Synthesis gene acnmt3 of benzyl isoquinoline alkaloid and derivative, protein, application and screening method

Abstract

The present application relates to the technical field of plant genetic engineering, and specifically discloses a benzylisoquinoline alkaloid and derivative synthesis gene AcNMT3, a protein, an application, and a screening method. The screening method of the present application can accurately and effectively screen out the coding gene of nitrogen methyltransferase AcNMT3 in the synthesis pathway of the benzylisoquinoline alkaloid and derivative aristolochic acid of aristolochia contorta, and determine the encoded protein and function. AcNMT3 has the functions of catalyzing nornuciferine to generate N-methylnornuciferine, catalyzing nuciferine to generate N-methylnuciferine, catalyzing 3'-hydroxynuciferine to generate 3'-hydroxy-N-methylnuciferine, catalyzing N-methylnuciferine to generate magnoflorine, and catalyzing laurifoline to generate fujimorine, wherein the conversion efficiency of the secondary amine nitrogen to the tertiary amine nitrogen is the highest, so the main function is AcCNMT. The present application discloses one of the key enzymes in the synthesis of the benzylisoquinoline alkaloid of aristolochia contorta, and lays a foundation for the analysis of the biosynthesis pathway of the benzylisoquinoline alkaloid and derivative, the biosynthesis of the medicinal components, and the molecular breeding of aristolochia contorta.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering technology, and more specifically to the gene AcNMT3 for the synthesis of benzyl isoquinoline alkaloids and their derivatives, the protein, its application, and screening methods. Background Technology

[0002] Northern Aristolochic ...

[0003] To ensure the safe and appropriate use of these medicinal materials, it is crucial to completely remove aristolochic acid. Currently, the main methods for reducing the toxicity of aristolochic acid-containing medicinal materials include processing, compounding, and traditional breeding. However, these methods all have drawbacks such as high cost, long processing time, difficulty in quantitative detection, and challenges in supervision. Molecular breeding is one of the effective ways to improve medicinal material varieties. Theoretically, once the biosynthetic pathway of aristolochic acid is fully understood, this component can be completely removed from medicinal materials.

[0004] Aristolochic acid belongs to the benzyl isoquinoline alkaloid derivatives, and its upstream synthetic pathway overlaps with that of benzyl isoquinoline alkaloids. Furthermore, benzyl isoquinoline alkaloids are a class of metabolites with significant medicinal value. Studying the function of key enzymes in their shared synthetic pathway will help in removing aristolochic acid from medicinal materials using molecular breeding techniques and enhancing the efficacy of benzyl isoquinoline alkaloids. It also lays the foundation for the synthetic biology applications of the active components of benzyl isoquinoline alkaloids in aristolochic acid-containing medicinal materials.

[0005] Therefore, how to provide the AcNMT3 gene for the synthesis of benzyl isoquinoline alkaloids and their derivatives and study its biological metabolic pathways for application in the reduction of toxicity of medicinal materials is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0006] In view of this, the present invention provides the gene AcNMT3 for the synthesis of benzyl isoquinoline alkaloids and their derivatives, the protein, its application, and a screening method.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] A gene for the synthesis of benzyl isoquinoline alkaloids and their derivatives, AcNMT3, has the nucleotide sequence shown in SEQ ID No. 1.

[0009] A gene AcNMT3 for the synthesis of benzyl isoquinoline alkaloids and their derivatives is disclosed, the amino acid sequence of which encodes a protein as shown in SEQ ID No. 2.

[0010] Furthermore, the full-length amplification primers for the AcNMT3 gene are as follows:

[0011]

[0012] The application of the AcNMT3 gene in any of the following: biosynthetic pathway analysis, biosynthesis of pharmacodynamic components, or molecular improvement breeding.

[0013] The screening method for the gene AcNMT3, which synthesizes benzyl isoquinoline alkaloids and their derivatives, is as follows:

[0014] 1) Based on the Aristolochia contorta genome, members of the Aristolochia contorta NMT gene family were screened, a phylogenetic tree was constructed, and the evolutionary relationship between NMT and other species was analyzed. Key genes of benzylisoquinoline alkaloid N-methyltransferase in Aristolochia contorta were preliminarily screened.

[0015] 2) Based on the transcriptome data and aristolochic acid content of roots, stems, leaves and flowers of Aristolochic Acid, the expression of differentially expressed genes was analyzed, and key genes of benzylisoquinoline alkaloid N-methyltransferase of Aristolochic Acid were further screened.

[0016] Further, the specific operation of step 1) is as follows: search the NCBI database for nine known functional protein sequences of CjCNMT, TfCNMT, TfPavNMT, PsRNMT, PsTNMT, PsNMT4, EcTNMT, GflNMT, and PbTNMT from poppy, Japanese coptis, ephedra, yellow pine grass, California poppy, yellow sea poppy, and big red poppy. Use the above nine enzyme protein sequences as the initial query sequences, and use the tBLASTn program to locally search the Aristolochia javanica nucleic acid database to obtain the Aristolochia javanica AcNMT sequence;

[0017] The AcNMT protein sequence was compared with the NMT protein sequences of other species related to the synthesis of benzyl isoquinoline alkaloids using MUSCLE to construct an NJ phylogenetic tree, with bootstrap selection repeated 1000 times.

[0018] We focused on 10 AcNMTs clustered with CNMT and RNMT, namely AcNMT1-10; based on transcriptomic data, we extracted the expression levels (FPKM values) of the 10 AcNMTs in different tissues and plotted a heatmap.

[0019] Further, in step 1), the NMT protein sequences of the other species include PsCNMT, AY217336; PsRNMT, KX369612; PsNMT4, KX369613; PsTNMT, Q108P1; CjCNMT, AB061863; EcTNMT, EU882977; TfCNMT, AY610508; TfPavNMT, EU883010; PbTNMT, EU882994.

[0020] Furthermore, the analysis of differential gene expression in step 2) is based on the comparison of transcriptome data and aristolochic acid content of roots, stems, leaves, and flowers of Aristolochic Acid in step 1) using the heatmap drawn in step 1).

[0021] Functional verification of AcNMT3, the gene for synthesizing benzyl isoquinoline alkaloids and their derivatives, was performed using the following method:

[0022] 1) The AcNMT3 encoding gene was cloned into a vector to form a recombinant expression vector, and the recombinant expression vector and the empty vector were transformed into Escherichia coli expression strains, respectively;

[0023] 2) Take the overnight activated bacterial culture and add it to LB liquid medium for culture. When the OD value reaches 0.6-0.8, add IPTG to make the final concentration 0.5mM and start induction.

[0024] 3) After induction for 20 hours, centrifuge to collect bacterial cells, sonicate to disrupt them, and collect the supernatant;

[0025] 4) Filter the supernatant through a 0.45 μm filter membrane, load it onto a well-equilibrated Ni column, wash three times with washing buffer, wash three times with elution buffer, collect the eluent, concentrate it to 500 μL using an ultrafiltration tube, and obtain purified protease solution.

[0026] 5) To verify the purified protease solution, the reaction system was prepared and incubated for 1 hour. The reaction was terminated by mixing with 50 μL of methanol, centrifuged, and filtered through a 0.22 μm filter membrane. The chemical composition was detected by UPLC, and the analysis was performed based on the chromatogram and mass spectrum.

[0027] Further, in step 5), the reaction system includes 25 mM Tris-HCl (pH 8.0), 25 mM sodium vitamin C, 0.1 mM S-adenosylmethionine, 10 μg of purified protein, and 100 mM substrate;

[0028] The substrates include (S)-norcodonine, (S)-codonine, (S)-3'-hydroxycodonine, (S)-N-methylcodonine, and (S)-codonine.

[0029] The UPLC detection was performed using an ACQUITY UPLC BEH C18 column (2.1 x 100 nm, 1.7 μm; Waters); detection wavelength: 280 nm; column temperature: 35 °C; flow rate: 0.3 mL / min. -1 Injection volume: 10 μL; Mobile phase: methanol (A), 0.1% formic acid water (B); Gradient elution conditions: 0-0.5 min, maintain 5% A; 0.5-4 min, increase 5% A to 95% A; 4-6 min, maintain 95% A; 6-6.1 min, decrease 95% A to 5% A; 6.1-8 min, maintain 5% A.

[0030] As can be seen from the above technical solutions, compared with the prior art, this invention discloses the screening, identification and application of the AcNMT3 gene for the synthesis of benzyl isoquinoline alkaloids and their derivatives, and provides the N-methyltransferase AcNMT3 encoding gene involved in the synthesis pathway of benzyl isoquinoline alkaloids and their derivative aristolochic acid in Aristolochic acid, as well as the protein it encodes, and the screening and functional verification methods, laying the foundation for the analysis of the biosynthetic pathway of benzyl isoquinoline alkaloids and their derivative aristolochic acid in Aristolochic acid, the biosynthesis of pharmacodynamic components, and the molecular improvement breeding of Aristolochic acid. Attached Figure Description

[0031] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0032] Figure 1 For the identification and evolutionary analysis of AcNMT based on the Aristolochia brevis genome;

[0033] Figure 2 To illustrate the differential expression of the AcNMT gene in different tissues of Aristolochia debilis;

[0034] Figure 3 The mechanism by which AcNMT3 encodes the protein that catalyzes norcodonine;

[0035] Figure 4 The mechanism by which AcNMT3 encodes the protein that catalyzes causative balsamine;

[0036] Figure 5 The mechanism by which AcNMT3 encodes the protein that catalyzes 3'-hydroxycodonine;

[0037] Figure 6 The mechanism by which AcNMT3 encodes the protein that catalyzes N-methylcorydaline;

[0038] Figure 7 The mechanism by which AcNMT3-encoded protein catalyzes bovine heart fruit alkaloids. Detailed Implementation

[0039] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0040] Example 1

[0041] The AcNMT3 gene has the following nucleotide sequence as shown in SEQ ID No. 1.

[0042] >SEQ ID No.1

[0043]

[0044] Example 2

[0045] The protein encoded by the AcNMT3 gene has the amino acid sequence shown in SEQ ID No. 2.

[0046] >SEQ ID No.2

[0047] MANSPAMAPEKHNNSDQMLRKLEEGSVPDNEIRRLMRIELERRLQWCRKPSYGEQAAEEVALVKYLRKQGICTESDSLNSQAYETPISSLKTIFGELLKGSPSYFKNDSMTLDEAEVAMLDLCCERAQLKDGQKLLDLGCGHGAFTLHVAQKFKNSHVTAVTNSVSQKMYIEDQCRILEL SNVEIILEDMAKLTMDTTFDRVIVIGLFEHMKNYGLLLQHISQWMTADGLLFVDHACHKSFAYLSEPLDEDDWFTEYIFPPGSLIIPSSSFLLYFQDDVCVVDHWILSGKHFSRTAAEWLKRLDGNVEMAKEILESSFGGREAVVKEINYWRGSCIYTEEFFGYNGGEEWMTSHLLFKKK*

[0048] Example 3: Screening and phylogenetic analysis of the AcNMT gene based on Aristolochic acid genome and transcriptome data.

[0049] Nine NMT protein sequences with known functions (CjCNMT, TfCNMT, TfPavNMT, PsRNMT, PsTNMT, PsNMT4, EcTNMT, GflNMT, and PbTNMT) from *Papaversomniferum*, *Coptis japonica*, *Ephedra sinica*, *Thalictrum flavum*, *Eschscholzia californica*, *Glaucium flavum*, and *Papaverbracteatum* were retrieved from the NCBI database. These nine enzyme protein sequences were used as initial query sequences, and the *Aristolochia contorta* nucleic acid database was searched locally using the tBLASTn program to obtain the *Aristolochia contorta* AcNMT sequence.

[0050] The AcNMT protein sequence was aligned with NMT protein sequences from other species associated with the synthesis of benzyl isoquinoline alkaloids (PsCNMT, AY217336; PsRNMT, KX369612; PsNMT4, KX369613; PsTNMT, Q108P1; CjCNMT, AB061863; EcTNMT, EU882977; TfCNMT, AY610508; TfPavNMT, EU883010; PbTNMT, EU882994) using Muscle alignment to construct an NJ phylogenetic tree. Bootstrap selection was repeated 1000 times. The results are shown in the attached figure. Figure 1 .

[0051] Pay close attention to the appendix Figure 1 Ten AcNMTs, AcNMT1-10, were clustered with CNMT and RNMT. Based on transcriptomic data, the expression levels (FPKM values) of these 10 AcNMTs in different tissues were extracted, and a heatmap was plotted. Figure 2 Comparative studies revealed that AcNMT3 expression was more significant in different tissues and positively correlated with aristolochic acid accumulation. It is speculated that this is likely related to aristolochic acid synthesis.

[0052] Example 4: Cloning of the AcNMT3 gene from Aristolochic acid

[0053] Primers for full-length amplification of the AcNMT3 gene were designed based on the open reading frame of the AcNMT3 sequence. Using cDNA from *Aristolochia debilis* as a template, PCR amplification yielded the nucleotide sequence of the AcNMT3 gene, as shown in SEQ ID No. 1 (1083 bp). The amino acid sequence of AcNMT3, comprising 360 amino acid residues, was deduced after translation, as shown in SEQ ID No. 2. The primers for full-length amplification of the AcNMT3 gene are as follows:

[0054]

[0055]

[0056] Example 5: AcNMT3 protein induction, expression, and purification

[0057] The AcNMT3 encoding gene was cloned into the pET30a vector to form the pET30a-AcNMT3 recombinant expression vector. This vector and the empty pET30a vector were then transformed into the BL21(DE3) *E. coli* expression strain. 1 mL of the overnight activated bacterial culture was added to 100 mL of LB liquid medium (containing 50 μg / mL kanamycin) and incubated at 37°C and 200 rpm for 3.5 h. When the OD value reached 0.6-0.8, IPTG was added to a final concentration of 0.5 mM, and induction was initiated at 16°C and 200 rpm. After 20 h of induction, the bacterial cells were collected by centrifugation, sonicated, and the supernatant was further purified.

[0058] The obtained solution was filtered through a 0.45 μm filter membrane and loaded onto a pre-equilibrated Ni column. It was washed three times with 10 mL of washing buffer (500 mM NaCl, 20 mM Tris-HCl pH 8.0, 100 mM imidazole). The eluent was then washed three times with 6 mL of elution buffer (500 mM NaCl, 20 mM Tris-HCl pH 8.0, 500 mM imidazole) and collected. The eluent was concentrated to 500 μL using an ultrafiltration tube, and the protein concentration was determined to obtain the purified protease solution.

[0059] Example 6: Key to the biosynthesis of aristolochic acid and benzyl isoquinoline alkaloids from Aristolochic acid

[0060] Functional verification of the enzymes AcCNMT and AcRNMT

[0061] The purified protease solution was functionally validated in vitro using the following reaction system (50 μL): 25 mM Tris-HCl (pH 8.0), 25 mM sodium vitamin C, 0.1 mM S-adenosylmethionine, 10 μg of purified protein, and 100 mM substrate, including (S)-norcoguline, (S)-coguline, (S)-3'-hydroxycoguline, (S)-N-methylcoguline, and (S)-caryophylline. The reaction was incubated at 37°C for 1 hour, terminated by mixing with 50 μL of methanol, centrifuged, and filtered through a 0.22 μm filter. UPLC was used to determine the chemical composition. An ACQUITY UPLC BEH C18 column (2.1 x 100 nm, 1.7 μm; Waters) was used; detection wavelength: 280 nm; column temperature: 35°C; flow rate: 0.3 mL / min. -1 Injection volume: 10 μL; Mobile phase: methanol (A), 0.1% formic acid water (B); Gradient elution conditions: 0-0.5 min, maintain 5% A; 0.5-4 min, increase 5% A to 95% A; 4-6 min, maintain 95% A; 6-6.1 min, decrease 95% A to 5% A; 6.1-8 min, maintain 5% A.

[0062] Based on the chromatogram and mass spectrum, i.e., attached Figure 3-7 Analysis showed that AcNMT3 has the functions of catalyzing the conversion of norcodonine to N-methylnorcodonine, catalyzing the conversion of caudonine to N-methylcaudonine, catalyzing the conversion of 3'-hydroxycaudonine to 3'-hydroxy-N-methylcaudonine, catalyzing the conversion of N-methylcaudonine to magnocurarine, and catalyzing the conversion of caudonine to styraxine. Among these, the conversion efficiency of secondary amine nitrogen to tertiary amine nitrogen is the highest. Therefore, AcNMT3 mainly has the function of AcCNMT (caudonine N-methyltransferase).

[0063] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0064] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A gene for synthesizing a benzyl isoquinoline alkaloid and its derivatives. AcNMT3 Its characteristics are, Its nucleotide sequence is shown in SEQ ID No.

1.

2. The gene-encoded protein according to claim 1, characterized in that, The amino acid sequence of the protein is shown in SEQ ID No.

2.

3. An amplification method for the gene according to claim 1 AcNMT3 The primer set is characterized by, The sequences are shown in SEQ ID NO.3~SEQ ID NO.

4.

4. The primer set according to claim 3 in the identification AcNMT3 Applications in genes.

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