GaUGT131 gene of gastrodia elata glycosyl transferase and application of GaUGT131 gene in preparation of gastrodin

Abstract

The invention relates to a gastrodia elata glycosyl transferase GaUGT131 gene and application thereof in preparation of gastrodin, and belongs to the technical field of biology. The nucleotide sequence of the gastrodia elata glycosyl transferase GaUGT131 gene is as shown in SEQ ID NO. 1, and the total length of the sequence is 1524 bp; the amino acid sequence of the encoded protein is shown as SEQ ID NO.2, and 508 amino acid residues are encoded. The gastrodia elata glycosyl transferase GaUGT131 gene can be used as a biosynthesis regulation gene of gastrodin, is applied to preparation of gastrodin, and is remarkable in application prospect and easy to popularize and apply.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and specifically relates to a gastrodia elata glycosyltransferase GaUGT131 Gene and its application in the preparation of gastrodin. Background Art

[0002] Gastrodia elata Gastrodia elata Bl . ) is from the genus Gastrodia of the family Orchidaceae ( Gastrodia ) The tuber of the plant Gastrodia elata is a precious Chinese medicinal material. It was first recorded in the "Shennong Bencao Jing" and has a medicinal history of more than 2,000 years. Gastrodia elata has the effects of calming wind and relieving spasms, calming liver yang, dispelling wind and unblocking meridians. It is often used to treat infantile convulsions, epilepsy, tetanus, headache and dizziness, limb paralysis, limb numbness, rheumatism and pain. Modern pharmacological studies have shown that Gastrodia elata has the effects of anti-depression, improving sleep, anticonvulsant, anti-inflammatory, analgesic, and regulating blood pressure. It has a wide range of clinical applications and is also an important product of medicinal and edible origin. The main active ingredient of Gastrodia elata is gastrodin, also known as gastrodin glycoside, chemical name: 4-hydroxymethylphenyl- β -D-pyranoglucoside is a phenolic glycoside with the molecular formula C 13 H 18 O7, molecular weight is 286.28 kDa.

[0003] Studies have shown that gastrodin can inhibit cell death and apoptosis induced by excitatory amino acids, has the ability to scavenge free radicals, can resist free radical-induced damage to PC12 cells, and has a neuroprotective effect. Gastrodin has a good therapeutic effect on patients with functional dyspepsia accompanied by anxiety and depression symptoms, can dilate blood vessels, reduce cardiac hypertension and fibrosis, and also has pharmacological effects on the central nervous system such as anti-anxiety, sedation and hypnosis, anticonvulsant, anti-epileptic, analgesic, intelligence-promoting, anti-aging and neuroprotection. Therefore, the market demand for gastrodia elata continues to expand. Although wild gastrodia elata has a high content of active ingredients and good efficacy, its yield is low and cannot meet actual demand. It is also on the verge of extinction due to excessive artificial collection. Now gastrodia elata is mainly cultivated artificially, and the use of bioengineering to produce gastrodin is the direction of the future industry.

[0004] The main methods for obtaining gastrodin early were direct extraction from Gastrodia elata tubers or chemical processing. However, the chemical synthesis of gastrodin is energy-consuming and environmentally unfriendly; the artificial cultivation and extraction method has problems such as the inability to guarantee the quality of Gastrodia elata, a long cultivation cycle of Gastrodia elata, and difficulty in extracting gastrodin with high purity. Therefore, the biosynthesis of gastrodin has attracted increasing attention in recent years. The biosynthesis of gastrodin uses 4-hydroxymethyl-phenol (HBA) as a substrate and uridine diphosphate glucose (UDP-glucose) as a glycosyl donor. Under the catalysis of glycosyltransferase, glycosylation occurs at the C hydroxyl group at the -4 position of 4-hydroxymethyl-phenol to generate gastrodin.

[0005] In recent years, with the rapid development of the field of synthetic biology, using synthetic biology technology to produce natural medicine monomers can effectively solve the above problems. However, to clarify the biosynthetic pathways of these active ingredients, it is necessary to identify the key genes related to these pathways, and exploring these catalytic enzyme genes has become a key link in studying the biosynthetic pathways of plant metabolites. Currently, the synthetic pathway for catalyzing the glycosylation reaction of 4-hydroxymethyl-phenol at the C hydroxyl group at the -4 position to form gastrodin has been clarified, but the function of the glycosyltransferase responsible for the synthesis of gastrodin mined from Gastrodia elata has not been verified, which has affected the progress of gastrodin biosynthesis work. Summary of the Invention

[0006] To solve the above problems, the present invention provides a Gastrodia elata glycosyltransferase GaUGT131 gene, which can be used as a biosynthetic regulatory gene for gastrodin.

[0007] To achieve the above object, the technical solution adopted by the present invention is: The first aspect of the present invention provides a Gastrodia elata glycosyltransferase GaUGT131 gene, and the Gastrodia elata glycosyltransferase GaUGT131 In the second aspect of the present invention, there is provided the above gastrodia glycosyltransferase GaUGT131 protein encoded by the gene, and the amino acid sequence of the protein is as follows SEQ ID NO.2, encoding 508 amino acid residues.

[0008] MDLPPFPCIPTSAPTKHRSSEGSEASMEIPSEGRRPHLAMVSSPGMGHLFPLAELAKILVNRHRFTVTFITFAASDNRAAHTFLSTLPSGISWLTLPPVPLDDIPPAATIETRMSLVSLRSLPAIRSALADLHSTTNLVAVVADFFATDAFAAARDIGVPYYMFIPTNLLFLSLLLELPALDAAITGRFQDLETPLRLSGCVPIPGSDILTPLLNRSSEEYRWMLHHARLYRQAQGILVNSFAAIEPGPTKSLSQNSLSRPPVYPVGPMVQSPKPGTSLCLAWLDRQPEGSVLFVSFGSGGTLTRTQLGELALGLEASKQRFLLVVRSPCDEDSSSTYFTAQTKDDPLLFLPEGFVDRTRDVGLVVPSWAPQIEVLAHRATGGFLSHCGWNSTLESVSHGVPLIAWPLFAEQFMNAVMLVEEARVAMRPKKADAAAAVDRAEVSRVAAALMEGEEGKALRRRAQQLKEAAHAALREGGSSYEALGLVADMWKEKSAGAGGAAAAVAPQ; (SEQ ID NO.2) In the third aspect of the present invention, there is provided a recombinant plasmid containing the above gastrodia glycosyltransferase GaUGT131 gene.

[0009] Preferably, the recombinant plasmid is obtained by homologous recombination of the above gastrodia glycosyltransferase GaUGT131 gene with the pET28a vector, and is named pET28a- GaUGT131 .

[0010] In the fourth aspect of the present invention, there is provided a genetically engineered bacterium containing the above recombinant plasmid, or, the genome of the genetically engineered bacterium integrates an exogenous above gastrodia glycosyltransferase GaUGT131 gene.

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

[0012] The fifth aspect of the present invention provides a gastrodia glycosyltransferase as described above GaUGT131 for use in the preparation of gastrodin.

[0013] Preferably, using 4-hydroxybenzyl alcohol and the glycosyl donor UDP-glucose as raw materials, under the catalysis of the gastrodia glycosyltransferase GaUGT131 encoded by the gene, glycosylation occurs at the hydroxyl group at the -4 position of 4-hydroxybenzyl alcohol to produce gastrodin. C

[0014] In the present invention, the target protein is obtained by in vitro expression through a recombinant plasmid, and after further catalyzing the substrate 4-hydroxybenzyl alcohol, gastrodin is directly produced.

[0015] The glycosyltransferase GaUGT131 gene of the present invention was identified by transcriptome sequencing and bioinformatics techniques from the tubers of Gastrodia elata, and was obtained by reverse transcribing the RNA of Gastrodia elata tubers using RNA reagents into cDNA and then performing PCR amplification. The amplification primers for the glycosyltransferase GaUGT131 gene are as follows: F: ATGGACCTTCCCCCTTTCC; (SEQ ID NO.3) R: CTACTGCGGCGCAACG; (SEQ ID NO.4) In addition, when performing homologous recombination with the vector pET28a, GaUGT131 the gene needs to be amplified and recovered using primers with homologous arms. The primers with homologous arms are as follows: F: gtggacagcaaatgggtcgcggatccATGGACCTTCCCCCTTTCC; (SEQ ID NO.5) R: tgtcgacggagctcgaattcggatccCTACTGCGGCGCAACG. (SEQ ID NO.6) The glycosyltransferase GaUGT131 gene isolated and identified from Gastrodia elata can be used as an important marker gene for molecular assisted breeding of Gastrodia elata, and can also be used as an important candidate gene for the production of gastrodin in the construction of yeast chassis cells.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) The present invention provides the gastrodia glycosyltransferase GaUGT131 gene, which can be used as a gene for regulating the biosynthesis of gastrodin and applied to the preparation of gastrodin.

[0017] (2) With the rapid development of bioinformatics technology, the excavation of key enzyme genes in the biosynthesis pathway of gastrodin has been greatly promoted. The biosynthesis regulatory gene of gastrodin in this invention, namely gastrodia glycosyltransferase GaUGT131 gene, was identified and successfully verified for the first time, opening up a new method for the biosynthesis of gastrodin. In this invention, the target product is obtained by the way of in vitro enzyme catalysis through heterologous expression of proteins in Escherichia coli. By using in vitro biosynthesis for directional production, it has the advantages of fewer by-products.

[0018] (3) This invention also provides a recombinant plasmid, a genetically engineered bacterium and a recombinant protein containing the glycosyltransferase GaUGT131 gene, laying a foundation for the large-scale synthesis of gastrodin by bioengineering methods and further for the research on constructing a cell factory for producing gastrodin.

[0019] (4) By in vitro biosynthesis of gastrodin, it has strong controllability, can reduce the demand for raw material planting, the production product is single, facilitating the separation and purification of gastrodin in the later stage, and can also reduce problems such as difficulties in chemical synthesis and complex synthesis pathways. The gastrodia glycosyltransferase GaUGT131 gene, as a key gene in the biosynthesis of gastrodin, can also be used for the breeding research of plants rich in gastrodin such as Mirabilis jalapa. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Schematic diagram of the deduced synthesis pathway for gastrodin; Figure 2 Schematic diagram of the construction of the recombinant expression plasmid pET28a- GaUGT131 ; Figure 3 is GaUGT131 Detection result of electrophoresis after recombination; Figure 4 is GaUGT131 SDS-PAGE protein electrophoresis map. Among them, M is the protein molecular weight standard; Figure 5 is the glycosylation effect of the glycosyltransferase GaUGT131 on p-hydroxybenzyl alcohol detected by HPLC. The abscissa is time, unit min, and the ordinate is the response value, unit mAU; among them, CK: control group (p-hydroxybenzyl alcohol + uridine diphosphate glucose + inactivated gastrodia glycosyltransferase GaUGT131 ) enzyme inactivation enzyme activity reaction result; standard product: p-hydroxybenzyl alcohol standard product + gastrodin standard product; GaUGT131 : experimental group (p-hydroxybenzyl alcohol + uridine diphosphate glucose + gastrodia glycosyltransferase GaUGT131 ) enzyme activity reaction result; Figure 6Fragment ion chromatogram of the reaction product gastrodin (theoretical molecular weight 331) (LC / MS / MS); Figure 7 Fragment ion chromatogram of the standard gastrodin (theoretical molecular weight 331) (LC / MS / MS). Detailed implementation manners

[0021] The present invention will be further described in detail below in conjunction with the embodiments.

[0022] Those skilled in the art will understand that the following embodiments are only used to illustrate the present invention and should not be construed as limiting the scope of the present invention. For those not specified in the embodiments regarding specific techniques or conditions, the techniques or conditions described in the literature in this field or according to the product specifications shall be followed. For those materials or equipment without indicating the manufacturer, they are all conventional products that can be obtained by purchase. Example 1

[0023] Based on the basic functional annotation information of Gastrodia elata transcriptome Unigene, UGT candidate genes were screened in the sequencing annotation results. At the same time, glycosyltransferases (UGTs) identified in plants were used for local BLAST analysis of sequences. Then, the screening results were sorted out and analyzed. Finally, 1 glycosyltransferase (UGT) gene was found. After a series of work such as cDNA preparation, amplification and recovery of candidate genes, homologous recombination, protein expression, in vitro enzyme activity reaction, and HPLC and LC / MS detection, the target candidate gene that can catalyze the glycosylation of the hydroxyl group at the C-4 position of 4-Hydroxybenzyl alcohol was finally identified GaUGT131 ( Figure 1 ) The operating steps of each stage of gastrodin synthesis are as follows (the reagents, raw materials, instrument equipment, etc. used in the following examples are all commercially available): (1) Preparation of cDNA template Take the fresh sample of Gastrodia elata tuber, quick-freeze it in liquid nitrogen after slicing, and extract RNA. The RNA extraction uses the HiPure Plant RNA Mini Kit of Magen (Guangzhou Meiji Biotechnology Co., Ltd.). Extract RNA according to the operating steps of the kit. After passing the detection, use the TAKARA reverse transcription kit to reverse transcribe RNA into cDNA and store it at -20 °C for standby.

[0024] (2) Gene amplification and recovery Use SnapGene software to design specific primers for candidate genes and entrust Beijing Tsingke Biotechnology Co., Ltd. The Kunming Branch synthesized and used 2×Phanta Max Master Mix polymerase to amplify the target gene. The reaction system and reaction program are as follows. The reaction system is 25 μl of 2×Phanta Max Master Mix, 1 μl each of the forward and reverse primers of the candidate gene, 1 μl of Bletilla striata tissue cDNA, and 22 μl of ddH2O. The PCR reaction program is as follows: Pre-denaturation at 95°C for 3 min; Denaturation at 95°C for 15 s, Annealing at 58°C for 15 s, Extension at 72°C for 1 min, for 35 cycles; Final extension at 72°C for 5 min; Incubation at 10°C.

[0025] After the PCR program ended, the target fragment was recovered using 1% agarose gel and the Tiangen agarose gel DNA recovery kit. After recovery, its recovery concentration was measured on a NanoReady ultra-micro ultraviolet-visible spectrophotometer, and finally it was stored at -20 °C in the refrigerator for later use. The Gastrodia elata glycosyltransferase GaUGT131 gene fragment was obtained. Its nucleic acid sequence was as shown in SEQ ID NO.1, and the amino acid sequence of the protein was as shown in SEQ ID NO.2.

[0026] Glycosyltransferase GaUGT131 The amplification primers for the gene are as follows: F: ATGGACCTTCCCCCTTTCC; (SEQ ID NO.3) R: CTACTGCGGCGCAACG; (SEQ ID NO.4) In addition, when performing homologous recombination with the vector pET28a, GaUGT131 the gene needs to be amplified and recovered using primers with homologous arms. The primers with homologous arms are as follows: F: gtggacagcaaatgggtcgcggatccATGGACCTTCCCCCTTTCC; (SEQ ID NO.5) R: tgtcgacggagctcgaattcggatccCTACTGCGGCGCAACG. (SEQ ID NO.6) (3) Construction and identification of the gene recombinant vector The schematic diagram of homologous recombination is shown in detail in Figure 2. First, linearize the vector pET28a. During homologous recombination, assemble according to the operation instructions of the homologous recombination enzyme. Then, calculate the dosage of each component based on the concentrations of the inserted fragment and the vector and in accordance with the recombination instructions. Finally, add each component to the PCR reaction tube on ice. After assembly, detect the result and send it to the company for sequencing. The electrophoresis detection result after assembly is shown in Figure 3 , indicating successful assembly.

[0027] (4) SDS-PAGE protein electrophoresis After small-scale protein expression experiments, it was determined that GaUGT131 the protein induction conditions are as follows: 16 °C, 0.2 mM IPTG, 180 r / min, induce for 12 h; then perform large-scale shaking, collect bacteria, break the cells, obtain the protein supernatant after high-speed centrifugation, and then detect it by SDS-PAGE protein electrophoresis. The detection result is shown in Figure 4 , indicating that the supernatant protein of the target gene was obtained.

[0028] (5) Enzyme activity reaction The enzyme activity reaction is carried out in a 1.5 mL centrifuge tube and prepared according to the components in Table 1. Then, add each component in the order shown in the table, mix well, and briefly centrifuge to collect the reaction solution to the bottom of the centrifuge tube. Place the centrifuge tube in a metal bath and react at 32 °C for 12 h. After completion, terminate the reaction with 100 μL of methanol, and finally perform product detection.

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

[0030] (6) Product detection The HPLC detection conditions are as follows: The instrument used for HPLC detection is the Agilent 1290 ultra-high performance liquid chromatograph. The liquid chromatography column is an Agilent ZORBAX SB-C18 column (250 mm × 4.6 mm, 5.0 μm). The mobile phase is: 0.01% formic acid solution (A) and acetonitrile (B). The gradient elution program is 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 is 0.6 mL / min. The column temperature is 30 °C. The injection volume is 10 μL. The absorption wavelength is 220 nm. The detection result is shown in Figure 5 , indicating 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) instrument. Mass spectrometry conditions: The ion source was operated in the negative ion mode, with a voltage of 3500 V; fragmentation voltage: 135 V; cone voltage: 60 V; RF voltage: 750 V, and the scanning range was 100 - 1000 m / z. Chromatography conditions: The column used was an Agilent ZORBAX SB-C18 column (250 mm × 4.6 mm, 5.0 μm), with a flow rate of 0.6 mL / min. The mobile phase was 0.01% formic acid (A) and acetonitrile (B), and 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 , and it can be seen from the results that the fragment ion spectrum of the reaction product gastrodin ( Figure 6 ) is consistent with the fragment ion spectrum of the standard gastrodin ( Figure 7 ), further confirming that the product generated is gastrodin.

[0032] The above shows and describes 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 by the above embodiments. What is described in the above embodiments and the specification only illustrates the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention will have various changes and improvements, and these changes and improvements all fall within the scope of the present invention claimed. The scope of protection claimed by the present invention is defined by the appended claims and their equivalents.

Claims

1. A gastrodia glycosyltransferase GaUGT131 gene, characterized in that The gastrodia elata glycosyltransferase GaUGT131 The nucleic acid sequence of the gene is shown in SEQ ID NO.

1.

2. The gastrodia glycosyltransferase according to claim 1 GaUGT131 The protein encoded by the gene, characterized in that The amino acid sequence of the encoded protein is shown in SEQ ID NO.

2.

3. A recombinant plasmid containing the gastrodia glycosyltransferase gene recited in claim 1 GaUGT131 gene.

4. The recombinant plasmid containing the gastrodia glycosyltransferase GaUGT131 gene according to claim 3, characterized in that The gastrodia glycosyltransferase GaUGT131 gene was obtained by homologous recombination with the pET28a vector to obtain the pET28a- GaUGT131 recombinant plasmid.

5. A genetically engineered bacterium, characterized in that, Containing the recombinant plasmid described in claim 3 or 4, or, the exogenous gastrodia glycosyltransferase described in claim 1 is integrated into the genome of the genetically engineered bacterium GaUGT131 gene.

6. The genetically engineered bacterium according to claim 5, wherein The genetically engineered bacterium is Escherichia coli BL21(DE3) strain.

7. Use of the gastrodia elata glycosyltransferase gene according to claim 1 GaUGT131 in the preparation of gastrodin.

8. Use of the gastrodia glycosyltransferase gene according to claim 7 GaUGT131 in the preparation of gastrodin, characterized in that Using p-hydroxybenzyl alcohol as a substrate and UDP-glucose as a glycosyl donor, under the catalysis of the gastrodia glycosyltransferase encoded by the gastrodia glycosyltransferase gene as described above, GaUGT131 glycosylation occurs at the hydroxyl group at the -4 position of p-hydroxybenzyl alcohol to produce gastrodin. C ​

Images