CN117604007A - Camphor tree eucalyptol synthetase gene CcCins, its expression protein and application - Google Patents
Camphor tree eucalyptol synthetase gene CcCins, its expression protein and application Download PDFInfo
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- CN117604007A CN117604007A CN202311645509.7A CN202311645509A CN117604007A CN 117604007 A CN117604007 A CN 117604007A CN 202311645509 A CN202311645509 A CN 202311645509A CN 117604007 A CN117604007 A CN 117604007A
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- 229960005233 cineole Drugs 0.000 title claims abstract description 80
- WEEGYLXZBRQIMU-UHFFFAOYSA-N 1,8-cineole Natural products C1CC2CCC1(C)OC2(C)C WEEGYLXZBRQIMU-UHFFFAOYSA-N 0.000 title claims abstract description 78
- WEEGYLXZBRQIMU-WAAGHKOSSA-N Eucalyptol Chemical compound C1C[C@H]2CC[C@]1(C)OC2(C)C WEEGYLXZBRQIMU-WAAGHKOSSA-N 0.000 title claims abstract description 78
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 73
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Abstract
The invention discloses a camphor tree eucalyptol synthase gene CcCins and an expression protein and application thereof, belonging to the technical field of plant genetic engineering. The nucleotide sequence of the camphor tree eucalyptol synthase gene CcCins is shown as SEQ ID NO. 1. The substrate GPP is added into the CcCins recombinant protein constructed by the invention to catalyze the activity of the recombinant protein, and the GC-MS detection result shows that the CcCins protein catalyzes the GPP to generate eucalyptol. The pCambia1300S-CcCins plant over-expression vector is transferred into tobacco leaves, and after 48 hours of transient transformation, ccCins genes are expressed in a large amount in the tobacco leaves, and the synthesis of eucalyptol is obviously promoted by over-expression of CcCins. The CcCins gene is shown to be a eucalyptol synthase gene and has important application value.
Description
Technical Field
The invention belongs to the technical field of plant genetic engineering, and particularly relates to a camphor tree eucalyptol synthase gene CcCins and an expression protein and application thereof.
Background
Terpenoid is a natural product with the most variety and the most abundant chemical structure change, widely exists in higher plants and microorganisms, and has important economic and medicinal values. Cyclic monoterpene 1, 8-eucalyptol (C) 10 H 18 O) is a colorless transparent liquid, is volatile, and has strong camphora smell and cool herbal taste. The natural 1, 8-eucalyptol is mainly extracted from branches and leaves of plants of Myrtaceae, namely Eucalyptus globulus and Lauraceae, is an important fine chemical product, has good antibacterial, anti-inflammatory, antiseptic, insecticidal and mosquito-repellent effects, is widely applied to the fields of essence and spice, sanitation, medicine and the like, and has wide market prospect. (1) Can be used as medicine for preventing and treating common cold, relieving asthma, eliminating phlegm, treating pulmonary tuberculosis and dermatoses, and cleaning skin ulcer, and fistula; is added into essential balm, ten drops of water, mosquito repellent oil and various external tincture, has effects of removing toxic substances and relieving inflammation, and can also be used as analgesic for patients with mental disorder. (2) Daily chemical industry is used for perfumed soap, bath soap, toothpaste and prickly heatPowders and additives in certain cosmetics. (3) The food such as candy has effects of refreshing, moistening throat, relieving gastrointestinal dyspepsia, expelling parasites and killing parasites. (4) Can be used as raw materials of terpineol and borneol in chemical industry. (5) Can be used as solvent diluted paint or used as flotation agent for ore floatation.
Camphor tree (Cinnamomum camphora) is a precious aromatic oil-based economic forest tree species in the world. According to the main components of leaf essential oil, camphor tree can be classified into 5 main chemical types of linalool type, camphor type, eucalyptol type, iso-nerolidol type, borneol type, etc. The main component of the essential oil of the camphor tree is monoterpene eucalyptol, and the content of the essential oil of the camphor tree is generally more than 55%. Compared with other eucalyptol plant resources (such as Eucalyptus citriodora, eucalyptus globulus and Eucalyptus robusta), eucalyptol has high content, and is one of the most ideal plant materials for producing natural eucalyptol essential oil. At present, the main components of camphor tree essential oil are extracted and identified, and no related research reports on eucalyptol synthase gene cloning and function identification exist in camphor tree essential oil biosynthesis research.
Disclosure of Invention
Aiming at the problems in the prior art, the invention aims at providing a camphor tree eucalyptol synthase gene CcCins for constructing transgenic plants. Another problem to be solved by the present invention is to provide an expression protein of camphor tree eucalyptol synthase gene CcCins. The invention also solves the technical problem of providing application of camphor tree eucalyptol synthase gene CcCins for screening new germplasm with enhanced eucalyptol production capability.
In order to solve the technical problems, the technical scheme adopted by the invention is as follows:
the nucleotide sequence of the camphor tree eucalyptol synthase gene CcCins is shown as SEQ ID NO. 1.
The amino acid sequence of the expression protein of camphor tree eucalyptol synthase gene CcCins is shown as SEQ ID NO. 2.
Application of camphor tree eucalyptol synthase gene CcCins in catalyzing tobacco or camphor tree to produce eucalyptol.
Application of camphor tree eucalyptol synthase gene CcCins in catalyzing tobacco to produce eucalyptol comprises the following steps:
1) Constructing an expression vector of a camphor tree eucalyptol synthase gene CcCins;
2) Transforming the constructed expression vector of the camphor tree eucalyptol synthase gene CcCins into tobacco leaves;
3) And culturing, screening and obtaining the transgenic tobacco plants with the content of eucalyptol remarkably improved.
The expression vector is a plant expression vector.
The plant expression vector is pCambia1300S-CcCins.
Application of an expression protein of camphor tree eucalyptol synthase gene CcCins in catalyzing GPP to generate eucalyptol.
Application of an expression protein of camphor tree eucalyptol synthase gene CcCins in catalyzing GPP to generate eucalyptol, comprising the following steps:
1) Constructing a prokaryotic expression vector of a camphor tree eucalyptol synthase gene CcCins;
2) The prokaryotic expression vector of the constructed camphor tree eucalyptol synthase gene CcCins is transformed into escherichia coli BL21 to construct a recombinant escherichia coli strain;
3) After culturing and screening recombinant escherichia coli strains, adding IPTG to induce protein expression, collecting and purifying the recombinant protein, and obtaining the recombinant protein of camphor tree eucalyptol synthase gene CcCins for catalyzing GPP to generate eucalyptol.
The prokaryotic expression vector is pET28a-CcCins.
The application of the expression protein of camphor tree eucalyptol synthase gene CcCins in catalyzing GPP to generate eucalyptol comprises the following specific steps:
1) The prokaryotic expression vector pET28a-CcCins of camphor tree eucalyptol synthase gene CcCins is constructed by utilizing a homologous recombination method, and the sequence of the used primer is shown as follows:
forward primer: 5'-tcagcagtcgaagagcCAAGTGCCCCAACCTACTCG-3';
reverse primer: 5'-ttagcgtgtgaagagcCTACATAAACTTGAAGGGC-3';
2) The prokaryotic expression vector of the constructed camphor tree eucalyptol synthase gene CcCins is transformed into escherichia coli BL21 to construct a recombinant escherichia coli strain;
3) Placing recombinant escherichia coli strains with correct sequencing in a shaking table, controlling the temperature at 37 ℃ and shake culturing at 200 r/min; when the OD value reaches 0.6-0.8, thiogalactoside with the final concentration of 0.2mM is added, and shake culture is continued for 6 hours at 37 ℃; taking 1mL of bacterial liquid, centrifuging at 5000rpm for 5min, discarding the supernatant, then adding 100uL of 1 XSDS-PAGE buffer, and blowing to resuspend the bacterial liquid; placing the bacterial liquid into a water bath kettle, boiling for 5 minutes at 99 ℃, then centrifuging for 1 minute at 5000 rpm; taking 30uL supernatant, dyeing with 0.25% coomassie brilliant blue staining solution for 2 hours, then decoloring, clearly observing, and selecting the bacterial solution with the highest expression level; adding 0.3mM IPTG into the bacterial liquid, culturing at 20deg.C for 12 hr to induce protein expression, and utilizingCollecting and purifying the recombinant protein by using the Ni-NTAResin protein purification reagent to obtain the recombinant protein of camphor tree eucalyptol synthase gene CcCins for catalyzing GPP to generate eucalyptol.
Compared with the prior art, the invention has the beneficial effects that:
1) The substrate GPP is added into the CcCins recombinant protein constructed by the invention, and the recombinant active protein catalytic detection is carried out, and the GC-MS detection result shows that the CcCins protein catalyzes GPP to generate eucalyptol. The CcCins recombinant protein provided by the invention is a key enzyme for synthesizing camphor tree eucalyptol and is used for analyzing the basic characteristics of camphor tree eucalyptol metabolism.
2) The invention uses injection method to transfer the agrobacterium which already contains the pCambia1300S-CcCins plant over-expression vector into the tobacco leaf of the receptor material, after 48 hours of transient transformation, the CcCins gene is expressed in a large amount in the tobacco leaf, and the over-expression of the CcCins significantly promotes the synthesis of eucalyptol. Therefore, the CcCins gene provided by the invention is a eucalyptol synthase gene, can be used as a gene resource, and has important application value in plant genetic engineering for improving the eucalyptol content of plants.
Drawings
FIG. 1 is a diagram of a GC-MS detection of a CcCins protein catalytic production product;
FIG. 2 is a graph of analysis of expression level of CcCins gene in tobacco transgenic lines (A) and a graph of detection of tobacco transgenic lines by GC-MS.
Detailed Description
The present invention will be further described with reference to specific embodiments for the purpose of making the objects, technical solutions and advantages of the present invention more apparent. In the following examples, unless otherwise indicated, all technical means used are conventional means well known to those skilled in the art.
Example 1
Based on camphor tree whole genome data, a specific primer is designed to amplify an open reading frame sequence of CcCins, and the primer sequence is as follows:
CcCins forward primer: 5'-ACAATCTCCCAGTCCGCA-3';
CcCins reverse primer: 5'-CATCACCCATTAACTTCTCG-3'.
The PCR amplification system is as follows: primerSTARMaxPRIMIX 25. Mu.L, forward primer (10 uM) 2. Mu.L, reverse primer (10 uM) 2. Mu. L, cDNA 1-3. Mu.L (50 ng), primerSTARMaxDNAxDamerase 1. Mu. L, ddH 2 O was made up to 50. Mu.L.
The PCR amplification reaction procedure was: pre-denaturation 94 ℃ for 2 minutes; 35 cycles: denaturation at 98℃for 10 seconds, annealing at 55℃for 30 seconds, and extension at 68℃for 2min; final extension at 72 ℃ for 5 min; preserving at 4 ℃. The CDS coding region of the CcCins gene has a length of 1749bp, the nucleotide sequence of which is shown as SEQ ID NO.1, and the total coding of which is 582 amino acids, and the amino acid sequence of which is shown as SEQ ID NO. 2.
Example 2
The prokaryotic expression vector pET28a-CcCins is constructed by utilizing a homologous recombination method, and the sequence of the used primer is as follows:
forward primer: 5'-tcagcagtcgaagagcCAAGTGCCCCAACCTACTCG-3';
reverse primer: 5'-ttagcgtgtgaagagcCTACATAAACTTGAAGGGC-3'.
The recombinant vector pET28a-CcCins is transformed into escherichia coli, and thenPositive clone detection and plasmid extraction were then performed and the plasmid was transformed into E.coli BL21 (DE 3) strain. Placing recombinant escherichia coli strains with correct sequencing in a shaking table, controlling the temperature at 37 ℃ and shake culturing at 200 r/min; when the OD value reaches 0.6-0.8, thiogalactoside (IPTG) with the final concentration of 0.2mM is added, and shake culture is continued for 6 hours at 37 ℃; taking 1mL of bacterial liquid, centrifuging at 5000rpm for 5min, discarding the supernatant, then adding 100uL of SDS-PAGE (1×) buffer, and blowing to resuspension the bacterial liquid; boiling the bacterial liquid in a water bath kettle at 99 ℃ for 5 minutes, centrifuging at 5000rpm for 1 minute, and taking supernatant for SDS-PAGE electrophoresis detection; taking 30uL supernatant, dyeing with 0.25% coomassie brilliant blue staining solution for 2 hours, then decoloring, clearly observing, and selecting the bacterial solution with the highest expression level for culturing and preserving for subsequent experiments; adding 0.3mM IPTG into the bacterial liquid, culturing at 20deg.C for 12 hr to induce protein expression, and utilizingCollecting and purifying recombinant protein by using a Ni-NTAResin (Transgen, china) protein purifying reagent; then, 1. Mu.g of recombinant protein was combined with 10mM substrate (GPP, sigma), 10mM MgCl 2 、10mMMnCl 2 10% (v/v) glycerol, 5mM DTT and 50mM Tris-buffer (pH 7.0) were mixed and incubated at 30℃for 1 hour.
The results are shown in FIG. 1, and GC-MS detection results show that the CcCins protein catalyzes the GPP production of eucalyptol.
Example 3
1. The amplification primer is designed according to the CDS sequence of the CcCins gene:
forward primer: 5'-tcagcagtcgaagagcATGGCATTGCAATTGCTTAC-3';
reverse primer: 5'-ttagcgtgtgaagagcCTACATAAACTTGAAGGGC-3'.
Amplifying CDS sequence of CcCins gene, constructing plant over-expression vector pCambia1300S-CcCins by using EXclone kit of hundred company, the specific method is as follows:
amplifying target DNA fragments, purifying and recovering DNA after electrophoresis detection, and carrying out EXIN reaction, wherein a PCR reaction system is as follows: 5 XEX-Buffer 2. Mu. L, EX-Vector 2. Mu.L, EXclonase Enzyme 1. Mu.LL, insert DNA Xul (30 ng), ddH 2 O was made up to 10. Mu.L.
The PCR reaction procedure was: incubation at 37 ℃ for 30 minutes; 15 minutes at 20 ℃.
Taking 5 mu L of reaction liquid, carrying out transformation, screening culture and fungus picking detection on 100 mu L of competent cells, and preserving positive strains for later use.
2. The constructed overexpression vector pCambia1300S-CcCins is transformed into LBA4404 agrobacterium competent cells, bacterial liquid with OD600 of 0.6-0.8 is prepared by taking overexpression idle load as a reference, the bacterial liquid is resuspended by suspension to prepare infection liquid with OD600 of 0.6 (10mM MES,10mM MgCl2, 20mg.L-1 As, pH 5.2), and the mixture is stood for 2-3 hours at 28 ℃. Tobacco with good state is selected, infection liquid is injected into tobacco leaves by using a needleless injector, and after culturing for 48-60 hours, GC-MS detection and qRT-PCR analysis are carried out, and each sample is biologically repeated for 3 times.
As shown in FIG. 2, ccCins was expressed in tobacco leaves in large amounts after 48 hours of transient transformation, and overexpression of CcCins significantly promoted eucalyptol synthesis.
Claims (10)
1. The nucleotide sequence of the camphor tree eucalyptol synthase gene CcCins is shown as SEQ ID NO. 1.
2. The expression protein of camphor tree eucalyptol synthetase gene CcCins as set forth in claim 1, which has the amino acid sequence shown in SEQ ID No. 2.
3. Use of the camphor tree eucalyptol synthase gene CcCins according to claim 1 for catalyzing tobacco or camphor tree production of eucalyptol.
4. Use of the camphor tree eucalyptol synthase gene CcCins according to claim 3, for catalyzing the production of eucalyptol from tobacco, comprising the steps of:
1) Constructing an expression vector of a camphor tree eucalyptol synthase gene CcCins;
2) Transforming the constructed expression vector of the camphor tree eucalyptol synthase gene CcCins into tobacco leaves;
3) And culturing, screening and obtaining the transgenic tobacco plants with the content of eucalyptol remarkably improved.
5. The use of the camphor tree eucalyptol synthase gene CcCins according to claim 4, for catalyzing tobacco to produce eucalyptol, wherein said expression vector is a plant expression vector.
6. The use of the camphor tree eucalyptol synthase gene CcCins according to claim 5, for catalyzing tobacco to produce eucalyptol, wherein said plant expression vector is pCambia1300S-CcCins.
7. Use of the expression protein of camphor tree eucalyptol synthase gene CcCins according to claim 2 for catalyzing GPP to produce eucalyptol.
8. The use of the expression protein of camphor tree eucalyptol synthase gene CcCins according to claim 7, for catalyzing the production of eucalyptol by GPP, comprising the steps of:
1) Constructing a prokaryotic expression vector of a camphor tree eucalyptol synthase gene CcCins;
2) The prokaryotic expression vector of the constructed camphor tree eucalyptol synthase gene CcCins is transformed into escherichia coli BL21 to construct a recombinant escherichia coli strain;
3) After culturing and screening recombinant escherichia coli strains, adding IPTG to induce protein expression, collecting and purifying the recombinant protein, and obtaining the recombinant protein of camphor tree eucalyptol synthase gene CcCins for catalyzing GPP to generate eucalyptol.
9. The use of camphor tree eucalyptol synthase gene CcCins according to claim 8, for catalyzing tobacco to produce eucalyptol, wherein said prokaryotic expression vector is pET28a-CcCins.
10. The use of the expression protein of camphor tree eucalyptol synthase gene CcCins according to claim 8, for catalyzing GPP to produce eucalyptol, comprising the specific steps of:
1) The prokaryotic expression vector pET28a-CcCins of camphor tree eucalyptol synthase gene CcCins is constructed by utilizing a homologous recombination method, and the sequence of the used primer is shown as follows:
forward primer: 5'-tcagcagtcgaagagcCAAGTGCCCCAACCTACTCG-3';
reverse primer: 5'-ttagcgtgtgaagagcCTACATAAACTTGAAGGGC-3';
2) The prokaryotic expression vector of the constructed camphor tree eucalyptol synthase gene CcCins is transformed into escherichia coli BL21 to construct a recombinant escherichia coli strain;
3) Placing recombinant escherichia coli strains with correct sequencing in a shaking table, controlling the temperature at 37 ℃ and shake culturing at 200 r/min; when the OD value reaches 0.6-0.8, thiogalactoside with the final concentration of 0.2mM is added, and shake culture is continued for 6 hours at 37 ℃; taking 1mL of bacterial liquid, centrifuging at 5000rpm for 5min, discarding the supernatant, then adding 100uL of 1 XSDS-PAGE buffer, and blowing to resuspend the bacterial liquid; placing the bacterial liquid into a water bath kettle, boiling for 5 minutes at 99 ℃, then centrifuging for 1 minute at 5000 rpm; taking 30uL supernatant, dyeing with 0.25% coomassie brilliant blue staining solution for 2 hours, then decoloring, clearly observing, and selecting the bacterial solution with the highest expression level; adding 0.3mM IPTG into the bacterial liquid, culturing at 20deg.C for 12 hr to induce protein expression, and utilizingCollecting and purifying the recombinant protein by using the Ni-NTAResin protein purification reagent to obtain the recombinant protein of camphor tree eucalyptol synthase gene CcCins for catalyzing GPP to generate eucalyptol. />
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