CN115247168A

CN115247168A - Oat oxidation squalene cyclase AsHS2 and coding gene and application thereof

Info

Publication number: CN115247168A
Application number: CN202111016897.3A
Authority: CN
Inventors: 薛哲勇; 梁苗苗
Original assignee: Northeast Forestry University
Current assignee: Northeast Forestry University
Priority date: 2021-08-31
Filing date: 2021-08-31
Publication date: 2022-10-28
Anticipated expiration: 2041-08-31
Also published as: CN115247168B

Abstract

The invention relates to the biosynthesis of triterpene, in particular to oat oxidosqualene cyclase AsHS2 and a coding gene and application thereof. The amino acid sequence of the oxidosqualene cyclase is shown as SEQ ID NO. 1, and the nucleotide sequence of the coding gene is shown as SEQ ID NO. 2. The invention discovers and clones oat oxidosqualene cyclase AsHS2 for the first time, identifies the product of the enzyme through tobacco in vitro transient expression, GC-MS and nuclear magnetic resonance, and lays a foundation for the biosynthesis of 17 (21) -olefine primary alcohol (hop- (17) 21-en-3 beta-ol).

Description

Oat oxidation squalene cyclase AsHS2 and coding gene and application thereof

Technical Field

The invention relates to the biosynthesis of triterpene, in particular to oat oxidosqualene cyclase AsHS2 and a coding gene and application thereof.

Background

Triterpenoids are the most diverse group of natural products, and more than 20000 triterpenoids have been found so far, and are mainly distributed in plants, wherein dicotyledonous plant triterpenoids are abundantly distributed, and are also distributed in animals and microorganisms, such as triterpenoids found in sea cucumber and starfish, more than 150 ganoderma triterpenoids found in ganoderma lucidum, and the bacteria can catalyze squalene (squalene) to synthesize triterpene hoprene (hopane).

Triterpenoids have important biological activity and medicinal value, and have been widely used in various fields such as food, industry and medicine. 2, 3-Oxysulene is a direct precursor to triterpene aglycone synthesis, and 2, 3-Oxysulene cyclase (OSC), also known as triterpene synthase, catalyzes a key step in the synthesis of this triterpene. The diversity of the triterpene backbone is due to the diversity of triterpene synthases in plants. The function of over 170 plant-derived oxidosqualene cyclases has been demonstrated, most of which were identified using heterologous expression from yeast strains. About one third of these oxidosqualene cyclases are sterol synthases (stero synthases), including cycloartenol synthase (CAS) and lanosterol synthase (LAS). Examples of oxidosqualene cyclases which catalyze the synthesis of triterpenes include β -balsamic alcohol synthase (bsas), lupeol synthase (LUS), dammarendiol Synthase (DS), and the like, among which β -balsamic alcohol synthase and Lupeol synthase (Thimmappa et al, 2014) are common.

In recent years, with the rapid development of genome sequencing, the whole genome sequence of some plants has been published, and many OSC genes have been cloned. OSC of dicotyledonous plants has been studied extensively, but there are few OSC enzymes found to have novel functions. Functional analysis of OSC in monocotyledonous plants of the Gramineae, rice and sorghum, revealed several unprecedented triterpene synthases, such as isoarborenol, fernenol and simiarenol synthases (Busta et al, 2021), respectively. The development of OSC enzyme with novel functions is helpful for deeply analyzing the triterpene biosynthesis pathway and lays a foundation for the biosynthesis of triterpene compounds.

Disclosure of Invention

Based on the current state of research in the above-mentioned fields, we speculate that OSC enzymes having novel functions are more likely to be mined from monocotyledons. According to the invention, through analyzing oat transcriptome data, oat oxidosqualene cyclase gene AsHS2 is found and cloned from oat, an AsHS2 tobacco heterologous expression vector is constructed by using Gateway technology and tobacco transient expression is carried out, extracts of transgenic tobacco and control tobacco are compared by GC-MS, a differential compound is found and is identified as 17 (21) -enoyl primary alcohol (hop- (17) 21-en-3 beta-ol) by using nuclear magnetic resonance technology, and the compound is a product of oat oxidosqualene cyclase AsHS2.

The amino acid sequence of the oxidosqualene cyclase AsHS2 is shown as SEQ ID NO. 1.

The coding gene of the oxidosqualene cyclase AsHS2 also belongs to the protection scope of the invention.

The nucleotide sequence of the gene is shown in SEQ ID NO. 2.

Expression cassettes, vectors and recombinant bacteria containing the genes of claim 2 or 3 are also within the scope of the present invention.

The present invention also provides a method for preparing a transgenic plant, comprising the steps of: introducing the gene into a starting plant to obtain a transgenic plant; the content of the compound hop- (17) 21-en-3 β -ol in the transgenic plants is altered compared to the starting plants.

Preferably, the gene is introduced into the starting plant by a recombinant expression vector; the recombinant expression vector is obtained by inserting the gene into a starting vector pEAQ-HT-DEST 1; the starting plant is control tobacco; the transgenic plant is transgenic tobacco.

The transgenic tobacco produced the compound hop- (17) 21-en-3 β -ol compared to the control tobacco.

The application of the oxidosqualene cyclase AsHS2 or the gene in hop- (17) 21-en-3 beta-ol biosynthesis also belongs to the protection scope of the invention.

The invention clones the oat oxidosqualene cyclase gene AsHS2 for the first time, verifies the function of the oat oxidosqualene cyclase gene, and lays a foundation for deeply analyzing the biosynthesis pathway of 17 (21) -olefine primary alcohol (hop- (17) 21-en-3 beta-ol).

Drawings

FIG. 1 is an electrophoresis picture of the recovered AsHS2 gene on agarose gel.

FIG. 2. Total ion current chromatogram (TIC) and extraction chromatogram (EIC 455.4) of tobacco expression product GC-MS; figure 1 in the figure indicates a differential compound produced by AsHS2 transgenic tobacco (compared to control tobacco), designated compound 1.tHMGR represents a control tobacco leaf sample into which the pEAQ-HT-DEST1-tHMGR vector was transferred; tHMGR-AsHS2 represents the AsHS2 transgenic tobacco leaf samples with pEAQ-HT-DEST1-tHMGR and pEAQ-HT-DEST1-AsHS2 expression vectors transferred into them, with three replicates per sample.

FIG. 3 is a mass spectrum of TMS derivative of Compound 1.

FIG. 4 carbon spectrum of Compound 1.

FIG. 5 shows the hydrogen spectrum of Compound 1.

FIG. 6 Structure of Compound 1.

Detailed Description

The present invention is described in detail below with reference to specific examples, it being understood that the following examples are only illustrative and illustrative of the present invention and do not limit the scope of the present invention in any way.

Biological material

Plant: the oat (Avena strigosa) variety used in the following examples was S75. Seeds of Nicotiana benthamiana (Nicotiana benthamiana) used in the following examples were from laboratory storage, a material that has been prepared in Ting H, et al, snare-RNAi results in higher depends emission from orthopedically expressed caryophylen synthsase in Nicotiana benthamiana molecular plant.2015;8, 454-466. Tobacco transient expression experiments were performed using 6 week old tobacco plants.

The strain is as follows: escherichia coli Trelief ^TM 5 α competent cells were purchased from Biotech, inc., of Okinsonidae, beijing. Agrobacterium tumefaciens GV3101 competent cells were purchased from Shanghai Diego Biotechnology, inc.

The above-mentioned biological material is also stored in the laboratory and the applicant states that it can be distributed to the public for the necessary verification tests within twenty years from the filing date.

Carrier: the intermediate vector pDONR207, which is a DONR vector of the Gateway system and has a size of 5585bp and resistance to gentamicin and chloramphenicol, was purchased from Invitrogen. The plant binary transient expression vector pEAQ-HT-DEST1 is purchased from Biovector NTCC plasmid vector strain cell gene collection center, the size of the vector is 11654bp, the gateway technology is compatible, the vector has a 35S promoter, a P19 gene silencing inhibition protein coding gene and Kan resistance.

Primer synthesis was entrusted to Beijing Ongke Biotechnology Inc. and Suzhou Jinzhi Biotechnology Inc.

DNA sequencing was entrusted to Ruibo biotechnology, inc. and Beijing optingke biotechnology, inc.

Main reagents and consumables:

TRIzon Total RNA extraction kit (cat # CW 0580S), agarose gel DNA recovery kit (cat # CW 2302) purchased from Kangji of century Biotechnology Ltd. SuperScript ^TM III reverse transcription kit, purchased from Invitrogen, cat no: 18080093.2 is good

Max Master Mix (cat # P515-01), 2 × Rapid Taq Master Mix (cat # P222-01) from Biotech, inc. of Kinzoka, nanjing.

BP

II Enzyme mix (cat # 11789021),

LR

II Enzyme mix (cat # 11791100) from Thermo Fisher scientific, inc., satemer Feishell technology (China). Plasmid miniprep kit (cat # D1100) was purchased from Beijing Solaibao Tech Co. MES purchased from Sigma-Aldrich, CAS number: 4432-31-9, cat #: m3671. Acetosyringone was purchased from Sigma-Aldrich, CAS No.: 2478-38-8, cat # I: D134406.coprostanol (copranol) was purchased from Sigma-Aldrich, CAS No.: 360-68-9, cargo number: C7578. ethyl acetate was purchased from Sigma-Aldrich, CAS No.: 141-78-6, item number: 270989.1- (Trimeth)ylsilyl) imidazole-Pyridine mixture, chinese name: 1- (trimethylsilyl) imidazole-pyridine mixture, purchased from Sigma-Aldrich, CAS No.: 8077-35-8, cat #: 92718.DNA Marker, available from Takara, inc., boehringer Bio Inc. Various antibiotics and agar powder were purchased from Nachuan Biotech. Yeast Extract (Yeast Extract) and peptone (Tryptone) were purchased from Oxid. Sodium chloride (analytically pure) and potassium hydroxide (analytically pure) were purchased from Yongda reagent. Deionized water was purchased from drochen.

Instruments and equipment:

unless otherwise specified, the reagents used in the following examples are conventional in the art, and are either commercially available or formulated according to conventional methods in the art; the experimental methods and conditions used are conventional in the art and can be found in the relevant experimental manuals, such as the Molecular cloning laboratory Manual (Sambrook J & Russell DW, molecular cloning: a laboratory Manual, 2001), the well-known literature or product instructions. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

Example 1 discovery and cloning of Avena sativa Oxysqualene cyclase Gene AsHS2

1. Discovery of oat AsHS2 gene

Based on oat transcriptome data (http:// db.ncgr.ac.cn/oat/RNAseq.php), 1 complete OSC gene sequence is found by using the OSC gene sequence of rice for comparison, and is named as AsHS2. The nucleotide sequence of the AsHS2 gene is shown as SEQ ID NO. 2, and the coded amino acid sequence is shown as SEQ ID NO. 1.

Amino acid sequence (755 aa) encoded by oat AsHS2 gene:

MWRLKVSEGGGPWLRSSNGFLGRQVWEFDADAGTPDERAQIERLRHNFTEHRFHRRESHDLLLRFQYAKLNNLPANPPLTKLEKSTEVTEEIITRSLRRALNQYSTLQAHDGHWPGDYSGILFLMPMFIFSLYVTRSLNIVLSSEHRREICRHIYNHQNEDGGWGIHVAGPSTMLGSCLNYVALRLLGEMLDDKNDALIKGQAWILSHGSATAVPQWGKIFLSIIGVYDWSGNNPIIPELWLVPYFLPIHPGRYWCFCRLVYMSMAYLYGKKFVGPITATILELREELYGTSYENIDWSKTRNTCAQEDLRRPRSKVLSVILDCVNKFVEPMLNCWPAEKLRERALNNVMEQIQYNNETTEYIGLCPVDKALSMICCWVQNPNSDSFRQHLPRVYDYFWLAEDGMKAKIADGCTGWDTSFIIQAFCSTDMISEFSSTIKKAHEFIKKSQVRSNFPSYEIFYRHRSKGSWPLSTVDIGWSSSDCTAEAVKTLMLLSNNSPKLVGDSIEEEKLYDAIDCLISFMNKDGSVSTYEPKRGYSWLEILNPTESFKNIVVDHPTVEVTASVLDALMSFRELYPQYHEKEIRQHTESSAMYIESEQRDDGSWYGSWAICFTYGTLFAVKGLVAAGRTYENSSYIRKACNFLLSKQQITGGWGESYLSVETEDYVDTGSPHAVNTAWAMLALIYAGQAEIDPVPLYRGARVLINMQLDTGEFPQQEYTGAANSAFFFNYSNYRNIYPIMALGELRRKLAASRK(SEQ ID NO:1)。

nucleotide sequence of oat AsHS2 gene (2268 bp):

ATGTGGCGGCTGAAGGTGAGCGAGGGTGGCGGGCCGTGGCTACGATCGAGCAACGGCTTCCTCGGCAGGCAGGTGTGGGAGTTCGACGCCGACGCCGGCACACCCGACGAGCGCGCCCAGATCGAGAGGCTGCGTCACAACTTCACCGAGCACCGCTTCCACAGGAGGGAGTCCCACGACCTTCTCCTACGCTTCCAGTATGCAAAGTTAAACAACCTTCCAGCCAATCCTCCATTAACGAAGCTTGAAAAGAGTACCGAAGTCACTGAAGAAATCATAACAAGATCATTGAGACGAGCTCTAAATCAATACTCCACTCTACAAGCACATGATGGCCATTGGCCCGGTGATTACAGCGGAATATTGTTCCTTATGCCAATGTTTATATTCTCATTATATGTTACTAGATCACTTAACATTGTTTTATCGTCTGAACATCGACGTGAAATATGTCGCCACATTTACAACCACCAGAATGAGGATGGTGGTTGGGGAATACATGTTGCAGGGCCTAGCACGATGCTTGGCTCATGCTTGAATTATGTTGCATTAAGGCTCCTTGGTGAGATGCTAGATGACAAAAATGACGCATTGATAAAAGGGCAAGCTTGGATTTTATCTCATGGAAGCGCAACTGCGGTACCCCAATGGGGAAAGATATTTCTCTCGATAATTGGTGTATATGATTGGTCAGGAAACAATCCAATTATTCCTGAACTGTGGTTAGTTCCTTATTTTCTTCCAATACACCCAGGACGATATTGGTGCTTTTGCCGACTAGTGTATATGTCAATGGCATATCTTTATGGGAAGAAATTCGTTGGGCCCATTACTGCAACTATATTGGAACTACGCGAAGAGCTATATGGGACATCGTACGAAAATATTGATTGGAGTAAGACACGCAATACTTGTGCCCAGGAAGACCTTCGTCGTCCCCGCTCAAAGGTGCTGAGTGTTATTTTGGATTGTGTTAACAAGTTTGTGGAGCCAATGTTAAATTGTTGGCCAGCAGAGAAGCTCAGAGAGAGAGCTTTGAATAATGTCATGGAGCAAATTCAGTACAATAATGAAACAACTGAATACATTGGTCTTTGTCCTGTGGACAAGGCATTGAGCATGATTTGTTGTTGGGTACAAAATCCAAATTCAGATTCTTTCCGCCAACATCTTCCTCGAGTTTATGATTACTTTTGGCTCGCTGAAGATGGCATGAAGGCAAAGATAGCTGATGGCTGCACTGGCTGGGATACATCATTTATAATTCAAGCATTTTGCTCAACGGACATGATTAGTGAGTTCAGTTCAACTATAAAAAAGGCTCATGAGTTTATAAAAAAATCACAGGTTCGTTCAAATTTCCCAAGTTATGAAATATTTTATCGCCATAGATCAAAAGGTTCATGGCCTCTTTCAACTGTGGACATTGGTTGGTCTTCGTCTGACTGCACAGCAGAAGCAGTTAAGACATTGATGTTGTTATCAAATAATTCCCCCAAACTTGTTGGTGATTCGATAGAGGAAGAGAAGTTGTATGATGCAATTGATTGCCTCATTTCCTTCATGAATAAAGATGGCTCTGTTTCTACATACGAACCCAAAAGAGGTTACTCATGGTTAGAGATTCTCAATCCGACAGAGAGTTTTAAGAACATTGTCGTCGATCATCCAACGGTTGAAGTTACAGCATCTGTACTTGACGCCCTTATGTCATTCAGAGAGCTATATCCACAGTATCACGAAAAAGAGATAAGACAACATACAGAAAGTTCTGCTATGTATATTGAGAGTGAACAACGCGATGATGGTTCTTGGTATGGATCTTGGGCAATTTGTTTTACTTACGGGACTTTATTTGCGGTAAAAGGATTAGTTGCCGCTGGAAGAACATACGAGAATAGTTCTTATATTAGGAAAGCATGCAACTTCCTCTTGTCAAAGCAACAAATAACGGGTGGATGGGGCGAAAGCTACCTTTCCGTGGAAACCGAGGATTATGTTGACACTGGTAGTCCTCATGCGGTCAACACTGCATGGGCAATGTTAGCTCTAATTTATGCTGGGCAGGCTGAAATTGATCCGGTACCACTGTATCGTGGAGCAAGAGTATTGATCAACATGCAGCTAGACACAGGAGAGTTTCCTCAGCAGGAATACACTGGAGCTGCTAACTCGGCTTTTTTCTTTAACTACTCCAACTATCGCAACATCTACCCCATTATGGCTCTTGGAGAGCTTCGGCGCAAACTTGCTGCGAGCAGAAAGTAA(SEQ ID NO:2)。

2. cloning primer design

According to the Primer design principle, primer Premier 5.0 and DNMAN software are used for designing the cloning Primer of the AsHS2 gene, and Suzhou Jinzhi Biotech limited is entrusted to complete the Primer synthesis. The nucleotide sequences of the primers are as follows:

AsHS2-F:5'-ATGTGGCGGCTGAAGGTGAGC-3'(SEQ ID NO:3)；

AsHS2-R:5'-TTACTTTCTGCTCGCAGCAAGTTTGCG-3'(SEQ ID NO:4)。

3. oat inflorescence total RNA extraction

Extracting oat inflorescence total RNA by using a TRIzon total RNA extraction kit (CW 0580S) of Kangji century according to a method recorded in a kit specification, and comprising the following steps of:

(1) Quickly grinding the avena sativa inflorescence into powder in liquid nitrogen, filling 50mg of powder sample in a centrifugal tube, adding 1ml of TRIzon, uniformly mixing, and standing at room temperature for 5 minutes to completely separate the protein-nucleic acid compound.

(2) Then 200. Mu.L of chloroform was added to the tube, and the tube was shaken for 15 seconds to mix well and left at room temperature for two minutes.

(3) After centrifugation at 12,000rpm for 15min at 4 ℃ the sample was divided into 3 layers, a lower red organic phase, an intermediate layer and an upper colorless aqueous phase in which RNA is mainly present, and the aqueous phase (about 600. Mu.L) was transferred to a new RNase-Free centrifuge tube to avoid touching or aspirating the intermediate and lower layers.

(4) The resulting aqueous phase was added with an equal volume of isopropanol, sufficiently inverted and mixed, and left at room temperature for 10 minutes.

(5) Centrifuge at 12,000rpm for 15min at 4 ℃ and discard the supernatant.

(6) The precipitate was washed by adding 1mL of 75% ethanol.

(7) Centrifuge at 12,000rpm for 3min at 4 ℃ and carefully discard the supernatant, taking care not to discard the RNA pellet.

(8) Standing at room temperature for 2-3min, air drying, adding 50 μ L RNase-free water, and dissolving RNA completely.

(9) And (3) RNA quality detection: the quality and concentration of RNA extraction were determined by 1% agarose gel and Nanodrop spectrophotometer, respectively.

Through the detection of a Nanodrop spectrophotometer, the oat inflorescence total RNA solution with the concentration of 120 ng/mu L is obtained.

4. Oat inflorescence total RNA reverse transcription

SuperScript from Invitrogen was used ^TM III reverse transcription kit (18080093), reverse transcription is carried out on the extracted oat inflorescence total RNA according to the method described in the kit instruction, and the steps are as follows:

(1) The following reagents were added to the PCR tube in order, gently mixed and centrifuged briefly.

The reaction was carried out at 65 ℃ for 5min, followed by immediate ice-bath for 3min.

(2) The following solutions were sequentially added to the PCR tube on ice to prepare a reverse transcription system:

the reaction is carried out for 1h at 50 ℃, and the time can be properly prolonged.

(3) Inactivating at 70 deg.C for 15min, terminating reaction to obtain cDNA solution, collecting 1 μ L, detecting reverse transcription effect with 1% agarose gel, and storing the rest solution at-20 deg.C.

5. Amplification of AsHS2 Gene

And (3) carrying out PCR by using the cDNA obtained by the reverse transcription as a template and adopting a cloning primer (AsHS 2-F/AsHS 2-R) according to the following reaction system and reaction program to amplify the oat AsHS2 gene segment.

And (3) PCR reaction system:

PCR reaction procedure:

after the PCR reaction, 1. Mu.L of the reaction product was collected and subjected to electrophoresis on 1% agarose gel to determine whether the size of the target band was correct.

6. Recovery of AsHS2 Gene fragments

Gel recovery of the correct band of interest was performed using the kang century agarose gel DNA recovery kit (CW 2302) as follows:

(a) Electrophoresis: the DNA bands were distinguished by 2% agarose gel electrophoresis.

(b) Cutting glue: after the strips had separated sufficiently, the strips were cut rapidly under a uv lamp and weighed.

(c) Sol: the sol solution was added according to W/V =1, 100, and water bath at 50 ℃ for 15min, with shaking, until the gel mass was completely dissolved. If the gel block can not be completely dissolved, the sol solution can be properly added or the sol time can be prolonged.

(d) DNA column adsorption: and after the melted gel blocks are cooled to room temperature, transferring the solution into a centrifugal adsorption column, standing for 2min, centrifuging at room temperature of 12,000rpm for 1min, discarding the waste liquid, and reinserting the adsorption column into a recovery tube.

(e) Rinsing: adding 600 μ L of rinsing solution into centrifugal adsorption column, centrifuging at room temperature of 12,000rpm for 1min, and discarding waste liquid. This was repeated once and the rinsing solution was thoroughly removed by centrifugation at 12,000rpm for 2min at room temperature.

(f) And (3) elution: the adsorption column was placed in a clean 1.5mL centrifuge tube, 40. Mu.L of elution buffer was added to the center of the adsorption column, and the resulting mixture was allowed to stand at room temperature for 5min, and centrifuged at 12,000rpm for 2min to obtain the AsHS2 gene fragment (FIG. 1).

Example 2 construction of oat AsHS2 tobacco in vitro transient expression vector

1. Cloning of AsHS2 Gene with recombinant linker

(1) Design of primers with recombinant linkers

Designing a primer with a recombination joint according to the gene sequence of the AsHS2, wherein the primer sequence is as follows:

G-AsHS2-F:

5'-GGGGACAAGTTTGTACAAAAAAGCAGGCTTAATGTGGCGGCTGAAGGTGAGC-3'(SEQ ID NO:5)；

G-AsHS2-R:

5'-GGGGACCACTTTGTACAAGAAAGCTGGGTATTACTTTCTGCTCGCAGCAAGTTTGCG-3'(SEQ ID NO:6)。

(2) Amplification of AsHS2 Gene with recombinant linker

The AsHS2 gene fragment obtained by recovering the glue is taken as a template, a primer (G-AsHS 2-F/G-AsHS 2-R) with a recombinant joint is adopted, PCR is carried out according to the following reaction system and reaction program, and the AsHS2 gene fragment with the recombinant joint is amplified.

And (3) PCR reaction system:

PCR reaction procedure:

after the PCR reaction, 1. Mu.L of the reaction product was collected and subjected to electrophoresis on 1% agarose gel to determine whether the size of the target band was correct. And recovering the target band to obtain the AsHS2 gene segment added with the recombination linker, namely the G-AsHS2 gene segment.

2. BP reaction and conversion

(1) BP reaction

Incubate at 25 ℃ for 10h.

Proteinase K 1μL

Incubation was carried out at 37 ℃ for 10min to obtain recombinant product pDONR207_ AsHS2, which was placed on ice.

(2) Transformation of

Taking out Escherichia coli competent cells Trelief preserved at-80 deg.C ^TM 5 alpha (Beijing Okame organisms) was melted on ice. mu.L of the recombinant product pDONR207_ AsHS2 was added, mixed gently, and left on ice for 30min. The mixture was heat-shocked at 42 ℃ for 45s and kept on ice for 2min. 500. Mu.L of LB liquid medium without any antibiotic was added and activated at 37 ℃ and 180rpm for 1h. Activated E.coli was centrifuged at 3,000rpm for 1min, 450. Mu.L of supernatant was removed, the remaining supernatant was used to resuspend the cells and the total amount was plated on Gen containing 50. Mu.g/ml ⁺ (gentamicin sulfate) on LB solid plate, 37 degrees C overnight culture.

(3) Positive colony identification

Single colonies were picked from the above LB solid plates in 500. Mu.L of 50. Mu.g/ml Gen ⁺ The liquid LB medium of (1) was activated at 37 ℃ and 200rpm for 3 hours. Then, PCR was carried out on the bacterial solution according to the following system and method to identify positive clones. The PCR primers were as follows:

pDONR-F：5’-TCGCGTTAACGCTAGCATGGATCTC-3’(SEQ ID NO:7)；

pDONR-R：5’-GTAACATCAGAGATTTTGAGACAC-3’(SEQ ID NO:8)。

and (3) PCR reaction system:

PCR reaction procedure:

after the PCR reaction, 5. Mu.L of the reaction product solution was taken, and 1% agarose gel was used to determine whether the band size of the target gene was correct, and a positive bacterial strain solution was selected and submitted to the Biotech Co., ltd, beijing Optimalaceae for sequencing.

(4) Extraction of plasmids

Selecting bacterial liquid with correct sequencing, taking 200 mu L of small shaking bacterial liquid in 10mL containing 50 mu g/mL Gen ⁺ The culture was carried out overnight at 200rpm in LB liquid medium (9). Mixing 600 μ L of the bacterial liquid with 600 μ L of 60% glycerol, and preserving bacteria. Extracting plasmids from the residual bacterial liquid by using a plasmid small-amount extraction kit (Beijing Soilebao, D1100) according to a method recorded in an instruction book of the kit, and specifically comprising the following steps:

collecting bacteria: 4mL of the bacterial solution was centrifuged at 12,000rpm for 1min to collect the bacteria, and the supernatant was discarded.

Resuspending: the residual bacteria solution in the centrifuge tube was aspirated away, 250. Mu.L of RNase-containing cell suspension was added, and the cells were thoroughly suspended by vortexing.

Cracking: 250 μ L of cell lysate was added and mixed by gently inverting the mixture upside down, at which time the solution was translucent.

Neutralizing: add 350. Mu.L of neutralization buffer, reverse gently upside down to mix well, centrifuge at room temperature at 12,000rpm for 15min.

DNA binding: carefully transfer the supernatant to a centrifugal adsorption column with a centrifuge tube, centrifuge at room temperature at 12,000rpm for 1min, discard the filtrate, and replace the centrifugal adsorption column into the centrifuge tube again.

Rinsing: adding 500 μ L of DNA rinsing solution into the adsorption column, centrifuging at 12,000rpm for 1min at room temperature, removing the filtrate, and putting the centrifugal adsorption column into the centrifuge tube again. This was repeated once and centrifuged at 12,000rpm for 2min to completely remove the residual liquid from the column.

And (3) elution: the centrifugation and adsorption column was transferred to a 1.5mL centrifuge tube, 80. Mu.L of elution buffer was added to the center of the column, the column was left at room temperature for 2min, and the column was centrifuged at 12,000rpm for 2min to collect plasmids.

And (3) gel detection: the mass of the extracted plasmid was checked by 1% agarose gel electrophoresis and the mass and concentration of the plasmid was determined by Nanodrop spectrophotometer.

3. LR reaction and transformation

(1) LR reaction system:

incubate at 25 ℃ for 10h.

Proteinase K 1μL

Incubation was carried out at 37 ℃ for 10min to obtain the recombinant product pEAQ-HT-DEST1-AsHS2, which was placed on ice.

(2) Transformation of

Taking the Escherichia coli Trelief stored at-80 DEG C ^TM 5 α competent cells were thawed on ice. Adding 5 μ L of the recombinant product pEAQ-HT-DEST1-AsHS2, mixing gently, standing on ice for 30min. The mixture was heat-shocked at 42 ℃ for 45s and kept on ice for 2min. 500. Mu.L of LB liquid medium without any antibiotic was added and activated at 37 ℃ and 180rpm for 1h. The activated E.coli was centrifuged at 3,000rpm for 1min, 450. Mu.L of supernatant was removed, the remaining supernatant was used to resuspend the cells and the whole was plated in a plate containing 100. Mu.g/ml Kan ⁺ On solid LB plates, the cells were cultured overnight at 37 ℃.

(3) Positive colony identification

Single colonies were picked from the solid LB plates described above in 500. Mu.L Kan ⁺ In (100. Mu.g/ml) liquid LB medium, activated at 37 ℃ for 3h at 200 rpm. Then, PCR of the bacterial liquid is carried out according to the following system and method to identify positive clones. The PCR primers were as follows:

pEAQ-F：5’-CTTGCTGAAGGGACGACCTGCTAAA-3’(SEQ ID NO:9)；

pEAQ-R：5’-TAGTGCGGCGCCATTAAATAACGTG-3’(SEQ ID NO:10)。

and (3) PCR reaction system:

PCR reaction procedure:

after the PCR reaction, 5. Mu.L of the reaction product solution was taken, and 1% agarose gel was used to detect whether the size of the target gene band was correct, and positive colony bacteria solution was selected and used to perform sequencing by Beijing Okagaku Biotechnology Co.

(4) Plasmid extraction

Selecting bacterial liquid with correct sequencing, taking 200 mu L of small shaking bacterial liquid in 10mL of medium containing 100 mu g/mL Kan ⁺ The culture was carried out overnight at 200rpm in LB liquid medium (9). Mixing 600 μ L of the bacterial liquid with 600 μ L of 60% glycerol, and preserving bacteria. The remaining bacterial solution was extracted with plasmid using a plasmid miniprep kit (Beijing Soilebao, D1100) according to the method described in the kit instructions.

Example 3 tobacco in vitro transient expression of oat AsHS2

1. Tobacco in vitro transient expression vector transformation

The pEAQ-HT-DEST1-AsHS2 expression vector is used for transforming Agrobacterium tumefaciens GV3101 competent cells.

The agrobacterium transformation method is as follows:

(1) Agrobacterium GV3101 competent cells preserved at-80 ℃ were placed at room temperature or on the palm of the hand for a while, after they were partially thawed, they were inserted into ice while the ice water was mixed.

(2) Add 0.5. Mu.g plasmid DNA to every 50. Mu.L competent cell, dial the tube bottom with the finger to mix with plasmid, stand on ice for 5 minutes, liquid nitrogen for 5 minutes, water bath at 37 ℃ for 5 minutes, ice bath for 5 minutes in sequence.

(3) Then, 700. Mu.L of LB liquid medium without antibiotics was added thereto, and cultured at 28 ℃ for 3 hours using a shaker at 200 rpm.

(4) Centrifuging the cultured bacterial solution at 5000rpm for 1min to collect thallus, collecting supernatant about 100 μ L, gently blowing and beating heavy suspended bacterial block, and coating on resistance screen (50 μ g/m)l Gent ⁺ ,50μg/ml Kan ⁺ ,20μg/ml Rif ⁺ ) The plates were inverted and cultured in an incubator at 28 ℃ for 2 days.

2. Positive identification of colonies

Single colonies were picked from the LB solid medium described above containing 50. Mu.g/ml Gent ⁺ 、50μg/ml Kan ⁺ 、20μg/ml Rif ⁺ The culture was carried out in LB liquid medium containing antibiotics at 28 ℃ and 200rpm for 1 day. Bacterial liquid PCR was performed according to the following system and procedure to identify whether the colonies were positive.

And (3) PCR reaction system:

PCR reaction procedure:

after the PCR reaction, 5. Mu.L of the reaction product solution was taken, and 1% agarose gel was used to determine whether the band size of the target gene was correct, and the bacterial solution of the positive colony was selected and submitted to the Biotech Co., ltd, beijing Optimalaceae for sequencing.

3. Bacterial liquid amplification culture and tobacco transient transfection

(1) The bacterial suspension of positive colonies with correct sequencing was placed in 10mL of 50. Mu.g/mL Gent ⁺ 、50μg/ml Kan ⁺ 、20μg/ml Rif ⁺ Culturing in LB liquid culture medium of antibiotics at 28 deg.C and 200rpm with shaking to OD ₆₀₀ And about = 0.6.

(2) An AgroMix buffer solution was prepared according to the following system, mixed well and stored at 4 ℃.

(3) And centrifuging the cultured bacterial liquid at 5000rpm for 5min, and collecting thalli. The supernatant was removed, the cells were resuspended in AgroMix buffer and OD adjusted ₆₀₀ =0.4, yield pEAQBacterial suspension for transfection of-HT-DEST 1-AsHS2-GV 3101. Meanwhile, a bacterial solution for pEAQ-HT-DEST1-tHMGR-GV3101 transfection was prepared. pEAQ-HT-DEST1-tHMGR-GV3101, which represents Agrobacterium GV3101 containing pEAQ-HT-DEST1-tHMGR expression vector, with tHMGR being a partial HMGR catalytic subunit (3-hydroxymethylglutaryl-CoA reductase, geneBank ID: KY 284573), is disclosed in the non-patent document James Reed et al, A translational synthetic biology plan for Rapid access to gram-scale quantities of novel drug-like molecules, metabolic Engineering 42 (2017) 185-193, the entire contents of which are incorporated herein by reference.

(4) pEAQ-HT-DEST1-tHMGR-GV3101 transfection is adopted to inject tobacco leaves with bacterial liquid, and control tobacco is obtained by culturing. And uniformly mixing the bacterial liquid for pEAQ-HT-DEST1-tHMGR-GV3101 transfection and the bacterial liquid for pEAQ-HT-DEST1-AsHS2-GV3101 transfection in equal volume, injecting tobacco leaves, and culturing to obtain the AsHS2 transgenic tobacco. The injection method comprises the following steps: sucking 1mL of bacterial liquid by using a disposable syringe, pricking 2-3 small holes on a tobacco leaf by using a needle head, pressing the syringe on the pinhole part, propping the lower part of the leaf by using a finger, pumping the bacterial liquid in the syringe and permeating the bacterial liquid into leaf tissues by applying force slightly, and marking the injection part by using a marking pen. The injected tobacco plants were cultured at 25 ℃ for seven days under 12h light and 12h dark photoperiod, and then leaf discs of control tobacco and AsHS2 transgenic tobacco were cut for compound determination.

Example 4 AsHS2 tobacco expression product analysis

1. Extraction of tobacco expression products

The compounds in the control tobacco and AsHS2 transgenic tobacco leaves obtained in example 3 were extracted separately according to the following procedure:

(1) Preparing saponification reagent ethanol: water: KOH (solid) =9ml:1ml:1g, coprotanol (Sigma, C7578) was added as an internal standard to a final concentration of 10ng/mL.

(2) Tobacco leaves were freeze-dried and ground to a powder, and 10mg of the powder was sampled in a new eppendorf tube and 1000. Mu.L of saponification reagent was added.

(3) Heating at 75 deg.C for 1h while intermittently vortexing, then opening the cover and heating for 1-2h to evaporate ethanol.

(4) To the dried sample was added 500 μ L of chromatographic grade ethyl acetate (Sigma, 270989) and the sample was resuspended by vortexing.

(5) An additional 500. Mu.L of water was added and vortexed again.

(6) The solution was separated by centrifugation at 8,000rpm for 1min (yellow upper layer and green lower layer), and the upper layer was transferred to a glass automated sample vial and allowed to remain at-20 ℃ for a long period of time.

2. Analysis of tobacco expression products

(1) Transfer 50 μ L ethyl acetate layer into sample bottle with liner tube, blow dry with nitrogen, add 50 μ L derivatization reagent 1- (Trimethylsilyl) imidazole-Pyridine mixture (Sigma, 92718), vortex to ensure complete redissolution of dried extract, react at 70 deg.C for 30min and machine analyze.

(2) Detecting and analyzing the derivatized sample by using a GC-QQQ-MS platform; the GC-QQQ-MS platform is a Thermo Scientific TM TRACE 1310 gas chromatograph and a Thermo Scientific TM ISQTM 7000 single quadrupole GC-MS system; the column was ZB-5HT (0.25 mm. Times.30m, thermo Fisher scientific); the carrier gas is He, and the flow rate is 1.0mL/min; the injector temperature was 280 ℃. The temperature-raising program is: the initial temperature is 170 ℃, the temperature is increased to 290 ℃ at 6 ℃/min, the temperature is kept for 4min, and the temperature is increased to 340 ℃ at 10 ℃/min. The sample volume is 1 μ L, and the split ratio is 20. The MS detector has a scanning interval of 60-800 under the condition of 70 eV.

(3) The results of GC-MS were analyzed using Dionex Chromeleon 7 chromatographic data System software.

From the GC-MS measurements, it can be seen that the AsHS2 transgenic tobacco produced a different compound, the product of the AsHS2 enzyme, designated Compound 1, compared to the control tobacco. The peak time of compound 1 was 19.37min (fig. 2) and the characteristic ions were 365.37, 455.41, 498.48 (fig. 3).

3. Separation and identification of AsHS2 tobacco expression product

(1) Isolation of Compounds

The bacterial liquid used for pEAQ-HT-DEST1-tHMGR-GV3101 transfection and the bacterial liquid used for pEAQ-HT-DEST1-AsHS2-GV3101 transfection are mixed with equal volume, and then are infected with about 200 tobacco in batch, tobacco leaves infected for 7 days are collected and freeze-dried, the dried leaves are weighed, saponification reagent (100 mL/g) is added according to proportion, and then the mixture is heated for 1 hour at 75 ℃. Adding n-hexane with the same volume for extraction for three times, collecting the upper n-hexane phase and spin-drying. The extract is separated by using a medium-low pressure separation system (Biotage), the specification of the silica gel column is 45g, the mobile phase is ethyl acetate/n-hexane (gradient elution is 0-50 percent), and the flow rate of the mobile phase is 60mL/min. The fractions were checked by TLC plate and the fraction of the desired compound was confirmed. Similar fractions were combined according to assay results, spun dry, dissolved in dichloromethane, and passed through a silica gel column again. The specification of the silica gel column is 5g, the mobile phase is ethyl acetate/n-hexane (gradient elution is 0-2 percent), and the flow rate of the mobile phase is 25mL/min. The fractions were checked by TLC plate and the fractions of the desired compound were confirmed. And (5) combining similar components according to the detection result, and performing spin drying. Further purifying the compound by high performance liquid chromatography using a semi-preparative column of Thermo Hypersil GOLD C18 column,250mm × 10mm,5 μm, mobile phase of water (A) and methanol (B), gradient program of 0-8min, 60-100% B; 8-22min, 100-100% of B; 22-25min, 100% -60%, the flow rate is 5mL/min, the column temperature is 30 ℃, the sample injection amount is 80 muL, and the detection wavelength is 210nm. Co-isolation gave 3mg of Compound 1.

(2) Compound identification

Subjecting a sample of the isolated compound to CDCl ₃ Dissolving, adding into a nuclear magnetic tube, and analyzing the purified compound by Bruker 500MHz nuclear magnetic resonance spectrometer in the laboratory of the university of capital medical science to obtain corresponding carbon spectrum and hydrogen spectrum. The hydrocarbon assignment of compound 1 is shown in table 1, and the carbon spectrum and the hydrogen spectrum are shown in fig. 4 and 5, respectively. The structure of the compound 1 was analyzed based on the carbon and hydrogen spectra, and the result showed that the compound 1 was hop- (17) 21-en-3. Beta. -ol, and the structure is shown in FIG. 6.

TABLE 1 Hydrocarbon assignment of Compound 1

NO.	δ _H	δ _C	NO.	δ _H	δ _C	NO.	δ _H	δ _C
									1	0.95m,1.70m	38.9	11	1.26m,1.53m	21.4	21	/	136.1
2	1.60(2H,m)	27.4	12	1.39(2H,m)	24.0	22	2.64,m	26.4
									3	3.21ddd(11.2,6.1,4.9)	79.0	13	1.41m	49.3	23	0.98,s	28.0
4	/	38.8	14	/	41.7	24	0.76,s	15.4
									5	0.66(d,11,4)	55.3	15	1.26(2H,m)	31.8	25	0.83,s	16.2
6	1.37m,1.51m	18.4	16	1.89m,2.26(3.7,14.2)	19.8	26	0.93,s	16.3
									7	1.29m,1.43m	33.5	17	/	139.9	27	1.03,s	15.0
8	/	42.0	18	/	49.8	28	0.84,s	19.1
									9	1.27m	50.9	19	1.30m,1.65m	41.6	29	0.92,d(6.5)	21.3
10	/	37.2	20	1.63m,2.14m	27.5	30	0.97,d(6.6)	21.9

Reference documents:

Thimmappa R,Geisler K,Louveau T,O'Maille P,Osbourn A.2014.Triterpene biosynthesis in plants.Annu Rev Plant Biol 65:225-257.

Busta L,Schmitz E,Kosma DK,Schnable JC,Cahoon EB.2021.A co-opted steroid synthesis gene,maintained in sorghum but not maize,is associated with a divergence in leaf wax chemistry.Proc Natl Acad Sci U S A 118(12):e2022982118.。

sequence listing

<110> northeast university of forestry

<120> oat oxidosqualene cyclase AsHS2, and coding gene and application thereof

<130> P210499-DBL

<160> 10

<170> SIPOSequenceListing 1.0

<210> 1

<211> 755

<212> PRT

<213> Avena strigosa

<400> 1

Met Trp Arg Leu Lys Val Ser Glu Gly Gly Gly Pro Trp Leu Arg Ser

1 5 10 15

Ser Asn Gly Phe Leu Gly Arg Gln Val Trp Glu Phe Asp Ala Asp Ala

20 25 30

Gly Thr Pro Asp Glu Arg Ala Gln Ile Glu Arg Leu Arg His Asn Phe

35 40 45

Thr Glu His Arg Phe His Arg Arg Glu Ser His Asp Leu Leu Leu Arg

50 55 60

Phe Gln Tyr Ala Lys Leu Asn Asn Leu Pro Ala Asn Pro Pro Leu Thr

65 70 75 80

Lys Leu Glu Lys Ser Thr Glu Val Thr Glu Glu Ile Ile Thr Arg Ser

85 90 95

Leu Arg Arg Ala Leu Asn Gln Tyr Ser Thr Leu Gln Ala His Asp Gly

100 105 110

His Trp Pro Gly Asp Tyr Ser Gly Ile Leu Phe Leu Met Pro Met Phe

115 120 125

Ile Phe Ser Leu Tyr Val Thr Arg Ser Leu Asn Ile Val Leu Ser Ser

130 135 140

Glu His Arg Arg Glu Ile Cys Arg His Ile Tyr Asn His Gln Asn Glu

145 150 155 160

Asp Gly Gly Trp Gly Ile His Val Ala Gly Pro Ser Thr Met Leu Gly

165 170 175

Ser Cys Leu Asn Tyr Val Ala Leu Arg Leu Leu Gly Glu Met Leu Asp

180 185 190

Asp Lys Asn Asp Ala Leu Ile Lys Gly Gln Ala Trp Ile Leu Ser His

195 200 205

Gly Ser Ala Thr Ala Val Pro Gln Trp Gly Lys Ile Phe Leu Ser Ile

210 215 220

Ile Gly Val Tyr Asp Trp Ser Gly Asn Asn Pro Ile Ile Pro Glu Leu

225 230 235 240

Trp Leu Val Pro Tyr Phe Leu Pro Ile His Pro Gly Arg Tyr Trp Cys

245 250 255

Phe Cys Arg Leu Val Tyr Met Ser Met Ala Tyr Leu Tyr Gly Lys Lys

260 265 270

Phe Val Gly Pro Ile Thr Ala Thr Ile Leu Glu Leu Arg Glu Glu Leu

275 280 285

Tyr Gly Thr Ser Tyr Glu Asn Ile Asp Trp Ser Lys Thr Arg Asn Thr

290 295 300

Cys Ala Gln Glu Asp Leu Arg Arg Pro Arg Ser Lys Val Leu Ser Val

305 310 315 320

Ile Leu Asp Cys Val Asn Lys Phe Val Glu Pro Met Leu Asn Cys Trp

325 330 335

Pro Ala Glu Lys Leu Arg Glu Arg Ala Leu Asn Asn Val Met Glu Gln

340 345 350

Ile Gln Tyr Asn Asn Glu Thr Thr Glu Tyr Ile Gly Leu Cys Pro Val

355 360 365

Asp Lys Ala Leu Ser Met Ile Cys Cys Trp Val Gln Asn Pro Asn Ser

370 375 380

Asp Ser Phe Arg Gln His Leu Pro Arg Val Tyr Asp Tyr Phe Trp Leu

385 390 395 400

Ala Glu Asp Gly Met Lys Ala Lys Ile Ala Asp Gly Cys Thr Gly Trp

405 410 415

Asp Thr Ser Phe Ile Ile Gln Ala Phe Cys Ser Thr Asp Met Ile Ser

420 425 430

Glu Phe Ser Ser Thr Ile Lys Lys Ala His Glu Phe Ile Lys Lys Ser

435 440 445

Gln Val Arg Ser Asn Phe Pro Ser Tyr Glu Ile Phe Tyr Arg His Arg

450 455 460

Ser Lys Gly Ser Trp Pro Leu Ser Thr Val Asp Ile Gly Trp Ser Ser

465 470 475 480

Ser Asp Cys Thr Ala Glu Ala Val Lys Thr Leu Met Leu Leu Ser Asn

485 490 495

Asn Ser Pro Lys Leu Val Gly Asp Ser Ile Glu Glu Glu Lys Leu Tyr

500 505 510

Asp Ala Ile Asp Cys Leu Ile Ser Phe Met Asn Lys Asp Gly Ser Val

515 520 525

Ser Thr Tyr Glu Pro Lys Arg Gly Tyr Ser Trp Leu Glu Ile Leu Asn

530 535 540

Pro Thr Glu Ser Phe Lys Asn Ile Val Val Asp His Pro Thr Val Glu

545 550 555 560

Val Thr Ala Ser Val Leu Asp Ala Leu Met Ser Phe Arg Glu Leu Tyr

565 570 575

Pro Gln Tyr His Glu Lys Glu Ile Arg Gln His Thr Glu Ser Ser Ala

580 585 590

Met Tyr Ile Glu Ser Glu Gln Arg Asp Asp Gly Ser Trp Tyr Gly Ser

595 600 605

Trp Ala Ile Cys Phe Thr Tyr Gly Thr Leu Phe Ala Val Lys Gly Leu

610 615 620

Val Ala Ala Gly Arg Thr Tyr Glu Asn Ser Ser Tyr Ile Arg Lys Ala

625 630 635 640

Cys Asn Phe Leu Leu Ser Lys Gln Gln Ile Thr Gly Gly Trp Gly Glu

645 650 655

Ser Tyr Leu Ser Val Glu Thr Glu Asp Tyr Val Asp Thr Gly Ser Pro

660 665 670

His Ala Val Asn Thr Ala Trp Ala Met Leu Ala Leu Ile Tyr Ala Gly

675 680 685

Gln Ala Glu Ile Asp Pro Val Pro Leu Tyr Arg Gly Ala Arg Val Leu

690 695 700

Ile Asn Met Gln Leu Asp Thr Gly Glu Phe Pro Gln Gln Glu Tyr Thr

705 710 715 720

Gly Ala Ala Asn Ser Ala Phe Phe Phe Asn Tyr Ser Asn Tyr Arg Asn

725 730 735

Ile Tyr Pro Ile Met Ala Leu Gly Glu Leu Arg Arg Lys Leu Ala Ala

740 745 750

Ser Arg Lys

755

<210> 2

<211> 2268

<212> DNA

<213> Avena strigosa

<400> 2

atgtggcggc tgaaggtgag cgagggtggc gggccgtggc tacgatcgag caacggcttc 60

ctcggcaggc aggtgtggga gttcgacgcc gacgccggca cacccgacga gcgcgcccag 120

atcgagaggc tgcgtcacaa cttcaccgag caccgcttcc acaggaggga gtcccacgac 180

cttctcctac gcttccagta tgcaaagtta aacaaccttc cagccaatcc tccattaacg 240

aagcttgaaa agagtaccga agtcactgaa gaaatcataa caagatcatt gagacgagct 300

ctaaatcaat actccactct acaagcacat gatggccatt ggcccggtga ttacagcgga 360

atattgttcc ttatgccaat gtttatattc tcattatatg ttactagatc acttaacatt 420

gttttatcgt ctgaacatcg acgtgaaata tgtcgccaca tttacaacca ccagaatgag 480

gatggtggtt ggggaataca tgttgcaggg cctagcacga tgcttggctc atgcttgaat 540

tatgttgcat taaggctcct tggtgagatg ctagatgaca aaaatgacgc attgataaaa 600

gggcaagctt ggattttatc tcatggaagc gcaactgcgg taccccaatg gggaaagata 660

tttctctcga taattggtgt atatgattgg tcaggaaaca atccaattat tcctgaactg 720

tggttagttc cttattttct tccaatacac ccaggacgat attggtgctt ttgccgacta 780

gtgtatatgt caatggcata tctttatggg aagaaattcg ttgggcccat tactgcaact 840

atattggaac tacgcgaaga gctatatggg acatcgtacg aaaatattga ttggagtaag 900

acacgcaata cttgtgccca ggaagacctt cgtcgtcccc gctcaaaggt gctgagtgtt 960

attttggatt gtgttaacaa gtttgtggag ccaatgttaa attgttggcc agcagagaag 1020

ctcagagaga gagctttgaa taatgtcatg gagcaaattc agtacaataa tgaaacaact 1080

gaatacattg gtctttgtcc tgtggacaag gcattgagca tgatttgttg ttgggtacaa 1140

aatccaaatt cagattcttt ccgccaacat cttcctcgag tttatgatta cttttggctc 1200

gctgaagatg gcatgaaggc aaagatagct gatggctgca ctggctggga tacatcattt 1260

ataattcaag cattttgctc aacggacatg attagtgagt tcagttcaac tataaaaaag 1320

gctcatgagt ttataaaaaa atcacaggtt cgttcaaatt tcccaagtta tgaaatattt 1380

tatcgccata gatcaaaagg ttcatggcct ctttcaactg tggacattgg ttggtcttcg 1440

tctgactgca cagcagaagc agttaagaca ttgatgttgt tatcaaataa ttcccccaaa 1500

cttgttggtg attcgataga ggaagagaag ttgtatgatg caattgattg cctcatttcc 1560

ttcatgaata aagatggctc tgtttctaca tacgaaccca aaagaggtta ctcatggtta 1620

gagattctca atccgacaga gagttttaag aacattgtcg tcgatcatcc aacggttgaa 1680

gttacagcat ctgtacttga cgcccttatg tcattcagag agctatatcc acagtatcac 1740

gaaaaagaga taagacaaca tacagaaagt tctgctatgt atattgagag tgaacaacgc 1800

gatgatggtt cttggtatgg atcttgggca atttgtttta cttacgggac tttatttgcg 1860

gtaaaaggat tagttgccgc tggaagaaca tacgagaata gttcttatat taggaaagca 1920

tgcaacttcc tcttgtcaaa gcaacaaata acgggtggat ggggcgaaag ctacctttcc 1980

gtggaaaccg aggattatgt tgacactggt agtcctcatg cggtcaacac tgcatgggca 2040

atgttagctc taatttatgc tgggcaggct gaaattgatc cggtaccact gtatcgtgga 2100

gcaagagtat tgatcaacat gcagctagac acaggagagt ttcctcagca ggaatacact 2160

ggagctgcta actcggcttt tttctttaac tactccaact atcgcaacat ctaccccatt 2220

atggctcttg gagagcttcg gcgcaaactt gctgcgagca gaaagtaa 2268

<210> 3

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

atgtggcggc tgaaggtgag c 21

<210> 4

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

ttactttctg ctcgcagcaa gtttgcg 27

<210> 5

<211> 52

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

ggggacaagt ttgtacaaaa aagcaggctt aatgtggcgg ctgaaggtga gc 52

<210> 6

<211> 57

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

ggggaccact ttgtacaaga aagctgggta ttactttctg ctcgcagcaa gtttgcg 57

<210> 7

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

tcgcgttaac gctagcatgg atctc 25

<210> 8

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

gtaacatcag agattttgag acac 24

<210> 9

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

cttgctgaag ggacgacctg ctaaa 25

<210> 10

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

tagtgcggcg ccattaaata acgtg 25

Claims

1. An oxidosqualene cyclase, whose amino acid sequence is shown in SEQ ID NO 1.

2.A gene encoding oxidosqualene cyclase as claimed in claim 1.

3. The gene of claim 2, wherein the nucleotide sequence is as shown in SEQ ID NO. 2.

4. An expression cassette comprising the gene of claim 2 or 3.

5. A vector comprising the gene of claim 2 or 3.

6. A recombinant bacterium comprising the gene according to claim 2 or 3.

7. A method of making a transgenic plant comprising the steps of: introducing the gene of claim 2 or 3 into a starting plant to obtain a transgenic plant; the content of the compound hop- (17) 21-en-3 β -ol in the transgenic plants is altered compared to the starting plants.

8. The method of claim 7, wherein: the gene is introduced into a starting plant through a recombinant expression vector; the recombinant expression vector is obtained by inserting the gene into a starting vector pEAQ-HT-DEST 1; the starting plant is control tobacco; the transgenic plant is transgenic tobacco.

9. The method of claim 8, wherein: the transgenic tobacco produced the compound hop- (17) 21-en-3 β -ol compared to the control tobacco.

10. Use of an oxidosqualene cyclase according to claim 1 or of a gene according to claim 2 or 3 for the biosynthesis of hop- (17) 21-en-3 β -ol.