Gastrodin glucosyltransferase GaUGT012 gene and application thereof in preparing gastrodin

Abstract

The application relates to a Gastrodia elata R. Br. sugar transferase GaUGT012 gene and application thereof in preparing gastrodin, and belongs to the technical field of biotechnology. The Gastrodia elata R. Br. sugar transferase GaUGT012 gene nucleotide sequence is shown as SEQ ID NO. 1, and the sequence has a full length of 1422 bp; the amino acid sequence of the coded protein is shown as SEQ ID NO. 2, and 474 amino acid residues are coded. The Gastrodia elata R. Br. sugar transferase GaUGT012 gene can be used as a biosynthesis regulation gene of gastrodin, and is applied to the preparation of gastrodin, has a significant application prospect, and is easy to popularize and apply.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a gastrodia glycosyltransferase. GaUGT012 Genes and their application in the preparation of gastrodin. Background Technology

[0002] Gastrodia elata ( Gastrodia elata Bl . Gastrodia elata, also known as Dingfengcao or Chijian, belongs to the genus Gastrodia elata in the family Orchidaceae. Gastrodia Gastrodia elata is a perennial parasitic herb. It has the effects of calming wind and stopping spasms, suppressing liver yang, dispelling wind and unblocking collaterals. It is often used to treat infantile convulsions, epilepsy, tetanus, headache and dizziness, hemiplegia, numbness of limbs, and rheumatic pain.

[0003] Gastrodin (GAS) is one of the main active ingredients of Gastrodia elata, also known as gastrodin, chemical name: 4-hydroxymethylphenyl- β -D-glucopyranoside is a phenolic glycoside with the molecular formula: C 13 H 18 O7, molecular weight: 286.28 kDa. Gastrodin has long been used clinically in my country for its analgesic and hypnotic effects with no obvious side effects, and is a central nervous system drug. It is mainly used for sedation, hypnosis, and analgesia. Gastrodin can inhibit excitatory amino acid-induced cell death and apoptosis, has the ability to scavenge free radicals, and can counteract free radical-induced damage to PC12 cells, exhibiting neuroprotective effects. Gastrodin has a significant effect in the treatment of sclerosis, presumably related to its promotion of angiogenesis. Gastrodin has a good therapeutic effect on patients with functional dyspepsia accompanied by anxiety and depression symptoms, and can dilate blood vessels, reduce cardiac hypertension, and reduce fibrosis, among other pharmacological effects on the central nervous system. Gastrodin enhances spinal cord synaptic activity and inhibits inflammatory pain. In addition, some studies have also found that gastrodin has various biological effects such as anti-obesity, anti-inflammation, memory improvement, and acetylcholinesterase inhibition.

[0004] Early methods for obtaining gastrodin mainly involved direct extraction from Gastrodia elata tubers or chemical processing. However, chemical synthesis of gastrodin is energy-intensive and environmentally unfriendly; artificial cultivation and extraction methods suffer from problems such as inconsistent Gastrodia elata quality, long cultivation cycles, and difficulty in achieving high-purity gastrodin. Therefore, the biosynthesis of gastrodin has attracted increasing attention in recent years. Gastrodin biosynthesis uses 4-hydroxymethyl-phenol (HBA) as a substrate and UDP-glucose as a glycosyl donor. Under the catalysis of glycosyltransferase, gastrodin is synthesized from 4-hydroxymethyl-phenol (HBA) using HBA as a substrate and UDP-glucose as a glycosyl donor. C Glycosylation is performed on the hydroxyl group at the -4 position to generate gastrodin.

[0005] In recent years, with the rapid development of synthetic biology, the production of natural drug monomers using synthetic biology techniques can effectively solve the aforementioned problems. However, to elucidate the biosynthetic pathways of these active ingredients, it is essential to identify the key genes involved in these pathways, and discovering these catalytic enzyme genes has become a crucial step in studying the biosynthetic pathways of plant metabolites. Currently, p-hydroxybenzyl alcohol... C The synthetic pathway for gastrodin, which involves glycosylation at the -4 hydroxyl group, has been clarified. However, the function of the glycosyltransferase responsible for gastrodin synthesis discovered in Gastrodia elata has not yet been verified, which has hindered the progress of gastrodin biosynthesis research. Summary of the Invention

[0006] To address the above problems, this invention provides a gastrodia glycosyltransferase. GaUGT012 This gene can serve as a regulator of the biosynthesis of gastrodin.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows: The first aspect of this invention provides a gastrodia glycosyltransferase. GaUGT012 Gene, the gastrodin glycosyltransferase GaUGT012 The second aspect of the present invention provides the above-mentioned gastrodin glycosyltransferase. GaUGT012 The gene encodes a protein whose amino acid sequence is shown in SEQ ID NO.2, encoding 474 amino acid residues.

[0008] MENSSSSSTGPHLALFPSLGIGHLNPFTELAKLLVDRHGFTVTIVLFSESWNPAQEALLSSLPPSITYLTLPPIPLSDIPRDSRPETLISSSRCVPSVRSLLHTLQSSTNLVAFV VDFLNAPACDVAKQLSLPCYVFMPITLHFCTLLLHLPTIDAGLTCDFWELDLPVCLPGCLPIPGPDIPSPLQDRKSDFYKWLLQHIKHFREAGGILMNTFDAVEPEASRLLKGEKESGAP MIYTVGPIIRSAAENGDRSHPLFEWLDLQPERSVIFVSFGSGGTLSADQLAELAMGLEDSRQRFLWVVRSPIGGSASGAYFTAGSEDDPLAYLPQGFLERTKGVGLVVPSWGPQVEILAH PATGGFLSHCGWNSTLESVTHGVPMIGWPLYAEQRMNAVLLAEGVKVMLRMKAREDGLFDRTEIARAVRELMEGEEGKQARKKARELQQEAAAALAEGGTSTTALAAVADRWKRV; (SEQ ID NO.2) The third aspect of the present invention provides a gastrodinyl transferase containing the above-mentioned gastrodinyl transferase. GaUGT012 Recombinant plasmids of genes.

[0009] Preferably, the recombinant plasmid is prepared by transfecting the above-mentioned gastrodin glycosyltransferase. GaUGT012 The gene was obtained through homologous recombination with the pET28a vector and named pET28a- GaUGT012 .

[0010] A fourth aspect of the present invention provides a transgenic engineered bacterium containing the recombinant plasmid described above, or wherein the genome of the transgenic engineered bacterium is integrated with an exogenous gastrodin glycosyltransferase as described above. GaUGT012 Gene.

[0011] Preferably, the genetically engineered bacteria is Escherichia coli. BL21 (DE3) strain.

[0012] The fifth aspect of this invention provides the above-mentioned gastrodia glycosyltransferase. GaUGT012Application of genes in the preparation of gastrodin.

[0013] Preferably, p-hydroxybenzyl alcohol and the glycosyl donor UDP-glucose are used as raw materials, and the above-mentioned gastrodin glycosyltransferase is used. GaUGT012 The gastrodia glycosyltransferase encoded by the gene catalyzes the reaction of 4-hydroxybenzyl alcohol. C Glycosylation of the hydroxyl group at the -4 position produces gastrodin.

[0014] This invention obtains the target protein by expressing it in vitro using a recombinant plasmid, and then directly generates gastrodin by catalyzing the substrate p-hydroxybenzyl alcohol.

[0015] The glycosyltransferase described in this invention GaUGT012 The gene was identified from the tubers of Gastrodia elata through transcriptome sequencing and bioinformatics techniques, after extensive experimental screening. RNA from the tubers was extracted using RNA reagents, reversed to cDNA, and then amplified by PCR. The glycosyltransferase mentioned... GaUGT012 The primers for gene amplification are shown below: F: ATGGAGAACAGCAGCAGCAG; (SEQ ID NO.3) R: CTACACACGCTTCCACCG; (SEQ ID NO.4) Furthermore, when homologous recombination occurs with the vector pET28a, GaUGT012 Genes need to be amplified and recovered using primers with homology walls. The primers with homology walls are as follows: F: gtggacagcaaatgggtcgcggatccATGGAGAACAGCAGCAGCAG; (SEQ ID NO.5) R: tgtcgacggagctcgaattcggatccCTACACACGCTTCCACCG. (SEQ ID NO.6) Glycosyltransferase isolated and identified from Gastrodia elata GaUGT012 The gene can serve as an important marker gene for molecular-assisted breeding of Gastrodia elata, and can also serve as an important candidate gene for the production of gastrodin in yeast chassis cell construction.

[0016] Compared with the prior art, the beneficial effects of this invention are as follows: (1) This invention provides gastrodia glycosyltransferase GaUGT012 The gene can serve as a regulator of the biosynthesis of gastrodin and can be used to prepare gastrodin.

[0017] (2) With the rapid development of bioinformatics technology, the discovery of key enzyme genes in the gastrodin biosynthesis pathway has been greatly promoted. In this invention, the gastrodin biosynthesis regulatory gene is gastroglycosyltransferase. GaUGT012 This invention, the first to identify and successfully verify a gene, opens up a new biosynthetic method for producing gastrodin. The invention obtains the target product through in vitro enzymatic catalysis of heterologous protein expression in *E. coli*, employing in vitro biosynthesis for targeted production, which has advantages such as fewer byproducts.

[0018] (3) The present invention also provides a glycosyltransferase containing this glycosyltransferase. GaUGT012 Recombinant plasmids, genetically engineered bacteria, and recombinant proteins lay the foundation for the large-scale synthesis of gastrodin through bioengineering methods, and further for the research on constructing cell factories that produce gastrodin.

[0019] (4) In vitro biosynthesis of gastrodin offers strong controllability, reduces the need for raw material cultivation, produces a single product, facilitates subsequent separation and purification of gastrodin, and reduces difficulties in chemical synthesis and complex synthetic pathways. The gastrodin glycosyltransferase mentioned... GaUGT012 As a key gene in the biosynthesis of gastrodin, it can also be used for breeding research of plants rich in gastrodin, such as Mirabilis jalapa. Attached Figure Description

[0020] Figure 1 A schematic diagram illustrating the synthetic pathway of gastrodin. Figure 2 A schematic diagram illustrating the construction of the recombinant expression plasmid pET28a-GaUGT012; Figure 3 The electrophoretic detection results are for GaUGT012 after recombination; Figure 4 This is an SDS-PAGE protein electrophoresis image of GaUGT012. M represents the protein molecular weight standard. Figure 5 For HPLC detection of glycosyltransferases GaUGT012 Glycosylation of p-hydroxybenzyl alcohol. The horizontal axis represents time (in minutes), and the vertical axis represents the response value (in milliseconds, mAU). CK: control group (p-hydroxybenzyl alcohol + uridine diphosphate glucose + inactivated gastrodin glycosyltransferase). GaUGT012 Enzyme inactivation reaction results; Standards: p-hydroxybenzyl alcohol standard + gastrodin standard; GaUGT012 Experimental group (p-hydroxybenzyl alcohol + uridine diphosphate glucose + gastrodinyl transferase) GaUGT012 The results of the enzyme activity reaction; Figure 6 Fragment ion diagram of the reaction product gastrodin (theoretical molecular weight 331) (LC / MS / MS); Figure 7 Fragment ion diagram of the standard gastrodin (theoretical molecular weight 331) (LC / MS / MS). Detailed Implementation

[0021] The present invention will now be described in further detail with reference to the embodiments.

[0022] Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be construed as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed in accordance with the techniques or conditions described in the literature in the field or according to the product instructions. Materials or equipment whose manufacturers are not specified are all conventional products that can be obtained by purchase. Example 1

[0023] Based on the basic functional annotation information of the Unigene transcriptome of Gastrodia elata, candidate UGT genes were screened from the sequencing annotation results. Simultaneously, glycosyltransferases (UGTs) identified in plants were analyzed using local BLAST sequence analysis. The screening results were then analyzed and compiled, ultimately identifying one glycosyltransferase (UGT) gene. Following a series of procedures including cDNA preparation, candidate gene amplification and recovery, homologous recombination, protein expression, in vitro enzyme activity reaction, and HPLC and LC / MS detection, the target candidate gene capable of catalyzing glycosylation at the C-4 position of 4-hydroxybenzyl alcohol was finally identified. GaUGT012 ( Figure 1 The steps for each stage of gastrodin synthesis are as follows (all reagents, raw materials, instruments, and equipment used in the following procedures are commercially available): (1) Preparation of cDNA template Fresh samples of Gastrodia elata tubers were collected, sliced, and flash-frozen in liquid nitrogen for RNA extraction. RNA extraction was performed using the Magen (Guangzhou Meiji Biotechnology Co., Ltd.) HiPure Plant RNA Mini Kit. RNA was extracted according to the kit's instructions, and after passing quality checks, the RNA was reverse transcribed into cDNA using the TAKARA reverse transcription kit and stored at -20 ℃ for later use.

[0024] (2) Gene amplification and recovery Specific primers for candidate genes were designed using SnapGene software, and Beijing Qingke Biotechnology Co., Ltd. was commissioned to do so. The Kunming branch synthesized the gene and used 2×PhantaMax Master Mix polymerase to amplify the target gene. The reaction system and procedure are as follows: 25 μL of 2×PhantaMax Master Mix, 1 μL each of the candidate gene pre- and post-selection, 1 μL of cDNA from each Bletilla striata tissue, and 22 μL of ddH2O. The PCR reaction procedure is as follows: Pre-denaturation at 95℃ for 3 minutes; Denaturation at 95℃ for 15 seconds Annealing at 58℃ for 15 seconds. 72℃, 1 min extension, 35 cycles; Final extension at 72℃ for 5 minutes; Keep warm at 10℃.

[0025] The target fragment was recovered using a 1% agarose gel and the Tiangen agarose gel DNA recovery kit. The recovered concentration was determined using a NanoReady ultra-micro UV-Vis spectrophotometer, and the fragment was stored at -20 °C for later use. Gastrodia elata glycosyltransferase was obtained. GaUGT012 The gene fragment, after sequencing, has the nucleic acid sequence shown in SEQ ID NO.1, and the protein amino acid sequence shown in SEQ ID NO.2.

[0026] Glycosyltransferase GaUGT012 The primers for gene amplification are shown below: F: ATGGAGAACAGCAGCAGCAG; (SEQ ID NO.3) R: CTACACACGCTTCCACCG; (SEQ ID NO.4) Furthermore, when homologous recombination occurs with the vector pET28a, GaUGT012 Genes need to be amplified and recovered using primers with homology walls. The primers with homology walls are as follows: F: gtggacagcaaatgggtcgcggatccATGGAGAACAGCAGCAGCAG; (SEQ ID NO.5) R: tgtcgacggagctcgaattcggatccCTACACACGCTTCCACCG. (SEQ ID NO.6) (3) Construction and identification of gene recombination vectors For a detailed diagram of homologous recombination, please see [link to diagram]. Figure 2First, the pET28a vector was linearized. For homologous recombination, assembly was performed according to the instructions for the homologous recombinase. Then, based on the concentrations of the insert fragment and vector, and following the recombination instructions, the amounts of each component were calculated. Finally, each component was added to the PCR reaction tube on ice. After assembly, the results were detected and sent to the company for sequencing. The electrophoresis results after assembly are shown below. Figure 3 This indicates that the assembly was successful.

[0027] (4) SDS-PAGE protein electrophoresis Determined after small-scale protein expression trials GaUGT012 The protein induction conditions were: 16 ℃, 0.2 mM IPTG, 180 r / min, for 12 h; then, the cells were shaken vigorously, harvested, and the cell walls were broken. The protein supernatant was obtained after high-speed centrifugation, and then detected by SDS-PAGE protein electrophoresis. The results are shown below. Figure 4 This indicates that the supernatant of the target gene was obtained.

[0028] (5) Enzyme activity reaction The enzyme activity reaction was carried out in 1.5 mL centrifuge tubes, prepared according to the components in Table 1. The components were then added sequentially in the order listed in the table, mixed thoroughly, and briefly centrifuged to collect the reaction solution at the bottom of the centrifuge tube. The centrifuge tubes were placed in a metal bath and reacted at 32°C for 12 h. The reaction was terminated with 100 μL of methanol, and the product was finally detected.

[0029] Table 1. Component ratio of the UGT enzyme activity reaction system

[0030] (6) Product testing The HPLC detection conditions are as follows: The instrument used for HPLC analysis was an Agilent 1290 ultra-high performance liquid chromatograph. The HPLC column was an Agilent ZORBAX SB-C18 column (250 mm × 4.6 mm, 5.0 μm). The mobile phase was 0.01% formic acid solution (A) and acetonitrile (B). The gradient elution program was as follows: 0–8 min, 99%A–95%A; 8–13 min, 95%A–90%A; 13–20 min, 90%A–80%A; 20–25 min, 80%A–55%A; 25–35 min, 55%A–10%A; 35–40 min, 10%A–10%A; 40–40.01 min, 10%A–99%A; 45 min Stop. The flow rate was 0.6 mL / min. The column temperature was 30 ℃. The injection volume was 10 μL. The absorption wavelength was 220 nm. The detection results are shown below. Figure 5 This indicates the production of gastrodin.

[0031] The LC-MS detection conditions are as follows: Detection was performed using an Agilent 1290 UPLC / 6540 Q-Tof liquid chromatography-mass spectrometry (LC / MS) system. Mass spectrometry conditions: negative ion source, voltage 3500 V; fragmentation voltage: 135 V; cone voltage: 60 V; radio frequency voltage: 750 V; scan range: 100-1000 m / z. Chromatographic conditions: an Agilent ZORBAX SB-C18 column (250 mm × 4.6 mm, 5.0 μm) was used, with a flow rate of 0.6 mL / min. The mobile phase was 0.01% formic acid (A) and acetonitrile (B). The gradient was as follows: 0 min, A:B = 99:1; 8 min, A:B = 95:5; 13 min, A:B = 90:10; 20 min, A:B = 80:20; 25 min, A:B = 55:45; 35 min, A:B = 10:90; 40 min, A:B = 10:90; 40.01 min, A:B = 99:1; 45 min, Stop. The detection results are shown in […]. Figure 6 , Figure 7 The results show the fragment ion diagram of the reaction product gastrodin ( Figure 6 Fragment ion diagrams of gastrodin and standard ( Figure 7 The results were consistent, further confirming that the generated product was gastrodin.

[0032] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of this invention is defined by the appended claims and their equivalents.

Claims

1. A gastrodia glycosyltransferase GaUGT012 The application of genes in the preparation of gastrodin is characterized by, Using p-hydroxybenzyl alcohol as a substrate and UDP-glucose as a glycosyl donor, the above-mentioned gastrodin glycosyltransferase... GaUGT012 Under the catalysis of the gene-encoded gastrodin glycosyltransferase, in the presence of p-hydroxybenzyl alcohol... C Glycosylation is performed on the hydroxyl group at the -4 position to generate gastrodin; the gastrodin glycosyltransferase GaUGT012 The nucleic acid sequence of the gene is shown in SEQ ID NO.

1.

2. The gastrodia glycosyltransferase as described in claim 1 GaUGT012 The application of genes in the preparation of gastrodin is characterized by, The amino acid sequence of the encoded protein is shown in SEQ ID NO.2.

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