CN117924513A - Blumea balsamifera terpene synthase AaTPS and coding gene and application thereof - Google Patents

Blumea balsamifera terpene synthase AaTPS and coding gene and application thereof Download PDF

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CN117924513A
CN117924513A CN202311013825.2A CN202311013825A CN117924513A CN 117924513 A CN117924513 A CN 117924513A CN 202311013825 A CN202311013825 A CN 202311013825A CN 117924513 A CN117924513 A CN 117924513A
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aatps
protein
mugwort
terpene synthase
myrcene
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马蕊
桂新景
陈随清
张宝
薛淑娟
练从龙
刘英子
朱晔
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Henan University of Traditional Chinese Medicine HUTCM
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Abstract

The invention relates to a mugwort terpene synthase AaTPS and a coding gene and application thereof, which provide an important basis for improving the content of active ingredient myrcene, (trans) -beta-farnesene in mugwort or directly producing myrcene, (trans) -beta-farnesene by utilizing a genetic engineering technology, and the technical scheme is that the mugwort terpene synthase AaTPS is protein of the following a) or b) or c): a) The amino acid sequence is a protein shown as SEQ ID No. 1; b) A fusion protein obtained by connecting a tag to the N-terminal and/or C-terminal of the protein shown in SEQ ID No. 1; c) The AaTPS protein with the same function is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown in SEQ ID No.1, can catalyze GPP to form myrcene, can catalyze FPP to form (trans) -beta-farnesene, and has an important effect on biosynthesis of myrcene, (trans) -beta-farnesene and other terpenoid compounds in mugwort leaves.

Description

Blumea balsamifera terpene synthase AaTPS and coding gene and application thereof
1. Technical field
The invention relates to the field of medicinal plant genetic engineering, in particular to a mugwort leaf terpenoid synthase AaTPS and a coding gene and application thereof.
2. Background art
The mugwort leaf is a dried leaf of Artemisia argyi ARTEMISIA ARGYI Levl et vant of Artemisia of Compositae, and is usually picked and dried in the sun when flowers are not blooming in summer and leaves are flourishing. Pungent and bitter taste, warm nature, small toxicity, and has the effects of regulating qi and blood, expelling cold and dampness, warming channel, stopping bleeding, preventing miscarriage, etc. In China Ai Zhu is produced in Henan, hubei, hunan, anhui, shandong, hebei and other provinces. "Tangyin North moxa", "south-yang moxa", hebei "Qihai" and Hubei "Qi" in Henan are known for their excellent quality. The volatile oil of the mugwort leaves is the main material foundation for the mugwort to exert the curative effect, and has obvious effects of resisting bacteria, resisting viruses, resisting inflammation, inducing resuscitation, relieving swelling, relieving pain, relieving cough and asthma, and the like.
Myrcene (Myrcene), also known as myrcene, is known for its pharmaceutical properties such as analgesic, antioxidant, anti-inflammatory, antibacterial, analgesic and melanin activity inhibiting and antioxidant effects. Myrcene is also an important industrial raw material, and is widely used in the perfume industry as a precursor of various high-value synthetic fragrances, and can also be used for synthesizing vitamin E. In addition, the laurene has high polymerization activity, can be polymerized to form a rubber material with excellent performance, and has important application value.
(Trans) -beta-farnesene ((E) -beta-FARNESENE), also known as (trans) -beta-farnesene, is the main component of mugwort leaf volatile oil. The (trans) -beta-farnesene is used as aphid alarm information element, which not only can attract aphids from habitat and actively contact with insecticide, reduce the usage amount of insecticide, but also can attract natural enemies of the aphids, and avoid the use of insecticide. Recent research progress shows that (trans) -beta-farnesene is a specific activator of mosquito odor receptors, can be used as a high-efficiency synergist for expelling and killing mosquitoes, and plays an important role in plant disease defense as insect pheromone.
Myrcene is an acyclic monoterpene compound, and (trans) -beta-farnesene belongs to acyclic sesquiterpenes. The general substrate of terpenes, isopentenyl pyrophosphate (Isopentenyl pyrophosphate, IPP) and its isomer DIMETHYLALLYL PYROPHOSPHATE (DMAPP), are produced by the cytoplasmic mevalonate pathway (mevalonic acid (MVA) pathway) and the plastid 2-methyl-D-erythritol-4-phosphate (MEP) pathway, from which the monoterpene precursor geranyl pyrophosphate (Geranyl diphosphate, GPP), the sesquiterpene precursor farnesyl pyrophosphate (farnesyl diphosphate, FPP) and the mugwort terpene synthase AaTPS are produced, which is capable of catalyzing both the formation of myrcene by GPP and the formation of (trans) - β -farnesene by FPP. The development of new drugs from the active ingredients of traditional Chinese medicines is a potential way, however, the development of the new drugs is greatly limited because the growth of plants is slow, the content of the active ingredients in the plants is not large, the extraction and separation processes are complicated and environmental pollution is easy to cause. By searching and explaining the biosynthesis way and the regulation and control mechanism of the terpenoid components in the mugwort leaf, the preparation method is beneficial to providing a theoretical basis for the formation of medicinal material quality, and simultaneously brings wide application space for improving the content of target components or directly producing effective components by using biotechnology. The gene clone and function verification of the artemisia argyi terpene synthase AaTPS provide an important basis for improving the content of active ingredient myrcene, (trans) -beta-farnesene in the artemisia argyi or directly producing myrcene, (trans) -beta-farnesene by utilizing a genetic engineering technology, and the artemisia argyi terpene synthase AaTPS gene and the amino acid sequence thereof are not disclosed or reported before the invention is disclosed.
3. Summary of the invention
Aiming at the situation, the invention aims to solve the defects in the prior art, and provides the mugwort leaf terpene synthase AaTPS, the coding gene and the application thereof, which provide an important basis for improving the content of active ingredient myrcene, (trans) -beta-farnesene in mugwort leaves or directly producing myrcene, (trans) -beta-farnesene by utilizing a genetic engineering technology.
The technical scheme is that the blumea balsamifera terpene synthase AaTPS is protein of the following a) or b) or c):
a) The amino acid sequence is a protein shown as SEQ ID No. 1;
b) A fusion protein obtained by connecting a tag to the N-terminal and/or C-terminal of the protein shown in SEQ ID No. 1;
c) The protein with the same function is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown in SEQ ID No. 1.
Wherein SEQ ID No.1 consists of 575 amino acid residues.
In order to facilitate purification of the protein of a), the amino-terminal or carboxyl-terminal linkage of the protein shown in SEQ ID No.1 of the sequence Listing may be provided with the tag shown in Table 1.
TABLE 1 sequence of tags
Label (Label) Residues Sequence(s)
Poly-Arg 5-6 (Usually 5) RRRRR
Poly-His 2-10 (Usually 6) HHHHHH
FLAG 8 DYKDDDDK
Strep-tag II 8 WSHPQFEK
c-myc 10 EQKLISEEDL
The protein of c) above, wherein the substitution and/or deletion and/or addition of one or several amino acid residues is a substitution and/or deletion and/or addition of not more than 10 amino acid residues.
The protein in the c) can be synthesized artificially or can be obtained by synthesizing the coding gene and then biologically expressing.
The coding gene of the protein in c) can be obtained by deleting one or more amino acid residues in the DNA sequence shown in the 1 st to 1728 th positions of SEQ ID No.2 and/or carrying out one or more base pair missense mutations and/or linking the coding sequences of the tags shown in the table 1 at the 5 'end and/or the 3' end.
It is a second object of the present invention to provide biological materials related to AaTPS proteins.
The biological material related to AaTPS protein provided by the invention is any one of the following A1) to A12):
A1 A nucleic acid molecule encoding AaTPS48,48 proteins;
A2 An expression cassette comprising A1) said nucleic acid molecule;
a3 A) a recombinant vector comprising the nucleic acid molecule of A1);
a4 A recombinant vector comprising the expression cassette of A2);
a5 A) a recombinant microorganism comprising the nucleic acid molecule of A1);
A6 A) a recombinant microorganism comprising the expression cassette of A2);
a7 A) a recombinant microorganism comprising the recombinant vector of A3);
a8 A) a recombinant microorganism comprising the recombinant vector of A4);
A9 A transgenic plant cell line comprising the nucleic acid molecule of A1);
a10 A transgenic plant cell line comprising the expression cassette of A2);
A11 A transgenic plant cell line comprising the recombinant vector of A3);
A12 A) a transgenic plant cell line comprising the recombinant vector of A4).
In the above biological material, the nucleic acid molecule of A1) is a gene as shown in the following 1), 2) or 3):
1) The coding sequence is cDNA molecule shown in SEQ ID No.2 at positions 1-1728;
2) A cDNA molecule or a genomic DNA molecule having 75% or more identity to the nucleotide sequence defined in 1) and encoding a AaTPS protein;
3) Hybridizing under stringent conditions to the nucleotide sequence defined in 1) or 2) and encoding a AaTPS protein, a cDNA molecule or a genomic DNA molecule.
Wherein the nucleic acid molecule may be DNA, such as cDNA, genomic DNA, or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA, etc.
The nucleotide sequence encoding AaTPS of the present invention may be readily mutated by one of ordinary skill in the art using known methods, such as directed evolution and point mutation. Those artificially modified nucleotides having 75% or more identity to the nucleotide sequence encoding AaTPS48 are derived from the nucleotide sequence of the present invention and are equivalent to the sequence of the present invention as long as they encode AaTPS and have the same function.
The term "identity" as used herein refers to sequence similarity to a native nucleic acid sequence. "identity" includes a nucleotide sequence having 75% or more, or 85% or more, or 90% or more, or 95% or more identity with the nucleotide sequence of the protein consisting of the amino acid sequence shown in SEQ ID No.1 of the present invention. Identity can be assessed visually or by computer software. Using computer software, the identity between two or more sequences can be expressed in percent (%), which can be used to evaluate the identity between related sequences.
The 75% or more identity may be 80%, 85%, 90% or 95% or more identity.
In the biological material, the stringent conditions are hybridization and washing the membrane 2 times at 68 ℃ in a solution of 2 XSSC, 0.1% SDS for 5min each time, and hybridization and washing the membrane 2 times at 68 ℃ in a solution of 0.5 XSSC, 0.1% SDS for 15min each time; alternatively, hybridization and washing of the membrane were performed at 65℃in a solution of 0.1 XSSPE (or 0.1 XSSC) and 0.1% SDS.
In the above biological materials, the expression cassette (AaTPS gene expression cassette) described in A2) containing the nucleic acid molecule encoding AaTPS and 48 refers to a DNA capable of expressing AaTPS in a host cell, which may include not only a promoter for initiating AaTPS transcription but also a terminator for terminating AaTPS transcription. Further, the expression cassette may also include an enhancer sequence.
In the above biological material, the vector may be a plasmid, cosmid, phage or viral vector.
In the above biological material, the microorganism may be yeast, bacteria, algae or fungi, such as agrobacterium.
In the above biological material, the transgenic plant cell line does not include propagation material.
A third object of the present invention is to provide a novel use of AaTPS protein.
The invention provides an application of AaTPS protein in terpene synthase or terpene compound synthesis.
The present invention provides the use of AaTPS protein as a catalyst for the formation of myrcene in geranyl pyrophosphate (GPP) or for the formation of (trans) -beta-farnesene in farnesyl pyrophosphate (FPP).
A fourth object of the present invention is to provide a novel use of the above-mentioned related biological material.
The invention provides application of the related biological material in preparing terpene synthase or synthesizing terpene compounds.
The invention provides application of the related biological material in catalyzing myrcenyl pyrophosphate (GPP) to form myrcene or catalyzing farnesyl pyrophosphate (FPP) to form (trans) -beta-farnesene.
In the above-mentioned application,
The terpenoid is a monoterpene and/or a sesquiterpene;
the monoterpene compound is myrcene, and the sesquiterpene compound is (trans) -beta-farnesene.
The last aim of the invention is to provide a method for synthesizing terpenoid.
The synthesis method of the terpenoid provided by the invention comprises the following steps: and uniformly mixing AaTPS protein, a substrate and an enzymatic buffer solution, and reacting to obtain the terpenoid.
In the above-mentioned method, the method comprises,
The mass ratio of AaTPS protein to substrate is 2:1, a step of;
in the above-mentioned method, the method comprises,
The enzymatic buffer solution consists of HEPES, mgCl 2, PMSF and DTT;
the concentration of HEPES in the enzymatic buffer is 50mM;
the concentration of MgCl 2 in the enzymatic buffer is 10mM;
the concentration of PMSF in the enzymatic buffer is 1mM;
the concentration of the DTT in the enzymatic buffer is 5mM;
The pH value in the enzymatic buffer is 7.0.
In the above-mentioned method, the method comprises,
The substrate is geranyl pyrophosphate or farnesyl pyrophosphate;
The terpenoid is a monoterpene and/or a sesquiterpene;
the monoterpene compound is myrcene, and the sesquiterpene compound is (trans) -beta-farnesene.
The AaTPS gene is cloned from the mugwort cDNA, and is a key enzyme gene which is obtained from mugwort for the first time and can catalyze GPP to form monoterpene components and FPP to form sesquiterpene components for synthesis. Experiments prove that: the AaTPS protein can catalyze GPP to form myrcene (Myrcene) and FPP to form (trans) -beta-farnesene ((E) -beta-FARNESENE), has important effects on biosynthesis of myrcene, (trans) -beta-farnesene and other terpenoids in mugwort leaves, and has important theoretical and practical significance on regulation and production of plant terpenoid and cultivation of high-quality mugwort leaves.
4. Description of the drawings
FIG. 1 shows agarose gel electrophoresis of the gene clone of Artemisia princeps argyi AaTPS of the invention. M represents Trans2K DNA MARKER (nucleic acid molecular weight standard, band from top to bottom 2000, 1000, 750, 500, 250, 100bp respectively).
FIG. 2 shows the AaTPS protein expressed in E.coli by polyacrylamide gel electrophoresis (SDS-PAGE) analysis according to the invention. Lane 1 shows the molecular weight of the protein, bands 180, 135, 100, 75, 63, 48, 35kDa from top to bottom; lane 2 is recombinant plasmid pET32a AaTPS expressing the protein of interest, arrow indicating the protein of interest; lane 3 is negative control empty plasmid pET32a.
FIG. 3 is a GC-MS analysis of the enzymatic reaction product of the invention AaTPS. FIG. 3A is an extracted ion flow diagram of a standard Myrcene and AaTPS48 catalytic substrate GPP formation product; FIG. 3B is an extracted ion flow diagram of the product of the catalytic substrate FPP formation of standard (E) -beta-FARNESENE and AaTPS 48; fig. 3C is a mass spectrum of standard Myrcene, and fig. 3D is a mass spectrum of AaTPS48 catalytic substrate GPP formed product; fig. 3E is a mass spectrum of standard (E) - β -FARNESENE, and fig. 3F is a mass spectrum of AaTPS for the catalytic substrate FPP to form the product.
FIG. 4 shows the fermentative production of the yeast strain MD-Aa48 of the invention (recombinant plasmid pESC-Leu:: aaTPS, obtained by introducing the yeast strain MD) Myrcene.
5. Detailed description of the preferred embodiments
The following describes the embodiments of the present invention in further detail with reference to the drawings.
The test methods used in the present invention, unless otherwise specified, are all common general knowledge to those skilled in the art, and various buffer solutions, detection reagents, etc., if otherwise specified, are commercially available or prepared by conventional experimental methods.
In the following testQ5 2X Master Mix, bamHI restriction endonuclease was a product of NEW ENGLAND Biolabs;
the rapid universal plant RNA extraction kit is a product of Beijing Hua Vietnam biotechnology Co., ltd;
Trans ScriptⅡTwo-Step RT-PCR Super Mix、pEASY-Blunt Zero Cloning Kit、Trans2K DNA Marker、pEASY-Basic Seamless Cloning and Assembly Kit、 Coli competent cells Trans1-T1 and TRANSETTA (DE 3), pET32a (+) vector, proteinIso Ni-NTA Resin are products of Beijing full gold biotechnology Co., ltd;
The rainbow 180 broad-spectrum protein Marker is a product of Solarbio company;
Geranyl pyrophosphate GPP is a product of Sigma company with catalog number G6772 and CAS number 763-10-0;
farnesyl pyrophosphate FPP is a product of Sigma company, catalog number F6892, CAS number 13058-04-3;
Myrcene (Myrcene) is a product from Sigma company, catalog number 6464643, CAS number 123-35-3;
(trans) -beta-farnesene is a product of Source leaf company under the catalog number S25161 and CAS number 18794-84-8.
1. Full-length cDNA sequence clone of blumea AaTPS gene
1. Extraction of Total RNA
The method is operated according to the instruction of a rapid universal plant RNA extraction kit of Beijing Hua Vietnam biotechnology Co Ltd, and total RNA of folium artemisiae argyi leaves is extracted.
2. Synthesis of first strand cDNA
The reverse transcription reaction system was operated according to the first strand cDNA synthesis kit TRANS SCRIPT II Two-Step RT-PCR Super Mix instruction of Beijing full gold Biotechnology Co., ltd, and shown in Table 1:
TABLE 1 reverse transcription reaction system
(1) The reverse transcription reaction was performed under the following conditions: reacting for 10min at 25 ℃ and 30min at 50 ℃; heating at 85 ℃ for 5s Zhong Shihuo;
(4) The cDNA samples were stored at-20 ℃.
3. Primer design
According to the transcriptome data of folium artemisiae argyi leaves, aaTPS gene Open Reading Frame (ORF) sequences are obtained, and clone primers AaTPS-48-F and AaTPS-R are designed based on the sequence, wherein the sequences of the primers are as follows:
AaTPS48-F：5’-ATGTCAACTACTATTCCTGTTTCTAGT-3’；
AaTPS48-R：5’-TTAGACAACCATAGGGTGAACGAAG-3’。
4. PCR amplification
Using the cDNA obtained in the step 2 as a template, and adopting high-fidelity enzymeThe primers Q5 2X Master Mix, aaTPS-F and AaTPS-R were PCR amplified to give PCR amplified products, the results of which are shown in FIG. 1, and the PCR amplified products were sequenced. The PCR reaction procedure was as follows:
PCR reaction procedure: pre-denaturation at 98℃for 1min;98℃for 20s,55℃for 20s,72℃for 90s,40 cycles; extending at 72℃for 10min.
Sequencing results showed that: the sequence of PCR amplified product is shown as SEQ ID No.2, the gene shown as SEQ ID No.2 is named AaTPS to code protein composed of 575 amino acid residues, the protein is named AaTPS to 48, and the amino acid sequence of the protein is shown as SEQ ID No.1.
2. Obtaining of Artemisia argyi AaTPS protein
1. Construction of recombinant vectors
The DNA fragment shown in the 1 st to 1728 th positions of SEQ ID No.2 is constructed to a BamHI enzyme cutting site of a pET32a (+) vector (full gold biotechnology Co., ltd.) by adopting pEASY-Basic Seamless Cloning and Assembly Kit of Beijing full gold biotechnology Co., ltd.) and other sequences of the pET32a (+) vector are kept unchanged, so that recombinant plasmid pET32a: aaTPS48 with the primer sequences as follows (the sequences shown by underlines are vector homologous regions) is obtained:
AaTPS48-32a-F：
5’-CCATGGCTGATATCGGAATGTCAACTACTATTCCTGTTTCTAGT-3’；
AaTPS48-32a-R：
5’-ACGGAGCTCGAATTCGGGACAACCATAGGGTGAACGAAG-3’。
2. Recombinant bacterium acquisition
Converting the recombinant plasmid pET32a AaTPS to colibacillus expression strain TRANSETTA (DE 3) (purchased from Beijing full-scale gold biotechnology Co., ltd.) to obtain pET32a AaTPS recombinant strain; simultaneously, a pET32a empty vector without a target gene is used for transforming an escherichia coli expression strain TRANSETTA (DE 3) as a control bacterium.
3. Obtaining recombinant protein AaTPS48
PET32a is picked up, aaTPS recombinant bacteria and control bacteria are respectively inoculated in 5mL of LB liquid medium (100 mg/L ampicillin) and cultured overnight at 37 ℃ in a shaking way. The following day is 1:40 dilution is added into 200mL LB liquid medium, shaking culture is carried out at 37 ℃ until OD 600 is 0.6-0.8, shaking is carried out at 16 ℃ for 1.5 hours, IPTG is added to the final concentration of 0.4mM, and shaking culture is continued at 16 ℃ for 18 hours to induce the target protein expression. Centrifuging the bacterial liquid with 8000g for 5min, discarding the supernatant, collecting pET32a (AaTPS) recombinant bacteria and control bacteria, and storing in a refrigerator at-80deg.C.
4. Extraction of recombinant protein AaTPS48
Extracting the protein in the recombinant bacteria of AaTPS and the control bacteria of pET32 a. The method comprises the following specific steps: the pET32a AaTPS recombinant bacteria and control bacteria were resuspended in pre-chilled 5mL HEPES buffer (50mM HEPES,1mM PMSF,5mM DTT,pH 7.0); placing the mixture into ice bath, and performing ultrasonic sterilization (10% power, ultrasonic treatment for 1s at intervals of 3s and lasting for 16min, repeating for 1 time) with 10000g and centrifuging at 4 ℃ for 30min to obtain pET32a (AaTPS) 48 recombinant strain supernatant and control strain supernatant, respectively, namely protein solution.
The results of SDS-PAGE of recombinant supernatants are shown in FIG. 2. As can be seen from the figure, aaTPS recombinant proteins were approximately 85kDa in size, which was consistent with expectations.
3. Analysis of enzymatic Activity of recombinant protein AaTPS48
1. Enzymatic Activity
(1) Preparation of enzymatic reaction System
Taking the protein solution in the step 4 for enzymatic reaction, and taking the supernatant of the control bacteria as a control group. The enzyme reaction systems of the two groups are as follows:
GPP group:
The total system of the enzymatic reaction is 300 mu L, 293 mu L of recombinant bacteria supernatant and control bacteria supernatant are respectively taken, 5.5 mu L of geranyl pyrophosphate (GPP) and 1.5 mu L of MgCl 2 are added and uniformly mixed, the total system of the enzymatic reaction is covered with 300 mu L of normal hexane and sealed, the reaction is stopped by vortex oscillation for 30s after the incubation is finished for 2 hours at 30 ℃, the normal hexane is extracted for 2 times, the normal hexane layer is combined and the extracting solution is blown dry by nitrogen, and 100 mu L of normal hexane is added for dissolution for GC-MS analysis.
FPP group:
The total system of the enzymatic reaction is 300 mu L, 293 mu L of recombinant bacteria supernatant and control bacteria supernatant are respectively taken, 5.5 mu L of geranyl pyrophosphate FPP serving as a substrate and 1.5 mu L of MgCl 2 are added and uniformly mixed, the total system of the enzymatic reaction is covered with 300 mu L of normal hexane and is sealed, the reaction is stopped by vortex oscillation for 30s after the incubation is finished for 2 hours at 30 ℃, the normal hexane is used for extraction for 2 times, the normal hexane layer is combined and the extracting solution is blown dry by nitrogen, and 100 mu L of normal hexane is added for dissolution for GC-MS analysis.
(2) GC-MS analysis
Detecting the target compound by using gas chromatography-mass spectrometry (GC-MS): the GC-MS analysis system is a Thermo TRACE 1310/TSQ 8000gas chromatograph,TG-5MS capillary column (30 m 0.25mm 0.25 μm); programming temperature: the initial temperature of the column is 50 ℃, the temperature is increased to 85 ℃ at a rate of 5 ℃ min -1, and the column is kept for 3min; raising the temperature to 100 ℃ at a rate of 2 ℃ min -1; raising the temperature to 200 ℃ at a rate of 4 ℃ min -1; the temperature was raised to 300℃at a rate of 20℃min -1 and maintained for 5min. The sample inlet temperature was 250 ℃, carrier gas: he, carrier gas flow rate 1mL min -1; and (3) split sample injection, wherein the sample injection amount is 1.0 mu L, the split ratio is 10:1, and the split flow rate is 10mL min -1. The mass spectrum conditions are ionization mode: EI, electron energy: 70eV; the ion source temperature was 250 ℃ and the transmission line temperature was 250 ℃. The scanning mode is full scanning, and the detection range is 40-400 m/z; the solvent was delayed for 3min.
The GC-MS analysis results are shown in FIG. 3: the AaTPS recombinant protein is capable of catalyzing both GPP to form myrcene (Myrcene) and FPP to form (trans) -beta-farnesene ((E) -beta-farnesene) compared to the empty vector enzymatic reaction control.
4. Introducing folium Artemisiae Argyi AaTPS and yeast strain for fermenting Myrcene
1. Strain construction
1. Construction of eukaryotic expression vectors
The DNA fragment shown in the 1 st to 1728 th positions of SEQ ID No.2 is constructed to the BamHI restriction site of a pESC-Leu vector (Agilent company) by adopting pEASY-Uni Seamless Cloning and Assembly Kit of Beijing full-type gold biotechnology Co., ltd, and other sequences of the pESC-Leu vector are kept unchanged, so that a recombinant plasmid pESC-Leu is obtained, wherein AaTPS is obtained, the primer sequence is as follows (the sequence shown by underline is a vector homologous region):
AaTPS48-Leu-F：5’-AAGGAGAAAAAACCCCGATGTCAACTACTATTCCTGTTTCTAGT-3’；
AaTPS48-Leu-R：5’-AGTGAGTCGTATTACGGGACAACCATAGGGTGAACGAAG-3’。
2. Yeast competent preparation
SD-Ura solid plate: SD-Ura+2% glucose+2% agar; the liquid culture medium (SD-Ura liquid culture medium) is prepared without adding agar;
SD-Ura-Leu solid plate: SD-Ura-Leu+2% glucose+2% agar; the corresponding liquid medium (SD-Ura-Leu liquid medium) was obtained without adding agar.
MD yeast (MD yeast is obtained by modifying yeast strain CEN.PK2-1D, CEN.PK2-1D genotype: MAT alpha, URA3-52, TRP1-289, LEU2-3112, HIS3Δ1, MAL2-8C, SUC2; MD yeast genotype ：CEN.PK2-1D,YPRC△15URA3-PGAL1-ERG10-TTPI1-PGAL10-ERG13-TPGI-PGAL1-tHMG1-TADH1-PGAL10-tHMG1-TCYC1-PGAL1-tHMG1-TFBA1-PGAL10-ERG12-TPDC1-PGAL1-ERG8-TRPS2-PGAL10-ERG19-TTDH1-PGAL1-IDI1-TCCW12-PGAL10-ERG20F96W/N127W-TRPL9A) is coated on SD-Ura solid plate, and culturing is performed upside down at 30 ℃ for 48-72h.
The ZYMO RESEARCH Frozen-EZ Yeast Transformation II kit is adopted as yeast competent cells:
(1) Picking a newly activated single colony from an SD-Ura flat plate, inoculating the single colony into 10mL of SD-Ura liquid culture medium, and carrying out shaking culture at 30 ℃ until OD 600 = 0.8-1.0;
(2) Centrifuging at room temperature for 4min at 500g, and removing supernatant;
(3) Adding 10mL of Frozen-EZ Solution 1 suspension thalli, centrifuging at room temperature for 4min at 500g, and removing the supernatant;
(4) Adding 1mL of Frozen-EZ Solution 2 suspension thalli, and subpackaging into sterilized 1.5mL EP tubes, wherein each tube is 50 mu L;
(5) The competent cells were prohibited from being frozen with liquid nitrogen and were slowly cooled to-70deg.C (4deg.C, 1 h), 20 deg.C, 1h, 40 deg.C, 1h, and 70 deg.C.
3. Construction of Yeast Strain MD-Aa48
(1) Mixing 0.2-1 μg recombinant plasmid pESC-Leu:: aaTPS (less than 5 μl) with 50 μLMD competent cells;
(2) Add 500. Mu.L of Frozen-EZ Solution 3 and mix vigorously;
(3) Incubating at 30 ℃ for 1-2h, and uniformly mixing for 2-3 times;
(4) Taking 50-150 mu L of incubated bacterial liquid, coating a corresponding defective SD plate (SD-Ura-Leu), airing, and then placing the bacterial liquid at 30 ℃ for inversion culture for 48-96h.
4. Fermentation
(1) Picking single colony growing on SD-Ura-Leu solid plate, placing in 10mL SD-Ura-Leu liquid culture medium, and 200g 48h at 30deg.C;
(2) The cells were collected by centrifugation at 5000g for 5min at room temperature and transferred to 20mL of an inducible SD-Ura-Leu liquid medium (SD-Ura-Leu+2% galactose) and subjected to induction culture at 30℃for 72h with 200 g.
5. Fermentation product extraction
The target component is terpenoid, fat-soluble and easy to dissolve in ethyl acetate, so that the ethyl acetate is selected as solvent to extract the target terpenoid. The extraction steps are as follows:
(1) Collecting fermentation finished bacterial liquid, and adding ethyl acetate with the same volume;
(2) Ultrasonic sterilization is carried out for 1h, and shaking and mixing are carried out for many times during the period;
(3) Taking an upper organic phase at room temperature of 5000g for 5min, adding a proper amount of anhydrous sodium sulfate (dried for 30min at 120 ℃), shaking while adding water, and removing water from an extract;
(4) Concentrating to near dryness on a rotary evaporator,
(5) Sucking the concentrated solution, filtering with 0.22 μm PTFE needle filter, storing the filtrate in liquid phase vial, sealing with sealing film, and storing in refrigerator at 4deg.C.
5. GC-MS detection of fermentation products
Detecting the target compound by using gas chromatography-mass spectrometry (GC-MS): the GC-MS analysis system is a Thermo TRACE 1310/TSQ 8000gas chromatograph,TG-5MS capillary column (30 m 0.25mm 0.25 μm); programming temperature: the initial temperature of the column is 50 ℃, the temperature is increased to 85 ℃ at a rate of 5 ℃ min -1, and the column is kept for 3min; raising the temperature to 100 ℃ at a rate of 2 ℃ min -1; raising the temperature to 200 ℃ at a rate of 4 ℃ min -1; the temperature was raised to 300℃at a rate of 20℃min -1 and maintained for 5min. The sample inlet temperature was 250 ℃, carrier gas: he, carrier gas flow rate 1mL min -1; and (3) split sample injection, wherein the sample injection amount is 1.0 mu L, the split ratio is 10:1, and the split flow rate is 10mL min -1. The mass spectrum conditions are ionization mode: EI, electron energy: 70eV; the ion source temperature was 250 ℃ and the transmission line temperature was 250 ℃. The scanning mode is full scanning, and the detection range is 40-400 m/z; the solvent was delayed for 3min.
The GC-MS analysis results are shown in FIG. 4: the yeast strain containing pESC-Leu AaTPS 48:48 recombinant plasmid can synthesize myrcene (Myrcene).
5. Conclusion(s)
The AaTPS gene is cloned from folium artemisiae argyi leaves, and is a key enzyme gene which is obtained from folium artemisiae argyi for the first time and can catalyze GPP to form monoterpene components and FPP to form sesquiterpene components for synthesis. Experiments prove that: the AaTPS protein can catalyze GPP to form monoterpene compound myrcene (Myrcene) and FPP to form sesquiterpene compound (trans) -beta-farnesene ((E) -beta-farnesene), has important effects on biosynthesis of myrcene, (trans) -beta-farnesene and other terpenes in mugwort leaves, and has important theoretical and practical significance on regulation and production of plant terpenes and cultivation of high-quality mugwort leaves.
The above description is not intended to limit the invention, nor is the invention limited to the examples described above. Variations, modifications, additions, or substitutions that would be within the spirit and scope of the invention are also within the scope of the invention, which is defined by the following claims.

Claims (10)

1. A mugwort terpene synthase AaTPS, characterized by being a protein of the following a) or b) or c):
a) The amino acid sequence is a protein shown as SEQ ID No. 1;
b) A fusion protein obtained by connecting a tag to the N-terminal and/or C-terminal of the protein shown in SEQ ID No. 1;
c) The protein with the same function is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown in SEQ ID No. 1.
2. A biomaterial associated with the mugwort terpene synthase AaTPS according to claim 1, characterized by being any one of the following A1) to a 12):
A1 A nucleic acid molecule encoding AaTPS48,48 proteins;
A2 An expression cassette comprising A1) said nucleic acid molecule;
a3 A) a recombinant vector comprising the nucleic acid molecule of A1);
a4 A recombinant vector comprising the expression cassette of A2);
a5 A) a recombinant microorganism comprising the nucleic acid molecule of A1);
A6 A) a recombinant microorganism comprising the expression cassette of A2);
a7 A) a recombinant microorganism comprising the recombinant vector of A3);
a8 A) a recombinant microorganism comprising the recombinant vector of A4);
A9 A transgenic plant cell line comprising the nucleic acid molecule of A1);
a10 A transgenic plant cell line comprising the expression cassette of A2);
A11 A transgenic plant cell line comprising the recombinant vector of A3);
A12 A) a transgenic plant cell line comprising the recombinant vector of A4).
3. The biological material related to the blumea balsamifera terpene synthase AaTPS according to claim 2, wherein the nucleic acid molecule A1) is a gene represented by the following 1), 2), or 3):
1) The coding sequence is cDNA molecule shown in SEQ ID No.2 at positions 1-1728;
2) A cDNA molecule or a genomic DNA molecule having 75% or more identity to the nucleotide sequence defined in 1) and encoding the protein of claim 1;
3) A cDNA molecule or a genomic DNA molecule which hybridizes under stringent conditions to the nucleotide sequence defined in 1) or 2) and which encodes the protein of claim 1.
4. Use of the mugwort leaf terpene synthase AaTPS of claim 1 in the manufacture of a terpene synthase or a terpene compound.
5. Use of the biological material related to the mugwort leaf terpene synthase AaTPS of claim 2 or 3 for preparing terpene synthases or terpene compounds.
6. Use of a mugwort terpene synthase AaTPS according to claim 1 for catalyzing the formation of myrcene from geranyl pyrophosphate (GPP) or for catalyzing the formation of (trans) - β -farnesyl pyrophosphate (FPP).
7. Use of a biological material related to the mugwort terpene synthase AaTPS of claim 2 or 3 for catalyzing the formation of myrcene by geranyl pyrophosphate (GPP) or for catalyzing the formation of (trans) - β -farnesene by farnesyl pyrophosphate (FPP).
8. A method for synthesizing a terpenoid is characterized in that the tarragon terpene synthase AaTPS, a substrate and an enzymatic buffer solution which are described in claim 1 are uniformly mixed and reacted to obtain the terpenoid.
9. The method for synthesizing terpenoids according to claim 8, wherein said substrate is geranyl pyrophosphate or farnesyl pyrophosphate; the terpenoid is monoterpene and/or sesquiterpene.
10. A method for synthesizing terpenoid, characterized in that the mugwort terpene synthase AaTPS of claim 1 is introduced into saccharomyces cerevisiae to synthesize terpenoid; the terpenoid is monoterpene and/or sesquiterpene.
CN202311013825.2A 2023-08-10 2023-08-10 Blumea balsamifera terpene synthase AaTPS and coding gene and application thereof Pending CN117924513A (en)

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