CN115927280B - Horse chestnut 2, 3-oxidation squalene cyclase and encoding gene and application thereof - Google Patents

Abstract

The invention discloses horse chestnut 2, 3-oxidation squalene 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 as a sequence 2 in a sequence table; b) And a protein which is derived from a) and has beta-amyrin synthase activity through substitution and/or deletion and/or addition of one or more amino acid residues of an amino acid sequence shown in a sequence 2 in a sequence table. The invention obtains the gene sequence of beta-amyrin alcohol synthetase mainly through the combined analysis of transcriptome and metabolome, and confirms that AcOSC6 can catalyze 2, 3-oxidation squalene to synthesize beta-amyrin alcohol through a tobacco transient transformation system. Can express beta-amyrin synthase gene in the living leaf of Nicotiana benthamiana, and heterologously synthesize beta-amyrin, thus providing a new idea for synthesizing aescin. The identification of horse chestnut 2, 3-oxidation squalene cyclase also greatly promotes the biosynthesis of aescin, and provides sufficient raw materials for the aescin to become a new medicine.

Description

Horse chestnut 2, 3-oxidation squalene cyclase and encoding gene and application thereof

Technical Field

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

Background

Semen Aesculi is Aesculus hippocastanum of AesculaceaeAesculus chinensisBge, zhejiang horse chestnut a.chinensis BgeVar chekiangensis (Hu et Fang) Fang or Tianshi chestnutA. wilsonii The dried mature seeds of Red are an important Chinese medicine recorded in Chinese pharmacopoeia. The Chinese medicine theory considers that semen aesculi has the functions of soothing liver, regulating qi, harmonizing stomach and relieving pain, and is used for treating liver and stomach qi stagnation, chest and abdominal distention, and stomach and Anhui pain. European horse chestnutA. hippocastanumL. seeds, also known as European chestnut, are European traditional folk herbs, and horse chestnut seed extracts are often used for the treatment of chronic venous insufficiency, hemorrhoids, postoperative oedema, etc. Aescin is the main active substance of semen Aesculi and semen Castaneae, and has antiinflammatory, repercussive, antitumor and antiviral effects. As the market demand is continuously increased, but the basic plant resources are limited, and the artificial synthesis cannot be performed at present, so that the aescin resources are tense. The aescin is the product of hydroxylation, glycosylation and acylation of beta-amyrin alcohol, and the beta-amyrin 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-amyrin alcohol has important medical significance and economic value.

Terpenoid is the largest family of plant secondary metabolites, widely distributed in nature, and beta-amyrin is an important pentacyclic triterpene in plants, with important physiological and pharmacological activities. Beta-amyrin alcohol cannot be synthesized artificially at present and completely depends on natural resources. Beta-amyrin is formed from (3S) -2, 3-oxidosqualene catalyzed by the family of oxidosqualene cyclase proteins. The catalytic process of a 2, 3-oxidosqualene cyclase can be divided into four stages, first of all, binding to a substrate and folding, then initiating the reaction by protonation of the epoxide, then cyclizing and carbonium rearrangement, and finally deprotonation to form the final product.

Analysis of key enzymes in the biosynthetic pathway of natural products may drive the study of the synthetic biology of natural products. For example, the university of California, berkeley division Jay Kealling research team constructed a Saccharomyces cerevisiae artificial cell that produced arteannuic acid efficiently, in yields of up to 25 g/L (Paddon C J, westfall P J, pitera D J, et al High-level semi-synthetic production of the potent antimalarial artemisinin [ J ]. Nature,2013,496 (7446); 528.). Team Zhou Zhihua over-expression of all genes of the mevalonate pathway in Saccharomyces cerevisiae chassis strains, including 7 upstream and 6 downstream modular genes, eventually increased ginsenoside Rh2 production to 2.25 g/L (Wang P, wei Y, fan Y, et al Production of bioactive ginsenosides Rh and Rg3 by metabolically engineered yeasts [ J ]. Metabolic engineering,2015,29; 97-105.). Ajikumar et al lay a foundation for the synthesis of taxol by introducing 4 key enzyme genes of the upstream module and GGPP synthase genes and taxadiene synthase genes of the downstream module into E.coli in amounts up to 1.02 g/L for the synthesis of taxol (Ajikumar P K, xiao W, tyo K E, et al Isoprenoid pathway optimization for Taxol precursor overproduction in Escherichia coli [ J ]. Science,2010,330 (6000); 70-74.). Wang Caixia et al increased the glycyrrhetinic acid content to 8.78 mg/L by introducing cytochrome gene GuCYB5 and 10 known mevalonate pathway genes into Saccharomyces cerevisiae using a novel integration strategy (Wang C, suX, sun M, et al Efficient production of glycyrrhetinic acid in metabolically engineered Saccharomyces cerevisiae via an integrated strategy [ J ]. Microbial cell factories,2019,18 (1); 95.).

Although aescin can be directly extracted from aesculus hippocastanum fruit, due to limited wild resources of aesculus hippocastanum and long artificial cultivation period, the supply of aescin resources is tension, so that analysis of aescin biosynthesis pathway is urgently needed, and a key gene element is provided for the study of aescin synthesis biology and the new aesculus hippocastanum variety with high yield of aescin.

Disclosure of Invention

The beta-amyrin alcohol synthetase catalyzes the key steps of the aescin synthesis path, and the identification of the enzyme can greatly promote the integral analysis of the aescin biosynthesis path and provide theoretical basis for the biosynthesis.

Accordingly, the present invention is directed to a hippocastanum 2, 3-oxidosqualene cyclase, and a coding gene and application thereof.

The technical scheme of the invention is as follows:

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

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

b) And a protein which is derived from a) and has beta-amyrin synthase activity through substitution and/or deletion and/or addition of one or more amino acid residues of an amino acid sequence shown in a sequence 2 in a sequence table.

The beta-amyrin synthase activity is to catalyze the synthesis of beta-amyrin by 2, 3-oxidation squalene.

The coding genes of the proteins also belong to the protection scope of the invention.

The coding gene is as follows 1) or 2) or 3):

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

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

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

The gene was namedAcOSC6，The protein encoded by this was designated AcOSC6. The specific information is as follows:AcOSC6the gene sequence is shown as a sequence 1 in a sequence table, contains 2298 nucleotides, encodes a protein shown as a sequence 2 in the sequence table, 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 invention.

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

The beta-amyrin synthase activity is to catalyze the synthesis of beta-amyrin by 2, 3-oxidation squalene.

The application of the protein and the coding gene in catalyzing the synthesis of beta-amyrin by using 2, 3-oxidation squalene also belongs to the protection scope of the invention.

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

The invention obtains the gene sequence of beta-amyrin alcohol synthetase mainly through the combined analysis of transcriptome and metabolome, and confirms that AcOSC6 can catalyze 2, 3-oxidation squalene to synthesize beta-amyrin alcohol through a tobacco transient transformation system. Can express beta-amyrin synthase gene in the living leaf of Nicotiana benthamiana, and heterologously synthesize beta-amyrin, thus providing a new idea for synthesizing aescin. The identification of horse chestnut 2, 3-oxidation squalene cyclase also greatly promotes the biosynthesis of aescin, and provides sufficient raw materials for the aescin to become a new medicine.

Drawings

For purposes of illustration and not limitation, the 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 formulae of 2, 3-oxidosqualene and beta-amyrin.

FIG. 2 is a graph showing the changes in the content of aescin metabolites at different tissue sites of Aesculus hippocastanum.

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

FIG. 4 is a GC-MS diagram for identification of AcOSC6 injected tobacco products.

Detailed Description

EXAMPLE 1 cloning of a Gene encoding a hippocastanum 2, 3-Oxidation squalene cyclase

1. Method and procedure for cloning of genes

Firstly, leaves, branches, flowers, epicarp and kernels of horse chestnut are ground into powder in liquid nitrogen, and the total content of aescin A, aescin B and aescin is detected by LC-QQQ-MS (Agilent 1290, 6470 QQ)) with a ratio of 1:10 by methanol extraction. The chromatographic column was Agilent Eclipse Plus C column (RRHD 1.8 μm, 2.1X150 mm), the mobile phase included chromatographic grade 0.1% acetic acid-acetonitrile (A) and 0.1% acetic acid-water (B), isocratic elution procedure, flow rate 0.3 mL/min, column temperature 35 ℃, detection wavelength 203 nm, and sample injection volume 1. Mu.L. The mass spectrometer was selected to be either in positive or negative ion mode with a sheath gas temperature of 250 ℃, gas flow of 11.0L/min, and nebulizer gas at 40 psi. The capillary voltage was 4000V, the nozzle voltage was 500 v, and the emv (+) was 200V. Metabolites were detected using multiplex reaction monitoring (multiple reaction monitoring, MRM) mode, with two precursor product ion MRM transfers (one for quantification and the other for identification) per compound selected for quantitative analysis using external standard methods. Data were acquired and analyzed using a MassHunter (version b.07.00). The total content of aescin A, aescin B and aescin in the flowers and kernels was found to be highest, especially the total content of the three aescins and aescin in the kernels was all highest (FIG. 2).

On the basis of hippocampus genome sequencing, 2, 3-oxidation squalene cyclase genes are annotated in the hippocampus genome by utilizing a bioinformatics technology, and RNAseq is carried out on different tissues of the hippocampus by utilizing an Illumina platform, and a transcriptome data gene expression analysis result shows that AcOSC6 is specifically and highly expressed in kernels and is consistent with a change rule of the content of aescin (table 1).

Collecting mature seeds of horse chestnut, extracting RNA in the horse chestnut seeds by using an RNA extraction kit (Aidelai, RN38 EASYspin plus), and carrying out reverse transcription after the quality detection of the RNA is qualified to obtain cDNA with qualified quality. The primer sequences (as in Table 2) were designed and cloned into AcOSC6 gene fragments using kernel cDNA as template and KOD-Plus-Neo Hi-Fi enzyme (total volume of KOD Hi-Fi enzyme PCR system was 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.L KOD enzyme and 32. Mu.L water, procedure as in Table 3). The AcOSC6 gene fragment was successfully ligated into the vector (ligation system total volume: 3. Mu.L: 0.5. Mu.L pEASY-Blunt vector and 2.5. Mu.L cDNA template, ligation reaction at 25℃for 1 h) using pEASY-Blunt vector (full gold, pEASY. Mu. -Blunt Cloning Vector). The ligation system directly transformed TransT1 competence (full gold, trans1-T1 Phage Resistant Chemically Competent Cell), positive clone sequencing (total volume of colony PCR system 12.5. Mu.L: 6.25. Mu.L 2 XTaq PCR Mix, 1. Mu.L template, 0.25. Mu.L forward primer, 0.25. Mu.L reverse primer and 4.75. Mu.L water, procedure as shown in Table 4, PCR detection results as shown in FIG. 3A) was selected, aligned with RNAseq sequence, and the actual sequencing nucleotide sequence was 100% similar to the original data.

Construction of a tobacco transient expression vector by referring to Invitrogen Gateway cloning technology, which comprises the following specific steps:

(1) Primers were designed, attB1 sequence was added to the 5 '-end and attB2 sequence was added to the 3' -end of the target fragment (Table 2), and KOD-PCR was performed using the plasmid of the target fragment obtained above as a template (KOD high-fidelity enzyme PCR system total volume was 50. Mu.L: 5. Mu.L 10X Buffer, 3. Mu.L MgSO) ₄ 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.L KOD enzyme and 32. Mu.L water, were used as in Table 3.

(2) BP reaction: 25ng of attB PCR recovery and 75ng of pDONR207 entry vector (Biovector NTCC collection) were mixed to 4. Mu.L with water, then L. Mu.L of BP Clonase II enzyme (Thermo Fisher, gateway ™ BP close ™ enzyme mixture) were added, mixed, incubated at 25℃for L h, 0.5. Mu.L of protease K was added, incubated at 37℃for 10min, transferred to TransT1 competence, positive clones were screened on 15 mg/L Kan resistant LB solid medium and PCR detection (PCR detection results are shown in FIG. 3B) and positive clones sequenced successfully were extracted.

(3) LR reaction: taking the pDONR207 recombinant plasmid of 75ng and 75ng pEAQ-HT-DEST vector plasmid (Biovector NTCC collection) and mixing with deionized water to 4. Mu.L, then adding 0.4. Mu.L of LR Clonase II enzyme (Thermo Fisher, gateway ™ LR close ™ enzyme mixture), mixing, incubating at 25℃for L h, adding 0.5. Mu.L of protease K, incubating at 37℃for 10min, transferring to TransT1 competence, screening positive clones on 50 mg/mL Kan resistant LB solid medium and PCR detection (Table 4), preserving and extracting plasmids from the sequenced positive clones (PCR detection results are shown in FIG. 3C), finally obtainingAcOSC6A plasmid having the gene linked to the pEAQ-HT-DEST vector.

2. Obtaining a gene sequence and a protein sequence encoded by the gene sequence

Sequencing results show that the gene amplified by using the primers in Table 1 contains 2298 nucleotides (shown as sequence 1 in the sequence table), and the coding sequence isThe 765 amino acid protein (shown as sequence 2 in the sequence table) is named by official authoritiesAcOSC6The protein encoded by this was designated AcOSC6.

3. Verification of Gene function

The test of the full gold plasmid extraction kit EasyPure HiPure Plasmid MiniPrep Kit is referred to for extracting plasmids in positive E.coli:

(1) The bacterial liquid cultured overnight at 2.2 mL was centrifuged at 10,000 g for 1 minute, and the supernatant was removed (as completely as possible).

(2) 250. Mu.L of colorless solution RB (containing RNase A) was added, and the bacterial pellet was suspended by shaking, leaving no small clumps.

(3) 250 mu L of blue solution LB is added, and the mixture is gently turned up and down and mixed for 4 to 6 times, so that the thalli are fully cracked, and a blue transparent solution is formed.

(4) 350. Mu.L of yellow solution NB was added and gently mixed 5-6 times until a compact yellow agglomerate formed and allowed to stand at room temperature for 2 minutes.

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

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

(7) Centrifugation at 12,000 g for 2 min, the residual WB was removed thoroughly.

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

(9) 10,000 g was centrifuged for 1 min and the DNA eluted for the next Agrobacterium transformation experiment.

The agrobacterium transformation was performed with reference to the experimental description of the local organism EH105 agrobacterium electrotransformation:

(1) Taking out the 0.1 cm electric shock cup and the cup cover from the storage liquid, pouring the electric shock cup and the cup cover on clean water-absorbing paper for 5 minutes, draining ethanol until the electric shock cup and the cup cover are drained, standing for 5 minutes, enabling the ethanol to volatilize fully, inserting the electric shock cup and the cup cover into ice immediately after the ethanol volatilizes cleanly, compacting the ice surface, enabling the top of the electrode cup to be away from the ice surface by 0.5 cm so as to cover the cup cover conveniently, standing in the ice for 5 minutes, and cooling fully.

(2) EH105 Agrobacterium competent cells were removed from the-80℃refrigerator and inserted into ice for 5 min and 0.01-1. Mu.g ligation was added to the iceAcOSC6The pEAQ-HT-DEST positive plasmid DNA (volume not more than 6. Mu.l) of the gene is stirred by hands at the bottom of a tube, mixed evenly, immediately inserted into ice, the competent-plasmid mixture is quickly moved into a electric shock cup by using a 200. Mu.l gun head, a cup cover is covered, and the hollow tube is reserved for standby.

(3) Starting an electrotransport device, rapidly placing an electric shock cup into an electrotransport tank, rapidly inserting electric shock into ice, adding 700 mu l of LB without antibiotics, transferring the LB without antibiotics into a competent empty tube, and culturing at 28 ℃ for 2-3 hours in an oscillating way.

(4) After centrifugation at 6000 rpm for one minute, about 100. Mu.l of supernatant was left to gently blow the resuspended pellet and spread on LB plates containing 50 mg/L Kan and 50 mg/L Rif antibiotics, and the pellet was placed upside down in a 28℃incubator for 2-3 days.

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

Tobacco transient expression and product identification were as follows:

(1) Agrobacterium EHA105 injection Benshi tobacco leaf

EHA105 containing recombinant plasmid was inoculated in 5 ml LB liquid medium containing 50 mg/L Rif and 50 mg/L Kan antibiotic, shake cultured at 28℃to OD600 of about 1.0;4000 g centrifuging for 5 min, discarding supernatant, collecting bacterial precipitate, and concentrating with MMA solution (10 mM MES,10 Mm MgCl concentration) ₂ 200 mu M mixed solution of acetosyringone) is resuspended to an OD600 of 0.8-1.2 and left at room temperature of 3 h; selecting good tobacco (Goodin MM, zaitlin D, naidu RA, lommel SA)(2008) Nicotiana benthamiana: its History and Future as a Model for Plant-Pathen interactions. Molecular Plant-Microbe Interactions:21:1015-1026. Small holes are first gently pierced in the blade surface with a syringe needle, facilitating injection of an Agrobacterium MMA suspension; dark culturing 1 d, taking out, culturing with light again 4 d, collecting leaves injected with Agrobacterium, and preserving at-80deg.C.

(2) Analysis and identification of catalytic products

Grinding the tobacco leaves into powder in liquid nitrogen, taking about 0.1 and g of the powder into a 500uL leaching agent (extraction reagent: methanol: water: potassium hydroxide=9:1:1, v: w), leaching for 2 hours at 65 ℃, vibrating for 1 time every half hour, then adding 250ul of water, vibrating and mixing uniformly, adding 500ul of normal hexane, vibrating and mixing uniformly, centrifuging for 1 min at 12000 g, taking 100ul of upper normal hexane solution, and concentrating to dryness by a vacuum centrifugal concentrator (Concentrator plus, eppendor); then 50uL of ammonium N-methyl-N-trimethylsilyl trifluoroacetate (aladine, china) was added for derivatization for 30 min at 80 ℃, diluted with 50uL ethyl acetate, and transferred to agilent sample bottles for GC-MS detection.

GC-MS detection was performed using Agilent 7890, gas chromatography column hb-5 (30 mX0.25 mm X0.25 um, aglient), temperature program: maintaining at 170deg.C for 2 min, heating to 300deg.C at 20deg.C/min, and maintaining at 300deg.C for 10min (total 20 min); carrier gas (He) flow rate 1mL/min; the sample injection amount is 1 mu L; the temperature of the gasification chamber is 250 ℃; the mass spectrum detector is an Agilent 7000C triple quaternary rod, and the mass spectrum scanning range is 60-800 u.

The GC-MS analysis results are shown in FIG. 4, usingAcOSC6Injecting tobacco, detecting a 218 characteristic peak at 11.404 min, wherein the peak time and the secondary mass spectrum are the same as those of the beta-amyrin standard, and the no-load injection tobacco does not detect beta-amyrin, which indicatesAcOSC6The coded protein has the activity of catalyzing the synthesis of beta-amyrin.

The above embodiments do not limit the scope of the present invention. It will be apparent to those skilled in the art that various modifications, combinations, sub-combinations and alternatives can occur depending upon design requirements and other factors. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the scope of the present invention.

Claims (7)

1. A protein has an amino acid sequence shown in SEQ ID NO. 2.

2. The protein-encoding gene of claim 1, wherein: the nucleotide sequence of the coding gene is shown as SEQ ID NO. 1.

3. An expression cassette, recombinant expression vector, transgenic cell line or recombinant microorganism comprising the coding gene of claim 2.

4. Use of the protein of claim 1 as β -amyrin synthase.

5. The use according to claim 4, characterized in that: the beta-amyrin synthase is an enzyme catalyzing the synthesis of beta-amyrin by 2, 3-oxidation squalene.

6. Use of the protein of claim 1 for catalyzing the synthesis of β -amyrin from 2, 3-oxidosqualene.

7. The use of the coding gene of claim 2 in catalyzing the synthesis of beta-amyrin from 2, 3-oxidation squalene.