Oxidized squalene cyclase MtBUTS, its encoding gene and application

Abstract

The present invention relates to the biosynthesis of triterpenoid compounds, and specifically to oxidized squalene cyclase MtBUTS, its encoding gene and application. Provided is an oxidized squalene cyclase, whose amino acid sequence is shown in SEQ ID NO:2. Also provided are genes encoding the oxidized squalene cyclase, and expression cassettes, vectors or host bacteria containing the genes. Also provided are uses of the oxidized squalene cyclase in oxidized squalene cyclization reactions, especially in the synthesis of butyrylcetol. The present invention lays a foundation for the analysis of the biosynthetic pathway of limonoid compounds and the development of limonoid green biopesticides using synthetic biology.

Description

Technical Field

[0001] The invention relates to the biosynthesis of triterpenoid compounds, and in particular to oxidosqualene cyclase MtBUTS, a coding gene thereof and application thereof. Background Art

[0002] Limonoids are a class of highly oxidized triterpenoids found primarily in Meliaceae and Rutaceae plants. These compounds are known for their unique structural and chemical diversity and exhibit a wide range of biological activities, such as anticancer, anti-inflammatory, antibacterial, antiviral, and antioxidant. In addition, they also have a wide range of anti-insect and antifeedant activities, making them an important resource for the development of green pesticides. However, with the exception of azadirone, the biosynthetic pathways of the remaining limonoids are largely unknown. According to the deduction of the biogenic pathway, limonoids are formed by the modification of the triterpene skeleton of the gansuline type (20S) or euphorbiline type (20R). Previous researchers have identified oxidosqualene cyclase (OSC) genes that catalyze the formation of gansuline-type triterpenoid skeletons in species of Meliaceae and Rutaceae, such as AiOSC1, MaOSC1, CsOSC1, and MtOSC1. The oxidosqualene cyclases encoded by them can catalyze 2,3-oxidosqualene to form tirucalla-7,24-dien-3β-ol (20S), which can be further modified to form the insecticide compound azadirachtin. The study of the biochemical functions of the OSC gene family in Meliaceae plants will lay the foundation for the analysis of the biosynthetic pathway of limonoid compounds and the ultimate use of microbial chassis to efficiently synthesize green insecticides. Summary of the invention

[0003] The invention aims to provide a squalene oxidase MtBUTS from Toosendan, a coding gene thereof and application thereof.

[0004] The present invention provides an oxidosqualene cyclase MtBUTS, whose amino acid sequence is shown in SEQ ID NO:2.

[0005] The present invention also provides a gene encoding the oxidosqualene cyclase MtBUTS.

[0006] In some embodiments, the nucleotide sequence of the gene is as shown in SEQ ID NO:1.

[0007] The invention also provides an expression box, a vector or a host bacterium containing the gene.

[0008] The expression cassette consists of a promoter capable of initiating the expression of the gene, the gene, and a transcription terminator.

[0009] The vector can be any suitable cloning vector, comprising the above-mentioned gene and other elements required for plasmid replication. In some embodiments, the cloning vector is a pTOPO-Blunt vector.

[0010] The vector may also be any suitable expression vector, comprising the above gene and other elements that enable successful protein expression. In some embodiments, the expression vector is pDEST1.

[0011] The host bacteria may be a host bacteria containing the above cloning vector. In some embodiments, the host bacteria is E. coli DH5α, and the above gene is replicated by culturing the host bacteria under appropriate conditions.

[0012] The host bacteria can also be a host bacteria comprising the above-mentioned expression vector. In certain embodiments, the host bacteria is Agrobacterium tumefaciens EHA105, and the expression vector is introduced into the host plant by the genetic transformation technology mediated by Agrobacterium tumefaciens so that the above-mentioned gene is expressed. In certain embodiments, the host plant is tobacco. In certain embodiments, the tobacco is Nicotiana benthamiana.

[0013] The invention also provides a method for cloning the gene, comprising: extracting RNA from Toosendan plant tissue and reversely transcribing it into cDNA; carrying out polymerase chain reaction with cDNA as a template to clone the gene.

[0014] The plant tissue of Toosendan can be the tissue of the root, stem, leaf, flower, fruit or seed of Toosendan. In some embodiments, the plant tissue of Toosendan is the phloem tissue of the root or stem of Toosendan.

[0015] In some embodiments, the nucleotide sequences of the primers used for the polymerase chain reaction are shown in SEQ ID NO:3 and SEQ ID NO:4.

[0016] The use of the oxidized squalene cyclase MtBUTS in the oxidized squalene cyclization reaction also belongs to the scope of the present invention.

[0017] The use of the oxidosqualene cyclase MtBUTS in the synthesis of butyrylcetyl alcohol also belongs to the scope of the present invention.

[0018] The present invention also provides a method for obtaining butyryl cetyl alcohol, comprising: simultaneously expressing the oxidized squalene cyclase MtBUTS and 3-hydroxy-3-methylglutaryl coenzyme A reductase (3-Hydroxy-3-methylglutarylcoenzyme Areductase, HMGR) in tobacco; collecting tobacco leaves that successfully express the two enzymes and extracting butyryl cetyl alcohol.

[0019] In the above method, the gene expression vector of MtBUTS and the gene expression vector of HMGR can be introduced into tobacco at the same time, so that MtBUTS and HMGR are co-expressed for 6-8 days, and then the tobacco leaves are collected, freeze-dried and ground into powder, and butyryl cetyl alcohol is extracted.

[0020] In the above method, the gene expression vector of MtBUTS and the gene expression vector of HMGR can be introduced into tobacco by genetic transformation technology mediated by Agrobacterium tumefaciens. The Agrobacterium tumefaciens can be any suitable Agrobacterium tumefaciens strain, such as EHA105. The tobacco can be any suitable tobacco species, such as Nicotiana benthamiana.

[0021] In the above method, butyryl cetyl alcohol can be extracted from tobacco leaves by any suitable compound extraction method, such as solvent extraction.

[0022] In some embodiments, tobacco leaves that successfully express MtBUTS and HMGR are freeze-dried and ground into powder, and the tobacco powder is thoroughly mixed with the lysate and placed in a 70°C water bath for 2 hours to obtain a sample lysate. The formula of the lysate (100 mL) is as follows: KOH, 10 g; anhydrous ethanol, 90 mL; ddH2O is made up to 100 mL. After the sample lysate is centrifuged at room temperature, the supernatant is aspirated, and extracted with an equal volume of n-hexane three times, and the three extracts are combined. The obtained extract is washed with an equal volume of saturated saline, the upper n-hexane phase is aspirated, and this step is repeated twice. The obtained n-hexane phase is blown dry with nitrogen or evaporated in a fume hood to obtain an extract. Add n-hexane to re-dissolve the extract, and filter out insoluble impurities with an organic filter membrane to obtain an extract.

[0023] In some embodiments, the nucleotide sequence of the 3-hydroxy-3-methylglutaryl-CoA reductase is as shown in SEQ ID NO:5.

[0024] In summary, we cloned a new oxidosqualene cyclase gene MtBUTS from Toosendan. The biochemical function of oxidosqualene cyclase MtBUTS was analyzed using tobacco expression system. The results showed that MtBUTS can specifically catalyze 2,3-oxidosqualene to form butyrospermol, a triterpenoid skeleton structure of euphorbia type, which is a key enzyme in the biosynthesis pathway of limonoid compounds. The present invention lays a foundation for the analysis of the biosynthesis pathway of limonoid compounds and the development of limonoid green biopesticides using synthetic biology. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1The basic pathway of the biosynthesis of limonoid compounds is shown; wherein MtBUTS is the oxidized squalene cyclase provided by the present invention, and AiOSC1, MaOSC1, CsOSC1 and MtOSC1 are the oxidized squalene cyclases that have been discovered.

[0026] Figure 2 Total ion chromatogram (TIC) of tobacco sample detected by GC-MS.

[0027] Figure 3 This is the extracted ion chromatogram (EIC) of the GC-MS detection characteristic ion m / z=393.3 of the tobacco sample.

[0028] Figure 2 and Figure 3 In the figure, DEST1 represents the empty vector control tobacco (tobacco injected with EHA105-pDEST1); AstHMGR represents the tobacco expressing only AstHMGR (tobacco injected with EHA105-pDEST1-AstHMGR); MtBUTS represents the tobacco expressing only MtBUTS (tobacco injected with EHA105-pDEST1-MtBUTS); AstHMGR+MtBUTS represents the tobacco expressing both AstHMGR and MtBUTS (tobacco injected with EHA105-pDEST1-AstHMGR and EHA105-pDEST1-MtBUTS).

[0029] Figure 4 It is the mass spectrum of the catalytic product of oxidized squalene cyclase MtBUTS; wherein RT represents retention time; TMS represents N-methyl-N-(trimethylsilyl)trifluoroacetamide; AstHMGR+MtBUTS represents tobacco simultaneously expressing truncated oat 3-hydroxy-3-methylglutaryl-CoA reductase (AstHMGR) and oxidized squalene cyclase MtBUTS.

[0030] Figure 5 The catalytic substrate (2,3-squalene oxide) and product (butyrylcetyl alcohol) of the squalene oxide cyclase MtBUTS are shown.

[0031] Figure 6 The yield of butyryl cetyl alcohol in tobacco plants expressing both AstHMGR and MtBUTS is shown; the left figure shows the yield of butyryl cetyl alcohol, where DW represents the dry weight of tobacco leaves; the right figure shows the peak area integral of the characteristic ion m / z = 393.3 and the concentration of butyryl cetyl alcohol standard (μg mL -1 ) relationship curve.

[0032] Figure 7 This is the 400 MHz hydrogen spectrum of butyrylcetyl alcohol, the catalytic product of squalene cyclase MtBUTS.

[0033] Figure 8 This is the 400MHz carbon spectrum of butyrylcetyl alcohol, the catalytic product of squalene cyclase MtBUTS.

[0034] Fig. 9 This is the 400 MHz HSQC spectrum of butyrylcetyl alcohol, the catalytic product of squalene cyclase MtBUTS.

[0035] Fig.10 This is the 400 MHz HMBC spectrum of butyrylcetyl alcohol, the catalytic product of squalene cyclase MtBUTS.

[0036] Description of Sequence Listing

[0037] The nucleotide and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for the nucleotide bases and the three letter codes for the amino acids. The nucleotide sequences follow the standard convention of starting at the 5' end and proceeding toward the 3' end. Only one strand of each nucleotide sequence is shown, it being understood that the complementary strand of the strand shown is also included. The amino acid sequences follow the standard convention of starting at the amino terminus of the sequence and proceeding toward the carboxyl terminus.

[0038] SEQ ID NO: 1 shows the coding sequence of the MtBUTS gene; the MtBUTS gene encodes the oxidosqualene cyclase provided by the present invention.

[0039] SEQ ID NO: 2 shows the amino acid sequence of oxidosqualene cyclase encoded by the MtBUTS gene.

[0040] SEQ ID NO: 3 shows the nucleotide sequence of primer MtBUTS-F.

[0041] SEQ ID NO: 4 shows the nucleotide sequence of primer MtBUTS-R.

[0042] SEQ ID NO:5 shows the coding sequence of the truncated oat HMGR gene; the HMGR gene encodes 3-hydroxy-3-methylglutaryl-CoA reductase.

[0043] SEQ ID NO:6 shows the nucleotide sequence of primer attB1-MtBUTS.

[0044] SEQ ID NO:7 shows the nucleotide sequence of primer attB2-MtBUTS.

[0045] SEQ ID NO: 8 shows the nucleotide sequence of primer pDONR-F.

[0046] SEQ ID NO:9 shows the nucleotide sequence of primer pDONR-R.

[0047] SEQ ID NO: 10 shows the nucleotide sequence of primer DEST1-F.

[0048] SEQ ID NO: 11 shows the nucleotide sequence of primer DEST1-R.

[0049] SEQ ID NO: 12 shows the nucleotide sequence of primer attB1-AstHMGR.

[0050] SEQ ID NO: 13 shows the nucleotide sequence of primer attB2-AstHMGR.

[0051] SEQ ID NO: 14 shows the nucleotide sequence of primer M13-F.

[0052] SEQ ID NO: 15 shows the nucleotide sequence of primer M13-R. DETAILED DESCRIPTION

[0053] The following implementation plans are provided:

[0054] 1. A squalene cyclase, the amino acid sequence of which is shown in SEQ ID NO: 2.

[0055] 2. A gene encoding the oxidosqualene cyclase described in Implementation Example 1.

[0056] 3. The gene described in Embodiment 2, whose nucleotide sequence is shown in SEQ ID NO:1.

[0057] 4. An expression cassette, vector or host bacteria containing the gene described in embodiment 2 or 3.

[0058] 5. A method for cloning the gene described in embodiment 2 or 3, comprising: extracting RNA from Toosendan plant tissue and reverse transcribing it into cDNA; and performing polymerase chain reaction using the cDNA as a template to clone the gene.

[0059] 6. The method according to embodiment 5, wherein the nucleotide sequences of the primers used for the polymerase chain reaction are shown in SEQ ID NO:3 and SEQ ID NO:4.

[0060] 7. Obtain the gene described in embodiment 2 or 3 by DNA chemical synthesis method.

[0061] 8. Use a prokaryotic protein expression system or a eukaryotic protein expression system to obtain the oxidized squalene cyclase described in Embodiment 1.

[0062] 9. Use of the oxidized squalene cyclase described in Embodiment 1 in the oxidized squalene cyclization reaction.

[0063] 10. Use of the oxidosqualene cyclase described in Embodiment 1 in the synthesis of butyryl cetyl alcohol.

[0064] 11. A method for obtaining butyryl cetyl alcohol, comprising: simultaneously expressing the oxidized squalene cyclase and 3-hydroxy-3-methylglutaryl-CoA reductase described in Embodiment 1 in tobacco; collecting tobacco leaves that have successfully expressed the two enzymes and extracting butyryl cetyl alcohol.

[0065] 12. The method described in embodiment 11, wherein the nucleotide sequence of the 3-hydroxy-3-methylglutaryl-CoA reductase is as shown in SEQ ID NO:5.

[0066] The present invention is further described below in conjunction with specific examples. It should be understood that the following examples are only used as explanations and illustrations of the present invention and do not limit the scope of the present invention in any way.

[0067] If not otherwise specified, the reagents used in the following examples are all conventional reagents in the art, which can be obtained commercially or prepared according to conventional methods in the art; the experimental methods and conditions used are conventional experimental methods and conditions in the art, and reference can be made to relevant experimental manuals, known documents or manufacturer specifications. If not otherwise specified, the quantitative tests in the following examples are set up for three repeated experiments, and the results are averaged. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by ordinary technicians in the field to which the present invention belongs.

[0068] The primer synthesis and sequencing in the following examples were completed by Sangon Biotech (Shanghai) Co., Ltd.

[0069] Example 1. Gene cloning

[0070] 1.1 Cloning of the MtBUTS gene

[0071] The plant material used in this experiment was the phloem tissue of Melia Toosendan, which was taken from the phloem of 2-3 year old branches of Melia Toosendan. The phloem tissue of Melia Toosendan was quickly frozen in liquid nitrogen, ground into powder in a mortar, and extracted using the RNAprep Pure Polysaccharide and Polyphenol Plant Total RNA Extraction Kit ( Total RNA was extracted according to the method described in the product manual. The RNA concentration was measured by spectrophotometer, and the RNA integrity was tested by 1% agarose gel electrophoresis. II First-Strand cDNA Synthesis SuperMix (Cat. No. AH301-02) was used to reverse transcribe the extracted total RNA from Toosendan to obtain the first-strand cDNA.

[0072] The first-strand cDNA obtained by reverse transcription was used as a template, and the primers MtBUTS-F and MtBUTS-R and Toyobo's high-fidelity enzyme KOD FX (Cat. No.: KFX-101) were used for polymerase chain reaction (PCR) to amplify the MtBUTS gene. The nucleotide sequences of the primers are as follows:

[0073] MtBUTS-F:5'-ATGTGGAGGCTTAAGCTCTTAGGGGAAGAGGAAG-3' (SEQ ID NO: 3);

[0074] MtBUTS-R: 5'-TTAAAATTGTTTTGGAAAATTCATCTTGCCAGGCAAC-3' (SEQ ID NO: 4).

[0075] The PCR reaction system was as follows: 2×KOD buffer, 15 μL; dNTPs (2 mM), 5 μL; MtBUTS-F (10 μM), 1 μL; MtBUTS-R (10 μM), 1 μL; cDNA (5×), 0.5 μL; KOD FX, 0.5 μL; ddH2O, 7 μL. The PCR reaction procedure was as follows: 94°C pre-denaturation for 3 min; 32 cycles (98°C denaturation for 30 sec; 56°C annealing for 30 sec; 68°C extension for 2 min 30 sec); 68°C extension for 10 min; 12°C incubation for 5 min. After the reaction, the PCR product was detected by 1% agarose gel electrophoresis. The band of about 2300 bp was cut off with a scalpel, and the target gene fragment was recovered using the Axygen DNA gel recovery kit (Cat. No.: AP-GX-250) according to the method described in the product manual.

[0076] use The company's EZ-Blunt zero-background pTOPOII cloning kit (Cat. No.: T186-20) was used to connect the recovered target gene fragment to the pTOPO-Blunt vector. The connection system is as follows: 10× Enhancer, 1 μL; pTOPO-Blunt Vector (ng·μL -1), 1μL; target gene fragment, 8μL. Incubate the ligation system at 25℃ for 5min to obtain the ligation product. Transform the ligation product into Escherichia coli DH5a competent cells by heat shock method. The transformation method is as follows: take out the DH5α competent cells from the -80℃ refrigerator, put them on ice, add the ligation product when they are just melted, pipette to mix, and put them in ice for 30min. After heat shock at 42℃ for 90sec, immediately place on ice for 2-3min. Add 1mL of SOC or LB medium equilibrated to room temperature, and culture at 37℃, 180rpm for 1h. Take 50-100μL of bacterial solution and evenly spread it on a plate containing 50mg·L -1 Amp (ampicillin) solid LB medium, incubate at 37°C in the dark for 12-16 hours. Pick a single clone and inoculate it in 500 μL of 50 mg·L -1 The culture medium was incubated in liquid LB medium at 37°C with a shaker at 200 rpm for 4 h.

[0077] The universal primers M13-F and M13-R were used for bacterial liquid PCR identification. The nucleotide sequences of the primers are as follows:

[0078] M13-F: 5'-GTTGTAAAACGACGGCCAG-3' (SEQ ID NO: 14);

[0079] M13-R: 5'-CAGGAAACAGCTATGAC-3' (SEQ ID NO: 15).

[0080] The PCR reaction system was: 2×Taq mix, 5μL; M13-F (10μM), 1μL; M13-R (10μM), 1μL; bacterial solution 1μL; ddH2O, 2μL. The PCR reaction program was: 94℃ pre-denaturation for 3min; 32 cycles (94℃ denaturation for 30sec; 55℃ annealing for 30sec; 72℃ extension for 2min 30sec); 72℃ extension for 10min; 12℃ incubation for 5min. After the reaction, 1% agarose gel electrophoresis was used to detect the PCR product and screen the positive clones.

[0081] Three positive clones identified by bacterial liquid PCR were selected for gene sequencing to obtain the coding sequence of the MtBUTS gene, as shown in SEQ ID NO: 1. The amino acid sequence of the MtBUTS protein encoded by the MtBUTS gene is shown in SEQ ID NO: 2. The Axygen plasmid miniprep kit (Cat. No.: AP-MN-P-250) was used to extract the plasmid according to the method described in the product manual to obtain the recombinant plasmid pTOPO-MtBUTS containing the MtBUTS gene coding sequence.

[0082] Coding sequence (2301bp) of the MtBUTS gene:

[0083] ATGTGGAGGCTTAAGCTCTTAGGGGAAGAGGAAGATCCATACTTGTGCAGCACAAACA

[0084] ATTTTGTAGGAAGGCAAACATGGGTGTATGATCCTGAAGCAGGGAGTCCTGAAGAAAG

[0085] AGCTGAGGTTGAAGAAGCTCATCAGAGTTTCTACAAAAATCGTTTTAAAGTCAGAACT

[0086] AACGCTGATCTCCTATGGCAATTGCAGTTTCTAAGAGAGAATAAATTCAAACAAACAA

[0087] TTCCACCAGTGAAGGTTGGTGAGGGAGAGGAGATAACATATGAAATGGCCACAACATC

[0088] GTTGAAGAGATCAGTCCACTTACTTTCAGCTTTGCAATCAAGCCATGGCCATTGGCCTG

[0089] CTGATAACTCTGGTCCTTTGTTTTACCATACCCCTTTGGTATTGTGTCTATATATTACAG

[0090] GAACTCTCAATGTTGTATTCTCTGCTGAACATCGCAAAGAGATGTTGCGTTACATATAC

[0091] TATCATCAGAATAAAGATGGCGGTTGGGGACTACACACTGAGGGCAACAGCACAATGT

[0092] TCTGTACGGTCTTTAACTATATTTGCATGCGTTTACTCGGAGAAGGACCCGACGGCGGC

[0093] GAGAACAGTGCTTGTACGAGAGCAAGAAAGTGGATTCTCGATCATGGTGGTGCAACAG

[0094] GCATCCCATCTTGGGGAAAGACTTGGCTTTCAATTCTTGGTGCGTTTGACTGGTCTGGA

[0095] TGCAACCCAGTACCTCCAGAATTTTGGTCTTTTCCTTCCTTTCTTCCAATCGGCCCAGCA

[0096] AATATGCTTTGCTATTCTCGACAGACTTACCTGCCCGATGTCATATTTGTATGGGAAACG

[0097] ATTTATCGGGTCCCATCACACCTCTTGTTTTAGAATTGAGAAAAGAGCTCTATACTCAAC

[0098] CTTATAATGAAATTAATTGGAGTAAAAATAAGACATGAAGTTGTTAAGGATGATCTCTA

[0099] CTTTCCTCATTCAAGATTACAAAACTTACTATGGGATTCTCTTCATAATATATGGAGC

[0100] CTCTTTTAACTCTTTGGCCTTTTAAAAAAATTGAGAGAGAAAACTCTCCAAAAAACTATG

[0101] AAACTTATTCATTATGAAGATGAAGCCAGTTGCTACTTTACTATTGGATGTATTGAAAA

[0102] GCCATTATTTATGCTTGCTTGCTGGATTGAAGATCCCAATGGTGATTATTTTAAGAAGC

[0103] ATCTTGCTCAGACTAGAGAATACTTTTGGATGGAGAAGATGGATTGAGAATTCAGAG

[0104] TTTTGGTAGTCAAACGTGGGACTGTTCCCTCGTTCTTCAGGCTTTGCTGGCTTGCAATTT

[0105] CGTCGATGAGCTTGGACCTGTGTTTTATGAAAGCACATGACTTCTTAAAAAAATCTCAGG

[0106] TAGCGGATAATCCTCCAGGAGACTTTAAAAGCATGTTTCGATACACATCCAAAGGAGC

[0107] GTGGACTTTCTCTATTCAAGATCATGGATGGCAAGTTTCTGATTGCACTGCTGAAAGCT

[0108] TACTGTGCTGTCTACATTTCTCAATGCTGACACCAGAAATCGTGGGTGAGAAAATGGA

[0109] ACCAGAAAGGTTTTTTGATGCTGTTAATTTCATACTTTCTATGCAGGGTAAAGATGGTG

[0110] GGATATCATGCTGGGAGCCAGCAGGTGCATCAAAATGGTTGGAGATGCTAAATCCCAT

[0111] GGAATTTCTAGAAGACATTGTCATTGAGCGTCCTTACGTTGAGTGTACTGCATCAACAT

[0112] TGAAGGTGATGTGTTTTTTCAAAAAATTATACCCAAATCACAGAGAAAAAGAGGTTAA

[0113] AAACTTCATCAGAAATTGTATCAGATTTATCGAAGAATCTCAAATGCCCGATGGTTCCT

[0114] GGTATGGAAATTGGGGAGTATGCTTCATATATGGTATATGGTGGGCACTTCAAGGACT

[0115] GTCTGTTGCTGGGAAGACATACGATAACTGTTTGGCAATACGCAGAGCTACCAATTTTC

[0116] TACTACCACTGCAGACAGATGATGGTGGATGGGGAGAGAGTTATCTTTCTTGTCCCAA

[0117] AAAGAAATATACACCCCTTGAAGGAAATCAACCAAATTTGGTAACAACTGCATGGGCT

[0118] ATGATGAGTTTAATTTATGCTGGCCAGATGGATAGAGACCCTACACCTATTCACCGTGC

[0119] TGCTAGATTGTTAATCAATTCACAACAGGAAAAGGGAGACTTCCCCCAACAGGAAATT

[0120] CATGGAGTTTACTATAGGAACTGTATGCATAATTACCCGATGTATAGAAATGTTTTTCC

[0121] AATCTGGGCTCTGGGAGAATATCAGTCAAAGGTTCTGTTGCCTGGCAAGATGAATTTTCCAAAACAATTTTAA(SEQ ID NO:1)

[0122] Amino acid sequence of MtBUTS protein (766 aa):

[0123] MWRLKLLGEEEDPYLCSTNNFVGRQTWVYDPEAGSPEERAEVEEAHQSFYKNRFKVRTN

[0124] ADLLWQLQFLRENKFKQTIPPVKVGEGEEITYEMATTSLKRSVHLLSALQSSHGHWPADNS

[0125] GPLFYHTPLVLCLYITGTLNVVFSAEHRKEMLRYIYYHQNKDGGWGLHTEGNSTMFCTVF

[0126] NYICMRLLGEGPDGGENSACTRARKWILDHGGATGIPSWGKTWLSILGAFDWSGCNPVPP

[0127] EFWSFPSFLPIGPANMLCYSRQTYLPMSYLYGKRFIGPITPLVLELRKELYTQPYNEINWSKI

[0128] RHEVVKDDLYFPHSRLQNLLWDSLHNIMEPLLTLWPFKKLREKTLQKTMKLIHYEDEASC

[0129] YFTIGCIEKPLFMLACWIEDPNGDYFKKHLAQTREYFWMGEDGLRIQSFGSQTWDCSLVLQ

[0130] ALLACNFVDELGPVFMKAHDFLKKSQVADNPPGDFKSMFRYTSKGAWTFSIQDHGWQVS

[0131] DCTAESLLCCLHFSMLTPEIVGEKMEPERFFDAVNFILSMQGKDGGISCWEPAGASKWLEM

[0132] LNPMEFLEDIVIERPYVECTASTLKVMCFFKKLYPNHREKEVKNFIRNCIRFIEESQMPDGS

[0133] WYGNWGVCFIYGIWWALQGLSVAGKTYDNCLAIRRATNFLLPLQTDDGGWGESYLSCPK

[0134] KKYTPLEGNQPNLVTTAWAMMSLIYAGQMDRDPTPIHRAARLLINSQQEKGDFPQQEIHGVYYRNCMHNYPMYRNVFPIWALGEYQSKVLLPGKMNFPKQF(SEQ ID NO:2)

[0135] 1.2 Cloning of AstHMGR gene

[0136] AstHMGR gene is a truncated oat HMGR gene, and its coding sequence is shown in SEQ ID NO: 5. HMGR gene encodes 3-hydroxy-3-methylglutaryl coenzyme A reductase (3-Hydroxy-3-methylglutaryl coenzyme Areductase, HMGR), which is the rate-limiting enzyme of mevalonate (MVA) pathway. Overexpression can significantly increase the production of 2,3-oxidosqualene for use by triterpene cyclase. Sangon Biotech Co., Ltd. was commissioned to fully synthesize the coding sequence of AstHMGR gene and connect it to pTOPO blunt-end vector to obtain the recombinant plasmid pTOPO-AstHMGR.

[0137] Coding sequence of AstHMGR gene (1260 bp):

[0138] ATGCCCGAGGAGGACGAGGAAATCGTCGCCGGGGTCGTCGCAGGGAAGATCCCCTCCT

[0139] ACGTGCTCGAGACCAGGCTAGGCGACTGCCGCAGGGCAGCCGGGATCCGCCGCGAGG

[0140] CGCTGCGCCGGATCACCGGCAGGGAGATCGACGGCCTTCCCCTCGACGGCTTCGACTA

[0141] CGACTCGATTCTCGGACAGTGCTGCGAGATGCCCGTCGGGTACGTGCAGCTGCCGGTC

[0142] GGCGTCGCGGGGCCGCTCGTCCTCGACGGCCGCCGCATATACGTCCCGATGGCCACCA

[0143] CGGAGGGCTGCCTAATCGCCAGCACCAACCGCGGATGCAAGGCCATTGCCGAGTCCGG

[0144] AGGCGCATCCAGCGTCGTGTACCGCGACGGGATGACCCGCGCCCCCGTAGCCCGCTTC

[0145] CCCTCCGCACGACGCGCCGCAGAGCTCAAGGGCTTCCTGGAGAATCCGGCCAACTACG

[0146] ACACCCTGTCCGTGGTCTTTAACAGATCAAGCAGATTTGCAAGGCTGCAGGGGGTCAA

[0147] GTGCGCCATGGCTGGGAGGAACTTGTACATGAGGTTCACCTGCAGCACCGGGGATGCC

[0148] ATGGGGATGAACATGGTCTCCAAGGGCGTCCAAAATGTGCTCGACTATCTGCAGGAGG

[0149] ACTTCCCTGACATGGACGTTGTCAGCATCTCAGGCAACTTTTGTTCCGACAAGAAATCA

[0150] GCTGCTGTAAACTGGATTGAAGGCCGTGGAAAGTCCGTGGTTTGTGAGGCAGTAATCA

[0151] GAGAGGAAGTTGTCCACAAGGTTCTCAAGACCAACGTTCAGTCACTCGTGGAGTTGAA

[0152] TGTGATCAAGAACCTTGCTGGCTCAGCAGTTGCTGGTGCTCTTGGGGGTTTCAACGCCC

[0153] ACGCAAGCAACATCGTAACGGCTATCTTCATTGCCACTGGTCAGGATCCTGCACAGAA

[0154] TGTGGAGAGCTCACAGTGTATCACTATGTTGGAAGCTGTAAATGATGGCAGAGACCTT

[0155] CACATCTCCGTTACAATGCCATCTATCGAGGTGGGCACAGTTGGTGGAGGCACGCAGC

[0156] TGGCCTCACAGTCGGCCTGCTTGGACCTACTGGGCGTCAAAGGCGCCAACAGGGAATC

[0157] TCCGGGGTCGAACGCTAGGCTGCTGGCCACGGTGGTGGCTGGTGCCGTCCTAGCTGGG

[0158] GAGCTGTCCCTCATCTCCGCCCAAGCTGCCGGCCATCTGGTCCAGAGCCACATGAAATACAACAGATCCAGCAAGGACATGTCCAAGATCGCCTGCTGA(SEQ ID NO:5)

[0159] Example 2. Construction of gene expression vector

[0160] 2.1 Construction of MtBUTS gene expression vector

[0161] The recombinant plasmid pTOPO-MtBUTS constructed in Example 1 was used as a template, and primers attB1-MtBUTS and attB2-MtBUTS and DNA polymerase KOD (Cat. No.: KFX-101) from Toyobo were used to perform PCR to amplify the MtBUTS gene fragment with a linker. The nucleotide sequences of the primers are as follows:

[0162] attB1-MtBUTS:

[0163] 5'-GGGGACAAGTTTGTACAAAAAAGCAGGCTACATGTGGAGGCTTAAGCTCTT-3' (SEQ ID NO: 6);

[0164] attB2-MtBUTS:

[0165] 5'-GGGGACCACTTTGTACAAGAAAGCTGGGTCTTAAAATTGTTTTGGAAAATT-3' (SEQ ID NO: 7).

[0166] The PCR reaction system was: 2×KOD buffer, 15 μL; dNTPs (2 mM), 5 μL; attB1-MtBUTS (10 μM), 1 μL; attB2-MtBUTS (10 μM), 1 μL; pTOPO-MtBUTS, 0.5 μL; KOD, 0.5 μL; ddH2O, 7 μL. The PCR reaction program was: 94°C pre-denaturation for 3 min; 32 cycles (98°C denaturation for 30 sec; 55°C annealing for 30 sec; 68°C extension for 2 min 30 sec); 68°C extension for 10 min; 12°C incubation for 5 min. After the reaction, the PCR product was detected by 1% agarose gel electrophoresis, and the gene fragment attB-MtBUTS was recovered using the Axygen DNA gel recovery kit (Cat. No.: AP-GX-250) according to the method described in the product manual.

[0167] Using Gateway TM BP Clonase TM The enzyme mixture (Cat. No. 11789020) was used to clone the recovered gene fragment attB-MtBUTS into the entry vector pDONR207 of the Gateway system through BP recombination reaction. The BP recombination reaction system was: attB-MtBUTS (10 ng·μL -1 ), 3μL; pDONR207 (150ng·μL -1), 1μL; 5×BP Clonase enzymemix, 1μL. Vortex the reaction system twice, 2sec / time, centrifuge briefly, and incubate at 25℃ for 1~16h (no more than 18h). Add 1μL of proteinase K and incubate at 37℃ for 20min to terminate the reaction. Take 1~5μL BP reaction solution and transform Escherichia coli DH5α by heat shock method. Pick a single clone and use primers pDONR-F and pDONR-R to perform bacterial liquid PCR identification. The nucleotide sequences of the primers are as follows:

[0168] pDONR-F: 5'-TCGCGTTAACGCTAGCATGGATCTC-3' (SEQ ID NO: 8);

[0169] pDONR-R: 5'-GTAACATCAGAGATTTTGAGACAC-3' (SEQ ID NO: 9).

[0170] The PCR reaction system was: 2×Taq mix, 5μL; 0.5μL each of pDONR-F (10μM) and pDONR-R (10μM); 1μL of bacterial solution; 3μL of ddH2O. The PCR reaction program was: 94℃ pre-denaturation for 3min; 32 cycles (94℃ denaturation for 30sec; 50℃ annealing for 30sec; 72℃ extension for 1min 10sec); 72℃ extension for 10min; 12℃ insulation for 5min. After the reaction, the positive clones identified by PCR were sequenced and verified. The plasmid of the positive clone with correct sequencing results was extracted using the Axygen plasmid miniprep kit (Cat. No.: AP-MN-P-250) according to the method described in the product manual to obtain the recombinant plasmid pDONR207-MtBUTS.

[0171] Using Gateway TM LR Clonase TM The enzyme mixture (Cat. No. 11791020) was used to clone the MtBUTS gene into the terminal vector pDEST1 of the Gateway system through LR recombination reaction. The LR recombination reaction system was: pDONR207-MtBUTS (50-150 ng), 3 μL; pDEST1 (150 ng·μL -1), 1μL; 5×LR Clonase enzyme mix, 1μL. Vortex the reaction system twice, 2sec / time, centrifuge briefly, and incubate at 25℃ for 1~16h (no more than 18h). Add 1μL of proteinase K and incubate at 37℃ for 20min to terminate the reaction. Take 1~5μL LR reaction solution and transform Escherichia coli DH5α by heat shock method. Pick a single clone and use primers DEST1-F and DEST1-R to perform bacterial liquid PCR identification. The nucleotide sequences of the primers are as follows:

[0172] DEST1-F: 5'-GCTCACCAAACATAGAAATGCAC-3' (SEQ ID NO: 10);

[0173] DEST1-R: 5'-GCTTCTGTATATTCTGCCCAAATTC-3' (SEQ ID NO: 11).

[0174] The PCR reaction system was: 2×Taq mix, 5μL; 0.5μL each of DEST1-F (10μM) and DEST1-R (10μM); 1μL of bacterial solution; 3μL of ddH2O. The PCR reaction program was: 94℃ pre-denaturation for 3min; 32 cycles (94℃ denaturation for 30sec; 50℃ annealing for 30sec; 72℃ extension for 1min 10sec); 72℃ extension for another 10min; 12℃ incubation for 5min. After the reaction, the positive clones identified by PCR were sequenced and verified. The plasmid of the positive clone with correct sequencing results was extracted using the Axygen plasmid miniprep kit (Cat. No.: AP-MN-P-250) according to the method described in the product manual to obtain the recombinant plasmid pDEST1-MtBUTS.

[0175] 2.2 Construction of AstHMGR gene expression vector

[0176] The recombinant plasmid pTOPO-AstHMGR constructed in Example 1 was used as a template, and primers attB1-AstHMGR and attB2-AstHMGR and DNA polymerase KOD (Cat. No.: KFX-101) from Toyobo were used to perform PCR to amplify the AstHMGR gene fragment with a linker. The nucleotide sequences of the primers are as follows:

[0177] attB1-AstHMGR:

[0178] 5'-GGGGACAAGTTTGTACAAAAAAGCAGGCTACatgcccgaggaggacgag-3' (SEQ ID NO: 12);

[0179] attB2-AstHMGR:

[0180] 5'-GGGGACCACTTTGTACAAGAAAGCTGGGTAtcagcaggcgatcttggac-3' (SEQ ID NO: 13).

[0181] The PCR reaction system was: 2×KOD buffer, 15 μL; dNTPs (2 mM), 5 μL; attB1-AstHMGR (10 μM), 1 μL; attB2-AstHMGR (10 μM), 1 μL; pTOPO-AstHMGR, 0.5 μL; KOD, 0.5 μL; ddH2O, 7 μL. The PCR reaction program was: 94°C pre-denaturation for 3 min; 32 cycles (98°C denaturation for 30 sec; 55°C annealing for 30 sec; 68°C extension for 1 min 30 sec); 68°C extension for 10 min; 12°C incubation for 5 min. After the reaction, the PCR product was detected by 1% agarose gel electrophoresis, and the gene fragment attB-AstHMGR was recovered using the Axygen DNA gel recovery kit (Cat. No.: AP-GX-250) according to the method described in the product manual.

[0182] Using Gateway TM BP Clonase TM The enzyme mixture (Cat. No. 11789020) was used to clone the recovered gene fragment attB-AstHMGR into the entry vector pDONR207 of the Gateway system through BP recombination reaction. The BP recombination reaction system is: attB-AstHMGR (10 ng·μL -1 ), 3μL; pDONR207 (150ng·μL -1), 1μL; 5×BP Clonase enzymemix, 1μL. Vortex the reaction system twice, 2sec / time, centrifuge briefly, and incubate at 25℃ for 1~16h (no more than 18h). Add 1μL of proteinase K and incubate at 37℃ for 20min to terminate the reaction. Take 1~5μL BP reaction solution and transform Escherichia coli DH5α by heat shock method. Pick a single clone and use primers pDONR-F (SEQ ID NO:8) and pDONR-R (SEQ ID NO:9) for bacterial liquid PCR identification. PCR reaction system: 2×Taq mix, 5μL; pDONR-F (10μM) and pDONR-R (10μM) 0.5μL each; bacterial liquid, 1μL; ddH2O, 3μL. The PCR reaction program was: 94°C pre-denaturation for 3 min; 32 cycles (94°C denaturation for 30 sec; 50°C annealing for 30 sec; 72°C extension for 50 sec); 72°C extension for 10 min; 12°C incubation for 5 min. After the reaction, the positive clones identified by PCR were sequenced and verified. The plasmids of the positive clones with correct sequencing results were extracted using the Axygen plasmid miniprep kit (Cat. No.: AP-MN-P-250) according to the method described in the product manual to obtain the recombinant plasmid pDONR207-AstHMGR.

[0183] Using Gateway TM LR Clonase TM The enzyme mixture (Cat. No. 11791020) was used to clone the AstHMGR gene into the terminal vector pDEST1 of the Gateway system through LR recombination reaction. The LR recombination reaction system was: pDONR207-AstHMGR (50-150 ng), 3 μL; pDEST1 (150 ng·μL -1), 1μL; 5×LR Clonase enzyme mix, 1μL. Vortex the reaction system twice, 2sec / time, centrifuge briefly, and incubate at 25℃ for 1~16h (no more than 18h). Add 1μL of proteinase K and incubate at 37℃ for 20min to terminate the reaction. Take 1~5μL LR reaction solution and transform Escherichia coli DH5α by heat shock method. Pick a single clone and use primers DEST1-F (SEQ ID NO:10) and DEST1-R (SEQ ID NO:11) for bacterial liquid PCR identification. PCR reaction system: 2×Taq mix, 5μL; DEST1-F (10μM) and DEST1-R (10μM) 0.5μL each; bacterial liquid, 1μL; ddH2O, 3μL. The PCR reaction program was: 94°C pre-denaturation for 3 min; 32 cycles (94°C denaturation for 30 sec; 50°C annealing for 30 sec; 72°C extension for 50 sec); 72°C extension for 10 min; 12°C incubation for 5 min. After the reaction, the positive clones identified by PCR were sequenced and verified. The plasmids of the positive clones with correct sequencing results were extracted using the Axygen plasmid miniprep kit (Cat. No.: AP-MN-P-250) according to the method described in the product manual to obtain the recombinant plasmid pDEST1-AstHMGR.

[0184] Example 3. Transient expression of genes in tobacco

[0185] To verify the function of the MtBUTS gene, the truncated oat HMGR gene (AstHMGR) was co-expressed with the MtBUTS gene from Toosendan in Nicotiana benthamiana. Since the content of 2,3-oxidosqualene in plants is usually very low, the AstHMGR gene was co-expressed in tobacco in order to synthesize enough 2,3-oxidosqualene as a substrate for the MtBUTS catalytic reaction.

[0186] The pDEST1 empty vector and the recombinant plasmids pDEST1-MtBUTS and pDEST1-AstHMGR constructed in Example 2 were used to transform Agrobacterium tumefaciens EHA105 competent cells respectively. The method is as follows: take out the EHA105 competent cells from the -80℃ refrigerator, add 2μL of plasmid, mix well and transfer to an electric rotating cup, quickly add 750μL of liquid LB medium after 1800V electric shock, transfer the bacteria to a 1.5mL centrifuge tube, and culture at 28℃, 200rpm in a shaker for 2 to 3h. Collect the bacteria by centrifugation at 4000rpm and apply to a tube containing 25mg·L -1 Rif (rifampicin) and 50mg·L -1Kan (kanamycin) solid LB medium, inverted culture in a 28 ° C constant temperature incubator for 2 to 3 days. Pick a single clone, use primers DEST1-F (SEQ ID NO: 10) and DEST1-R (SEQ ID NO: 11) and the corresponding PCR reaction system and PCR reaction program for PCR verification, and obtain recombinant Agrobacterium EHA105-pDEST1 carrying pDEST1 empty vector, recombinant Agrobacterium EHA105-pDEST1-MtBUTS carrying pDEST1-MtBUTS, and recombinant Agrobacterium EHA105-pDEST1-AstHMGR carrying pDEST1-AstHMGR.

[0187] Single clones of EHA105-pDEST1, EHA105-pDEST1-MtBUTS and EHA105-pDEST1-AstHMGR were inoculated into 1 mL of 25 mg·L -1 Rif and 50mg·L -1 Kan's liquid LB medium was cultured overnight at 28°C and 200 rpm in a shaking incubator. 100 μL of the overnight cultured Agrobacterium was inoculated into 50 mL of 25 mg·L -1 Rif and 50mg·L -1 Kan's liquid LB medium was incubated at 28°C and 200 rpm for 24 h. The cells were collected by centrifugation at 4°C and 5,000 rpm for 10 min. The cells were resuspended in MMA solution to an OD of 600nm =0.8A, and incubate for 2 hours at room temperature in the dark to obtain the infection solution. The formula of MMA solution (1L) is as follows: 0.5M MES (2-[N-morpholinyl]ethanesulfonic acid) pH=5.6, 20mL; 1M MgCl2, 10mL; 200mM acetosyringone (AS), 500μL; distilled water to 1L.

[0188] Set up the following four experimental groups:

[0189] 1) Empty vector control tobacco (denoted by DEST1): Injected with EHA105-pDEST1 infection solution.

[0190] 2) Tobacco expressing only AstHMGR (indicated by AstHMGR): Injection of EHA105-pDEST1-AstHMGR infection solution.

[0191] 3) Tobacco expressing only MtBUTS (indicated by MtBUTS): Injection of EHA105-pDEST1-MtBUTS infection solution.

[0192] 4) Tobacco expressing both AstHMGR and MtBUTS (denoted as AstHMGR+MtBUTS): EHA105-pDEST1-MtBUTS infection fluid and EHA105-pDEST1-AstHMGR infection fluid were mixed in equal volumes, and the resulting mixed infection fluid was injected into tobacco.

[0193] The injection method is as follows: use a 1mL syringe to absorb the infection solution, then remove the needle, and infiltrate the infection solution into the tobacco leaves from the back through pressure until the entire leaf is infiltrated by the bacterial solution. Three leaves are injected per tobacco plant. The tobacco leaves are harvested after 6 to 8 days and freeze-dried in a freeze dryer. The freeze-dried tobacco leaves are ground into powder using a ball mill to obtain tobacco powder for compound extraction.

[0194] Example 4. Detection of MtBUTS catalytic products

[0195] 4.1 GC-MS detection of MtBUTS catalytic products

[0196] Weigh the tobacco powder samples of the four test groups (DEST1, AstHMGR, MtBUTS and AstHMGR+MtBUTS) obtained in Example 3, 100 mg of each sample, add 1 mL of lysis solution, mix thoroughly with a vortex instrument, and lyse the sample in a 70°C water bath for 2 hours to obtain a sample lysate. The formula of the lysate (100 mL) is as follows: KOH, 10 g; anhydrous ethanol, 90 mL; ddH2O is made up to 100 mL. The sample lysate is centrifuged at room temperature and 4,000 rpm for 20 min, the supernatant is aspirated, and extracted 3 times with an equal volume of n-hexane, and the 3 extracts are combined. Wash the obtained extract with an equal volume of saturated saline, aspirate the upper n-hexane phase, and repeat this step 2 times. The obtained n-hexane phase is blown dry with nitrogen or evaporated in a fume hood. Add 1 mL of n-hexane to dissolve again, and filter out insoluble impurities with a 0.22 μm organic filter membrane to obtain an extract.

[0197] 100 μL of the extract was added to a sample injection bottle equipped with an inner tube, placed in a fume hood to evaporate overnight, and then 100 μL of the silanization reagent MSTFA (Thermo Scientific TM) into the inner tube, derivatized at 60℃ for 2h, and detected by gas chromatography-mass spectrometer (GC-MS) after cooling. The GC-MS detection method is as follows: the GC detector model is Agilent 7890B, the MS detector model is Agilent 5977A, the chromatographic column used is gas phase high temperature capillary column DB-5HT [30×320 (inner diameter)×0.1μm (film thickness), Agilent 123-573], the carrier gas is helium (He), and the flow rate is 1mL·min -1 The injection port temperature was 250°C, the ion source temperature was 230°C, the spectrum acquisition started from 15 min, the Scan mode was selected, the scanning range was 60-800, and the scanning speed was 3125 μ·s -1 , EI electron bombardment energy was 70 eV, detection voltage was 1000 V. The initial furnace temperature was 170 °C, maintained for 2 min, and then 6 °C min -1 Raise the temperature to 290℃, maintain for 4min, and then increase the temperature by 10℃·min -1 The temperature was raised to 340°C and maintained for 0 min. The injection volume was set to 1 μL and no splitting was performed. The running time of all programs was 31 min, and the obtained data were qualitatively analyzed using Agilent analysis software Qualitative Analysis B.06.00. Figure 2-4 The GC-MS detection results are shown, where DEST1 represents empty vector control tobacco, AstHMGR represents tobacco expressing only AstHMGR, MtBUTS represents tobacco expressing only MtBUTS; AstHMGR+MtBUTS represents tobacco expressing both AstHMGR and MtBUTS.

[0198] The GC-MS total ion chromatogram (TIC) of the catalytic product of MtBUTS is shown in Figure 2 As shown in Figure 2, at a retention time of 21.42 min, the AstHMGR+MtBUTS sample showed a significant difference peak compared with the DEST1 sample ( Figure 2 The mass spectrum of the difference peak is as shown in FIG. Figure 4 As shown in FIG. 1 , the characteristic ion peak is m / z=393.3. The extracted ion chromatogram (EIC) of the characteristic ion peak m / z=393.3 is as shown in FIG. Figure 3As shown, only the MtBUTS sample and the AstHMGR+MtBUTS sample produced a new compound peak, while the compound peak did not exist in the DEST1 sample and the AstHMGR sample. Therefore, the compound peak was a new product formed by MtBUTS-catalyzed 2,3-oxidosqualene.

[0199] 4.2 NMR Identification of MtBUTS Catalytic Products

[0200] After mixing the infection solution of EHA105-pDEST1-MtBUTS and the infection solution of EHA105-pDEST1-AstHMGR in equal volumes, the whole tobacco leaves were injected by the injection method in Example 3, and a total of 100 tobacco plants were injected. After 6 days of expression, the injected leaves were collected and dried with a freeze dryer. After the leaves were completely dried, they were ground into powder with a medicinal material grinder, and the lysis solution was added according to a mass / volume ratio (g / ml) of 1:10. The sample was lysed in a water bath at 70°C for 2h to obtain a sample lysate. The formula of the lysate (100mL) is as follows: KOH, 10g; anhydrous ethanol, 90mL; ddH2O is made up to 100mL. The sample lysate was filtered with a suction flask to filter out the solid residue to obtain a crude extract. The crude extract was extracted 5 times with an equal volume of n-hexane, and the n-hexane phase on the upper layer was retained each time, and the 5 extracts were combined. The combined extracts were concentrated using a rotary evaporator to obtain a paste-like crude extract, and the weight was recorded.

[0201] Use a small amount of n-hexane to redissolve the above paste crude extract, and add silica gel at a ratio of 2g silica gel to 1g paste crude extract, mix the sample and silica gel thoroughly, and then use a rotary evaporator to evaporate the solvent for later use. SNAP320g silica gel column was filled with 200-300 mesh silica gel, tapped and filled, and at least 5 column volumes of 100% n-hexane were used at 50 mL min -1 Elute the equilibrated column at a speed of 50 mL / min until the silica gel is completely and evenly soaked. Open the top cover of the equilibrated silica gel column and fill the silica gel-mixed sample on the surface of the silica gel column and level it. Use n-hexane: ethyl acetate = 19:1 (v / v) as eluent at a rate of 50 mL / min. -1 The first 500-1000 mL of fractions were not collected, and the fractions thereafter were collected at a rate of 50 mL / tube.

[0202] The collected fractions were placed in a fume hood to evaporate for 2-3 days until about 2 mL was left, and then qualitative analysis was performed by thin layer chromatography (TLC). The developing solvent used for TLC was n-hexane: ethyl acetate = 5:1 (v / v). The TLC plate was a Merck silica gel 60 aluminum plate (Cat. No.: 1.05554.0001), which was cut into a suitable size for use. Draw a horizontal line with a pencil 1 cm from the bottom of the TLC plate, and gently mark a point every 0.5 cm on the line as the loading point. Dip a small amount of the blank control DEST1, the negative control AstHMGR, the positive control AstHMGR+MtBUTS, and the collected fractions with a capillary, and dot them on the loading points in turn. Place the loaded TLC plate in the above-mentioned developing solvent, wait until the solvent is developed to 1 cm from the top of the TLC plate, take out the TLC plate, and evaporate it in a fume hood. The evaporated TLC plate was evenly sprayed with a color developer having the following formula: anhydrous ethanol (McLean, CAS: 64-17-5): concentrated sulfuric acid (CAS: 7664-93-9): anisaldehyde (sigma, CAS: 123-11-5) = 674:25:18 (v / v / v), and then placed at 140°C for high temperature color development to observe the fractions containing the target component.

[0203] The fractions containing the target component were combined, dried with a nitrogen blower, redissolved with 1 ml of n-hexane, filtered through a 0.22 μm organic filter membrane, and then separated and purified using a normal phase preparative chromatograph. The separation conditions were as follows: set the injection volume to 200 μL, elute with a prepared 95% n-hexane / acetone chromatographic pure solution, and collect the target peak. The collected target compounds were concentrated, and the purity of the compounds was tested using GC-MS. If the test result is a single peak, the structure of the compound is determined by nuclear magnetic resonance spectroscopy. After enrichment and purification, a total of 5.8 mg of monomers with a purity greater than 90% were obtained, namely the MtBUTS catalytic product. Identified by nuclear magnetic resonance spectroscopy, the structure of the MtBUTS catalytic product is butyryl cetyl alcohol, and its hydrogen spectrum, carbon spectrum, HSQC spectrum, and HMBC spectrum are as follows: Figure 7-10 shown.

[0204] The hydrogen spectrum data of the MtBUTS catalytic product are as follows:

[0205] 1H NMR(400MHz,in CDCl3)δH:1.68(1H,m,H-1a),1.13(1H,m,H-1b),1.64(2H,m,H-2),3.25(1H,dd, J=4.4,11.2Hz,H-3),1.30(1H,m,H-5),2.11(1H,m,H-6a),1.97(1H,m,H-6b),5.2 5(1H,m,H-7),2.20(1H,m,H-9),1.49(1H,m,H-11a),0.96(1H,m,H-11b),1.64(1H ,m,H-12a),1.44(1H,m,H-12b),1.78(1H,m,H-15a),1.48(1H,m,H-15b),1.92(1H ,m,H-16a),1.27(1H,m,H-16b),1.48(1H,m,H-17),0.80(3H,s,H-18),0.74(3H, s,H-19),1.39(1H,m,H-20),0.84(3H,s,H-21),1.59(1H,m,H-22a),1.28(1H,m,H -22b),1.68(1H,m,H-23a),0.96(1H,m,H-23b),5.10(2H,m,H-24),1.68(3H,s,H- 26),1.60(3H,s,H-27),0.97(3H,s,H-28),0.86(3H,s,H-29),0.97(3H,s,H-30).

[0206] The carbon spectrum data of the MtBUTS catalytic product are as follows:

[0207] 13 C NMR (100MHz, in CDCl3) δC:37.3(C-1),27.8(C-2),79.4(C-3),39.1(C-4),50.8(C-5),24.1(C-6),117.9(C-7),146.0(C- 8),49.1(C-9),35.1(C-10),18.3(C-11),34.0(C-12),43.7(C-13),51.4(C-14),34.1(C-15),28. 6(C-16),53.4(C-17),22.2(C-18),13.3(C-19),35.9(C-20),18.7(C-21),35.3(C-22),25.5(C-2 3),125.3(C-24),131.1(C-25),25.9(C-26),17.8(C-27),27.8(C-28),14.9(C-29),17.8(C-30).

[0208] 4.3 Determination of butyryl cetyl alcohol production

[0209] Take 1 mg of purified butyryl cetyl alcohol monomer (structure confirmed by NMR) powder, add 1 mL of chloroform, vortex mix to obtain a 1 mg / mL mother solution. Dilute the mother solution with n-hexane to prepare concentrations of 250 μg mL -1 , 200 μg·mL -1 , 150μg·mL -1 , 100 μg·mL -1 , 50μg·mL -1 , 25 μg·mL -1 , 10μg·mL -1 , 1μg·mL -1 Butyryl cetyl alcohol standard solution.

[0210] 100 μL of each concentration of butyryl cetyl alcohol standard solution was taken out and dried with a nitrogen blower. Then 100 μL of silanization reagent MSTFA (Thermo Scientific TM ), derivatized at 70℃ for 2h and then GC-MS detected. The detection method was the same as the GC-MS detection method in 4.1 above. The data after being taken off the machine were processed by Agilent analysis software Qualitative Analysis B.06.00 software as follows: extract the characteristic ion peak of m / z=393.3 and integrate the peak area. Draw a scatter plot between the characteristic ion peak area integral (y) and the butyryl cetyl alcohol concentration (x), and fit the curve to obtain the standard curve equation: y=6701.8x+21013, R 2 =0.9938( Figure 6 ).

[0211] 100 μL of the AstHMGR+MtBUTS sample extract from 4.1 was taken and dried with a nitrogen blower. Then, 100 μL of the silanization reagent MSTFA (Thermo Scientific TM ), after derivatization at 70°C for 2h, GC-MS detection was performed in the same manner, the characteristic ion peak m / z=393.3 was extracted and the peak area was integrated, and then the characteristic ion peak area integral was substituted into the above curve equation y=6701.8x+21013, and the corresponding butyryl cetyl alcohol concentration was calculated to be: 146.65 μg·mL -1 The total mass of AstHMGR+MtBUTS sample used to extract compounds was 100 mg. According to the formula: W = butyryl cetyl alcohol concentration (146.65 μg mL -1 )×total volume of extract (1 mL) / total mass of sample (100 mg) to calculate the yield of butyrylcetol. The results showed that in tobacco expressing both AstHMGR and MtBUTS, the yield of butyrylcetol was 1.4665 μg / mg DW (dry weight of tobacco leaves).

Claims

1. A squalene cyclase MtBUTS, whose amino acid sequence is shown in SEQ ID NO:

2.

2. A gene encoding the oxidosqualene cyclase MtBUTS according to claim 1.

3. The gene according to claim 2, characterized in that: The nucleotide sequence of the gene is shown in SEQ ID NO:

1.

4. An expression cassette, vector or host bacteria containing the gene according to claim 2 or 3.

5. A method for cloning the gene according to claim 2 or 3, comprising: RNA was extracted from Toosendan plant tissues and reverse transcribed into cDNA; Using the cDNA as a template, polymerase chain reaction was performed to clone the gene; The nucleotide sequences of the primers used for the polymerase chain reaction are shown in SEQ ID NO:3 and SEQ ID NO:

4.

6. Use of the oxidosqualene cyclase MtBUTS according to claim 1 in the synthesis of butyryl cetyl alcohol in tobacco.

7. A method for obtaining butyryl cetyl alcohol, comprising: The oxidosqualene cyclase MtBUTS and 3-hydroxy-3-methylglutaryl coenzyme A reductase described in claim 1 are simultaneously expressed in tobacco; tobacco leaves successfully expressing the two enzymes are collected and butyryl cetyl alcohol is extracted.

8. The method according to claim 7, characterized in that: The nucleotide sequence of the 3-hydroxy-3-methylglutaryl-CoA reductase is shown in SEQ ID NO:5.

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