Hainan Cephalotaxus Ch2OGD1 gene and its application

Abstract

This invention discloses Hainan corn husk. Ch2OGD1 Genes and their applications belong to the field of genetic engineering technology. Ch2OGD1 The gene encodes a 2-oxoglutarate-dependent dioxygenase (2OGD) type Pictet-Spengler enzyme. Its expression was confirmed using a tobacco transient expression system and in vitro enzyme activity assays. Ch2OGD1 It can specifically catalyze the condensation of dopamine and 4-hydroxyphenylpropionaldehyde to generate 1-phenylethylisoquinoline. This invention provides a method based on... Ch2OGD1 The method for directional preparation of 1-phenylethyl isoquinoline from the gene provides a key enzyme element for the biosynthesis of precursor compounds of Cephalotaxine alkaloids, provides a technical basis for expanding the application of this gene in plant genetic engineering, and provides a potential technical path for alleviating the shortage of Cephalotaxine alkaloid resources.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, and specifically relates to Hainan Torreya grandis. Ch2OGD1 Genes and their applications. Background Technology

[0002] Cephalotaxus sinensis is a slow-growing coniferous tree belonging to the Cephalotaxaceae family. It produces over 70 structurally diverse alkaloids, among which holoharpin and its esters are noteworthy for their antitumor and anticancer activities. Holoharpin esters, such as holoharpinine, isoholoharpinine, deoxyholoharpinine, and homoharpinine, have shown potent antileukemic activity in vivo experiments. Holoharpinine, in particular, exhibits nanomolar inhibitory activity against myeloid and lymphocytic leukemia cell lines and was approved by the U.S. Food and Drug Administration in 2012 for the treatment of chronic myeloid leukemia.

[0003] Despite their significant biomedical value, cephalotaxine and its ester alkaloids are present in extremely low concentrations in plants of the Cephalotaxus genus, leading to a long-standing shortage of medicinal resources and challenges in the conservation of raw materials. (Hainan Cephalotaxus fortunei) Cephalotaxus hainanensis Cephalotaxus sinensis is the plant in the genus that contains the most diverse and abundant alkaloids of cephalotaxus sinensis and its esters. However, due to over-harvesting and its inherently slow growth rate, wild resources have been listed as a Class II protected plant in China. Current commercial production mainly relies on a semi-synthetic route: first, cephalotaxus sinensis is extracted from its needles, and then chemically esterified using synthetic side chains. Although there are reports on the total synthesis of cephalotaxus sinensis and homocephalotaxus sinensis alkaloids, these methods are limited by numerous steps and low yields, making them commercially uncompetitive.

[0004] Cephalotaxine and its ester alkaloids belong to the phenylethylisoquinoline alkaloid class. Their biosynthesis begins with dopamine and 4-hydroxydihydrocinnamaldehyde (4-HDCA). In the synthetic pathway, Pictet-Spengler enzymes catalyze the condensation reaction of these two substrates to generate the precursor compound of phenylethylisoquinoline alkaloids—1-phenylethylisoquinoline. Currently, genes encoding Pictet-Spengler enzymes are mainly divided into two categories: one belongs to the PR10 / Bet v1-like gene family, and the other belongs to the 2-oxoglutarate-dependent dioxygenase (2OGD) gene family. However, to date, no 2OGD-type Pictet-Spengler enzyme gene has been found that catalyzes the condensation of dopamine and 4-HDCA to generate 1-phenylethylisoquinoline; its gene sequence and protein structure remain to be elucidated.

[0005] To address this technological gap, this invention isolates and identifies the source of Hainan corn nut. Ch2OGD1 The gene encodes a protein that, experimentally confirmed, specifically catalyzes the reaction of dopamine and 4-HDCA to produce 1-phenylethylisoquinoline, providing a key enzyme element for elucidating the biosynthetic pathway of Cephalotaxine alkaloids. Therefore, developing an application of a Pictet-Spengler enzyme gene with a clearly defined catalytic function is of great significance for addressing the shortage of Cephalotaxine alkaloid resources. Summary of the Invention

[0006] The purpose of this invention is to provide Hainan cornmeal. Ch2OGD1 Genes and their applications: a technical solution to the shortage of Cephalotaxine alkaloids.

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

[0008] This invention provides a method for preparing 1-phenylethyl isoquinoline using Ch2OGD1 protein, comprising the following steps:

[0009] The purified Ch2OGD1 protein was mixed with 4 mM dopamine and 2 mM 4-hydroxyphenylpropionaldehyde in a reaction buffer containing 50 mmol / L N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, 5 mmol / L L-ascorbic acid, and pH 7.0, and incubated in the dark for 3 hours to generate 1-phenylethylisoquinoline.

[0010] The amino acid sequence of the Ch2OGD1 protein is shown in SEQ ID NO:1, specifically: MYNSIKLPNAKIFSIPVVQEFAAKDHEALPERYIREEEETPLSIAKDSWHSSSIPTIDMELLSDHHECRPKMMENLASACQEWGFFQVVNHGIPGSVLEQMKRVARGFFELPLEEKLKYAMQDNDGYGQAFVFSDEQKLDWSDIFALTTLPEDIRNMDFWPTEPMDFRGSANEFAIETKNLSDTLLSLIAENLGIKANSFIYPQGKWKHGIKLNYYPRCPKPDLVLGISPHSDSTSITILLQDNEIIGLHILKDEQWIPVPPIPGALVINIGDIVEVMSNGRYKSIEHLVLPDKNLDRISIVAGTYHTEDVEIGPHPDLIDDTHLALYRTFKRGDYDKKYMENKLEVGKKTLKAYKIK.

[0011]

[0012] Furthermore, the conditions for the light-protected incubation are 37°C and 160 rpm.

[0013] Furthermore, the Ch2OGD1 protein is obtained through the following steps:

[0014] (1) A nucleic acid molecule containing the nucleotide sequence shown in SEQ ID NO:2 was ligated into the pET28(a) vector and transformed into Escherichia coli;

[0015] (2) Ch2OGD1 protein expression was induced at 18℃ for 24 hours with 1 mmol / L propyl-β-D-thiogalactoside;

[0016] (3) Ch2OGD1 protein was obtained by purification using a Ni-NTA affinity column.

[0017] The present invention also provides a method for producing 1-phenylethyl isoquinoline in plants, comprising the following steps:

[0018] (1) A nucleic acid molecule encoding the Ch2OGD1 protein is introduced into plant cells for expression, wherein the nucleic acid molecule contains the nucleotide sequence shown in SEQ ID NO:2;

[0019] (2) Inject 10 μmol / L 4-hydroxyphenylpropionaldehyde and 12 μmol / L dopamine into plant cells expressing Ch2OGD1 protein;

[0020] (3) After 24 hours of cultivation, 1-phenylethylisoquinoline was obtained from the plant cells.

[0021] Furthermore, the plant in question is Nicotiana benthamiana.

[0022] Furthermore, the nucleic acid molecules are introduced into plant cells through the following steps:

[0023] (1) A nucleic acid molecule containing the nucleotide sequence shown in SEQ ID NO:2 was ligated into the pCAMBIA1304 vector and transformed into Agrobacterium GV3101;

[0024] (2) The transformed Agrobacterium was injected into the leaves of Tobacco Benedict and cultured for 3 days to obtain plant cells expressing Ch2OGD1 protein. Beneficial effects

[0025] The Hainan corn husk provided by this invention Ch2OGD1 Genes and their applications, through the isolation and identification of genes encoding 2-oxoglutarate-dependent dioxygenases. Ch2OGD1The gene (SEQ ID NO:2) was confirmed to specifically catalyze the synthesis of 1-phenylethyl isoquinoline from dopamine and 4-hydroxyphenylpropionaldehyde. The transient expression of this gene in tobacco and the in vitro protein catalysis system confirmed its key role in the synthesis of precursors of Cephalotaxine alkaloids, providing a technical basis for expanding the application of this gene in plant genetic engineering and providing a potential technical path to alleviate the shortage of Cephalotaxine alkaloid resources. Attached Figure Description

[0026] Figure 1 A schematic diagram of the Ch2OGD1-catalyzed reaction of dopamine and 4-hydroxyphenylpropionaldehyde to produce 1-phenylethylisoquinoline.

[0027] Figure 2 This is a chromatogram showing the results of liquid chromatography-mass spectrometry (LC-MS) detection of Ch2OGD1-catalyzed reaction products. In the figure, A represents the products overexpressed in tobacco. Ch2OGD1 A represents the detection results of substrate injection; B represents the detection results of the Ch2OGD1-catalyzed reaction of purified protein in vitro.

[0028] Figure 3 This is the mass spectrum of the Ch2OGD1 catalytic product. In this spectrum, A represents the product overexpressed in tobacco. Ch2OGD1 A) Mass spectrum of catalytic products; B) Mass spectrum of purified protein Ch2OGD1 catalytic products in vitro; C) Mass spectrum of standard products. Detailed Implementation

[0029] To enable those skilled in the art to better understand the technical solutions of this invention, the present application will be further described in detail below with reference to embodiments.

[0030] The Chinese correspondence of the reagents used in the embodiments of this invention is as follows:

[0031] (1) Dopamine;

[0032] (2) 4-HDCA: 4-hydroxyphenylpropionaldehyde;

[0033] (3) MES: 2-(N-morpholino)ethanesulfonic acid;

[0034] (4) MgCl2: Magnesium chloride;

[0035] (5) AS: Acetyleugenol;

[0036] (6) IPTG: Propyl-β-D-thiogalactoside;

[0037] (7) HEPES: N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid;

[0038] (8) L-Ascorbic acid: L-ascorbic acid.

[0039] Example 1: Hainan Torreya grandis Ch2OGD1 Cloning of genes

[0040] Bioinformatics analysis was performed on the full-length transcriptome sequencing results of *Torreya grandis* hainanensis. Specific primers P1 (5′-ATGTATAATTCAATCAAATTACCCA-3′, SEQ ID NO:3) and P2 (5′-TTATTTGATTTTGTAAGCTTTCAGA-3′, SEQ ID NO:4) were designed, using the first-strand cDNA from *Torreya grandis* leaves as a template for sequencing. Ch2OGD1 The coding region sequence of the gene was amplified by PCR to obtain... Ch2OGD1 The full-length coding sequence of the gene is 1077 bp (SEQ ID NO:2). Based on the open reading frame sequence, the amino acid sequence of Ch2OGD1, consisting of 358 amino acid residues (SEQ ID NO:1), was deduced. The specific steps are as follows:

[0041] 1. Take 50 mg of Hainan Torreya leaf powder ground with liquid nitrogen and extract total RNA using the Piant Total RNA Isolation Kit Plus (Chengdu Fuji Biotechnology Co., Ltd.) kit.

[0042] 2. The extracted total RNA was reverse transcribed into cDNA using the MonScript™ RTIII All-in-One Mix with dsDNase kit (Mona Biotechnology Co., Ltd.).

[0043] 3. Using primers P1 and P2 as a template, amplify the synthesized cDNA. Ch2OGD1 The coding region sequence of the gene was amplified under the following conditions: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 sec, 55℃ annealing for 30 sec, 70℃ extension for 1 min, for a total of 35 cycles; 70℃ extension for 5 min.

[0044] 4. The amplified PCR products were ligated into pET28(a) and pCAMBIA1304 vectors, transformed into E. coli competent cells, positive clones were screened and sequenced, and those with correctly sequenced DNA were identified. Ch2OGD1 The recombinant vectors for the genes were named pET28(a)-Ch2OGD1 and pCAMBIA1304-Ch2OGD1, respectively.

[0045] Example 2: Ch2OGD1 Identifying its function through transient overexpression in tobacco

[0046] To verify Ch2OGD1 The function of genes in plants was studied using Agrobacterium-mediated transformation. Ch2OGD1 The gene was overexpressed in tobacco leaves after transformation. Subsequently, tobacco leaves were co-infected with Dopamine and 4-HDCA as reaction substrates. The function of Ch2OGD1 was verified by detecting product synthesis using LC-MS. The specific steps are as follows:

[0047] (1) Transform the pCAMBIA1304-Ch2OGD1 plasmid from Example 1 into Agrobacterium GV3101 competent cells, pick a single colony and culture it in 2 ml of kanamycin-containing medium for 18-24 hours.

[0048] (2) Take 1 ml of bacterial culture and inoculate it into 50 ml of liquid LB medium until the OD600 value reaches between 0.6 and 0.8. Centrifuge at 6000 rpm for 10 min at room temperature to collect the bacterial cells and discard the supernatant; add 100 mL of buffer (10 mmol / L MES + 10 mmol / L MgCl2 + 200 μmol / L AS, pH 5.8) to resuspend the bacterial cells and let it stand at room temperature for 2 h.

[0049] (3) Then, using a 2 mL syringe, the resuspended solution was injected into 6-8 week old tobacco leaves of similar growth, ensuring the entire leaf was completely wetted with the bacterial solution. The same amount of tobacco leaves was injected into each treatment, and the leaves were co-cultured in the tissue culture room for 3 days to obtain tobacco leaf cells expressing the Ch2OGD1 protein. The substrate solution required for the reaction (10 μmol / L 4-hydroxydihydrocinnamaldehyde and 12 μmol / L Dopamine) was prepared and injected into the above-mentioned tobacco leaves expressing the Ch2OGD1 protein for co-infection and culture for 24 h. The tobacco leaves were then harvested for the extraction of the reaction product.

[0050] (4) After 3 days of cultivation, the infected leaves were collected, freeze-dried and ground into fine powder. Then, 2g of leaf powder was mixed with 20 mL of methanol and shaken for 30 minutes. Ultrasonic extraction was performed twice (45 minutes each time) to obtain the filtrate.

[0051] (5) Concentrate the filtrate to dryness under reduced pressure using a rotary evaporator at room temperature; add an appropriate amount of methanol and transfer the sample to a centrifuge tube. After the methanol has completely evaporated, perform solid-phase extraction by passing a 15% methanol-water solution through a solid-phase extraction column, followed by elution with a 70% methanol-water solution. After concentrating and drying the 70% methanol eluent under reduced pressure, dissolve it in 500 μL of chromatographic grade methanol, filter it through a 0.22 μm filter membrane, and then perform liquid chromatography-mass spectrometry (LC-MS) analysis.

[0052] LC-MS analysis conditions: High-performance liquid chromatograph (Dionex 3000 series), LC column model: COSMOSIL 38020-41 5C18-MS-II 4.6 mm*250 mm. Mobile phase: methanol (A)-ultrapure water (B), gradient elution (0~40 min, 10%A~100%A), elution flow rate: 0.4 ml / min, detection wavelength: 273 nm, injection volume: 10 μL.

[0053] Ch2OGD1 protein-catalyzed reactions such as Figure 1 As shown, the molecular weight of the target product is approximately 285.

[0054] LC-MS analysis results showed that it contained Ch2OGD1 In the extract co-infected with gene and substrate (pCambia1304-Ch2OGD1 + Dopamine + 4-HDCA), the target compound showed an absorption peak at 15.3 min. Figure 2 A), and the mass spectrum also detected the target product with the same molecular weight as the standard ([M+H]+ = 286.1437). Figure 3 A); while in all other control extracts, no absorption peak was observed at 15.3 min, further verification in tobacco showed that Ch2OGD1 can catalyze the reaction of Dopamine and 4-HDCA to generate 1-phenylethylisoquinoline.

[0055] Example 3: In vitro enzyme activity verification of Ch2OGD1 from Hainan Torreya grandis

[0056] For further analysis Ch2OGD1 To verify the function of the gene-encoded protein, this example expresses and purifies the Ch2OGD1 protein in *E. coli*. The product synthesis is then detected by LC-MS after in vitro feeding with the precursor compound Dopamine and 4-HDCA. The specific steps are as follows:

[0057] (1) The pET28(a)-Ch2OGD1 plasmid in Example 1 was transformed into Escherichia coli BL21(DE3) and protein expression was induced. The induction conditions were: IPTG concentration of 1 mmol / L, induction temperature of 18℃, and induction time of 24 hours.

[0058] (2) The soluble protein obtained by induction was purified by Ni-NTA affinity column.

[0059] (3) The purified protein was subjected to in vitro enzyme activity identification. The reaction volume was 1 ml, containing 5 μg of recombinant Ch2OGD1 protein, 4 mM Dopamine and 2 mM 4-HDCA, and the reaction buffer (50 mmol / L HEPES, 5 mmol / L L-Ascorbic acid, pH 7.0) was added to make up to 1 ml. The mixture was incubated in a constant temperature shaker at 37 °C at 160 rpm for 3 h in the dark. After the reaction, the mixture was extracted three times with an equal volume of chloroform, and the chloroform layers were combined and concentrated to dryness at room temperature. 200 μL of the sample was dissolved in chromatographic methanol, filtered through a 0.22 μm filter membrane, and analyzed by LC-MS according to the method in Example 2 above.

[0060] LC-MS results showed that the product of the condensation reaction between Ch2OGD1 and Dopamine and 4-HDCA had the same absorption peak as the standard 1-phenylethylisoquinoline at elution time of 15.3 min. Figure 2 B), and the mass spectrum shows that the molecular weight of the target product is consistent with that of the standard ([M+H]+ = 286.1468). Figure 3 B) indicates that Ch2OGD1 has the function of catalyzing the formation of the cephalotaxine precursor compound 1-phenylethylisoquinoline from Dopamine and 4-HDCA.

[0061] The Hainan corn husk provided by this invention Ch2OGD1 The gene and its application provide key enzyme elements for the biosynthesis of precursor compounds of Cephalotaxine alkaloids. The targeted preparation of 1-phenylethyl isoquinoline can be achieved through in vitro catalysis or in vivo expression in plants, providing a potential technical path to alleviate the shortage of Cephalotaxine alkaloid resources.

Claims

1. A method for preparing 1-phenylethyl isoquinoline via Ch2OGD1 protein, characterized in that, Includes the following steps: The purified Ch2OGD1 protein was mixed with 4 mM dopamine and 2 mM 4-hydroxyphenylpropionaldehyde in a reaction buffer containing 50 mmol / L N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, 5 mmol / L L-ascorbic acid, and pH 7.0, and incubated in the dark for 3 hours to generate 1-phenylethylisoquinoline. The amino acid sequence of the Ch2OGD1 protein is shown in SEQ ID NO:

1.

2. The method according to claim 1, characterized in that, The Ch2OGD1 protein is encoded by the nucleotide sequence shown in SEQ ID NO:

2.

3. The method according to claim 1, characterized in that, The conditions for incubation in the dark were 37°C and 160 rpm.

4. The method according to claim 1, characterized in that, The Ch2OGD1 protein was obtained through the following steps: (1) Link a nucleic acid molecule with the nucleotide sequence shown in SEQ ID NO:2 to the pET28(a) vector and transform it into Escherichia coli; (2) Ch2OGD1 protein expression was induced at 18℃ for 24 hours with 1 mmol / L propyl-β-D-thiogalactoside; (3) Ch2OGD1 protein was obtained by purification using a Ni-NTA affinity column.

5. A method for producing 1-phenylethyl isoquinoline in plants, characterized in that, Includes the following steps: (1) The nucleic acid molecule encoding the Ch2OGD1 protein is introduced into plant cells for expression, wherein the nucleic acid molecule is the nucleotide sequence shown in SEQ ID NO:2; (2) Inject 10 μmol / L 4-hydroxyphenylpropionaldehyde and 12 μmol / L dopamine into plant cells expressing Ch2OGD1 protein; (3) After culturing for 24 hours, 1-phenylethylisoquinoline was obtained from plant cells; The plant in question is Nicotiana benthamiana.

6. The method according to claim 5, characterized in that, The nucleic acid molecules are introduced into plant cells through the following steps: (1) A nucleic acid molecule with the nucleotide sequence shown in SEQ ID NO:2 was ligated into the pCAMBIA1304 vector and transformed into GV3101 Agrobacterium; (2) The transformed Agrobacterium was injected into the leaves of Tobacco Benedict and cultured for 3 days to obtain plant cells expressing Ch2OGD1 protein.

Images