CN110777157A - CYP450 Gene Catalyzing the C-28 Oxidation of Arsinol and Its Encoded Product and Application - Google Patents

Abstract

The invention discloses a CYP450 gene IaAO2 that catalyzes the C-28 oxidation of balsam, encodes a protein that catalyzes the C-28 oxidation of balsam, and induces expression in engineering strains that can catalyze α-arosinol and β-aromatic Resin alcohols were oxidized to ursolic acid and oleanolic acid, respectively. Its nucleotide sequence is shown in SEQ ID No.1; its amino acid sequence is shown in SEQ ID No.2. The invention also discloses its expression vector and host bacteria, which are used for preparing triterpenoid compounds, preparing catalytic pentacyclic triterpene C-28 oxidation compounds, and preparing catalytic pentacyclic triterpenes α-arosinol and β-arosinol oxidation compounds applications in . The invention also discloses a method for homologous gene modification based on the sequence characteristics of the gene IaAO2 and the obtained new gene tIaAO1. The IaAO2 gene provided by the invention has a shorter nucleotide sequence compared with the homologous gene, can construct transgenic organisms by utilizing this gene, synthesize pentacyclic triterpenoids with important economic value, and has good application prospects .

Description

CYP450 Gene Catalyzing the C-28 Oxidation of Arsinol and Its Encoded Product and Application

technical field

The invention relates to a gene that catalyzes the oxidation of C-28 position of arasinol, its encoded product and application, and belongs to the field of genetic engineering of medicinal plants, in particular to a CYP450 gene that catalyzes the C-28 oxidation of balsinol, its encoded product and application.

Background technique

Cytochrome P450 monooxygenase (CYP450) is a family of heme proteases with oxidative functions that are widely present in nature and participate in various metabolic reactions in microorganisms, animals and plants. In plants, CYP450 plays important physiological functions in the biosynthesis of terpenoids, flavonoids, alkaloids, plant hormones and other substances. In the downstream biosynthetic pathway of triterpenoid saponins, CYP450 plays a role in modifying the triterpenoid skeleton, enriching the structural diversity, and is one of the factors that plants contain a wide variety of triterpenoids. Therefore, the identification of CYP450 is the key to reveal the mechanism of triterpenoid biosynthesis.

Gangmei (Ilex asprella) is a traditional Chinese medicinal material with Lingnan characteristics. Its main active ingredients are triterpenes and triterpenoid saponins, and most of them are arbutane (α-aromatic resin alcohol derivatives), and a few are olean seeds. Alkyl (β-aromatic resin alcohol derivatives). Although triterpenoids exist widely in plants, their specific biosynthetic pathways are not fully understood. The biosynthetic process of triterpenoids can be roughly divided into three steps: precursor formation, skeleton construction and skeleton modification.

The biosynthesis of triterpenoids starts from the common terpenoid precursors IPP (isopentenylpyrophosphate) and DMAPP (dimethylallyl diphosphate), which undergo a series of enzymatic reactions to generate 2,3-oxysqualene (2,3- oxidosqualene). 2,3-oxysqualene is cyclized by different 2,3-oxysqualene cyclase OSCs, such as lupeol synthase, dammarenediol Resin synthase (amyrin synthase), etc., generate different types of triterpenoid backbones. The triterpene backbone undergoes post-modification processes such as CYP450 oxidation, glycosyltransferase UGT glycosylation, etc., and finally forms a wide variety of triterpenoid saponins.

Although a large number of CYP450 related to the oxidative modification of triterpene backbone have been identified so far, no report has disclosed the protein encoded by the natural short-sequence cytochrome P450 enzyme gene that can catalyze the C-28 oxidation of aryl alcohol.

SUMMARY OF THE INVENTION

In order to overcome the deficiencies of the prior art, the object of the present invention is to provide a special cytochrome P450 gene IaAO2 that catalyzes the C-28 oxidation of balsamic resin, and the protein encoded by it can catalyze α- α-amyrin and β-amyrin are oxidized to ursolic acid and oleanolic acid, respectively, which have shorter nucleotide sequences compared with the homologous genes.

Another object of the present invention is to provide the above-mentioned protein encoded by the special cytochrome P450 gene IaAO2, which catalyzes the oxidation of the C-28 position of aryl alcohol, and its application.

Another object of the present invention is to provide a genetic modification method for truncating the 5'-end of a homologous gene and the gene tIaAO1 obtained therefrom.

In order to achieve the above object, the present invention adopts the following technical solutions:

The CYP450 gene IaAO2 that catalyzes the C-28 oxidation of balsamic resin provided by the invention, the nucleotide sequence is shown in any one of the following 1)-5):

1) The nucleotide sequence shown in SEQ ID No. 1; or

2) A nucleotide sequence encoding the amino acid sequence shown in SEQ ID No. 2; or

3) a nucleotide sequence capable of hybridizing with the complementary sequence of SEQ ID No. 1, and the protein encoded by the nucleotide sequence still has the function of catalyzing the oxidation of balsamic resin at the C-28 position; or

4) A nucleotide sequence having at least 75% or more homology with the nucleotide sequence shown in SEQ ID No. 1; or

5) A nucleotide sequence variant in which one or more bases are deleted, substituted or inserted on the basis of the nucleotide sequence shown in SEQ ID No. 1, and the nucleotide sequence variant encoded The protein still has the function or activity of catalyzing the C-28 oxidation of balsamic resin.

The present invention provides a protein that catalyzes the oxidation of balsamic resin at the C-28 position, and the amino acid sequence is shown in either 1) or 2):

1) the amino acid residue sequence shown in SEQ ID No.2; or

2) A protein with the function or activity of catalyzing the C-28 oxidation of balsamyl alcohol derived from the amino acid sequence shown in SEQ ID No.2 by replacement, deletion or/and insertion of one or more amino acid residues Variants.

The present invention provides a specific primer for amplifying CYP450 gene IaAO2 that catalyzes the C-28 oxidation of balsamic resin. The upstream primer sequence of the specific primer is shown in SEQ ID No. 3; the downstream primer sequence is shown in SEQ ID No. 3. ID No.4.

The present invention provides expression vectors containing the nucleotide sequences described above.

Further, the expression vector includes but is not limited to pET, pESC, pYES-DEST; the expression vector is inserted into the vector by but not limited to the nucleotide sequence shown in SEQID No.1 by DNA ligase or homologous recombination .

The present invention provides host bacteria containing the nucleotide sequences described above.

Further, the preparation method of this host bacteria is as follows:

The SEQ ID No.1 gene was cloned into a eukaryotic expression vector, and a recombinant expression vector with the IaAO2 gene was constructed; the recombinant expression vector was transferred into the engineering bacteria capable of producing α-arosinol and β-arosinol to obtain recombinant yeast.

Further, the eukaryotic expression vector used includes but is not limited to pESC, pYES-DEST; the eukaryotic expression vector is pESC-TRP.

Furthermore, the gene of SEQ ID No. 1 is cloned into the eukaryotic expression vector pESC-TRP between the restriction endonucleases BamH I and Xho I sites.

Further, the used engineering strain includes but is not limited to Saccharomyces cerevisiae; the engineering strain is Saccharomyces cerevisiae engineering strain WAT11tfAX.

The present invention provides the use of the above-mentioned nucleotide sequence in the preparation of triterpenoids.

The present invention provides the application of the above-mentioned nucleotide sequence in the preparation of a compound that catalyzes the oxidation of pentacyclic triterpenes at C-28 position.

The present invention provides the application of the above-mentioned nucleotide sequence in the preparation of catalyzing pentacyclic triterpenes α-arosinol and β-arosinol oxidation compounds.

The present invention provides a genetic modification method for truncation of the 5'-end of a homologous gene based on the above-mentioned nucleotide sequence characteristics.

Further, the genetic modification method is to obtain a new gene with a truncated 5'-end by, but not limited to, DNA restriction endonuclease or PCR on the basis of sequence alignment.

The present invention provides the gene tIaAO1 obtained by the genetic modification method of homologous gene 5'-end truncation;

The nucleotide sequence of the tIaAO1 gene is shown in SEQ ID No.5; its amino acid sequence is shown in SEQ ID No.6;

The specific primer sequences are: the upstream primer tIaAO1F is shown in SEQ ID No.7; the downstream primer tIaAO1R is shown in SEQ ID No.8.

Further, the preparation method of the new gene tIaAO1 is as follows:

Sequence alignment of the target gene and SEQ ID No. 1 gene was carried out to determine the overlapping sequence; specific primers were designed, and PCR amplification was performed to obtain a new gene with a truncated 5'-end.

Further, by but not limited to PCR amplification, restriction endonuclease digestion, and artificial synthesis methods, a new gene with a 5'-end truncated is obtained.

Compared with the prior art, the present invention has the following beneficial effects:

The invention provides a CYP450 gene capable of catalyzing the C-28 oxidation of balsamic resin. The gene is cloned from Gangmei for the first time. Oxidized to ursolic acid and oleanolic acid. Compared with the homologous CYP450 gene that catalyzes the C-28 oxidation of balsamic resin in plants, it has a shorter nucleotide sequence.

The invention also obtains a Saccharomyces cerevisiae engineering strain with a CYP450 gene recombinant expression vector, and the analysis results show that the induced expression protein can oxidize the substrates α-arosinol and β-arosinol into ursolic acid and Leanolic acid.

Description of drawings

Fig. 1 is balsamic resin alcohol structural formula and atomic number;

Figure 2 shows the GC-MS analysis of recombinant yeast metabolites; in the figure:

A is the total ion chromatogram of recombinant yeast metabolites;

B is the mass spectrum of oleanolic acid, ursolic acid standard and recombinant yeast metabolites Peak 3 and Peak 4.

Detailed ways

The invention discloses a CYP450 gene that catalyzes the C-28 oxidation of balsamic resin, its encoded product and its application. The gene was cloned from Plum for the first time, and the encoded protein can catalyze the oxidation of pentacyclic triterpenes α-arosinol and β-arosinol to ursolic acid and oleanolic acid, respectively.

The invention relates to the screening of CYP450 genes involved in the metabolic pathway of triterpenoids in gangmei from the transcriptome data of gangmei. Cloning a gene and using the method of heterologous expression in Saccharomyces cerevisiae to analyze the functional activity of the encoded protein of the gene, involving the CYP450 gene and its encoded product and application in the biosynthetic pathway of triterpenoids, the main active component of Radix Plum.

Our laboratory cloned and identified a multifunctional balsam resin synthase gene IaAS1 in the early stage, and obtained a yeast engineering strain capable of stabilizing high-yield α-amyrin and β-amyrin Bacterium WAT11tfAX, the present invention relates to a key cytochrome P450 enzyme gene that can catalyze pentacyclic triterpenes α-amyrinol and β-amyrinol to generate ursolic acid and oleanolic acid respectively, and is named IaAO2. Before the disclosure of the present invention, there has not been any report that disclosed the IaAO2 gene and its encoded amino acid sequence involved in this patent application. Compared with the currently identified homologous CYP450 genes that catalyze the C-28 oxidation of balsamic resin in plants, such as CYP716A1 and CYP716A2 in Arabidopsis, CYP716A15 and CYP716A17 in grapes, and CYP716A75 in basil, IaAO2 gene has more The short nucleotide sequence is a specific CYP450 gene that catalyzes the oxidation of balsamic resin at the C-28 position.

The structural formulas and atomic numbers of α-amyrin, β-amsinol, ursolic acid and oleanolic acid are shown in Figure 1.

The IaAO2 gene provided by the present invention has the nucleotide sequence shown in SEQ ID No. 1, which consists of 1239 bases, and the protein encoded by the gene has the amino acid residue sequence of SEQ ID No. 2, which consists of 412 amino acids. residue composition.

A kind of special cytochrome P450 gene provided by the invention and catalyzing the C-28 oxidation of balsamic resin alcohol: IaAO2, it is one of the following nucleotide sequences:

1) the DNA sequence in SEQ ID No.1 of the sequence listing;

2) A DNA sequence having one or several base mutations with the DNA sequence defined in SEQ ID No. 1 of the Sequence Listing and encoding the same functional protein.

The protein encoded by the above-mentioned gene IaAO2 provided by the present invention has the amino acid residue sequence in SEQ ID No. 2 of the sequence table or the amino acid residue sequence of SEQ ID No. 2 is substituted by one or several amino acid residues and has A protein derived from SEQ ID No. 2 having the same activity as the amino acid residue sequence of SEQ ID No. 2.

The expression vector host bacteria containing the gene IaAO2 of the present invention and the application of the gene in the production of triterpenoids also fall within the protection scope of the present invention.

The gene of SEQ ID No.1 was cloned into the restriction endonuclease BamH I and XhoI sites of the eukaryotic expression vector pESC-TRP, and the recombinant expression vector pESC-TRP-IaAO2 with the IaAO2 gene was constructed; Saccharomyces cerevisiae engineered strains WAT11tfAX (WAT11, trp1::PGAP1-SctHMGR1-TCYC1, ura3::PGAP1-ScERG20-TCYC1, leu2::PGAP1-SeACSL641P-TCYC1, his3::PTEF1-IaAS1-TCYC1) to obtain recombinant yeast and induce protein expression with galactose. After the recombinant yeast was cultured, the bacterial cells were collected, and their metabolites were extracted and detected by GC-MS after silanization. The results of GC-MS analysis showed that the IaAO2 protein could oxidize the substrates α-arosinol and β-arosinol to ursolic acid and oleanolic acid, respectively.

The research results show that the gene IaAO2 with the characteristic structural domain of the cytochrome P450 gene involved in the present invention is closely related to the biosynthesis of the triterpenoids of fenugreek. C-28 oxidation function.

The IaAO2 gene provided by the invention has a shorter nucleotide sequence compared with the homologous gene, and can construct a transgenic organism by utilizing the gene to synthesize pentacyclic triterpenoids with important economic value, and has a good application prospect .

The present invention also includes a method for truncating the 5'-end of the homologous gene based on the sequence characteristics of the IaAO2 gene.

The present invention will now be described in further detail with reference to the accompanying drawings and specific embodiments, but the present invention is not limited to the following embodiments.

The cloning of embodiment 1 Gangmei IaAO2 gene

Extract the total RNA from the young leaves of Prunus chinensis with a plant RNA extraction kit (Magen), and reverse-transcribe the cDNA with a reverse transcription kit (Full Gold), design specific primers according to the transcriptome data, and perform PCR with the cDNA as a template Amplification, agarose gel electrophoresis showed that a specific fragment appeared at about 1200bp, the target fragment was recovered by agarose gel recovery kit (Magen), cloned into pEASY vector (full gold), and positive clones were identified and verified by sequencing , for the construction of recombinant expression vectors.

1. Construction of yeast expression vector

By designing primers with BamH I and Xho I restriction sites, the cloned IaAO2 gene was inserted between the BamH I and Xho I restriction sites of the yeast expression vector pESC-TRP (Stratagene) by PCR. , the recombinant plasmid pESC-TRP-IaAO2 was obtained and verified by sequencing.

The nucleotide sequence of the IaAO2 gene is shown in SEQ ID No.1; the encoded amino acid sequence is shown in SEQ ID No.2.

The specific primer sequences are: the upstream primer IaAO2F is shown in SEQ ID No.3; the downstream primer IaAO2R is shown in SEQ ID No.4.

2. Induced expression

The recombinant plasmid pESC-TRP-IaAO2 was transformed into Saccharomyces cerevisiae WAT11tfAX (WAT11, trp1::PGAP1-SctHMGR1-TCYC1, ura3::PGAP1-ScERG20-TCYC1, leu2::PGAP1-Se ACSL641P-TCYC1, his3 ::PTEF1-IaAS1-TCYC1), recombinant yeast were obtained. The positive recombinants were inoculated into 15mL SC-T liquid medium (containing glucose), incubated at 30°C and 225rpm for 36h, then replaced with SC-T sugar-free medium for 4h, and then a certain amount of yeast was transferred to the In 100 mL of SC-T liquid medium (containing galactose), the OD600 value of the initial bacterial solution was 0.1, and the induction culture was carried out, and the protein expression was induced with galactose.

Example 2 Homologous gene IaAO1 and 5'-terminal truncated new gene tIaAO1

In our laboratory, another balsinol C-28 oxidase gene IaAO1 was cloned and identified from the medicinal plant Gangmei, and a Saccharomyces cerevisiae expression vector pESC-TRP-IaAO1 was constructed. Comparing the sequences of IaAO2 and the homologous gene IaAO1, the latter has an extra nucleotide fragment at the 5'-end than the former. According to the overlapping sequences of IaAO2 and IaAO1, specific primers were designed, and pESC-TRP-IaAO1 was used as a template for PCR amplification. Agarose gel electrophoresis showed that a specific fragment appeared at about 1200bp, and agarose gel recovery kit ( Magen) recovered the target fragment, cloned it into the pEASY vector (full gold), identified positive clones and performed sequencing verification for the construction of recombinant expression vectors.

1. DNA sequence alignment

Comparing the sequences of IaAO2 and the homologous gene IaAO1, it was confirmed that the partial sequences overlapped. In addition, there was an extra nucleotide fragment of 198 bp at the 5'-end of IaAO1.

2. Construction of yeast expression vector

According to the overlapping sequences of the two genes, primers with BamH I and Xho I restriction sites were designed, and the cloned truncated gene tIaAO1 gene was inserted into the BamHI of the yeast expression vector pESC-TRP (Stratagene) by PCR. The recombinant plasmid pESC-TRP-tIaAO1 was obtained and verified by sequencing.

The nucleotide sequence of tIaAO1 gene is shown in SEQ ID No.5; its amino acid sequence is shown in SEQ ID No.6.

The specific primer sequences are: the upstream primer tIaAO1F is shown in SEQ ID No.7; the downstream primer tIaAO1R is shown in SEQ ID No.8.

3. Induced expression

The recombinant plasmid pESC-TRP-tIaAO1 was transformed into Saccharomyces cerevisiae WAT11tfAX (WAT11, trp1::PGAP1-SctHMGR1-TCYC1, ura3::PGAP1-ScERG20-TCYC1, leu2::PGAP1-Se ACSL641P-TCYC1, his3 ::PTEF1-IaAS1-TCYC1), recombinant yeast were obtained. The positive recombinants were inoculated into 15mL SC-T liquid medium (containing glucose), incubated at 30°C and 225rpm for 36h, then replaced with SC-T sugar-free medium for 4h, and then a certain amount of yeast was transferred to the In 100 mL of SC-T liquid medium (containing galactose), the OD600 value of the initial bacterial solution was 0.1, and the induction culture was carried out, and the protein expression was induced with galactose.

The following is the content of the effect verification test example.

Test Example 1 Eukaryotic expression and functional analysis of IaAO2 gene

Functional identification of IaAO2 protein:

Collect the yeast cells that have been cultured for 7 days, add the extract (40% KOH: anhydrous ethanol = 1:1), heat and reflux to extract the metabolites, extract the extract three times with an equal volume of n-hexane, and separate the n-hexane layer, It was evaporated to dryness, and the residue was derivatized with BSTFA silylation reagent and detected by GC-MS.

The GC-MS instrument was 7890B-5977B (Agilent), and the gas phase conditions were that the inlet temperature was 250 °C; the injection volume was 1 μL, splitless; the chromatographic column was HP-5MS (30m×0.25mm×0.25μm, Agilent, USA) ; The heating program of the column oven starts at 150 °C, maintains for 1 min, rises to 300 °C at 10 °C/min, and holds for 20 min; the carrier gas is He, and the column flow rate is 0.7 mL/min. Mass spectrometry conditions: EI ion source, electron energy 70 eV, ion scanning range 50-600 m/z, quadrupole temperature 150 °C, ion source temperature 230 °C, GC-MS interface temperature 280 °C, and the compound search library was NIST14. L.

The GC-MS analysis results are shown in Figure 2. The analysis results show that the metabolites of yeast cells transformed with pESC-TRP empty load have a chromatographic peak of α-amyrin (Peak 1) when the retention time is 13.8min; Among the recombinant yeast metabolites, in addition to the chromatographic peak of the substrate α-amyrin (Peak 1) at the retention time of 13.8min, four new peaks appeared at the retention times of 15.7min, 16.2min, 17.0min and 17.3min. The chromatographic peaks Peak 2, Peak 3, Peak 4 and Peak 5 of , through spectral library search, four new compounds were preliminarily confirmed to be arbutol (94% matching degree), oleanolic acid (93% matching degree), Ursolic acid (95% matching degree) and ursolic aldehyde (99% matching degree), and the retention times of Peak 3 and Peak 4 are basically the same as those of oleanolic acid and ursolic acid standards, respectively. Mass spectrometry ion fragmentation Also consistent with the standard. It can be determined that the IaAO2 gene is a triterpene C-28 oxidase gene, and its encoded protein can oxidize the substrates α-amyrin and β-amyrin to ursolic acid and oleanolic acid, respectively.

Test Example 2 Eukaryotic expression and functional analysis of tIaAO1 gene obtained by 5'-terminal truncation

Functional identification of tIaAO1 protein obtained by 5'-terminal truncation:

Collect the yeast cells, add the extract (40% KOH: anhydrous ethanol = 1:1), heat and reflux to extract the metabolites, extract the extract three times with an equal volume of n-hexane, separate the n-hexane layer, evaporate to dryness, and the residue Analysis was performed by GC-MS after derivatization with BSTFA silylating reagent.

The GC-MS instrument was 7890B-5977B (Agilent), and the gas phase conditions were that the inlet temperature was 250 °C; the injection volume was 1 μL, splitless; the chromatographic column was HP-5MS (30m×0.25mm×0.25μm, Agilent, USA) ; The heating program of the column oven starts at 150 °C, maintains for 1 min, rises to 300 °C at 10 °C/min, and holds for 20 min; the carrier gas is He, and the column flow rate is 0.7 mL/min. Mass spectrometry conditions: EI ion source, electron energy 70 eV, quadrupole temperature 150 °C, ion source temperature 230 °C, GC-MS connection port temperature 280 °C, the compound search library is NIST14.L, SIM mode detection, specific qualitative and quantitative The ions are listed in Table 1 below.

Table 1 Qualitative and quantitative ions detected by GC-MS

compound Quantitative ion (m/z) Qualitative ion (m/z) β-Aryl Resinol 218.2 189.2, 203.2 Alpha-aromatic resin alcohol 218.2 189.2, 203.2 Oleanolic acid 203.2 189.2, 320.2 Ursolic acid 203.2 189.2, 320.2

Agilent MassHunter Quantitative Analysis quantitative software was used to calculate the contents of α-aromaticresinol, β-aromaticresinol, ursolic acid and oleanolic acid in the mixed standard and yeast extract. The results are shown in Table 2 below, showing that the transformed Saccharomyces cerevisiae with either IaAO1 or tIaAO1 gene produced ursolic acid and oleanolic acid, indicating that the truncated tIaAO1 gene based on the IaAO2 sequence retained the original function.

Table 2 Changes in the content of transgenic Saccharomyces cerevisiae (μg/L)

strain Alpha-aromatic resin alcohol β-Aryl Resinol Ursolic acid Oleanolic acid WAT11tfAX/pESC-TRP-IaAO1 129.76 24.72 15.97 731.70 WAT11tfAX/pESC-TRP-tIaAO1 272.85 45.63 23.21 421.16

The present invention is not limited to the above-mentioned embodiments. If various changes or modifications of the present invention do not depart from the spirit and scope of the present invention, if these changes and modifications belong to the claims of the present invention and the equivalent technical scope, the present invention also It is intended to include these changes and variations.

sequence listing

<110> Guangzhou University of Traditional Chinese Medicine

<120> CYP450 Gene Catalyzing the C-28 Oxidation of Arsinol and Its Encoded Product and Application

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Thr Phe Trp Leu Ala Cys Arg Val Phe Val Ser Ile Glu Asp Pro Asn

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Ile Ser Ile Pro Ile Asp Leu Pro Gly Thr Pro Phe His Arg Ala Ile

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Lys Ala Ser Asn Phe Ile Arg Lys Glu Leu Val Arg Ile Ile Lys Gln

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Arg Lys Val Asp Leu Ala Glu Gly Lys Ala Ser Pro Thr Gln Asp Ile

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Leu Ser His Met Leu Leu Thr Ser Asp Glu Asn Gly Lys Phe Met Ala

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Asp Thr Ala Ser Ser Ala Cys Cys Phe Ile Val Lys Tyr Leu Ala Glu

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Asn Ser Lys Ala Pro Gly Glu Leu Leu Asn Trp Glu Asp Ile Gln Lys

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Met Lys Tyr Ser Trp Asn Val Ala Cys Glu Val Leu Arg Leu Ala Pro

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Pro Leu Gln Gly Ala Phe Arg Glu Ala Leu Thr Asp Phe Met Tyr Asn

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Gly Phe Ser Ile Pro Lys Gly Trp Lys Ile Tyr Trp Ser Ala Asn Ser

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Thr His Arg Asn Pro Glu Phe Phe Pro Glu Pro Leu Lys Phe Asp Pro

325 330 335

Ser Arg Phe Glu Gly Ser Gly Pro Ala Pro Tyr Thr Phe Val Pro Phe

340 345 350

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

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Ile Leu Val Phe Met His His Leu Val Lys Arg Phe Arg Trp Glu Lys

370 375 380

Leu Ile Pro Asp Glu Lys Ile Val Val Asn Pro Met Pro Ile Pro Glu

385 390 395 400

Lys Gly Leu Pro Val Arg Leu Tyr Pro His Lys Ala

405 410

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aaggcctcca acttcatcag aaaagagctt gtttctatca tcaagcaaag aaagattgat 540

ttggcagagg gaaaggcatc gccaacacaa gatattatgt cacatatgtt attgacgagc 600

gatgagaatg gcaagttcat gactgagttg gatgtagctg ataagatttt gggtttgttg 660

attggtggcc atgacactgc tagctctgcc tgcactttcg tcgtcaagtt tcttgctgaa 720

ctgcctgaag tctacgaggg tgtctacaag gagcaaatcg aaatcgcgaa ctcgaaagct 780

ccaggtgaac tgttgaattg ggatgacatt caaaagatga ggtattcatg gaacgtagca 840

tgtgaagttc taagacttgc tccacccctc cagggagctt tcagagaagc cctagttgat 900

ttcatgtata atggtttctc tattccaaaa gggtggaaga tatattggag cgcaaactca 960

acacatagaa atcctgaaaa ttttcccgag cccttaaaat ttgatccctc gagatttgat 1020

gggaatggac ctgctcctta cacatttgtg cctttcggtg gaggacctag gatgtgtccc 1080

ggaaaagagt atgctagatt ggaaatactt gttttcatgc accatattgt gaaaaggttc 1140

aggtgggaga aagtgattcc ggatgagaag attgttgttg atcccatgcc tatcccagcc 1200

aagggacttc cagttcgtct ctatccgcac aaagcagcag cttaa 1245

<210> 6

<211> 415

<212> PRT

<213> Artificial Sequence

<400> 6

Met Ala Lys Tyr Ser Ser Thr Val Phe Lys Thr Ser Leu Leu Gly Glu

1 5 10 15

Gln Ala Ala Val Phe Cys Gly Ser Gly Gly Asn Lys Phe Leu Phe Ser

20 25 30

Asn Glu Asn Lys Leu Val Gln Ala Trp Trp Pro Asn Ser Val Asn Lys

35 40 45

Val Phe Pro Ser Ser Gln Gln Thr Ser Ser Lys Glu Glu Ala Ile Lys

50 55 60

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

65 70 75 80

Tyr Ile Gly Ile Met Asp His Ile Ala Gln Arg His Phe Val Ser Ala

85 90 95

Trp Glu Asn Lys Asp Glu Val Val Val Phe Pro Leu Ala Lys Asn Tyr

100 105 110

Thr Phe Trp Leu Ala Ala Arg Leu Phe Val Ser Val Glu Asp Pro Ile

115 120 125

His Val Ala Lys Leu Gly Asp Pro Phe Ala Val Leu Ala Ser Gly Leu

130 135 140

Ile Ser Ile Pro Ile Asp Leu Pro Gly Thr Pro Phe Asn Arg Ala Ile

145 150 155 160

Lys Ala Ser Asn Phe Ile Arg Lys Glu Leu Val Ser Ile Ile Lys Gln

165 170 175

Arg Lys Ile Asp Leu Ala Glu Gly Lys Ala Ser Pro Thr Gln Asp Ile

180 185 190

Met Ser His Met Leu Leu Thr Ser Asp Glu Asn Gly Lys Phe Met Thr

195 200 205

Glu Leu Asp Val Ala Asp Lys Ile Leu Gly Leu Leu Ile Gly Gly His

210 215 220

Asp Thr Ala Ser Ser Ala Cys Thr Phe Val Val Lys Phe Leu Ala Glu

225 230 235 240

Leu Pro Glu Val Tyr Glu Gly Val Tyr Lys Glu Gln Ile Glu Ile Ala

245 250 255

Asn Ser Lys Ala Pro Gly Glu Leu Leu Asn Trp Asp Asp Ile Gln Lys

260 265 270

Met Arg Tyr Ser Trp Asn Val Ala Cys Glu Val Leu Arg Leu Ala Pro

275 280 285

Pro Leu Gln Gly Ala Phe Arg Glu Ala Leu Val Asp Phe Met Tyr Asn

290 295 300

Gly Phe Ser Ile Pro Lys Gly Trp Lys Ile Tyr Trp Ser Ala Asn Ser

305 310 315 320

Thr His Arg Asn Pro Glu Asn Phe Pro Glu Pro Leu Lys Phe Asp Pro

325 330 335

Ser Arg Phe Asp Gly Asn Gly Pro Ala Pro Tyr Thr Phe Val Pro Phe

340 345 350

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

355 360 365

Ile Leu Val Phe Met His His Ile Val Lys Arg Phe Arg Trp Glu Lys

370 375 380

Val Ile Pro Asp Glu Lys Ile Val Val Asp Pro Met Pro Ile Pro Ala

385 390 395 400

Lys Gly Leu Pro Val Arg Leu Tyr Pro His Lys Ala Ala Ala Glu

405 410 415

<210> 7

<211> 49

<212> DNA

<213> Artificial Sequence

<400> 7

cgtcaaggag aaaaaaaccc cggatccatg gccaagtact cgtccaccg 49

<210> 8

<211> 70

<212> DNA

<213> Artificial Sequence

<400> 8

tagccgcggt accaagctta ctcgaggtta atgatgatga tgatgatgag ctgctgcttt 60

gtgcggatag 70

Claims (10)

1. CYP450 gene IaAO2 of catalyzing balsamic resin alcohol C-28 oxidation, it is characterized in that: nucleotide sequence is shown in any one of following 1)-5): 1) The nucleotide sequence shown in SEQ ID No. 1; or 2) A nucleotide sequence encoding the amino acid sequence shown in SEQ ID No. 2; or 3) a nucleotide sequence capable of hybridizing with the complementary sequence of SEQ ID No. 1, and the protein encoded by the nucleotide sequence still has the function of catalyzing the oxidation of balsamic resin at the C-28 position; or 4) A nucleotide sequence having at least 75% or more homology with the nucleotide sequence shown in SEQ ID No. 1; or 5) A nucleotide sequence variant in which one or more bases are deleted, substituted or inserted on the basis of the nucleotide sequence shown in SEQ ID No. 1, and the nucleotide sequence variant encoded The protein still has the function or activity of catalyzing the C-28 oxidation of balsamic resin. 2. a protein that catalyzes the C-28 oxidation of balsamic resin, characterized in that: amino acid sequence is shown in any one of 1) or 2): 1) the amino acid residue sequence shown in SEQ ID No.2; or 2) A protein with the function or activity of catalyzing the C-28 oxidation of balsamyl alcohol derived from the amino acid sequence shown in SEQ ID No.2 by replacement, deletion or/and insertion of one or more amino acid residues Variants. 3. a specific primer for the CYP450 gene IaAO2 of the C-28 oxidation of catalyzed balsamic resin, is characterized in that: the upstream primer sequence IaAO2F of the specific primer is as shown in SEQ ID No.3; downstream The primer sequence IaAO2R is shown in SEQ ID No.4. 4. An expression vector comprising the nucleotide sequence of claim 1. 5. A host bacterium comprising the nucleotide sequence of claim 1. 6. The application of the nucleotide sequence of claim 1 in the preparation of triterpenoids. 7. The application of the nucleotide sequence of claim 1 in the preparation of a compound that catalyzes the oxidation of pentacyclic triterpenes at C-28 position. 8. The application of the nucleotide sequence according to claim 1 in the preparation of compounds that catalyze the oxidation of pentacyclic triterpenes α-arosinol and β-arosinol. 9. Based on the nucleotide sequence feature according to claim 1, carry out the genetic modification method of homologous gene 5 '-end truncation, and described genetic modification method is based on sequence comparison, by but not limited to DNA restriction Endonuclease or PCR to obtain new genes with 5'-end truncated. 10. The gene tIaAO1 obtained by the genetic modification method of homologous gene 5'-end truncated, is characterized in that: The nucleotide sequence of the tIaAO1 gene is shown in SEQ ID No.5; the encoded amino acid sequence is shown in SEQ ID No.6; The specific primer sequences are: the upstream primer tIaAO1F is shown in SEQ ID No.7; the downstream primer tIaAO1R is shown in SEQ ID No.8.

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