Application of BnTDC protein and coding gene thereof in catalysis of tryptophan and tyrosine

Abstract

The invention belongs to the field of plant genetic engineering, and particularly relates to a BnTDC protein and application of a coding gene of the BnTDC protein in catalysis of tryptophan and tyrosine, and the BnTDC protein is BnaA01. TDC protein or BnaC01. TDC protein. The BnTDC protein with the TDC function is identified in rape for the first time, the BnTDC protein comprises BnaA01. TDC protein and BnaC01. TDC protein, in-vitro enzyme activity verification shows that the BnaA01. TDC protein and the BnaC01. TDC protein can catalyze tryptophan into tryptamine, and the BnTDC protein has important value in synthesis of tryptamine or in-vitro synthesis of melatonin and research on key enzymes for endogenous synthesis of melatonin in rape; in addition, in further functional verification, the BnaA01. TDC protein and the BnaC01. TDC protein are found to have the function of catalyzing tyrosine into tyramine, which is found for the first time in the rape body, and the BnaA01. TDC protein and the BnaC01. TDC protein are of great significance to research on the utilization approach of amino acid in the rape body.

Description

Technical Field

[0001] The invention belongs to the field of plant genetic engineering, and specifically relates to the application of BnTDC protein and its encoding gene in catalyzing tryptophan and tyrosine. Background Art

[0002] Melatonin (MT) is a derivative of tryptophan, chemically named N-acetyl-5-methoxytryptamine, and belongs to the small molecule indoleamine substances that are widely found in the animal and plant kingdoms. In plants, melatonin has the functions of regulating seed germination and root system architecture, regulating circadian rhythm, photoperiod, and tolerance to biotic and abiotic stresses, and is an emerging plant hormone.

[0003] As the research on plant melatonin continues to deepen, the synthesis route and key enzymes of melatonin in plants have gradually become clear. The synthesis of melatonin in plants starts with tryptophan, with 5-hydroxytryptamine as an intermediate, and finally synthesizes melatonin. In the process of converting tryptophan into 5-hydroxytryptamine, it must first be decarboxylated into tryptophan by tryptophan decarboxylase (TDC), and then tryptamine is converted into 5-hydroxytryptamine under the catalysis of tryptamine-5-hydroxylase (T5H). 5-hydroxytryptamine is acetylated into 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).

[0004] Tryptophan decarboxylase (TDC) is the first key enzyme in the melatonin biosynthesis pathway, which can catalyze the synthesis of tryptophan from tryptophan to tryptamine, but it has not yet been identified in many species, such as rapeseed. Summary of the invention

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

[0006] In a first aspect, the present invention provides the use of BnTDC protein in catalyzing the synthesis of tryptamine from tryptophan, wherein the BnTDC protein is BnaA01.TDC protein or BnaC01.TDC protein; the amino acid sequence of the BnaA01.TDC protein is shown in SEQ ID NO.1, and the amino acid sequence of the BnaC01.TDC protein is shown in SEQ ID NO.2.

[0007] In a second aspect, the present invention provides an application of a BnTDC gene in catalyzing the synthesis of tryptamine from tryptophan, wherein the BnTDC gene is a BnaA01.TDC gene or a BnaC01.TDC gene; the nucleotide sequence of the BnaA01.TDC gene is shown in SEQ ID NO.3, and the nucleotide sequence of the BnaC01.TDC gene is shown in SEQ ID NO.4.

[0008] In a third aspect, the present invention provides an application of BnTDC protein in catalyzing the synthesis of tyramine from tyrosine, wherein the BnTDC protein is BnaA01.TDC protein or BnaC01.TDC protein; the amino acid sequence of the BnaA01.TDC protein is shown in SEQ ID NO.1, and the amino acid sequence of the BnaC01.TDC protein is shown in SEQ ID NO.2.

[0009] In a fourth aspect, the present invention provides an application of a BnTDC gene in catalyzing the synthesis of tyramine from tyrosine, wherein the BnTDC gene is a BnaA01.TDC gene or a BnaC01.TDC gene; the nucleotide sequence of the BnaA01.TDC gene is shown in SEQ ID NO.3, and the nucleotide sequence of the BnaC01.TDC gene is shown in SEQ ID NO.4.

[0010] The present invention obtains tryptophan decarboxylase (TDC) with gene numbers BnaC01T0074100WE and BnaA01T0004400WE in the BnPIR library by screening the decarboxylase domain dependent on pyridoxal phosphate. The two genes and their expression products are TDC proteins identified for the first time in rapeseed, which are of great significance for the study of rapeseed melatonin synthesis. The two proteins are named BnaA01.TDC protein and BnaC01.TDC protein. In further functional detection of BnaA01.TDC protein and BnaC01.TDC protein, it is found that the two proteins also have the function of tyrosine decarboxylase. It is generally believed that tyrosine decarboxylase cannot catalyze tryptophan, and tryptophan decarboxylase cannot catalyze tyrosine. Therefore, the discovery of BnaA01.TDC protein and BnaC01.TDC protein having both tyrosine decarboxylase and tryptophan decarboxylase functions is of great significance for the study of amino acid utilization pathways in rapeseed.

[0011] In a fifth aspect, the present invention provides an application of a recombinant vector in catalyzing the decarboxylation of tryptophan and / or tyrosine, the recombinant vector comprising a BnTDC gene; the BnTDC gene is a BnaA01.TDC gene or a BnaC01.TDC gene; the nucleotide sequence of the BnaA01.TDC gene is shown in SEQ ID NO.3, and the nucleotide sequence of the BnaC01.TDC gene is shown in SEQ ID NO.4; the tryptophan is decarboxylated to produce tryptamine, and the tyrosine is decarboxylated to produce tyramine.

[0012] In a sixth aspect, the present invention provides an application of a genetically engineered bacterium in catalyzing the decarboxylation of tryptophan and / or tyrosine, wherein the genetically engineered bacterium comprises a BnTDC gene; the BnTDC gene is a BnaA01.TDC gene or a BnaC01.TDC gene; the nucleotide sequence of the BnaA01.TDC gene is shown in SEQ ID NO.3, and the nucleotide sequence of the BnaC01.TDC gene is shown in SEQ ID NO.4; the tryptophan is decarboxylated to produce tryptamine, and the tyrosine is decarboxylated to produce tyramine.

[0013] In a seventh aspect, the present invention provides a method for synthesizing tryptamine, wherein tryptamine is synthesized by using tryptophan as a substrate and catalyzed by BnTDC protein in the presence of pyridoxal phosphate;

[0014] The BnTDC protein is BnaA01.TDC protein or BnaC01.TDC protein; the amino acid sequence of the BnaA01.TDC protein is shown in SEQ ID NO.1, and the amino acid sequence of the BnaC01.TDC protein is shown in SEQ ID NO.2.

[0015] In an eighth aspect, the present invention provides an application of BnTDC protein in promoting tryptamine synthesis in rapeseed plants, wherein the BnTDC protein is BnaA01.TDC protein or BnaC01.TDC protein; the amino acid sequence of the BnaA01.TDC protein is shown in SEQ ID NO.1, and the amino acid sequence of the BnaC01.TDC protein is shown in SEQ ID NO.2.

[0016] In a ninth aspect, the present invention provides the use of BnTDC protein in promoting melatonin synthesis in rapeseed plants, wherein the BnTDC protein is BnaA01.TDC protein or BnaC01.TDC protein; the amino acid sequence of the BnaA01.TDC protein is shown in SEQ ID NO.1, and the amino acid sequence of the BnaC01.TDC protein is shown in SEQ ID NO.2.

[0017] Furthermore, in the synthesis of melatonin in rapeseed plants, tryptophan is converted into tryptamine under the catalysis of BnTDC protein.

[0018] As the first key enzyme in the rapeseed melatonin synthesis pathway, BnaA01.TDC protein and BnaC01.TDC protein can convert tryptophan into tryptamine for subsequent reactions, and they play an important role in the synthesis of endogenous melatonin in rapeseed.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] The present invention identifies BnTDC proteins with TDC function in rapeseed for the first time, including BnaA01.TDC protein and BnaC01.TDC protein. After in vitro enzyme activity verification, it is confirmed that BnaA01.TDC protein and BnaC01.TDC protein can catalyze tryptophan into tryptamine, which is of great value in the synthesis of tryptamine or in vitro synthesis of melatonin and the study of key enzymes for endogenous synthesis of melatonin in rapeseed. In further functional verification, it is also found that BnaA01.TDC protein and BnaC01.TDC protein have the function of catalyzing tyrosine into tyramine, which is the first discovery in rapeseed, and is of great significance for the study of amino acid utilization pathways in rapeseed. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The figure is a bar graph showing the amount of tryptamine produced by the in vitro reaction of GST-BnaA01.TDC fusion protein and GST-BnaC01.TDC fusion protein. The data are expressed as mean + standard deviation, with 2 replicates each. Among them, CK represents a buffer solution without protein, BnaA01.TDC represents GST-BnaA01.TDC fusion protein, and BnaC01.TDC represents GST-BnaC01.TDC fusion protein.

[0022] Figure 2 This is a liquid chromatography detection chart of the amount of tyramine generated by the in vitro reaction of GST-BnaA01.TDC fusion protein and GST-BnaC01.TDC fusion protein, with two replicates each; wherein CK represents a buffer solution without protein, BnaA01.TDC represents GST-BnaA01.TDC fusion protein, BnaC01.TDC represents GST-BnaC01.TDC fusion protein, and Tryamine-10 μg represents 10 μg tyramine. DETAILED DESCRIPTION

[0023] 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.

[0024] 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.

[0025] 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.

[0026] 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.

[0027] In the following examples, the rapeseed variety used was Westar.

[0028] 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.

[0029] The amino acid sequence of BnaA01.TDC protein is shown in SEQ ID NO.1, the amino acid sequence of BnaC01.TDC protein is shown in SEQ ID NO.2, the nucleotide sequence of BnaA01.TDC gene is shown in SEQ ID NO.3, and the nucleotide sequence of BnaC01.TDC gene is shown in SEQ ID NO.4.

[0030] SEQ ID NO.1:

[0031] MEYGNSTENGIGKSNGIGNGHGIGNGYVNGHGICNGHGNGHGIGNGHGNDNGYVNGNGKVNGEKMKKMKPMDSEMLREQGHIMVDFIADYYKNLEDSPQDFPVLSQVQPGYLRDILPDSAPDHPEPLKELLHDVSKKIIPGLTHWQSPSYFAYYASSTSVAGFLGEMLNAGLSVVGFTWLTSPAATELEVIVLDWLAKLLQLPDHFLSTGRGGGVIQGTGCEAVLVVVLAARDRIMKKAGKNSLSQLVVYASDQTHSSCRKACLIGGIHEENIRLLKTDSSTNYGMPPKSLEEAISSDLAKGFIPFFICATVGTTSSAAVDPLVPLGKIAKSYGIWMHVDAAYAGNACICPEFRKHIDGVENADSFNINAHKWLFANLTCSPLWVKDRYSLIDALKTNPEYLEYKVSKRDEVVNYKDWQISLSRRFRSLKLWMVLRLYGAENLRNFIRDHVNLAKHFEDYVAQDAHFEVVTTRYFSLVCFRLAPVDGDEDKCNERNRELLATVNSTGKIFISHTALSGKFILRFAVGAPLTEEKHVTEAWRIIQKHATKFIHNKKI。

[0032] SEQ ID NO.2:

[0033] MEYGNSTENGIGKSNGISNGYGIGNGYVNGHGIGNEHGNGNGDVYGNGRVNGEKVKKMKPMDSEMLREQGHIMVDFIADYYKNLEDSPQDFPVLSQVQPGYLRDILPDSAPDHPEPLKELLHDVSKKIIPGLTHWQSPSYFAYYASSTSVAGFLGEMLNAGLSVVGFTWLASPAATELEVIVLDWLAKLLQLPDHFLSTGNGGGVIQGTGCEAVLVVVLAARDRIMKKVGKNSLSQLVVYASDQTHSSFRKACLIGGIHEENIRLLKTDSSTNYGMPPKSLEEAISSDLAKGFIPFFICATVGTTSSAAVDPLFPLGKIAESYGIWMHVDAAYAGNACICPEFREHIDGVENADSFNINAHKWLFANLTCSPLWVKDRYSLINALKTNPEYLEYKVSKREEVVNYKDWQISLSRRFRSLKLWMVLRLYGAENLRNFIRDHVNLAKHFEDCVAQDAHFEVVTTRYFSLVCFRLAPIDGDEDKCNDQNRELIATVNSTGKIFISHTALSGKFILRFAVGAPLTEEKHVTEAWQIIQKHATKFIRNKKN。

[0034] SEQ ID NO.3:

[0035]

[0036] SEQ ID NO.4:

[0037]

[0038] Example 1 Screening of target genes

[0039] 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 rapeseed variety "westar" were downloaded from the BnPIR website (Brassicanapus pan-genome Information Resource (BnPIR) (hzau.edu.cn)), and the Pyridoxal_deC.hmm hidden Markov model (PF00282) was used to search the protein sequences of Arabidopsis thaliana, and the 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 CDD and Interpro databases of NCBI were used to confirm that the candidate genes had the conserved domain of pyridoxal phosphate-dependent decarboxylase.

[0040] The genes with gene numbers BnaC01G0074100WE and BnaA01G0004400WE in the BnPIR library were screened to have the conserved domain of pyridoxal phosphate-dependent decarboxylase and may have TDC function. They were named BnaC01.TDC gene (BnaC01G0074100WE) and BnaA01.TDC gene (BnaA01G0004400WE), and their expression products were named BnaC01.TDC protein and BnaA01.TDC protein.

[0041] Example 2 Functional Detection of BnaA01.TDC Protein and BnaC01.TDC Protein

[0042] 1. Prokaryotic expression and purification of BnaA01.TDC and BnaC01.TDC proteins

[0043] 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.

[0044] Table 1

[0045]

[0046] 2) The steps of protein prokaryotic system induction and purification are as follows:

[0047] (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;

[0048] (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.

[0049] (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.

[0050] (4) Place in a shaker at 23°C and 180 rpm and continue culturing for 12-14 h;

[0051] (5) After the culture is completed, the cells are collected by centrifugation at 4°C and suspended in 60 mL of PBS buffer;

[0052] (6) Use a high-pressure homogenizer to break the protein, centrifuge at 9000 rpm at 4°C, and collect the supernatant;

[0053] (7) Add 1 mL of equilibrated GST purification medium to the supernatant and incubate at 4°C for 1-2 h;

[0054] (8) Pour the supernatant containing the GST purification medium after incubation into the affinity chromatography column. After the liquid is drained, wash the GST purification medium with PBS buffer for 3-5 times;

[0055] (9) After washing, elute five times with 1 mL of GST elution buffer (50 mM Tris-HCl, 10 mM glutathione, pH = 8.0);

[0056] (10) The eluted protein solution was stored at -20°C until use;

[0057] (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;

[0058] (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.

[0059] 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.

[0060] Depend on Figure 1 It can be seen that the GST-BnaA01.TDC fusion protein and the GST-BnaC01.TDC fusion protein have the function of decarboxylating tryptophan to tryptamine, indicating that both BnaA01.TDC protein and BnaC01.TDC protein are tryptophan decarboxylases in rapeseed.

[0061] 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.

[0062] Depend on Figure 2 It can be seen that GST-BnaA01.TDC fusion protein and GST-BnaC01.TDC fusion protein also have the function of decarboxylating tyrosine to tyramine.

Claims

1. The use of BnTDC protein in catalyzing the synthesis of tryptamine from tryptophan, characterized in that: The BnTDC protein is BnaA01.TDC protein or BnaC01.TDC protein; the amino acid sequence of the BnaA01.TDC protein is shown in SEQ ID NO.1, and the amino acid sequence of the BnaC01.TDC protein is shown in SEQ ID NO.

2.

2. The use of the BnTDC gene in catalyzing the synthesis of tryptamine from tryptophan, characterized in that: The BnTDC gene is the BnaA01.TDC gene or the BnaC01.TDC gene; the nucleotide sequence of the BnaA01.TDC gene is shown in SEQ ID NO.3, and the nucleotide sequence of the BnaC01.TDC gene is shown in SEQ ID NO.

4.

3. The use of BnTDC protein in catalyzing the synthesis of tyramine from tyrosine, characterized in that: The BnTDC protein is BnaA01.TDC protein or BnaC01.TDC protein; the amino acid sequence of the BnaA01.TDC protein is shown in SEQ ID NO.1, and the amino acid sequence of the BnaC01.TDC protein is shown in SEQ ID NO.

2.

4. The application of BnTDC gene in catalyzing the synthesis of tyramine from tyrosine, characterized in that: The BnTDC gene is the BnaA01.TDC gene or the BnaC01.TDC gene; the nucleotide sequence of the BnaA01.TDC gene is shown in SEQ ID NO.3, and the nucleotide sequence of the BnaC01.TDC gene is shown in SEQ ID NO.

4.

5. Use of a recombinant vector in catalyzing the decarboxylation of tryptophan and / or tyrosine, characterized in that: The recombinant vector comprises a BnTDC gene; the BnTDC gene is a BnaA01.TDC gene or a BnaC01.TDC gene; the nucleotide sequence of the BnaA01.TDC gene is shown in SEQ ID NO.3, and the nucleotide sequence of the BnaC01.TDC gene is shown in SEQ ID NO.4; the tryptophan is decarboxylated to generate tryptamine, and the tyrosine is decarboxylated to generate tyramine.

6. Use of a genetically engineered bacterium in catalyzing the decarboxylation of tryptophan and / or tyrosine, characterized in that: The genetically engineered bacteria comprises a BnTDC gene; the BnTDC gene is a BnaA01.TDC gene or a BnaC01.TDC gene; the nucleotide sequence of the BnaA01.TDC gene is shown in SEQ ID NO.3, and the nucleotide sequence of the BnaC01.TDC gene is shown in SEQ ID NO.4; the tryptophan is decarboxylated to generate tryptamine, and the tyrosine is decarboxylated to generate tyramine.

7. A method for synthesizing tryptamine, characterized in that: In the presence of pyridoxal phosphate, tryptophan is used as a substrate to catalyze the synthesis of tryptamine using BnTDC protein; The BnTDC protein is BnaA01.TDC protein or BnaC01.TDC protein; the amino acid sequence of the BnaA01.TDC protein is shown in SEQ ID NO.1, and the amino acid sequence of the BnaC01.TDC protein is shown in SEQ ID NO.

2.

8. Application of BnTDC protein in promoting tryptamine synthesis in rapeseed plants, characterized in that: The BnTDC protein is BnaA01.TDC protein or BnaC01.TDC protein; the amino acid sequence of the BnaA01.TDC protein is shown in SEQ ID NO.1, and the amino acid sequence of the BnaC01.TDC protein is shown in SEQ ID NO.

2.

9. Application of BnTDC protein in promoting melatonin synthesis in rapeseed plants, characterized in that: The BnTDC protein is BnaA01.TDC protein or BnaC01.TDC protein; the amino acid sequence of the BnaA01.TDC protein is shown in SEQ ID NO.1, and the amino acid sequence of the BnaC01.TDC protein is shown in SEQ ID NO.

2.

10. The use according to claim 9, characterized in that: In the synthesis of melatonin in rapeseed plants, tryptophan is converted into tryptamine under the catalysis of BnTDC protein.

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