CN111411099A - Hemsleya amabilis acetyl transferase, coding gene thereof and application of hemsleya amabilis acetyl transferase in preparation of cucurbitacin - Google Patents
Hemsleya amabilis acetyl transferase, coding gene thereof and application of hemsleya amabilis acetyl transferase in preparation of cucurbitacin Download PDFInfo
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Abstract
The invention discloses a cucurbitacin acetyltransferase, a coding gene thereof and application thereof in preparation of cucurbitacin, wherein the cucurbitacin acetyltransferase has an amino acid sequence shown as SEQ ID No. 1. The invention identifies and obtains key enzyme in the synthetic route of the cucurbitacin, namely cucurbitacin acetyltransferase, for the first time; the cucurbitacin acetyltransferase can convert C-25 hydroxyl of cucurbitacin into acetyl, thereby obtaining cucurbitacin; based on the method, a recombinant vector containing a gene encoding cucurbitacin acetyltransferase is constructed, genetic engineering bacteria are further constructed, the transgenic engineering bacteria are used for expressing the cucurbitacin acetyltransferase in vitro, the substrate cucurbitacin is further catalyzed, the cucurbitacin is directly obtained, and the cucurbitacin is not required to be extracted after planting cucurbitacin plants; provides an important way for preparing the cucurbitacin in a large quantity and rapidly.
Description
Technical Field
The invention belongs to the technical field of biotechnology, and particularly relates to cucurbitacin acetyltransferase, a coding gene thereof and application thereof in preparation of cucurbitacin.
Background
Hemsleya chinensis Cogn is a plant of Hewsleya Cogn of Cucurbitaceae, 31 plants of Hewsleva are available worldwide, and except 2 plants produced in India and Vietnam, other plants are mainly distributed in southwest areas of Yunnan, Sichuan and Guizhou provinces in China.
Researches show that tubers of the cucurbitacin plants are rich in cucurbitacin and other triterpenoid saponin components, particularly cucurbitacin (a mixture of cucurbitacin and cucurbitacin B, mainly cucurbitacin) extracted from the cucurbitacin plants is developed into a cucurbitacin tablet, has multiple effects of clearing heat and removing toxicity, resisting bacteria and diminishing inflammation and the like, and is clinically and frequently used for treating various diseases such as bacillary dysentery, enteritis, bronchitis, acute tonsillitis and the like.
In addition, scholars at home and abroad also find that the Cucurbitacin (Cucurbitacin IIa) has obvious cytotoxicity, and the Cucurbitacin can destroy actin cytoskeleton by inhibiting the Survivin (Survivin) at the downstream of JAK2/STAT3, guide cells to generate PARP mediated apoptosis, thereby inhibiting the proliferation of tumor cells, and enabling the Cucurbitacin to become a novel anti-cancer drug.
At present, the cucurbitacin is mainly extracted from the tubers of the cucurbita plants. In recent years, due to the reasons of disordered mining and digging of human beings and the like, wild resources of hemsleya amabilis medicinal materials are increasingly reduced. In order to relieve the pressure of wild resources, people gradually develop the artificial cultivation of the hemsleya plants, but the planting period of the hemsleya plants is long, and the requirements on planting plots and planting technology are high, so that the artificial cultivation work of the hemsleya plants is not smoothly developed.
How to obtain the useful secondary metabolites efficiently is a problem for researchers to think and research. For natural products with high added values, homologous or heterologous expression systems established by adopting modern biotechnology for efficiently producing medicinal active ingredients are widely regarded as important technical means for solving the shortage of medicinal resources in the future. However, in order to understand the biosynthetic pathways of these active ingredients, it is necessary to identify key genes related to these pathways, and the discovery of these catalytic enzyme genes has become a key link in the study of biosynthetic pathways of plant metabolites.
The academia generally considers that the cucurbitacin is obtained by acetylating hydroxyl at C25 position of cucurbitacin, however, the synthetic route of the cucurbitacin obtained from the cucurbitacin is not clear, the function of acetyltransferase responsible for acetylating is not verified, and the promotion of the biosynthesis work of the cucurbitacin is influenced.
Disclosure of Invention
The invention aims to provide a Cucurbitacin acetyltransferase, a coding gene thereof and application thereof in preparation of Cucurbitacin, wherein the Cucurbitacin acetyltransferase can convert C-25 hydroxyl of Cucurbitacin (IIb) in vitro of a plant into acetyl to obtain the Cucurbitacin, and provides an important way for large-scale preparation of the Cucurbitacin.
In order to achieve the purpose, the technical scheme of the invention is as follows:
the hemsleya amabilis acetyl transferase has an amino acid sequence shown as SEQ ID No. 1.
The invention identifies and obtains the key enzyme in the synthetic route of the cucurbitacin in the cucurbitacin for the first time: the cucurbitacin acetyltransferase can convert hydroxyl at C-25 position of cucurbitacin into acetyl, thereby obtaining cucurbitacin.
Furthermore, the invention identifies and obtains the coding gene of the hemsleya amabilis acetyl transferase in the hemsleya amabilis for the first time, and the coding gene has a nucleotide sequence shown as SEQ ID No. 2.
In order to improve the yield of the cucurbitacin and reduce the dependence of the produced cucurbitacin on cucurbitacin plants, the invention provides a recombinant vector containing the coding gene of the cucurbitacin acetyltransferase; and a transgenic engineering bacterium for expressing the hemsleya amabilis acetyl transferase.
In the invention, the coding gene of the hemsleya amabilis acetyl transferase exists in the transgenic engineering bacteria in two ways, one of which is as follows: the transgenic engineering bacteria comprise the recombinant vector containing the coding gene of the hemsleya amabilis acetyl transferase; the second step is as follows: the genome of the transgenic engineering bacterium is integrated with the coding gene of the hemsleya amabilis acetyl transferase.
The invention also provides application of the transgenic engineering bacterium for expressing the cucurbitacin acetyltransferase in preparation of the cucurbitacin.
Preferably, the application comprises: the transgenic engineering bacteria are used for producing the cucurbitacin acetyltransferase, and the cucurbitacin is prepared by using the cucurbitacin acetyltransferase.
The invention also provides application of the cucurbitacin acetyltransferase in preparation of cucurbitacin.
Preferably, the application comprises: reacting at 28-35 deg.C for 20-40min with hemsleyadin and acetyl donor as raw materials and hemsleyadin acetyltransferase as catalyst to obtain cucurbitacin.
The method can utilize transgenic engineering bacteria to express the cucurbitacin acetyltransferase in vitro, and further catalyze the substrate cucurbitacin to directly obtain the cucurbitacin without planting cucurbitacin plants and then extracting the cucurbitacin. The invention also provides application of the coding gene of the hemsleya amabilis acetyl transferase in serving as a molecular marker for assisted breeding of hemsleya amabilis.
Because the gene encoding the cucurbitacin is a key gene for synthesizing the cucurbitacin by plants, the gene encoding the cucurbitacin can be used as an important marker gene for molecular assisted breeding of the cucurbitacin and also can be used as an important candidate gene for producing the cucurbitacin in the construction of yeast underpan cells.
Compared with the prior art, the invention has the beneficial effects that:
the invention identifies and obtains the key enzyme in the synthetic route of the cucurbitacin in the cucurbitacin for the first time: the cucurbitacin acetyltransferase can convert hydroxyl at C-25 position of the cucurbitacin into acetyl, thereby obtaining cucurbitacin; based on the method, a recombinant vector containing a gene encoding cucurbitacin acetyltransferase is constructed, genetic engineering bacteria are further constructed, the transgenic engineering bacteria are used for expressing the cucurbitacin acetyltransferase in vitro, the substrate cucurbitacin is further catalyzed, the cucurbitacin is directly obtained, and the cucurbitacin is not required to be extracted after planting cucurbitacin plants; provides an important way for preparing the cucurbitacin in a large quantity and rapidly.
Drawings
FIG. 1 is a schematic diagram of the synthesis of cucurbitacin from cucurbitacin B catalyzed by cucurbitacin acetyltransferase;
wherein Cucurbitacin IIb represents hemsleyadin, Cucurbitacin IIa represents hemsleyadin, HCAT1 represents hemsleyadin acetyltransferase of the invention, the same applies below;
FIG. 2 is a diagram showing the result of electrophoresis detection of recombinant plasmid pET32a-HCAT1 containing the gene encoding cucurbitaceyltransferase of the present invention;
m represents a Marker protein standard substance, and the same is carried out below;
FIG. 3 is a schematic structural diagram of a recombinant plasmid pET32a-HCAT1 containing the gene encoding cucurbitacin acetyltransferase of the present invention;
FIG. 4 is a diagram showing the results of protein electrophoresis detection of hemsleya amabilis acetyl transferase HCAT1 prepared by the present invention;
FIG. 5 is a Western blot analysis result of hemsleya amabilis acetyl transferase HCAT1 prepared by the present invention;
FIG. 6 is a graph showing the results of enzyme activity reaction HP L C of hemsleya amabilis acetyl transferase HCAT1 of the present invention;
wherein, Control represents blank Control, and standard represents standard;
FIG. 7 is a characteristic ion peak diagram of hemsleyadin;
wherein Mass-to-Charge (m/z) represents a Mass-to-Charge ratio (m/z), as follows;
FIG. 8 is a characteristic ion peak diagram of cucurbitacin;
FIG. 9 is a diagram showing the result of FC-MS detection of the enzyme activity reaction of hemsleya amabilis acetyltransferase HCAT1 according to the present invention;
wherein Substrate represents a reaction Substrate, Product represents a reaction Product, and Retention time (min) represents a peak time (min).
Detailed Description
The technical solution of the present invention will be further described in detail with reference to the accompanying drawings and the detailed description.
In this example, based on basic functional annotation information of cucurbit transcriptome Unigene, candidate genes of BAHD type acetyltransferase (BAHD-AT) were screened in sequencing annotation results, and meanwhile, using the identified BAHD-AT gene in cucurbitaceae plant, mainly using CmACT identified in csct and melon (Cucumismelo) identified in cucumber (Cucumis sativus) as clues, performing sequence local B L AST analysis, then performing sorting analysis on the screening results, and finally finding 2 BAHD-AT genes in the cucurbit transcriptome, and then performing a series of operations such as cDNA preparation, candidate gene amplification and recovery, homologous recombination, protein expression, in vitro enzyme activity reaction, and HP L C and L C/MS detection, and finally identifying acetyl groups capable of catalyzing hydroxyl groups AT C-25 position of Cucurbitacin (cubitacin IIb), and generating target candidate acetyltransferase of Cucurbitacin 1 gene (fig. 1).
The method specifically comprises the following steps:
(1) preparation of cDNA template
A fresh sample of hemsleya tuber is taken, sliced and quickly frozen in liquid nitrogen, and RNA extraction is carried out by using a HiPure Plant RNA Mini Kit of magenta (Guangzhou Meiji Biotech Co., Ltd.). After the extracted RNA is qualified by detection, the TAKARA reverse transcription kit is used for reverse transcription of the RNA into cDNA, and the cDNA is stored at the temperature of-20 ℃ for later use.
(2) Gene amplification and recovery
Using primer Design software (CE Design) v1.04, amplification primers were designed for the acetyltransferase HCAT1 gene:
an upstream primer: 5'-ATGGAGATGCCATTGAAAGTG-3' (SEQ ID No. 3);
a downstream primer: 5'-CTAAACTTGAATGACATTAGGATTTATGG-3' (SEQ ID No. 4);
in order to facilitate homologous recombination of the acetyl transferase HCAT1 gene obtained by amplification and a vector pET32a, a homology arm (Escherichia coli pET32a is used as the homology arm) is added on a primer; namely:
an upstream primer: 5'-ccatggctgatatcggatccATGGAGATGCCATTGAAAGTG-3' (SEQ ID No. 5);
a downstream primer:
5'-tgtcgacggagctcgaattcCTAAACTTGAATGACATTAGGATTTATGG-3'(SEQ ID No.6)；
and performing gene amplification by using KOD high fidelity enzyme.
The PCR reaction system is as follows: 94 ℃ for 5 min; 94 ℃, 30S, 62 ℃, 50S, 72 ℃, 1min, 35 cycles; 72 deg.C, 7 min.
After the PCR is finished, the gel is run, and the target band is recovered after the successful amplification is confirmed. Gene cutting recovery Using EasyPure Quick Gel Extraction Kit from Beijing all-terrain gold Biotechnology Ltd, recovery of the target gene was performed. After recovery, the concentration of the recovered water is measured on a NanoReady ultramicro ultraviolet-visible spectrophotometer, and finally the water is stored in a refrigerator at the temperature of-20 ℃ for later use.
(3) Construction and identification of Gene recombination vector
Firstly, carrying out single enzyme digestion by using a BamH I enzyme to obtain a linearized pET32a vector; then homologous recombination is carried out according to the reaction system shown in the following table, and all components are added into a PCR reaction tube on ice:
wherein X (base number of 0.02 × pET32a) ng/linearized pET32a concentration ng/μ L, Y (base number of 0.02 × pET32a) ng/HCAT1 recovery concentration ng/μ L;
after the recombination is finished, detecting the result and sending the result to a company for sequencing, wherein the assembled electrophoresis detection result is shown in figure 2, which shows that the assembly is successful; the structural schematic diagram of the obtained recombinant plasmid pET32a-HCAT1 is shown in FIG. 3.
(4) SDS-PAGE protein electrophoresis and immunoblot detection
The induction conditions for the acetyltransferase HCAT1 were determined after protein expression experiments to be: inducing for 12h at 17 ℃ with 0.1mM IPTG and 220 r/min; and then shaking greatly, collecting bacteria, breaking the wall, centrifuging at high speed to obtain protein supernatant, and performing SDS-PAGE protein electrophoresis and detection. The results are shown in FIG. 4.
In order to further confirm the target protein, immunoblot detection was performed. As the N-terminal of the acetyltransferase HCAT1 is provided with a His Tag, a high-purity mouse-derived monoclonal antibody His-Tag antibody (GenStar, Shenzhen, China) is required to be used for blocking and incubating the antibody according to WB detection requirements.
The primary antibody is diluted by 1:2000 dilution and 5% w/v skimmed milk in PBST, a membrane carrying the transfer protein is inoculated, the membrane is incubated for 1h at room temperature or is kept overnight at 4 ℃, a primary antibody mixed solution is recovered, then the membrane is added into PBST buffer solution and is subjected to shaking culture for 20min (40rpm) at room temperature in a shaking table, then a rabbit anti-mouse immunoglobulin G-horseradish peroxidase (IgG-HRP) coupled secondary antibody (GenStar, China Shenzhen) is diluted by PBST and 5% w/v skimmed milk at 1:5000 dilution and is subjected to shaking table for 1h at room temperature, finally, the secondary antibody mixed solution is discarded, the membrane is subjected to 10min shaking table by 20m L1 × PBST, repeated for 3 times, a luminescent substrate provided by a StarSignal hypersensitivity chemiluminescence detection kit (GenStar, China Shenzhen) is used, and then the membrane is exposed to an X-ray film to perform visual luminescence detection of the coupled secondary antibody, and the detection result is shown in figure 5, and the supernatant protein of the target.
(5) Enzyme activity reaction
The activity of the acetyltransferase HCAT1 was determined by the acetylation of cucurbitacin IIa (i.e. cucurbitacin) in 1.5m L centrifuge tubes.
The reaction system contained 40mM acetyl CoA, 400. mu.M substrate hemsleyadin, 50mM sodium phosphate buffer (pH7.5), and 40. mu.g purified acetyltransferase HcAT1, and the total volume of the reaction system was 100. mu. L.
After incubation at 31 ℃ for 30min, the reaction mixture was extracted 3 times with an equal volume of ethyl acetate, followed by brief centrifugation, supernatant was taken, concentrated, the product was dissolved in methanol and finally analyzed by HP L C and L C-MS for detection of the reaction product.
(6) Product detection
The HP L C detection conditions were as follows:
an instrument used for HP L C detection is an Agilent 1290 ultra-high performance liquid chromatograph, a chromatographic column is a phenomenex Kinetex C18 analytical column (4.6 × 100mm,2.6 mu m), the mobile phase for measuring the cucurbitacin (comprising the cucurbitacin and the cucurbitacin) is 0.2% phosphoric acid aqueous solution (A) -acetonitrile (B), gradient elution is carried out for 0-15 min, 25-33% of B, 15-20 min, 33-40% of B, 20-24 min, 40-60% of B, 24-28 min, 60-90% of B, the detection wavelength is 212nm, and the detection result is shown in figure 6, which shows that the cucurbitacin exists in a reaction product.
L C-MS detection conditions were as follows:
to further confirm the reaction product detected by HP L C, Agilent 1290 UP L C/6540Q-TOF liquid chromatography-mass spectrometer (L C/MS) was used for detection as follows:
mass spectrum conditions: the ion source adopts a positive ion mode, and the voltage: 3500V; fragmentation voltage is 135V; the taper hole voltage is 60V; radio frequency voltage 750V, scanning range: 100-1000 m/z;
the chromatographic conditions comprise that a chromatographic column is Phenomenex Kinetex C18 analytical column (4.6 × 100mm,2.6 mu m), the mobile phase of the hemsleyadin is measured to be 0.2 percent of phosphoric acid aqueous solution (A) -acetonitrile (B), gradient elution is carried out for 0-15 min, 25-33 percent of B, 15-20 min, 33-40 percent of B, 20-24 min, 40-60 percent of B, 24-28 min and 60-90 percent of B, the detection wavelength is 212nm, and the detection result is shown in figure 7, figure 8 and figure 9.
As can be seen from the results of fig. 9, the peak-off times of the substrate Cucurbitacin (Cucurbitacin IIb) and the reaction product Cucurbitacin (Cucurbitacin IIa) in the reaction solution coincide with the peak-off times of the Cucurbitacin standard product and the Cucurbitacin standard product; further confirming that the product is cucurbitacin.
Sequence listing
<110> Yunnan university of agriculture
<120> cucurbitacin acetyltransferase, coding gene thereof and application thereof in preparation of cucurbitacin
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Val Gly Gly Gly Asp Cys Glu Asp Ala Asn Glu Arg Ser Arg Ser Leu
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Arg Val Lys Glu Asp Asn Glu Ser Ile Phe Cys Asn Asp Glu Gly Ala
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Ile Tyr Val Glu Ala Lys Ala Asn Cys Leu Leu Ser Asp Phe Leu Asn
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Gln Leu Glu Ile Asp Ser Leu Asn Asp Phe Leu Pro Phe Asp Ser Ala
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Met Ala Ile Gly Leu Leu Met Ser His Lys Ile Ser Asp Ala Ser Ser
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Leu Ser Glu Ser Glu Leu Pro Lys Phe Ile Gly Ala Ser Val Leu Pro
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Pro Pro Gln Asp Phe Pro Ile Ser Thr Pro Thr Ser Asp Ser Gly Ile
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His Ala Lys Gly Val Thr Lys Arg Leu Val Phe Glu Ala Ser Lys Ile
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Ile Glu Leu Lys Ala Lys Ala Thr Ser Ala Thr Val Lys Gln Pro Thr
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Arg Val Glu Ala Val Thr Gly Leu Ile Trp Lys Cys Ala Ile Ala Ala
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Ser Lys Ser Thr Ser Gly Ile Ser Lys Pro Ser Val Val Gly Gln Ala
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Val Asn Leu Arg Lys Arg Leu Val Pro Thr Leu Pro Asp Thr Ser Ile
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Gly Asn Leu Leu Gly Phe Ile Thr Pro Glu Thr Lys Thr Glu Ala Gly
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Glu Ile Glu Leu Gln Gly Leu Val Gly Leu Leu Arg Glu Gly Ile Thr
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Glu Phe Asn Glu Asn Gly Phe Lys Lys Tyr Gln Asp Thr Glu Ala Tyr
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Asp Lys Asn Phe Tyr Leu Cys Ser Ser Trp Ser Arg Phe Gln Phe Tyr
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<211>41
<212>DNA
<213> artificially synthesized sequence (Unknown)
<400>5
ccatggctga tatcggatcc atggagatgc cattgaaagt g 41
<210>6
<211>49
<212>DNA
<213> artificially synthesized sequence (Unknown)
<400>6
tgtcgacgga gctcgaattc ctaaacttga atgacattag gatttatgg 49
Claims (10)
1. A hemsleya amabilis acetyl transferase is characterized by having an amino acid sequence shown as SEQ ID No. 1.
2. The gene encoding cucurbitaceyltransferase of claim 1.
3. The gene encoding cucurbitacin acetyltransferase of claim 2, having a nucleotide sequence shown as SEQ id No. 2.
4. A recombinant vector comprising a gene encoding the cucurbitaceyltransferase of claim 2 or 3.
5. A genetically engineered bacterium expressing the cucurbitacin acetyltransferase of claim 1.
6. The genetically engineered bacterium expressing cucurbitaceyltransferase of claim 5, comprising the recombinant vector containing the gene encoding cucurbitaceyltransferase of claim 4;
alternatively, the gene encoding the cucurbitacin acetyltransferase of claim 2 or 3 is integrated into the genome.
7. The use of the genetically engineered bacterium of claim 5 or 6 expressing cucurbitaceyltransferase in the preparation of cucurbitacin.
8. The use of the cucurbitacin acetyltransferase of claim 1 in the preparation of cucurbitacin.
9. The use of cucurbitacin acetyltransferase of claim 8 in the preparation of cucurbitacin, comprising:
reacting at 28-35 deg.C for 20-40min with hemsleyadin and acetyl donor as raw materials and hemsleyadin acetyltransferase as catalyst to obtain cucurbitacin.
10. The use of the gene encoding cucurbitacin acetyltransferase of claim 2 or 3 as a molecular marker for assisted breeding of cucurbitacin.
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| CN116334106A (en) * | 2022-09-15 | 2023-06-27 | 云南农业大学 | Application of hemsleyadin cytochrome oxidase gene and transgenic engineering bacteria thereof in preparation of cucurbitacin intermediate |
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