTDARFNSVFNEAMKQHSVII TKKLLELYTGFDGIGTLVDVAGGVGAVVHTITKKYPSIKGINFDLPHVISDAQPCPGVEHVGGDMFEKVPTGDAILMKWILNCFSDDECAILLKNCYDALPAHGKVINVECILPVNPDATNSTQGLISVDVSLLAYSPGGKERYLRDLEKLARAAGFTGVKATYIYADFWAMEYTK.

[0062] SEQ ID NO. 2

[0063] MANEEALMFALQLASSAVLPMTLRTCIELGLLETLVGAGGKTLTPEEVAAKLPSKAESNPDAASMVDRLLRVLATYKVVSCLVDECADGSLSRRYGAEPVCKWLTPNEDGVSMAPFCLLAQNKLFMEAWCHMKDAVLEGGSAFTKAFGASWFDYAGTDDHFNHLFNEAMKDHSVIITKKLLELYTGFDGIDTLVDLAGGVGAVIHAITKKYPSIKGINFDLPHVISDAQPYPGVEHVGGDMFEMVPSGDAILMKWILPCFSDDECAVLLKNCYDALPAHGKVINVECILPVNPDATNNAQGLICVDASLLAYSPGGKERNLRDFEKLAKAAGFTGVKASYIFANFWAMEYTK。

[0064] SEQ ID NO. 3

[0065] MADEEQACMQAVQLALSSVLPMTLKTAIELGLLETLVGAGGKLLTPVEVAEMLPSKDNPDAPSMVDRMLRVLASHNVVSCVVEDGKDGSLSRRYGAAPVCKWLTPNEDGVSMAPWALSAQDRVFMETWCYMKDAVLTGGSPFHKAYGMTWFEYAGTDIRFNRLFNEAMKHQSVIITNKLLELYSGFDGVGTLVDVGGGVGATINAIISKYPSIKGINFDLPHVISEATPCFPGVHVQHVGGNMLDKVPSGDAILMKWILNCFSDQECARLLKNCYDALPAHGKLISVECVLPVIPEATPSAQGMTQIDMSLLAYSDGGKERYHGELEELAKAAGFAGVKSTTYIYANFWAMEYTKTK。

[0066] Example 4: Transient expression of HvHNMT in Nicotiana benthamiana leaves and detection of hordenine content

[0067] 1. Construction of recombinant plant expression vector

[0068] The nucleotide sequences encoding the HvHNMT1 and HvHNMT2 genes were artificially synthesized (nucleotide sequences shown in SEQ ID NO. 9 and SEQ ID NO. 10), and inserted into the NcoI and BglII restriction endonuclease sites of the pCAMBIA1305 vector, respectively, to construct recombinant expression vectors expressing HvHNMT1 and HvHNMT2, respectively; simultaneously, a dual-gene recombinant expression vector expressing HvHNMT1 and HvHNMT2 was constructed. These three recombinant expression vectors were introduced into Agrobacterium EHA105 via heat shock to obtain recombinant Agrobacterium strains, denoted as HvHNMT1, HvHNMT2, and HvHNMT1 / HvHNMT2. An Agrobacterium strain transformed with the empty pCAMBIA1305 vector was constructed as a control strain, denoted as 1305EV.

[0069] 2. Preparation of standardized Agrobacterium infection solution

[0070] The recombinant Agrobacterium and control strains constructed in step 1 were inoculated into LB liquid medium containing 50 μg / mL kanamycin and 50 μg / mL rifampin, respectively, and cultured at 28℃ and 200 rpm for 36 h until the end of the logarithmic growth phase. Then, the Agrobacterium cells were collected by centrifugation at 4,000g for 10 min, and the cells were resuspended in a prepared plant infection buffer (buffer composition shown in Table 1, solvent: ultrapure water). The absorbance of each recombinant bacterial suspension was adjusted to achieve the desired OD value. 600 The concentration is approximately 1.0. Then, the mixture is placed at room temperature in the dark for 1 hour to allow acetylsuccinone to fully induce the expression of the Vir gene in Agrobacterium, thereby activating the T-DNA transfer system. The resulting bacterial suspension is the Agrobacterium infection solution.

[0071] Table 1 Infection buffer formulation

[0072] 3. Determination of plant infection and maltine accumulation

[0073] Select *Nicotiana benthamiana* plants that are 4–6 weeks old and have 5–6 fully expanded true leaves for infection. Using a 1 mL disposable needle-free sterile syringe, draw up the *Agrobacterium* infection solution prepared in step 2 and gently inject it into the flat area on the underside of the tender green leaves of *Nicotiana benthamiana*. Inject 1–1.5 mL of *Agrobacterium* infection solution into the underside of each leaf until the infection solution completely penetrates and covers the intercellular spaces of the entire target leaf. After injection, transfer the tobacco to an artificial climate chamber (25°C, 16 h light / 8 h dark) for continued normal culture for 3 days. On the 3rd day, weigh 100 mg of leaves from the infected area, flash-freeze in liquid nitrogen, and then crush them using a high-frequency grinder. Extract the secondary metabolites using a 50% methanol / water solution at a material-to-liquid ratio of 0.1 g / mL in a vortex mixer at 800 rpm for 2 h at room temperature. Analyze the extract using UPLC-MS / MS as described in Example 3. Calculate the maltolamine content using a standard curve of maltolamine standard concentration versus peak area. The results are as follows: Figure 4 As shown: leaves injected with the empty control (1305EV) contained no maltine at all; while tobacco leaves transformed with the HvHNMT1 and / or HvHNMT2 encoding genes of this invention achieved heterologous and efficient synthesis of maltine, with the highest accumulation of maltine in the dual-gene co-expression system.

[0074] Example 5: Bioassay of insect resistance and antifeedant activity of transiently expressed HvHNMT in tobacco leaves

[0075] Tobacco leaves of similar size, having been cultured for 3 days in Example 4 at 1305EV (empty control), HvHNMT1, HvHNMT2, and HvHNMT1 / HvHNMT2, were respectively infected and cultured in round sterile petri dishes (90 mm in diameter). Eight biological replicates were set up for each treatment, and 10 third-instar Spodoptera litura moths of similar body condition, starved for 4 h, were placed in each petri dish. Each treatment group had 8 parallel biological replicates, and each petri dish contained 10 third-instar Spodoptera litura moths of uniform body condition that had been starved for 4 h. Spodoptera litura Larvae were observed. Petri dishes were placed in a 25℃ constant temperature and light incubator. At 6 h and 18 h after feeding, photographs of the leaf remnants after larval feeding were taken using a digital camera with a fixed focus. ImageJ image processing software was used to accurately calculate the percentage (%) of the leaf surface area that had been bitten out relative to the initial total leaf area. Results are shown below. Figure 5 At 6 h of feeding, the bitten area in each treatment group incorporating the barley synthase gene of this invention was significantly lower than that in the control group. At 18 h of feeding, the bitten area in each treatment group incorporating the barley synthase gene of this invention was extremely significantly lower than that in the control group, with the bitten area in the dual-gene co-expression group being only 12.4%. The experimental results conclusively demonstrate, at the level of intact living plants, that the HvHNMT gene-mediated barley malt alkaloid biosynthesis can confer plants with extremely strong insect resistance and strong broad-spectrum antifeedant activity.

[0076] Example 6: Construction of stably inherited HvHNMT transgenic rice lines and detection of maltine content

[0077] 1. Construction and identification of transgenic plants

[0078] Embryogenic callus tissue of "Nipponbare," a major japonica rice variety in my country, was transformed using Agrobacterium-mediated transformation. The specific transformation procedure was as follows: Agrobacterium infection solution obtained in Example 4 was co-cultured with Nipponbare callus at 25 °C for 3 days; subsequently, the callus was transferred to a solid selection medium (N6 basal medium, supplemented with 30 g / L sucrose, 1.5-3.0 mg / L 2,4-D (2,4-dichlorophenoxyacetic acid), 3 g / L plant gel, pH 5.8) containing 50 mg / L hygromycin B and 250 mg / L cefotaxime, for 14 days of strict resistance selection at 28 °C and 16 h light / 8 h dark; surviving resistant callus was transferred to differentiation medium (MS basal medium, supplemented with 30 g / L sucrose, 2 mg / L 6-BA (6-benzyladenine), 0.5 mg / L NAA (naphthaleneacetic acid), 0.5 mg / L KT (6-furanmethyladenine), and 3 g / L plant gel, pH 5.8) at 28 °C for 16 h light / 8 h dark. Shoots were induced to differentiate under 16 h light / 8 h dark conditions; finally, they were transferred to rooting medium (1 / 2 MS medium, with 20 g / L sucrose and 3 g / L plant gel added, pH 5.8) at 28℃ and induced to develop into regenerated rice plants with complete root systems under 16 h light / 8 h dark conditions (T0 generation).

[0079] To accurately identify whether the target gene has been successfully integrated into the rice genome, genomic DNA was extracted from regenerated rice leaves as a template, and the following target gene-specific and highly specific primers were designed and used for PCR molecular detection: HvHNMT1 specific identification primers: Forward primer (F): 5'-gtgggttttactgatgcatatacatgatgg-3', Reverse primer (R): 5'-ctggtgtgagaaccttaccacca-3' HvHNMT2 specific identification primers: Forward primer (F): 5'-gatggatggaaatatcgatctaggataggtatacat-3', Reverse primer (R): 5'-tggagacaccgtcctcgtt-3' Positive plants that were correctly amplified by PCR and sequenced by electrophoresis were named the stable expression lines OE-HvHNMT1, OE-HvHNMT2, and the dual gene overexpression line OE-HvHNMT1 / HvHNMT2, respectively.

[0080] 2. Detection of maltine content

[0081] T1 generation seeds from PCR-positive plants were harvested and germinated concurrently with wild-type Nipponbare seeds. After germination, seedlings were transplanted into a modified Yoshida rice nutrient solution (Coolaber, NS1040) and cultured at 26–28 °C, 50% relative humidity, and 14 h light / 10 h dark conditions until tillering stage, for subsequent determination of barley malt alkaloid content and evaluation of insect resistance.

[0082] 100 mg of rice leaves at the tillering stage were collected, quick-frozen and ground in liquid nitrogen, and then extracted with 50% methanol / water at a material-to-liquid ratio of 0.1 g / mL at 800 rpm for 2 h. The endogenous maltamine content in the leaves was detected by UPLC-MS / MS as described in Example 3, with wild-type Nipponbare leaves transfected with 1305EV as a negative control (CK). The results are as follows. Figure 6 As shown, maltol accumulation was detected in the leaves of OE-HvHNMT1, OE-HvHNMT2, and OE-HvHNMT1 / HvHNMT2 rice varieties, while no maltol was detected in the negative control leaves. Among these, the highest maltol content was found in the leaves of the co-transgenic OE-HvHNMT1 / HvHNMT2 rice variety (1335.1 μg / g), while the lowest was found in the leaves of the OE-HvHNMT2 rice variety (445.8 μg / g).

[0083] Example 7: Detection of resistance and growth inhibition of HvHNMT transgenic rice to rice leaf folder

[0084] To further evaluate the practical insect-control effect of the gene of this invention in stable field crop breeding, this embodiment conducted in vivo insect resistance bioassays.

[0085] The transgenic rice plants obtained in Example 6 and the negative control were cultured to the peak tillering stage, and plants with uniform growth were selected for bioassays. Twelve independent biological replicates were carefully set up for each treatment group, and one rice leaf roller was inoculated onto each rice plant. Cnaphalocrocis medinalis Newly hatched larvae were cultured and fed for 14 days at a temperature of 26-28℃, an air humidity of 50%, and a 14-hour light / 10-hour dark environment. After 14 days of feeding, the weight gain of the larvae was recorded. Figure 7It can be seen that, compared with the control larvae that fed on the negative control, the rice leaf folder larvae that fed on the rice lines (OE-HvHNMT1, OE-HvHNMT2 and OE-HvHNMT1 / HvHNMT2) transformed with the gene of this invention showed a highly significant decrease in body weight gain. This indicates that the N-methyltransferase-mediated accumulation of maltine identified for the first time in this invention can significantly improve the resistance of rice to rice leaf folder.

[0086] Example 8: Construction, identification, and maltine content detection of Arabidopsis thaliana lines stably overexpressing HvHNMT1 and HvHNMT2 genes.

[0087] 1. Construction and identification of transgenic plants

[0088] To further verify the function and application potential of the HvHNMT1 and HvHNMT2 genes in dicotyledonous plants, the HvHNMT1 and HvHNMT2 plant dual expression vectors and the pCAMBIA1305 empty vector obtained in Example 4 were transformed into Arabidopsis thaliana using Agrobacterium-mediated inflorescence inoculation method. Arabidopsis thaliana ,Col-0) wild-type plants.

[0089] The specific procedure is as follows: Agrobacterium carrying the HvHNMT1 / HvHNMT2 dual expression vector or the pCAMBIA1305 empty vector is cultured until the bacterial culture reaches OD. 600 The OD value was 0.8–1.0. After centrifugation to collect the bacterial cells, they were resuspended in the infection solution (5% sucrose, 0.03% Silwet L-77, and sterile deionized water) to achieve an OD value of 0.8–1.0. 600 The value is 1.0, which represents the Agrobacterium infection solution. Early-flowering Arabidopsis inflorescences are immersed in the Agrobacterium infection solution for 1-2 minutes. After infection, the plants are placed in darkness and incubated at 22°C for 24 hours, then returned to normal culture conditions (22°C, 16 h light / 8 h dark, light intensity 100–150 μmol·m⁻¹). -2 ·s -1 (The relative humidity of the air is 50% to 60%) until the seeds mature.

[0090] T1 generation seeds were harvested, surface-sterilized with 75% ethanol and 2% sodium hypochlorite, and then sown on 1 / 2 MS selection medium (containing 1% sucrose, 0.8% agar, pH 5.8) containing 25 mg / L hygromycin B and 50 mg / L timentin. The seeds were cultured at 22℃ under 16 h light / 8 h dark conditions for 10–14 days. The resistant seedlings obtained through screening were transplanted into nutrient soil and cultured until maturity. Continuous screening yielded genetically stable homozygous T3 lines.

[0091] Genomic DNA and total RNA were extracted from leaves of the Arabidopsis thaliana T3 homozygous line and subjected to PCR detection (primers as in Example 6). PCR amplification products were detected by agarose gel electrophoresis, and positive amplified fragments were sequenced for verification. The double-gene overexpression line with correct sequencing results and stable expression was named OE-HvHNMT1 / HvHNMT2. Simultaneously, the empty vector-transformed line was denoted as EV.

[0092] 2. Detection of maltine content in transgenic plants

[0093] Empty vector transformed lines (EV) and OE-HvHNMT1 / HvHNMT2 lines were sown simultaneously and cultured at 22 ℃, 50%–60% relative humidity, and 16 h light / 8 h dark conditions until the 5–6 true leaf stage. 100 mg of leaves were taken, quick-frozen and ground in liquid nitrogen, and 1 mL of 50% (v / v) methanol aqueous solution was added. Extraction was performed by shaking at 800 rpm for 2 h. The maltidine content in the leaves was detected according to the UPLC-MS / MS method described in Example 3.

[0094] The test results show that ( Figure 8 No maltine was detected in the leaves of Arabidopsis thaliana with the empty vector, while significant accumulation of maltine was detected in the leaves of Arabidopsis thaliana with the OE-HvHNMT1 / HvHNMT2 vector, indicating that HvHNMT1 and HvHNMT2 can synergistically catalyze the biosynthesis of maltine in dicotyledonous plants.

[0095] Example 9: Evaluation of resistance of Arabidopsis thaliana with double gene overexpression of HvHNMT1 and HvHNMT2 to diamondback moth

[0096] To further verify the broad-spectrum insect-resistant effects of HvHNMT1 and HvHNMT2 in plants, a stable genetically inherited Arabidopsis thaliana line was used to investigate the diamondback moth (Prunella vulgaris) activity. Plutella xylostella ) In vivo biological assay.

[0097] OE-HvHNMT1 / HvHNMT2 Arabidopsis thaliana plants with consistent growth and at the 5-6 true leaf stage, as well as empty vector Arabidopsis thaliana (EV), were selected as experimental materials. Each group had 12 independent biological replicates, with one Arabidopsis thaliana plant placed in each replicate and inoculated with one newly hatched diamondback moth larva. The larvae were reared continuously for 14 days at 22 ℃, 50%-60% relative humidity, and 16 h light / 8 h dark conditions, and the larval weight gain was observed and recorded.

[0098] The results are as follows Figure 9As shown, larvae feeding on empty Arabidopsis leaves can grow and develop normally, and their weight gain is significantly higher than that of diamondback moths feeding on OE-HvHNMT1 / HvHNMT2 Arabidopsis leaves, while diamondback moth larvae feeding on OE-HvHNMT1 / HvHNMT2 Arabidopsis leaves have slow weight gain.

[0099] The above results indicate that the key enzymes HvHNMT1 and HvHNMT2 for malt alkaloid biosynthesis identified for the first time in this invention can efficiently catalyze the accumulation of malt alkaloid in Arabidopsis thaliana and endow the plant with significant insect resistance to diamondback moth, inhibiting the feeding of pests. This suggests that the genes of this invention have the potential for broad-spectrum resistance to lepidopteran pests and can be used to cultivate transgenic crops with excellent insect resistance.

[0100] Example 10: Construction and identification of tomato lines stably overexpressing HvHNMT1 and HvHNMT2 genes

[0101] To further verify the application potential of the HvHNMT1 and HvHNMT2 genes of this invention in economic crops, the HvHNMT1 and HvHNMT2 dual-expression plant vector and the pCAMBIA1305 empty vector obtained in Example 4 were transformed into tomatoes using Agrobacterium-mediated transformation. Solanum lycopersicum )variety.

[0102] The specific procedure is as follows: Agrobacterium carrying the HvHNMT1 / HvHNMT2 dual expression vector or the pCAMBIA1305 empty vector is cultured until the bacterial culture reaches OD. 600 The explants were selected from aseptically germinated tomato cotyledons and hypocotyls 7–10 days after germination. After infection with Agrobacterium for 10–15 min, they were co-cultured at 25 °C in the dark for 2 days. Subsequently, the explants were transferred to selection medium (MS basal medium, supplemented with 30 g / L sucrose, 2 mg / L 6-BA, 0.1 mg / L IAA, 3 g / L plant gel, pH 5.8) containing 50 mg / L hygromycin B and 250 mg / L cefotaxime for two consecutive rounds of resistance selection, each round lasting 14 days, at 25 °C.

[0103] The resistant shoots obtained through screening were transferred to elongation medium (MS medium supplemented with 30 g / L sucrose, 0.1 mg / L 6-BA, 20 mg / L hygromycin B, 300 mg / L Timentin, 3 g / L plant gel, pH 5.8) for further culture. When the seedlings grew to 3-5 cm, they were transferred to rooting medium (1 / 2 MS medium supplemented with 20 g / L sucrose, 3 g / L plant gel, pH 5.8) and induced to form complete root systems at 25 ℃ to obtain regenerated tomato plants (T0 generation).

[0104] Genomic DNA and total RNA were extracted from leaves of T0 generation tomatoes and subjected to PCR detection (primers same as in Example 6). PCR amplification products were detected by agarose gel electrophoresis, and positive amplified fragments were sequenced for verification. The dual-gene overexpression line with correct sequencing results and stable expression was named OE-HvHNMT1 / HvHNMT2.

[0105] T1 generation seeds were harvested, and genetically stable homozygous T2 lines were obtained through resistance selection. OE-HvHNMT1 /

[0106] HvHNMT2 tomatoes were sown simultaneously with empty vector transformed lines (EV) and wild-type tomatoes (WT), and cultured at 25 ℃, relative humidity of 50%–60%, and 16 h light / 8 h dark conditions until the 6–8 true leaf stage.

[0107] Take 100 mg of leaves, freeze and grind them in liquid nitrogen, add 1 mL of 50% (v / v) methanol aqueous solution, and extract by shaking at 800 rpm for 2 h. The content of maltine in the leaves is detected by UPLC-MS / MS method described in Example 3.

[0108] The test results show that ( Figure 10 No maltine was detected in the leaves of the empty vector strain, while significant accumulation of maltine was detected in the leaves of OE-HvHNMT1 / HvHNMT2 tomato strains, indicating that HvHNMT1 and HvHNMT2 can be stably expressed in tomatoes and catalyze the biosynthesis of maltine.

[0109] Example 11: Evaluation of resistance of tomatoes overexpressing HvHNMT1 and HvHNMT2 genes to cotton bollworm.

[0110] To further evaluate the insect-resistant application effects of HvHNMT1 and HvHNMT2 of the present invention in economic crops, the stable genetic OE-HvHNMT1 / HvHNMT2 tomato line obtained in Example 10 was used to investigate the bollworm (… Helicoverpa armigera Bioassay.

[0111] OE-HvHNMT1 / HvHNMT2 tomatoes and empty vector tomatoes (EVs) with uniform growth and at the 6-8 true leaf stage were selected as experimental materials. Bioassays were performed using a feeding method. One leaf each of OE-HvHNMT1 / HvHNMT1 and EV tomatoes was placed in a petri dish, and one third-instar bollworm larva was released from the center of each dish. Ten independent biological replicates were set up for each group. The plants were cultured at 25 ℃, 50%–60% relative humidity, and under 16 h light / 8 h darkness conditions. The weight gain of the larvae on the leaves under different treatments was recorded after 48 h.

[0112] The results are as follows Figure 11 As shown, compared with the larvae feeding on empty tomato leaves, the cotton bollworm larvae feeding on OE-HvHNMT1 / HvHNMT2 tomato leaves consumed significantly less food, their growth was significantly inhibited, and they exhibited obvious refusal to feed.

[0113] The above results further demonstrate that HvHNMT1 and HvHNMT2 provided by this invention can stably catalyze the accumulation of malt alkaloids in tomatoes and significantly enhance the repellency of tomatoes against bollworms, reducing the damage caused by pest feeding. This indicates that the genes of this invention have good cross-species application potential and can be widely used in the genetic improvement of insect resistance in food crops, cash crops, and horticultural crops.

[0114] Example 12: Screening and identification of candidate HNMTs from millet, wheat, oats and rye (Gramineae plants)

[0115] Using the HvHNMT1 amino acid sequence as a template, BLAST was performed on the GrainGenes website. Data sources included wheat pangenome, Rye Protein Collections, and Oat Protein Collections. For millet, BLAST was performed in the Phytozome plant genome database. Genes with high similarity were obtained (Table 2).

[0116] The gene obtained from BLAST was constructed into the pET28a vector using the method in Example 1, and the protein was expressed and purified using the method in Example 2. The purified protein was then subjected to in vitro enzyme activity detection using the method in Example 3.

[0117] Table 2. Candidate HNMTs in different Poaceae species

[0118] The results are as follows Figure 12As shown, the gene expression products of wheat GWHPEQUM058572, rye GWHPASIY030355, GWHPASIY013245, oat AVINS.12001a.r2.6Dg0000737, and millet AJ88BG11210.1 can catalyze the production of N-methyltyramine from tyramine, and further catalyze the production of maltine from N-methyltyramine. No N-methyltyramine or maltine was detected in the blank control CK. Therefore, the wheat GWHPEQUM058572 gene is designated as TaHNMT1, and the amino acid sequence of the encoded protein is shown in SEQ EQ ID NO.4, while the nucleotide sequence of the encoded gene is shown in SEQ EQ ID NO.12. The rye genes GWHPASIY030355 and GWHPASIY013245 are designated as SCHNMT1 (encoding protein amino acid sequence as shown in SEQ EQ ID NO. 5) and SCHNMT2 (encoding protein amino acid sequence as shown in SEQ EQ ID NO. 6), respectively. The oat gene AVINS.12001a.r2.6Dg0000737 is designated as AsHNMT1, and its encoded protein amino acid sequence is shown in SEQ EQ ID NO. 7. The millet gene AVINS.12001a.r2.6Dg0000737 is designated as PmHNMT1, and its encoded protein amino acid sequence is shown in SEQ EQ ID NO. 8. The nucleotide sequence of the encoding gene is shown in SEQ EQ ID NO. 13.

[0119] The eight N-methyltransferases obtained above were sequence aligned. Figure 13 The study revealed a highly conserved catalytic active site in the catalytic center region of the N-methyltransferase exhibiting maltine synthesis activity: tryptophan at position 255, leucine at position 257, and aspartic acid at position 262. The Trp, Leu, and Asp residues remained unchanged throughout the sequence, indicating that they are absolutely conserved and may play a crucial role in enzyme structural stability or catalytic function. Furthermore, the N-methyltransferase possesses an amino acid sequence with 46.15% or more homology to the conserved sequence shown in SEQ ID NO.14 (AILM KWIL NCFSD).

[0120] The above embodiments are for illustrative purposes only and are not intended to limit the scope of protection of the present invention. For those skilled in the art, any modifications, equivalent substitutions, and improvements made to the present invention without departing from its spirit and essence should be included within the scope of protection of the present invention.

Claims

1. An N-methyltransferase for the synthesis of maltine, characterized in that, The N-methyltransferase has conserved catalytic active site residues and has the activities of catalyzing the formation of N-methyltyramine from tyramine and catalyzing the formation of maltine from N-methyltyramine; the catalytic active site residues include tryptophan at position 255, leucine at position 257, and aspartic acid at position 262.

2. The N-methyltransferase according to claim 1, characterized in that, The N-methyltransferase has an amino acid sequence with 46.15% or more homology to the conserved sequence shown in SEQ ID NO.

14.

3. The N-methyltransferase according to claim 1, characterized in that, The N-methyltransferase has an amino acid sequence with 42.24% or more homology to SEQ ID NO.

1.

4. The N-methyltransferase according to claim 1, characterized in that, The amino acid sequence of the N-methyltransferase is shown in one of SEQ ID NO.1-SEQ ID NO.

8.

5. A recombinant genetically engineered bacterium comprising a nucleic acid molecule encoding the N-methyltransferase of claim 1.

6. The application of the N-methyltransferase of claim 1 in the biosynthesis of maltine.

7. The application as described in claim 6, characterized in that, The application involves a methylation reaction in the presence of S-adenosylmethionine, using tyramine as a substrate, under the action of the N-methyltransferase or an organism expressing the N-methyltransferase, ultimately producing maltine.

8. The use of the N-methyltransferase of claim 1 in the preparation of plant insecticides.

9. The application as described in claim 8, characterized in that, The application involves transferring the N-methyltransferase into the plant genome using Agrobacterium infection to obtain transgenic plants with increased malt alkaloid content, thereby enhancing the host plant's insect resistance and antifeedant activity.

10. The use of the N-methyltransferase of claim 1 in the cultivation of insect-resistant transgenic crops.

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