CN115927280A - Horse chestnut 2,3-oxidosqualene cyclase and coding gene and application thereof - Google Patents

Abstract

The invention discloses horse chestnut 2,3-oxidosqualene cyclase and a coding gene and application thereof. The protein is the protein of the following a) or b): a) A protein consisting of an amino acid sequence shown in a sequence 2 in a sequence table; b) Protein which is derived from a) and has beta-balsamic alcohol synthetase activity and is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown in the sequence 2 in the sequence table. The gene sequence of beta-amyrin synthase is obtained mainly through the combined analysis of transcriptome and metabolome, and the synthesis of beta-amyrin by 2,3-oxidized squalene under the catalysis of AcOSC6 is confirmed by a tobacco instantaneous conversion system. Can express beta-balsamic alcohol synthetase gene in the living tobacco leaf of Ben's tobacco, heterologously synthesizes beta-balsamic alcohol, and provides new thought for the synthesis of aescin. The identification of the aesculus 2,3-oxidosqualene cyclase can greatly advance the biosynthesis of aesculin and provide sufficient raw materials for the aesculin to become a new medicine.

Description

Horse chestnut 2,3-oxidosqualene cyclase and coding gene and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to horse chestnut 2,3-oxidosqualene cyclase and a coding gene and application thereof.

Background

Semen Aesculi is Aesculus hippocastanum of Aesculus familyAesculus chinensisBge., zhejiang horse chestnut a.chinensis BgeVar. Chekiangensis (Hu et Fang) Fang or chestnutA. wilsonii The dry mature seeds of Rehd are an important Chinese medicine recorded in the Chinese pharmacopoeia. Chinese medicine theory considers that buckeye has the functions of soothing liver, regulating qi, harmonizing stomach and relieving pain, and is used for treating qi stagnation of liver and stomach, chest and abdomen swelling and stuffiness, stomach and wan pain. Aesculus hippocastanumA. hippocastanumL. seed also known as Aesculus hippocastanumAs a traditional folk herb in europe, horse chestnut seed extract is often used for the treatment of chronic venous insufficiency, hemorrhoids, postoperative edema, and the like. Aescin is the main active matter of buckeye seed and European horse chestnut and has antiphlogistic, repercussive, antitumor, antiviral and other pharmacological activity. As the market demand is continuously increased, but the basic plant resources are limited and cannot be artificially synthesized at present, the aescin resource is in shortage. The aescin is a product of hydroxylation, glycosylation and acylation of beta-balsamic alcohol, and the beta-balsamic alcohol provides a skeleton structure for the formation of a plurality of pharmacological activities of the aescin, so that the research on the enzyme for catalyzing the synthesis of the beta-balsamic alcohol has important medical significance and economic value.

Terpenoids are the largest family of plant secondary metabolites, widely distributed in nature, and β -coumarol is an important pentacyclic triterpene in plants, and has important physiological and pharmacological activities. At present, beta-balsamic alcohol cannot be artificially synthesized and completely depends on natural resources. Beta-balsamic alcohol is formed from (3S) -2,3-oxidosqualene catalyzed by the oxidosqualene cyclase protein family. 2,3-oxidosqualene cyclase is catalyzed by a specific process that can be divided into four stages, first binding to a substrate and folding, then initiating a reaction by protonation of the epoxide, then undergoing cyclization and carbocation rearrangement, and finally deprotonation to form the final product.

The analysis of key enzymes in the biosynthetic pathway of natural products can promote the synthetic biology research of natural products. For example, the Jay Keasling research team at the university of California at Berkeley, university, california constructed an efficient artemisinic acid-producing Saccharomyces cerevisiae artificial cell with a yield of 25 g/L (Paddon C J, westfall P J, pitera D J, et al, high-level semi-synthetic production of the potential anti-industrial aromatic synthesis [ J ]. Nature,2013,496 (7446); 528.). 3238 Zxft 3238 team overexpressed mevalonate pathway in Saccharomyces cerevisiae chassis strains including 7 upstream modular genes and 6 downstream modular genes, ultimately increasing ginsenoside Rh2 Production to 2.25 g/L (Wang P, wei Y, fan Y, et al, production of bioactive enzymes Rh2 and Rg3 by Metabolic engineering yeases [ J ]. Metabolic engineering,2015, 29. Ajikumar et al established the foundation for paclitaxel synthesis by introducing 4 key enzyme genes of the upstream module and GGPP synthase gene and taxadiene synthase gene of the downstream module into E.coli with a paclitaxel diene synthesis amount of up to 1.02 g/L (Ajikumar P K, xiao W, tyo K E, et al. Isoprost pathway optimization for Taxol precursor over production in Escherichia coli [ J ] Science,2010,330 (6000); 70-74.). Wang Caixia et al used a novel integration strategy to increase the content of glycyrrhetinic acid to 8.78 mg/L (Wang C, su X, sun M, et al, effective production of glycyrrhetinic acid in metabolic engineered Saccharomyces cerevisiae via an integrated strain [ J ]. Microbial cell factors, 2019,18 (1); 95.) by introducing the cytochrome gene GuCYB5 and 10 known mevalonate pathway genes into s.cerevisiae.

Although aescin can be directly extracted from buckeye of aesculus fruit, the aescin resource supply is tense due to limited wild aesculus resource and long artificial cultivation period, so that the aescin biosynthesis path needs to be analyzed urgently, and key genetic elements are provided for the synthetic biology research of aescin and new aesculus varieties with high aesculus saponin yield.

Disclosure of Invention

The beta-amyrin synthase catalyzes the key steps of the aescin synthesizing path, and the identification of the beta-amyrin synthase can promote the integral analysis of the aescin synthesizing path and provide theoretical basis for the biosynthesis.

In view of the above, the main objective of the present invention is to provide horse chestnut 2,3-oxidosqualene cyclase and its coding gene and application.

The technical scheme of the invention is as follows:

the invention provides a protein which is the protein of a) or b) as follows:

a) A protein consisting of an amino acid sequence shown in a sequence 2 in a sequence table;

b) Protein which is derived from a) and has beta-amyrin alcohol synthetase activity, and the protein is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown in the sequence 2 in the sequence table.

The activity of the beta-balsamic alcohol synthase is to catalyze 2,3-oxidosqualene to synthesize beta-balsamic alcohol.

The coding gene of the protein also belongs to the protection scope of the invention.

The coding gene is shown as the following 1) or 2) or 3):

1) The nucleotide sequence is a DNA molecule shown in a sequence 1 in a sequence table;

2) A DNA molecule which hybridizes with the DNA molecule defined in 1) under stringent conditions;

3) DNA molecules having more than 90% homology with the DNA molecules defined in 1) or 2).

This gene was namedAcOSC6，The protein encoded by the protein was named AcOSC6. The specific information is as follows:AcOSC6the gene sequence is shown as sequence 1 in the sequence table, which contains 2298 nucleotides, and the protein shown as sequence 2 in the sequence table is coded, and the sequence 2 consists of 765 amino acids.

Expression cassettes, recombinant expression vectors, transgenic cell lines or recombinant microorganisms containing the coding genes also belong to the scope of protection of the present invention.

The application of the protein as beta-amyrin synthase also belongs to the protection scope of the invention.

The activity of the beta-balsamic alcohol synthase is to catalyze 2,3-oxidation of squalene to synthesize beta-balsamic alcohol.

The protein and the application of the coding gene in catalyzing 2,3-oxidosqualene to synthesize beta-balsamic alcohol also belong to the protection scope of the invention.

Based on the high-throughput sequencing of different tissue parts of aesculus chinensis leaves, branches, flowers, epicarp and kernels and related results of aesculus chinensis secondary metabolites, the invention finds and identifies the key enzyme catalyzing the synthesis of beta-turpentine by using a gene co-expression analysis method, and provides key genes and protein coding sequences for the biosynthesis of aesculus chinensis and the cultivation of new varieties of aesculus chinensis with high yield of aesculus chinensis.

The invention mainly obtains the gene sequence of beta-balsamic alcohol synthetase through the combined analysis of transcriptome and metabolome, and then confirms that AcOSC6 can catalyze 2,3-oxidized squalene to synthesize beta-balsamic alcohol through a tobacco instantaneous conversion system. Can express beta-amyrin synthase gene in the living leaves of the Nicotiana benthamiana, and can synthesize beta-amyrin in a heterologous way, thereby providing a new idea for the synthesis of aescin. The identification of the aesculus 2,3-oxidosqualene cyclase can greatly advance the biosynthesis of aesculin and provide sufficient raw materials for the aesculin to become a new medicine.

Drawings

For purposes of illustration and not limitation, the present invention will now be described in accordance with its preferred embodiments, particularly with reference to the accompanying drawings, in which:

FIG. 1 shows the molecular structural formula of 2,3-oxidosqualene and β -balsamic alcohol.

FIG. 2 is a graph showing the content change of metabolites of escin in different tissue parts of escin.

FIG. 3 is a schematic view ofAcOSC6Gene cloning and vector construction pectin binding maps.

FIG. 4 is a graph of the GC-MS identification of AcOSC6 injected tobacco products.

Detailed Description

Example 1 cloning of a Gene encoding Aesculus 2,3-Oxysqualene cyclase

1. Methods and procedures for gene cloning

Firstly, leaves, branches, flowers, epicarp and kernels of aesculus hippocastanum are ground into powder in liquid nitrogen, extracted by methanol at a ratio of 1 to 10, and the total content of aescin a, aescin B and aescin is detected by LC-qq-MS (Agilent 1290, 6470 QQQ)). The chromatographic column is Agilent Eclipse Plus C18 column (RRHD 1.8 μm, 2.1X 50 mm), the mobile phase comprises chromatographic grade 0.1% acetic acid-acetonitrile (A) and 0.1% acetic acid-water (B), the isocratic elution program has the flow rate of 0.3 mL/min, the column temperature is 35 ℃, the detection wavelength is 203 nm, and the sample feeding amount is 1 μ L. The mass spectrometer was selected in positive or negative ion mode with a sheath gas temperature of 250 ℃, a gas flow of 11.0L/min, and a nebulizer gas at 40 psi. The capillary voltage was 4000V, the nozzle voltage was 500V, and EMV (+) was 200V. Metabolites were detected using a Multiple Reaction Monitoring (MRM) mode, with two MRM transfers of precursor product ions selected for each compound (one for quantification and the other for identification), and quantified using an external standard method. Data were obtained and analyzed using MassHunter (version b.07.00). The total content of aescin a, aescin B and aescin in the flowers and kernels is found to be the highest, and particularly the total content of the three types of aescin and aescin in the kernels is the highest (fig. 2).

On the basis of aesculus hippocastanum genome sequencing, a bioinformatics technology is utilized to inject 2,3-oxidosqualene cyclase gene into aesculus hippocastanum genomes, an Illumina platform is utilized to carry out RNAseq on different tissues of aesculus hippocastanum, and the gene expression analysis result of transcriptome data shows that AcOSC6 is specifically and highly expressed in kernels and is consistent with the content change rule of aescin (Table 1).

Collecting mature seeds of the horse chestnut, extracting RNA in the horse chestnut seeds by using an RNA extraction kit (Edley, RN38 EASYspin plus), and carrying out reverse transcription after the RNA quality is qualified to obtain cDNA with qualified quality. Primer sequences (see Table 2) were designed and cloned into AcOSC6 gene fragment using KOD-Plus-Neo high fidelity enzyme using the kernel cDNA as template (total volume of KOD high fidelity enzyme PCR system 50. Mu.L: 5. Mu.L 10 XBuffer, 3. Mu.L MgSO4, 5. Mu.L dNTP (2 mM), 1.5. Mu.L forward primer (10. Mu.M), 1.5. Mu.L reverse primer (10. Mu.M), 1. Mu.L template, 1. Mu.LKOD enzyme and 32. Mu.L water, program as in Table 3). AcOSC6 gene fragments were successfully ligated into the vectors using pEASY-Blunt Vector (all-type gold, pEASY-Blunt Cloning Vector) (total ligation system volume of 3. Mu.L: 0.5. Mu.L pEASY-Blunt Vector and 2.5. Mu.L cDNA template, ligation reaction at 25 ℃ for 1 h). The ligation system directly transforms TransT1 competence (gold full-scale, trans1-T1 Phage resist chemical Complex Cell), selects positive clones for sequencing (the total volume of colony PCR system is 12.5. Mu.L: 6.25. Mu.L 2 XTaq PCR Mix, 1. Mu.L template, 0.25. Mu.L positive primer, 0.25. Mu.L reverse primer and 4.75. Mu.L water, program is shown in Table 4, PCR detection result is shown in A in FIG. 3), and compares with RNAeq sequence, and the similarity between the actually sequenced nucleotide sequence and the original data is 100%.

The tobacco transient expression vector construction is carried out by referring to the Invitrogen Gateway cloning technology, and the specific steps are as follows:

(1) Designing a primer, adding an attB1 sequence at the 5 'end of the target fragment, adding an attB2 sequence at the 3' end (Table 2), and performing KOD-PCR (the total volume of a KOD high fidelity enzyme PCR system is 50 muL: 5 muL 10X Buffer,3 muL MgSO 2) by using the obtained target fragment plasmid as a template ₄ 5. Mu.L dNTP (2 mM), 1.5. Mu.L forward primer (10. Mu.M), 1.5. Mu.L reverse primer (10. Mu.M), 1. Mu.L template, 1. Mu.LKOD enzyme and 32. Mu.L water, as per the protocol in Table 3.

(2) BP reaction: 25ng of attB PCR recovery product and 75ng of pDONRR 207 entry vector (Biovector NTCC type culture collection center) were mixed with water to 4. Mu.L, then, L of BP clone II enzyme (Thermo Fisher, gateway BP clone enzyme mixture) was added, mixed, incubated at 25 ℃ for L h, added with 0.5. Mu.L of Protease K, incubated at 37 ℃ for 10min, transferred to TransT1 competence, and positive clones were selected on 15 mg/L Kan-resistant LB solid medium and tested by PCR (the result of PCR is shown in B in FIG. 3), and recombinant plasmids were extracted from the successfully sequenced positive clones.

(3) LR reaction: taking 75ng pDONR207 recombinant plasmid and 75ng pEAQ-HT-DEST vector plasmid (Biovector NTCC type culture collection), adding deionized water, mixing to 4 μ L, adding 0.4 μ L LRclonase II enzyme (Thermo Fisher, gateway [. LR clone) mixture, mixing, incubating at 25 deg.C for L h, adding 0.5 μ L Proteinase K, incubating at 37 deg.C for 10min, transferring to TransT1 competence, screening positive clone on 50 mg/mL Kan resistant LB solid medium and detecting by PCR (Table 4), sequencing successful positive clone and extracting plasmid (the result of PCR is shown in C in FIG. 3), and obtaining the final productAcOSC6The gene was ligated into a plasmid of the pEAQ-HT-DEST vector.

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2. Obtaining gene sequences and protein sequences encoded thereby

The sequencing result shows that the gene obtained by amplification by using the primers in the table 1 contains 2298 nucleotides (shown as a sequence 1 in a sequence table) and encodes 765 amino acid proteins (shown as a sequence 2 in the sequence table), and the gene is named by official namesAcOSC6The protein encoded by it was named AcOSC6.

3. Verification of Gene function

The Plasmid in the positive escherichia coli is extracted according to the test specification of the full-type gold Plasmid extraction Kit EasyPure HiPure Plasmid MiniPrep Kit:

(1) The overnight culture broth of 2 mL was centrifuged at 10,000 g for 1 min and the supernatant removed (as complete as possible).

(2) Add 250. Mu.L of colorless solution RB (containing RNase A) and shake the suspended bacterial pellet without leaving small clumps.

(3) Adding 250 μ L of blue solution LB, and gently mixing by turning upside down for 4-6 times to fully crack the thallus to form a blue transparent solution.

(4) Add 350. Mu.L of yellow solution NB and mix gently 5-6 times until a compact yellow aggregate forms, and let stand at room temperature for 2 minutes.

(5) Centrifuge at 12,000 g for 5 minutes and carefully pipette the supernatant into a spin column. Centrifuge at 12,000 g for 1 min and discard the effluent.

(6) 650. Mu.L of WB solution was added, centrifuged at 12,000 g for 1 min, and the effluent was discarded.

(7) Centrifugation at 12,000 g for 2 minutes completely removed residual WB.

(8) The column was placed in a clean centrifuge tube, 40. Mu.L of deionized water was added to the center of the column, and the column was allowed to stand at room temperature for 1 minute.

(9) The DNA was eluted by centrifugation at 10,000 g for 1 minute and used for the next Agrobacterium transformation experiment.

Agrobacterium transformation was performed with reference to the experimental description of agrobacterium electrotransformation of the geoonly organism EH 105:

(1) Taking the 0.1 cm electric shock cup and the cup cover out of the storage liquid, inversely placing the electric shock cup and the cup cover on clean absorbent paper for 5 minutes, draining off ethanol, rightly placing for 5 minutes to fully volatilize the ethanol, immediately inserting the electric shock cup into ice after the ethanol is completely volatilized, compacting the ice surface, enabling the top of the electrode cup to be separated from the ice surface by 0.5 cm to conveniently cover the cup cover, and standing for 5 minutes in the ice to fully cool.

(2) Taking out EH105 Agrobacterium competent cells from-80 deg.C refrigerator, inserting into ice for 5 min, thawing, adding 0.01-1 μ g of ligationsAcOSC6The pEAQ-HT-DEST positive plasmid DNA (the volume is not more than 6 mu l) of the gene is dialed by hands to the bottom of the tube and is evenly mixed, the tube is immediately inserted into ice, the competence-plasmid mixture is quickly moved into an electric shock cup by a 200 mu l gun head, the cup cover is covered, and the empty tube is reserved for standby.

(3) Starting the electric rotating instrument, quickly placing the electric shock cup into an electric rotating groove, quickly inserting the electric shock cup into ice after the electric shock is finished, adding 700 mu l of antibiotic-free LB, transferring the electric shock cup into a competent empty tube, and carrying out shaking culture at 28 ℃ for 2~3 hours.

(4) The strain is collected after being centrifuged at 6000 rpm for one minute, about 100 mu L of supernatant is left and is lightly blown to beat the heavy suspension strain block, the heavy suspension strain block is coated on an LB flat plate containing 50 mg/L Kan and 50 mg/L Rif antibiotics, and the LB flat plate is inversely placed in an incubator at 28 ℃ for 2 to 3 days.

(5) Single colonies on LB plates were picked with sterilized toothpicks, and cultured with shaking at 28 ℃ for 8-12 hours in 500. Mu.L of liquid LB medium containing 50 mg/L Kan and 50 mg/L Rif antibiotics, and PCR was performed using the forward primer of pEAQ-HT-DEST and the reverse primer of the target fragment (Table 5), respectively (the results are shown in D in FIG. 3), and the positive strains were subjected to the next tobacco transient expression test.

Tobacco transient expression and product identification were as follows:

(1) Agrobacterium EHA105 injection Ben's tobacco leaf

The EHA105 containing the recombinant plasmid was inoculated into LB liquid medium containing 50 mg/L Rif and 50 mg/L Kan antibiotics in 5 ml and shake-cultured at 28 ℃ to OD600 of about 1.0;4000 g centrifugation for 5 min, discarding supernatant, collecting pellet, adding equal volume of MMA solution (10 mM MES,10 mM MgCl) ₂ 200 mu M acetosyringone mixed solution) is suspended until the OD600 is 0.8 to 1.2, and the mixture is placed at room temperature for 3 h; selecting good native tobacco (Goodin MM, zaitlin D, naidu RA, lommel SA (2008): nicotiana benthamiana: its History and Future as a Model for Plant-Patholoen Interactions. Molecular Plant-Microbe Interactions 21: 1015-1026.), and lightly puncturing pores on the surface of the leaves by using a syringe needle so as to be beneficial to the injection of the MMA suspension of the agrobacterium tumefaciens; dark culturing 1 d, taking out, light culturing 4 d, collecting Agrobacterium-injected leaves, and storing at-80 deg.C.

(2) Analysis and characterization of catalytic products

Grinding the injection tobacco leaves into powder in liquid nitrogen, taking about 0.1 g powder, leaching the powder in a 500uL extracting agent (extracting agent: methanol: water: potassium hydroxide = 9); then 50uL of N-methyl-N-trimethyl silane ammonium trifluoroacetate (Aladdin, china) is added for derivatization at 80 ℃ for 30 min, 50uL ethyl acetate is added for dilution, and the solution is transferred to an Agilent sample bottle for GC-MS detection.

GC-MS detection was performed using Agilent 7890, the gas chromatography column was hb-5 (30 m × 0.25mm × 0.25um, aglient), the temperature program: keeping at 170 deg.C for 2 min, heating to 300 deg.C at 20 deg.C/min, and keeping at 300 deg.C for 10min (20 min in total); the flow rate of carrier gas (He) is 1mL/min; the sample volume is 1 mu L; the temperature of the gasification chamber is 250 ℃; the mass spectrum detector is an Agilent 7000C triple quadrupole, and the mass spectrum scanning range is 60-800 u.

The results of GC-MS analysis are shown in FIG. 4, usingAcOSC6Injecting tobacco, detecting a characteristic peak 218 at 11.404 min, the peak time and the secondary mass spectrogram are the same as those of the beta-resinol standard, and no beta-resinol is detected in the no-load injection tobacco, which indicates thatAcOSC6The encoded protein has the activity of catalyzing the synthesis of beta-balsamic alcohol.

The above-described embodiments should not be construed as limiting the scope of the invention. It should be understood by those skilled in the art that various modifications, combinations, sub-combinations and substitutions may occur depending on design requirements and other factors. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims (8)

1. A protein which is a protein of a) or b) as follows:

a) A protein consisting of an amino acid sequence shown in a sequence 2 in a sequence table;

b) Protein which is derived from a) and has beta-balsamic alcohol synthetase activity and is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown in the sequence 2 in the sequence table.

2. The protein of claim 1, wherein: the activity of the beta-balsamic alcohol synthase is to catalyze 2,3-oxidation of squalene to synthesize beta-balsamic alcohol.

3. A gene encoding the protein of claim 1 or 2.

4. The coding gene according to claim 3, characterized in that: the coding gene is shown as the following 1) or 2) or 3):

1) The nucleotide sequence is a DNA molecule shown in a sequence 1 in a sequence table;

2) A DNA molecule which hybridizes with the DNA molecule defined in 1) under stringent conditions;

3) A DNA molecule having a homology of 90% or more with the DNA molecule defined in 1) or 2).

5. An expression cassette, recombinant expression vector, transgenic cell line or recombinant microorganism comprising the encoding gene of claim 3 or 4.

6. Use of the protein of claim 1 or 2 as a β -amyrin synthase.

7. The protein of claim 6, wherein: the activity of the beta-balsamic alcohol synthase is to catalyze 2,3-oxidation of squalene to synthesize beta-balsamic alcohol.

8. Use of the protein of claim 1 or 2, or the gene encoding the protein of claim 3 or 4, for catalyzing the synthesis of β -amyrin from 2,3-oxidosqualene.

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