CN117004628B - Saussurea involucrata flavonol synthase gene and its coding product and use - Google Patents
Saussurea involucrata flavonol synthase gene and its coding product and use Download PDFInfo
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- CN117004628B CN117004628B CN202310983256.8A CN202310983256A CN117004628B CN 117004628 B CN117004628 B CN 117004628B CN 202310983256 A CN202310983256 A CN 202310983256A CN 117004628 B CN117004628 B CN 117004628B
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- 241001145025 Saussurea involucrata Species 0.000 title claims abstract description 42
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- 108090000623 proteins and genes Proteins 0.000 claims abstract description 45
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
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- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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- C12Y114/00—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14)
- C12Y114/11—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14) with 2-oxoglutarate as one donor, and incorporation of one atom each of oxygen into both donors (1.14.11)
- C12Y114/11023—Flavonol synthase (1.14.11.23)
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Abstract
The invention relates to a saussurea involucrata flavonol synthase gene, a coding product and application thereof, belonging to the technical field of plant genetic engineering and gene editing, wherein the nucleotide sequence of the saussurea involucrata flavonol synthase gene is shown as SEQ ID NO. 1. The invention also provides a protein coded by the saussurea involucrata flavonol synthase gene, the amino acid sequence of which is shown as SEQ ID NO.2, and a recombinant vector pET32a-SiFLS containing the gene. The gene can regulate and control the application of the dihydromyricetin serving as a substrate to generate myricetin of the saussurea involucrata flavonol active substance.
Description
Technical Field
The invention belongs to the technical field of medicinal plant genetic engineering, and relates to saussurea involucrata flavonol synthase gene (SiFLS), and a coding product and application thereof.
Background
The dry aerial parts of saussurea involucrata (Saussurea involucrata) can be used as medicines, called "saussurea involucrata", which is a precious Chinese herbal medicine resource. The saussurea involucrata is used as a common medicine for Uygur nationality, has the effects of nourishing nerves, tonifying kidney, promoting blood circulation, regulating abnormal body fluid, strengthening tendons and bones and the like, and is mainly used for treating irregular menstruation, lower abdomen pain, wind-cold-dampness arthralgia and the like clinically. The saussurea involucrata has special growth environment and is mainly distributed in mountains such as Tianshan, alexandrite and Kunshan in Xinjiang of China; meanwhile, the natural reproduction rate of the saussurea involucrata is low, the saussurea involucrata grows slowly, and the wild resources are endangered due to the destructive digging and unreasonable utilization of people.
The main active components in saussurea involucrata are flavonoid substances, saussurea involucrata polysaccharide and alkaloid, wherein the flavonoid substances are used as main effective components. Flavonols are used as important members of flavonoid compounds, and are synthesized by starting flavonol synthase (flavonol synthase, FLS), and flavonols such as Dihydrokaempferol (DHK), dihydroquercetin (DHQ), dihydromyricetin (DHM) and the like are used as substrates to generate flavonols such as kaempferol, myricetin (Myricetin) and quercetin (quercetin) and the like; these products are further modified by the activity of enzymes such as methyltransferases, glycosyltransferases and acyltransferases to produce a variety of flavonol derivatives. The prior art does not disclose a catalytic enzyme of dihydromyricetin in saussurea involucrata.
Disclosure of Invention
The technical problem to be solved by the invention is to provide saussurea involucrata flavonol synthase (SiFLS) gene and its coding product, and the application of the product in medicine.
The saussurea involucrata flavonol synthase (SiFLS) gene provided by the invention is one of the following nucleotide sequences:
(1) Has a nucleotide sequence shown as SEQ ID NO. 1;
(2) The nucleotide sequence shown in SEQ ID NO.1 is a homologous sequence in which one or more nucleotides are added, substituted, inserted or deleted or an allele thereof and a nucleotide sequence derived therefrom.
The invention also provides the saussurea involucrata flavonol synthase coded by the SiFLS gene, wherein the amino acid of saussurea involucrata flavonol is one of the following sequences:
(1) Has an amino acid sequence shown in SEQ ID NO. 2;
(2) SEQ ID NO.2 adds, substitutes, inserts or deletes a homologous sequence of one or more amino acids.
The invention also provides a recombinant vector pET32a-SiFLS containing the gene.
The invention also provides an application of the gene in regulating and controlling the activity of the saussurea involucrata flavonols to generate myricetin by taking dihydromyricetin as a substrate.
The invention also provides an application of the recombinant vector pET32a-SiFLS in regulating and controlling the production of myricetin by taking dihydromyricetin as a substrate from saussurea involucrata flavonol active substances.
Compared with the prior art, the invention has the following beneficial effects:
the SiFLS gene obtained in the invention is a key gene for regulating and controlling the synthesis of the flavonols of saussurea involucrata, which has important significance for obtaining saussurea involucrata plants with high flavonols through a molecular breeding means; the SiFLS gene is subjected to molecular regulation, so that the content of the Chinese medicinal components in saussurea involucrata can be increased, and the method has important reference significance for improving the content of the effective active components in medicinal plants. The dihydromyricetin is used as a substrate to generate the myricetin product by using the Tianshan saussurea involucrata flavonol synthase coded by the SiFLS gene.
Drawings
Fig. 1: the detection result of SiFLS gene cDNA agarose gel electrophoresis;
fig. 2: predicting analysis results of SiFLS functional domains;
fig. 3: SDS-PAGE gel electrophoresis of pET32a-SiFLS recombinant protein:
m, marker,1, recombinant plasmid supernatant, 2, recombinant plasmid precipitation, 3, recombinant plasmid purified protein, 4, empty control supernatant, 5, empty control precipitation, 6, empty control purified protein;
fig. 4: and (5) in-vitro enzyme activity detection results of SiFLS. (a) is 288nm lower mixed standard sample (all components of reaction system and product myricetin), (b) is 368nm control group enzyme activity product, and (c) is 368nm experiment group enzyme activity product.
Detailed Description
Materials, reagents, vectors, E.coli, etc., used in the examples described below, were purchased commercially from companies, the PCR amplification reaction markers were purchased from the whole gold organism company, the PCR amplification reaction enzymes were purchased from the TaKaRa company, and the markers used in the protein electrophoresis experiments were purchased from the whole gold organism company, unless otherwise specified.
Implementation example 1: construction of full-Length cDNA library of saussurea involucrata
Extracting tender stem total RNA from tender leaf of saussurea involucrata seedling by using TransZol method (full golden biology company), detecting total RNA content and purity by using nucleic acid analyzer, and performing reverse transcription reaction on total RNA with Easy kitOne-Step gDNA Removal and cDNA Synthesis SuperMix (full gold biosystems). The method comprises the following specific steps:
1.1 homogenization: tissue samples of saussurea involucrata stored at-80℃were taken, transferred to a mortar pre-cooled with liquid nitrogen, and the plant samples were ground to a powder with a pestle. Taking 50-100mg (about 1/5 of the tube) of ground sample, placing the ground sample into a 2mL fully pre-cooled centrifuge tube (liquid nitrogen in the tube is required to be uniformly removed to prevent the cover from collapsing), and adding 1mL of TransZoL for uniform shaking and mixing.
1.2 layering: the homogenized sample was placed on ice for 5min (complete separation of nucleoprotein material). 200 μl of chloroform was added, the tube cap was closed, shaken vigorously for 3s, and placed on ice for 3min. The mixture was centrifuged at 4℃for about 15min with a refrigerated centrifuge to separate the layers, giving a colorless supernatant, a white middle layer protein and a red lower organic phase.
1.3 precipitation of RNA: about 550-650 μl of supernatant (pipetted as much as possible on the premise of quality assurance) was transferred to a new 2mL centrifuge tube (this tube required sufficient pre-cooling) at room temperature. 500. Mu.L of isopropanol was added and mixed gently upside down and left at-20℃for 10min. The supernatant was discarded (isopropanol was removed as much as possible) by high-speed centrifugation at 4℃for 10min.
1.4RNA rinsing: at least 1mL of 75% ethanol was added, and after vortexing, 7500g was centrifuged at 4℃for 5min.1.5RNA resolubilization: the supernatant was discarded, ethanol was sucked dry, and the mixture was left open and dried at room temperature for 20min. 30-40. Mu.L of RNase-free ddH was added 2 O (stored at 4 ℃ C., placed on ice) dissolves RNA for 2-3min.
1.6 detection of total RNA content and purity using agarose gel electrophoresis and a nucleic acid analyzer.
1.7 reverse transcription of RNA: the reverse transcription system is as follows:
RNA template, anchored Oligo (dT) and RNase-free Water were mixed well, incubated at 65℃for 5min, ice-bath for 2min, and then other reaction components were added. Mix gently and incubate at 42℃for 15min. Heating at 85deg.C for 5s to inactivate TransScript RT and gDNA remote, to obtain saussurea involucrata cDNA library.
Example 2: siFLS gene cloning and prokaryotic expression vector construction
2.1 Using the saussurea involucrata cDNA as a template, degenerate primers were used:
F：5'-ATGGAGGTBGNDAGDGTBCA-3'，
R：5'-TCACNGGAAGBTTRTTKARCT-3'，
the PCR amplification reaction was carried out in a reaction system of 20. Mu.L:
after mixing, the mixture was centrifuged to the bottom of the tube, and the PCR amplification procedure was as follows:
the PCR amplified products were subjected to agarose gel electrophoresis detection, photographed using a gel imager, and the gel electrophoresis pattern was observed, and amplified fragments were about 1000bp (FIG. 1). The reaction system is enlarged, and the gene fragment is recovered by using a gel recovery kit.
2.2 sequencing and verifying the amplified fragment to obtain a nucleic acid sequence shown as SEQ ID NO.1, wherein the coded amino acid sequence is shown as SEQ ID NO. 2. Functional domain analysis of the encoded amino acid sequence revealed that it belongs to the PLN02704 superfamily, which is a typical flavonol synthase (fig. 2).
Expanding system, designing pET32a joint at the upstream and downstream of the SiFLS gene cDNA sequence of saussurea involucrata, and carrying out PCR amplification by taking the PCR product obtained by 2.1 as a template, wherein the system is 50 mu L.
The PCR procedure was set to 2.1.
After the reaction, electrophoresis was performed with macroporous gel. The SiFLS fragment with pET32a linker was recovered using a DNA gel rapid purification kit, for specific procedures reference kit instructions.
Bacterial solutions containing pET32a empty vector stored at-80℃were inoculated into 50mL of carbenicillin (Car) resistant LB liquid medium, and cultured overnight at 37℃in shake flasks. The plasmid miniprep kit is used for extracting the pET32a empty vector, and specific operation steps refer to the kit instruction.
pET32a empty vector was stored at-20 ℃. The extracted pET32a empty vector was subjected to single cleavage with EcoRI, the system being as follows:
the components are sucked and blown by a pipette, centrifuged to the bottom of the tube, reacted for 1h at 37 ℃, taken out and immediately placed on ice, and the reaction is terminated. The digested product was separated by macroporous agarose gel electrophoresis, and the whole plasmid was used as a negative control.
pET32a linear vector was recovered using a DNA gel rapid purification kit.
The SiFLS gene fragment of the saussurea involucrata with the linker is connected with a linear vector pET32a by using an information method, the usage amount of the SiFLS linker fragment and the vector is referred to an Exnase specification, and the connection system is as follows:
note that: the calculation method of X, Y, Z is as follows:
vector optimum = [0.01 cloning vector base pair number (pET 32a is 5900 bp) ] ng
＝0.01*5900ng＝59ng，
X=59 ng/carrier concentration.
Fragment optimum = [0.02 base pair number of inserts (e.g. 1500 bp) ] ng=0.02 1500 ng=30 ng
Y = 30 ng/fragment concentration;
Z＝7-X-Y。
the components are sucked, blown and mixed evenly by a pipette, centrifuged to the bottom of the tube and reacted for 1h at 37 ℃. The ligation product was transferred into E.coli DH 5. Alpha. By heat shock, and screened with LB plates supplemented with Car.
2.3, picking up the monoclonal and culturing in LB culture solution for 4-6h, and carrying out bacterial liquid PCR detection. The 20. Mu.L reaction system was as follows:
the PCR reaction procedure was:
and (3) performing agarose gel electrophoresis detection on the PCR product, sending positive monoclonal bacteria liquid to sequence, and ensuring correct sequencing, wherein the expression vector is named pET32a-SiFLS.
Example 3: engineering bacteria induced expression
3.1 BL21 Strain containing recombinant plasmid pET32a-SiFLS induced expression of recombinant protein SiFLS comprises the following steps:
3.1.1 conversion: one BL21 competent cell was added with 5. Mu.L of recombinant plasmid pET32a-SiFLS, and the transformation method was the same as 2.3;
3.1.2 small shaking: single colonies on the plates were picked up in 600. Mu.LCar resistant LB liquid medium (at least 3 single colonies were picked up) and incubated at 37℃for 4-6h;
3.1.3 big shaking: step bacterial liquid (multiple monoclonals can be combined) is transferred into 100mL of Car resistant according to the ratio of 1:100Shake flask culturing in LB liquid medium at 37deg.C for 4-6 hr to OD 600 ＝0.4-0.8；
3.1.4 induced expression: and adding filtered and sterilized IPTG to the bacterial liquid to a final concentration of 0.5mmol/L, wherein the induction condition is 16 ℃ and 150rpm for 12-16h.
3.2 the whole process of extraction and purification of the recombinant protein SiFLS is carried out at 4 ℃ or on ice, and the method is as follows:
3.2.1 collecting the cells: taking out the bacterial liquid, placing the bacterial liquid on ice to stop induction, centrifuging at 5000rpm and 4 ℃ for 10min, and collecting bacterial bodies;
3.2.2 disruption of cells: resuspension of the cells with 15mL 50mM Tris-HCl (containing 20mM imidazole, pre-cooling at 4 ℃) and suction-blowing with a pipettor until the cells are sterile, transferring the cells to a 50mL centrifuge tube, adding PMSF (final concentration about 300 mu M), and working for 99 times by an ultrasonic cytoclasis instrument according to the mode of ultrasonic treatment for 5s and resting for 5 s;
3.2.3 centrifugation: after the ultrasonic treatment is finished, subpackaging the powder into 2mL centrifuge tubes, namely 8 tubes, centrifuging at 12000rpm and 4 ℃ for 30min; tube 1 was reserved (supernatant was removed and placed in another tube, pellet was resuspended in 2mL distilled water and labeled for sample retention);
3.2.4 washing of Ni-NTA column: filling the Ni-NTA column with 20% ethanol solution, repeating twice when the liquid flows out, and washing off all proteins;
3.2.5 equilibrated Ni-NTA column: filling the Ni-NTA column with 50mM Tris-HCl (containing 20mM imidazole) solution, repeating for three times when the liquid flows out, and finally keeping a little, and closing the lower cover;
3.2.6 adding the rest supernatant into the column, covering the upper cover, turning upside down, shaking up the solid in the column, and placing into an incubator to rotate for 30min;
3.2.7 taking out the column, standing for 5min, flushing the hybrid protein with 50mM Tris-HCl (containing 20mM imidazole), then adding 5mL 50mM Tris-HCl (containing 250mM imidazole) solution, closing the upper cover, reversing upside down, shaking up the solid in the column, and placing into an incubator for 30min;
3.2.8 taking out, standing for 5min, centrifuging to obtain target protein solution, washing all proteins with 20% ethanol, and keeping a small amount of solution to protect the column;
3.2.9 50. Mu.L of the purified protein solution was aspirated for SDS-PAGE gel electrophoresis; about 1.5mL glycerol was added to the remaining protein solution and stored at-80 ℃.
3.3SDS-PAGE gel electrophoresis
3.3.1 compounding glue: assembling a vertical electrophoresis plate, referring to a 10% color SDS-PAGE gel rapid kit instruction book by a gel configuration method, inserting a comb, and standing for 1h until the gel is completely solidified;
3.3.2 sample treatment: adding 4 μl of 6 Xprotein loading buffer solution into each 20 μl of protein sample, mixing, centrifuging to bottom of tube, decocting at 98deg.C for 10min, centrifuging, and collecting supernatant.
3.3.3 spotting: after the rubber plate is taken out and installed in the electrophoresis tank, 0.5 Xprotein electrophoresis buffer solution is added until the liquid level of the inner tank does not drop, and the comb is pulled out to sample;
3.3.4 electrophoresis: at the beginning, maintaining constant voltage of 80V to bromophenol blue to run through the concentrated glue, then maintaining constant voltage of 120V to bromophenol blue to run out of the gel, and turning off the power supply;
3.3.5 staining photographs: stripping gel from an electrophoresis plate, placing into a plate, adding appropriate amount of coomassie brilliant blue staining solution (reusable), shaking and staining for 30min, washing with clear water for 3 times each for 30min, and photographing to obtain a gel diagram.
As can be seen from an examination of the electrophoresis gel diagram (FIG. 3), the recombinant plasmid group expresses a specific protein band after IPTG induction, the relative molecular mass is about 60kDa, and the empty control does not express the protein, but only the fusion tag protein band. The fusion tag size of the empty vector pET32a is between 17 and 20kDa, the tag size is removed, and the actual size of the specific protein is consistent with the prediction.
Example 4: siFLS gene biological activity detection
The SiFLS function of saussurea involucrata prepared in example 3 was identified in vitro, dihydromyricetin was used as a substrate, the reaction system was 1mL, water was used to make up to 1mL, and a mixed standard sample group and a control group were set at the same time, and specific ingredients are shown in the following table.
Water was used instead of protein as a negative control. Mixing, placing in a water bath kettle at 30deg.C, reacting for 30min, extracting with equal volume of ethyl acetate for three times, drying by nitrogen blowing, dissolving with 600 μl methanol, filtering with 0.22 μm filter membrane, adding into sample bottle, and detecting by high performance liquid chromatography, see fig. 4.
As shown in FIG. 4, siFLS can react with dihydromyricetin and the product myricetin is produced.
Based on the newly discovered SiFLS gene, the protection scope of the invention also comprises DNA fragments homologous to the genes and DNA fragments of which the encoded proteins are functionally equivalent to the proteins shown in SEQ ID NO. 2. The expression "functionally equivalent to the protein shown in SEQ ID No. 2" as used herein means that the protein encoded by the target DNA fragment is identical or similar to the protein shown in SEQ ID No.2 of the present invention in terms of biological function, physiological and biochemical characteristics, etc. The present invention found that the typical biological function of the protein shown in SEQ ID No.2 is to react with dihydromyricetin and that the product myricetin is produced.
These DNA fragments homologous to the SiFLS gene include alleles, homologous genes, mutant genes and derivative genes corresponding to the nucleotide sequence (SEQ ID NO. 1) of the present invention; the protein encoded by the polypeptide is similar to the protein shown in SEQ ID NO.2 of the present invention, or the substitution, deletion or insertion phenomenon of one, a plurality or dozens of amino acids exists, which belongs to the content of the present invention.
Saussurea involucrata (saussure album) flavonol synthase gene
ATGGAGGTTGTGAGTGTTCAAGAAATAGCCTCACTTTCAAACCTAAATGGCACAATCCCAACTGAGTACATAAGATCATTGGGTGAGCAACCAGCAACCACCACCATCCATGGGGTGGTGCTGGAGGTTCCGGTGATCGATCTCAGCCACCCCGATGCCGGAAAACTTGTGGCTTCCATCTCAGAAGCCAGCAGAGAATGGGGAATCTTTCAAGTGGTAAACCATGGGATACCAAATGAACTCATAAGCAAGTTACAGAAAGTAGGAAAAGAGTTCTTTGAATTGCCACAAGAAGAGAAGGAAGCCATAGCCAGACCTGAAAATATTAATGAGGGTGTTGAAGGTTATGGAACCAAGCTTCAGAAGGAGGTGGAAGGGAAGAAAGGGTGGGTGGATCACTTGTTTCATAGGGTTTGGCCACCTTCTGCCATTAACTATCACTTTTGGCCCAAGAATCCTCCTTCTTACAGAGAGATAAATGAGCAATACACACAAATGTTGATAGGGGTGGCAAACAAATTGTTTGGATTTCTATCAAAAGGACTTGAACTAGAAGAGAATGCAATGAAAGAAGGGTTGGGTGGTGAAGACTTAACCTACATGATGAAAATAAACTACTACCCACCATGCCCATGTCCGGAGCTAGCTCTTGGGGTGGTACCCCATACTGATATGTCTTCCCTCACCATTCTTGTCCCAAATGAAGTCCAAGGTCTACAAGTTTTCAAAGATGATCATTGGTATGATGTTGCATACATCCCTAATGCTCTCATTATTCACATTGGTGATCAAATTGAGATATTGAGCAATGGGAAGTATAAGAGTGTGTACCACAGAACAACAGTGAATAAGGAGAAGACAAGGATGTCTTGGCCAATGTTCTTGGAGCCACCACCAGAGTTTGAGGTTGGACCAATTCCACAGCTCATCAATCAAGATAATCCACCAAAATTCAAGACTAAGAAGTTCAAAGATTATCTCTATTGCAAGTTAAACAAGCTTCCACAGTGA.
Saussurea involucrata flavonol synthase (saussure ai volcanic nolsynthase)
MEVVSVQEIASLSNLNGTIPTEYIRSLGEQPATTTIHGVVLEVPVIDLSHPDAGKLVASISEASREWGIFQVVNHGIPNELISKLQKVGKEFFELPQEEKEAIARPENINEGVEGYGTKLQKEVEGKKGWVDHLFHRVWPPSAINYHFWPKNPPSYREINEQYTQMLIGVANKLFGFLSKGLELEENAMKEGLGGEDLTYMMKINYYPPCPCPELALGVVPHTDMSSLTILVPNEVQGLQVFKDDHWYDVAYIPNALIIHIGDQIEILSNGKYKSVYHRTTVNKEKTRMSWPMFLEPPPEFEVGPIPQLINQDNPPKFKTKKFKDYLYCKLNKLPQ。
Claims (5)
1. The saussurea involucrata flavonol synthase gene is abbreviated as SiFLS, and is characterized in that the nucleotide of the gene is a nucleotide sequence shown as SEQ ID NO. 1.
2. The saussurea involucrata flavonol synthase encoded by the SiFLS gene of claim 1, wherein the amino acid of the saussurea involucrata flavonol synthase is the amino acid sequence shown in SEQ ID No. 2.
3. A recombinant vector pET32a-SiFLS containing the saussurea involucrata flavonol synthase gene of claim 1.
4. The use of the saussurea involucrata flavonol synthase gene of claim 1 for catalyzing the production of dihydromyricetin.
5. Use of the recombinant vector pET32a-SiFLS of claim 3 for catalyzing the production of dihydromyricetin.
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| CN101942420A (en) * | 2010-08-05 | 2011-01-12 | 中国科学院植物研究所 | Protein related to synthesis of flavone compounds, coding gene and application thereof |
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