Application of ZmTDC protein and coding gene thereof in catalyzing tryptophan to synthesize tryptamine

Abstract

The invention belongs to the field of plant genetic engineering, and particularly relates to application of ZmTDC protein and a coding gene thereof in catalyzing tryptophan to synthesize tryptamine, and the amino acid sequence of the ZmTDC protein is shown as SEQ ID NO.1. The tryptophan decarboxylase-ZmTDC protein is identified in the corn body for the first time, and in-vitro enzyme activity reaction confirms that the ZmTDC protein has the function of converting tryptophan into tryptamine, is extremely high in activity and is a key enzyme for the first step of a melatonin synthesis pathway in the corn body. The identification of the ZmTDC protein fills up the blank of research on key enzymes for synthesizing melatonin in corn at present, and has important significance on research on endogenous synthesis of melatonin in corn.

Description

Technical Field

[0001] The invention belongs to the field of plant genetic engineering, and in particular relates to the application of ZmTDC protein and its encoding gene in catalyzing tryptophan to synthesize tryptamine. Background Art

[0002] Melatonin, as an emerging plant hormone, plays a key role in guiding seed germination, shaping root structure, regulating stomatal activity, synchronizing biological rhythms, controlling flowering and aging, etc. In addition, melatonin can stimulate the antioxidant defense mechanism of plants and effectively remove harmful reactive oxygen species (ROS), thereby alleviating oxidative stress, osmotic stress, protein denaturation, cell damage and other problems, and significantly improving the resistance of plants to various biological and abiotic stresses.

[0003] In plants, tryptophan is first decarboxylated to tryptamine by tryptophan decarboxylase (TDC), and then converted to 5-hydroxytryptamine by tryptamine-5-hydroxylase (T5H). 5-hydroxytryptamine is acetylated to N-acetyl-5-hydroxytryptamine by 5-hydroxytryptamine-N-acetyltransferase (SNAT), and then synthesized into melatonin by N-acetyl-5-hydroxytryptamine methyltransferase (ASMT) and caffeic acid-0-methyltransferase (COMT). In addition, 5-hydroxytryptamine can also be catalyzed by ASMT / COMT to 5-methoxytryptamine, and then synthesized into melatonin by SNAT.

[0004] Tryptophan decarboxylase (TDC) can convert tryptophan into tryptamine and is the first key enzyme in the melatonin synthesis pathway. However, tryptophan decarboxylase and the gene encoding tryptophan decarboxylase have not yet been identified in corn. Summary of the invention

[0005] In view of the deficiency of the prior art in the research on TDC in corn, the present invention provides the application of ZmTDC protein and its encoding gene in catalyzing the synthesis of tryptophan into tryptamine. The specific technical scheme is as follows:

[0006] In a first aspect, the present invention provides the use of a ZmTDC protein in catalyzing the synthesis of tryptamine from tryptophan, wherein the amino acid sequence of the ZmTDC protein is shown in SEQ ID NO.1.

[0007] In a second aspect, the present invention provides the use of the ZmTDC gene in catalyzing the synthesis of tryptamine from tryptophan, and the nucleotide sequence of the ZmTDC gene is shown in SEQ ID NO.2.

[0008] In a third aspect, the present invention provides an application of a recombinant vector in catalyzing the synthesis of tryptamine from tryptophan, wherein the recombinant vector comprises a ZmTDC gene; the nucleotide sequence of the ZmTDC gene is shown in SEQ ID NO.2.

[0009] In a fourth aspect, the present invention provides an application of a genetically engineered bacterium in catalyzing the synthesis of tryptophan from tryptophan, wherein the genetically engineered bacterium comprises a ZmTDC gene; the nucleotide sequence of the ZmTDC gene is shown in SEQ ID NO.2.

[0010] In a fifth aspect, the present invention provides a method for synthesizing tryptamine, wherein tryptamine is synthesized using a catalyst in the presence of pyridoxal phosphate using tryptophan as a substrate;

[0011] The catalyst is ZmTDC protein or its immobilized enzyme, or a genetically engineered bacterium containing the ZmTDC gene, or a crude enzyme solution or wet bacteria after fermentation of the genetically engineered bacterium;

[0012] The amino acid sequence of the ZmTDC protein is shown in SEQ ID NO.1, and the nucleotide sequence of the ZmTDC gene is shown in SEQ ID NO.2.

[0013] In a sixth aspect, the present invention provides the use of ZmTDC protein in promoting tryptamine synthesis in corn plants, wherein the amino acid sequence of the ZmTDC protein is shown in SEQ ID NO.1.

[0014] In a seventh aspect, the present invention provides the use of ZmTDC protein in promoting melatonin synthesis in corn plants, wherein the amino acid sequence of the ZmTDC protein is shown in SEQ ID NO.1.

[0015] Furthermore, in the synthesis of melatonin in corn plants, tryptophan is converted into tryptamine under the catalysis of ZmTDC protein.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] The present invention identified a tryptophan decarboxylase, ZmTDC protein, in maize for the first time. The in vitro enzyme activity reaction confirmed that ZmTDC protein has the function of converting tryptophan into tryptamine and has extremely strong activity. It is the key enzyme in the first step of the melatonin synthesis pathway in maize. The identification of ZmTDC protein fills the current research gap on the key enzymes for melatonin synthesis in maize, and is of great significance for the research on endogenous synthesis of melatonin in maize. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1It is a liquid chromatography detection spectrum of tryptamine in the in vitro reaction solution of GST-ZmTDC fusion protein; wherein, ZmTDC represents GST-ZmTDC fusion protein, CK represents protein-free buffer, and 500ng-Tryptamine represents 500ng tryptamine standard.

[0019] Figure 2 It is a liquid chromatography detection spectrum of tyramine in the in vitro reaction solution of GST-ZmTDC fusion protein; wherein, ZmTDC represents GST-ZmTDC fusion protein, Zm00029ab007000 represents GST-Zm00029ab007000 protein, CK represents protein-free buffer, and 10μg-Tryptamine represents 10μg tyramine standard. DETAILED DESCRIPTION

[0020] In order to make those skilled in the art better understand the scheme of the present invention, the technical scheme of the present invention is clearly and completely described below in conjunction with specific embodiments. It should be noted that the following detailed descriptions are all exemplary and are only embodiments of a part of the present invention, rather than all embodiments.

[0021] Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative work should fall within the scope of protection of the present invention.

[0022] Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those of ordinary skill in the art to which this application belongs. The experimental materials used in the examples of the present invention are all conventional experimental materials in the art and can be purchased through commercial channels. Experimental methods without specifying detailed conditions were performed according to conventional experimental methods or according to the operating instructions recommended by the supplier.

[0023] Competent Escherichia coli Overexpress C43 (DE3) was purchased from Shanghai Weidi Biotechnology Co., Ltd. Tryptophan and tryptamine standards, PBS buffer, and Tris-HCl buffer were purchased from Shanghai Bioengineering; pyridoxal phosphate was purchased from Shanghai Yuanye Biotechnology.

[0024] In the following examples, the corn variety used was Ki3.

[0025] In the following examples, the tryptamine or tyramine content in the in vitro reaction solution was detected using a high performance liquid chromatography-fluorescence detector (Shimadzu LC-20). The specific liquid chromatography conditions were as follows: The chromatographic column model was Yuexu XB-C18, specifications are chromatographic column filler particle diameter 5μm, chromatographic column inner diameter 4.6mm, chromatographic column length 150mm. Mobile phase composition is A is 0.1% formic acid aqueous solution, mobile phase B is methanol, using gradient elution method (Gradient Elution Procedures): 0min A / B (95:5, V / V), 8min A / B (40:60, V / V), 8.5min A / B (40:60, V / V), 9.5min A / B (95:5, V / V). Set the flow rate to 1mL / min, the injection volume to 5μL; the column oven is set to 35℃. The excitation wavelength of the detection is 280nm, and the emission wavelength is 348nm.

[0026] The amino acid sequence of the ZmTDC protein is shown in SEQ ID NO.1, and the nucleotide sequence of the ZmTDC gene is shown in SEQ ID NO.2.

[0027] SEQ ID NO.1

[0028] .

[0029] SEQ ID NO.2

[0030]

[0031] Example 1 Screening of target genes

[0032] Pyridoxal phosphate acts as a coenzyme in the decarboxylation reaction, so many decarboxylases including tryptophan decarboxylase often contain a pyridoxal phosphate-dependent decarboxylase (Pyridoxal_deC) domain. Therefore, the protein sequence library and annotation files of the maize variety "Ki3" were downloaded from the MaizeGDB website (Welcome to MaizeGDB), and the protein sequences of Arabidopsis and tobacco were searched using the Pyridoxal_deC.hmm hidden Markov model (PF00282), and candidate genes were screened with an evalue<0.01 threshold. The amino acid sequences of the candidate genes were aligned using CLUSTALW 2.0, and the species-specific Pyridoxal_deC.hmm hidden Markov model was reconstructed using the alignment results. The protein sequences of the species were re-searched, and the evalue<0.01 was also used as the threshold. The new candidate genes screened were the TDC family genes of these species. The candidate genes were confirmed to have a pyridoxal phosphate-dependent decarboxylase conserved domain using the NCBI CDD and Interpro databases.

[0033] The genes with gene numbers Zm00029ab171870 and Zm00029ab007000 in the MaizeGDB library were screened to have the conserved domain of pyridoxal phosphate-dependent decarboxylase and may have TDC function. These two genes were selected as candidate genes.

[0034] Example 2 Functional detection of candidate gene expression products

[0035] 1. Prokaryotic expression and purification of candidate gene expression products

[0036] 1) The constructed prokaryotic expression plasmids containing GST tags were transformed into the E. coli expression strain OverExpresss C43 (DE3) respectively. The transformation method was referred to the strain manual. The primers used are shown in Table 1.

[0037] Table 1

[0038]

[0039] 2) The steps of protein prokaryotic system induction and purification are as follows:

[0040] (1) The OverExpresss C43(DE3) strain transformed with the plasmid was coated with LB solid medium containing 50 μg / mL kanamycin and cultured at 37°C overnight;

[0041] (2) Pick a single clone with good growth from the LB plate and place it in 5 mL of LB liquid medium containing 50 μg / mL kanamycin. Culture it overnight at 37°C and 200 rpm until it becomes turbid.

[0042] (3) 3 mL of the turbid bacterial solution from the overnight culture was taken out and placed in 300 mL of LB liquid medium containing 50 μg / mL kanamycin. The culture was incubated at 37°C and 200 rpm until the OD value was about 0.6-0.8. Then, 100 μM IPTG was added to induce protein expression.

[0043] (4) Place in a shaker at 23°C and 180 rpm and continue culturing for 12 to 14 h;

[0044] (5) After the culture is completed, the cells are collected by centrifugation at 4°C and suspended in 60 mL of PBS buffer;

[0045] (6) Use a high-pressure homogenizer to break the protein, centrifuge at 9000 rpm at 4°C, and collect the supernatant;

[0046] (7) Add 1 mL of equilibrated GST purification medium to the supernatant and incubate at 4°C for 1-2 h;

[0047] (8) Pour the incubated supernatant containing the GST purification medium into the affinity chromatography column. After the liquid is drained, wash the GST purification medium with PBS buffer for 3-5 times;

[0048] (9) After washing, elute five times with 1 mL of GST elution buffer (50 mM Tris-HCl, 10 mM glutathione, pH = 8.0);

[0049] (10) The eluted protein solution was stored at -20°C until use;

[0050] (11) Take 10 μL of the eluted protein, add 2.5 μL of 5*SDS-PAGE loading buffer, heat at 98°C for 10 min, put on ice for 5 min, and run the SDS-PAGE gel at 200 V for about 30 min;

[0051] (12) The SDS-PAGE gel after electrophoresis was placed in FastBlue protein rapid staining solution for 1-2 hours, then washed in water overnight, observed, and photographed for preservation.

[0052] 2. In vitro reaction was performed using tryptophan as substrate and pyridoxal phosphate as coenzyme. The specific in vitro reaction solution was: 100 μL Tris-HCl (pH=7.4) buffer containing 0.5 mM tryptophan, 0.1 mM pyridoxal phosphate and 25 μg of fusion protein, and a buffer without protein was used as a blank control. After the reaction was completed at 30°C, 200 μL acetonitrile was added to precipitate the protein, centrifuged at 10,000 rpm for 2 minutes, and filtered with a 0.22 μM needle filter. The reaction solution was tested for tryptamine content using HPLC, and the results were as follows: Figure 1 shown.

[0053] Depend on Figure 1 It can be seen that the expression product of the gene with the gene number Zm00029ab171870 in the MaizeGDB library has a strong activity of decarboxylating tryptophan to tryptamine, and the expression product of the blank control and the gene with the gene number Zm00029ab007000 in the MaizeGDB library is not displayed in the spectrum. Therefore, the gene with the gene number Zm00029ab171870 in the MaizeGDB library is named ZmTDC gene, and its expression product is named ZmTDC protein.

[0054] 3. In vitro reaction was performed using tyrosine as substrate and pyridoxal phosphate as coenzyme. The specific in vitro reaction solution was: 100 μL Tris-HCl (pH=7.4) buffer containing 0.5 mM tyrosine, 0.1 mM pyridoxal phosphate and 25 μg of fusion protein, and a buffer without protein was used as a blank control. After the reaction was completed at 30°C, 200 μL acetonitrile was added to precipitate the protein, centrifuged at 10,000 rpm for 2 minutes, and filtered with a 0.22 μM needle filter. The obtained reaction solution was tested for tyramine content using HPLC, and the results were as follows: Figure 2 shown.

[0055] Depend on Figure 2 It can be seen that the ZmTDC protein has a weak activity of decarboxylating tyrosine to tyramine, while the expression product of the gene with the gene number Zm00029ab007000 in the MaizeGDB library has a strong activity of decarboxylating tyrosine to tyramine, indicating that it is a tyrosine decarboxylase.

Claims

1. Application of ZmTDC protein in catalyzing the synthesis of tryptamine from tryptophan, characterized in that: The amino acid sequence of the ZmTDC protein is shown in SEQ ID NO.

1.

2. Application of ZmTDC gene in catalyzing the synthesis of tryptamine from tryptophan, characterized in that: The nucleotide sequence of the ZmTDC gene is shown in SEQ ID NO.

2.

3. Use of a recombinant vector in catalyzing the synthesis of tryptamine from tryptophan, characterized in that: The recombinant vector comprises the ZmTDC gene; the nucleotide sequence of the ZmTDC gene is shown in SEQ ID NO.

2.

4. Use of a genetically engineered bacterium in catalyzing the synthesis of tryptamine from tryptophan, characterized in that: The genetically engineered bacteria comprises the ZmTDC gene; the nucleotide sequence of the ZmTDC gene is shown in SEQ ID NO.

2.

5. A method for synthesizing tryptamine, characterized in that: In the presence of pyridoxal phosphate, tryptamine is synthesized using a catalyst with tryptophan as substrate; The catalyst is ZmTDC protein or its immobilized enzyme, or a genetically engineered bacterium containing the ZmTDC gene, or a crude enzyme solution or wet bacteria after fermentation of the genetically engineered bacterium; The amino acid sequence of the ZmTDC protein is shown in SEQ ID NO.1, and the nucleotide sequence of the ZmTDC gene is shown in SEQ ID NO.

2.

6. Application of ZmTDC protein in promoting tryptamine synthesis in corn plants, characterized in that: The amino acid sequence of the ZmTDC protein is shown in SEQ ID NO.

1.

7. Application of ZmTDC protein in promoting melatonin synthesis in corn plants, characterized in that: The amino acid sequence of the ZmTDC protein is shown in SEQ ID NO.

1.

8. The use according to claim 7, characterized in that: In the synthesis of melatonin in corn plants, tryptophan is converted into tryptamine under the catalysis of ZmTDC protein.

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