CN117004628B - Saussurea involucrata flavonol synthase gene and its coding product and use - Google Patents

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

Tianshan Snow Lotus flavonol synthase gene and its encoded products and applications

Technical field

The invention belongs to the technical field of medicinal plant genetic engineering and relates to the Tianshan Snow Lotus flavonol synthase gene (SiFLS) and its encoding products and applications.

Background technique

The dry above-ground part of Tianshan Snow Lotus (Saussurea involucrata) can be used as medicine and is called "Snow Lotus". It is a precious Chinese herbal medicine resource. Tianshan Snow Lotus is a commonly used medicine among the Uighurs. It has the functions of nourishing nerves, nourishing the kidneys and activating blood circulation, regulating abnormal body fluids, and strengthening muscles and bones. It is mainly used clinically to treat irregular menstruation, lower abdominal pain, wind-cold-damp arthralgia, etc. The Tianshan Snow Lotus has a special growth environment and is mainly distributed in the Tianshan, Altay and Kunshan mountains in Xinjiang, my country. At the same time, due to the low natural reproduction rate and slow growth of the Tianshan Snow Lotus, coupled with people's destructive mining and irrational utilization, its wild resources have been reduced. about to extinct.

The main active ingredients in Tianshan Snow Lotus are flavonoids, Snow Lotus polysaccharide and alkaloids, among which flavonoids are the main functional ingredients. Flavonols, as important members of flavonoids, are synthesized by flavonol synthase (FLS), with dihydroflavonols, such as dihydrokaempferol (DHK), dihydroquercetin ( dihydroquercetin (DHQ), dihydromyricetin (DHM), etc. are used as substrates to generate flavonols such as kempferol, myricetin, and quercetin; these products are passed through methyltransferase The activities of enzymes such as glycosyltransferase and acyltransferase are further modified to generate a variety of flavonol derivatives. The catalytic enzyme for dihydromyricetin in Snow Lotus Tianshan has not been disclosed in the prior art.

Contents of the invention

The technical problem to be solved by the present invention is to provide the Tianshan Snow Lotus flavonol synthase (SiFLS) gene and its encoded product, as well as the application of the product in medicine.

The Tianshan Snow Lotus flavonol synthase (SiFLS) gene provided by the present invention is one of the following nucleotide sequences:

(1) Having the nucleotide sequence shown in SEQ ID NO.1;

(2) The homologous sequence of one or more nucleotides added, substituted, inserted or deleted to the nucleotide sequence shown in SEQ ID NO. 1 or its allele and its derived nucleotide sequence.

The present invention also provides a flavonol synthase encoded by SiFLS gene, and the amino acid synthesized by the flavonol synthase of Snowdrop is one of the following sequences:

(1) Having the amino acid sequence shown in SEQ ID NO.2;

(2) A homologous sequence in which one or more amino acids are added, substituted, inserted or deleted in SEQ ID NO.2.

The invention also provides the recombinant vector pET32a-SiFLS containing the gene.

The invention also provides the application of the gene in regulating the production of myricetin from the flavonol active substances of Snow Lotus Tianshan using dihydromyricetin as a substrate.

The present invention also provides the application of the recombinant vector pET32a-SiFLS in regulating the production of myricetin from the flavonol active substances of Snow Lotus Tianshan using dihydromyricetin as a substrate.

Compared with the prior art, the beneficial effects of the present invention are as follows:

The SiFLS gene obtained in the present invention is a key gene for regulating the synthesis of Tianshan Snow Lotus flavonols, which is of great significance for obtaining high flavonol Tianshan Snow Lotus plants through molecular breeding means; molecular regulation of the SiFLS gene can increase the number of Tianshan Snow Lotus traditional Chinese medicines. The content of medicinal ingredients has important reference significance for improving the content of effective active ingredients in medicinal plants. The flavonol synthase encoded by the SiFLS gene uses dihydromyricetin as the substrate to generate the product myricetin.

Description of the drawings

Figure 1: SiFLS gene cDNA agarose gel electrophoresis detection results;

Figure 2: SiFLS functional domain prediction analysis results;

Figure 3: SDS-PAGE gel electrophoresis pattern 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;

Figure 4: SiFLS in vitro enzyme activity test results. (a) is the mixed standard sample at 288nm (all components of the reaction system and the product myricetin), (b) is the enzyme activity product of the control group at 368nm, (c) is the enzyme activity product of the experimental group at 368nm.

Detailed ways

The materials, reagents, vectors and E. coli used in the following examples can all be purchased from the company through commercial channels unless otherwise specified. The PCR amplification reaction Marker was purchased from Quanshijin Biotechnology Co., Ltd., and the PCR amplification reaction The enzyme was purchased from TaKaRa Company, and the marker used in the protein electrophoresis experiment was purchased from Quanjin Biotechnology Company.

Implementation example 1: Construction of full-length cDNA library of Snow Lotus from Tianshan Mountains

Take the young leaves of Tianshan Snow Lotus seedlings, use the TransZol method (Quanshijin Biotechnology Co., Ltd.) to extract the total RNA of the young stems, use a nucleic acid analyzer to detect the content and purity of the total RNA, and take the total RNA for reverse transcription reaction. The kit used is Easy One-Step gDNA Removal and cDNA Synthesis SuperMix (Quanshijin Biotechnology Co., Ltd.). Specific steps are as follows:

1.1 Homogenization: Take the Tianshan Snow Lotus tissue sample stored at -80°C, transfer it to a mortar pre-cooled with liquid nitrogen, and grind the plant sample to powder with a pestle. Take 50-100mg (approximately 1/5 of the tube) of the ground sample and put it into a 2mL fully pre-cooled centrifuge tube (the liquid nitrogen in the tube must be removed evenly to prevent the cap from collapsing), then add 1mL of TransZoL and shake to mix.

1.2 Stratification: Place the homogenized sample on ice for 5 minutes (to completely separate the nuclear protein material). Add 200 μL chloroform, cap the tube tightly, shake vigorously for 3 seconds, and place on ice for 3 minutes. Centrifuge at high speed for about 15 minutes in a refrigerated centrifuge at 4°C to separate into layers to obtain a colorless supernatant, white middle layer protein and red lower organic phase.

1.3 Precipitation of RNA: At room temperature, take about 550-650 μL of the supernatant (as much as possible while ensuring quality) and transfer it to a new 2mL centrifuge tube (this centrifuge tube needs to be fully pre-cooled). Add 500 μL isopropyl alcohol, mix gently by inverting, and place at -20°C for 10 minutes. Centrifuge at high speed for 10 minutes at 4°C and discard the supernatant (remove as much isopropyl alcohol as possible).

1.4 RNA rinse: Add at least 1 mL of 75% ethanol, vortex to mix, and centrifuge at 7500g for 5 minutes at 4°C. 1.5 Re-dissolve RNA: Discard the supernatant, absorb ethanol, open the lid and let dry at room temperature for 20 minutes. Add 30-40 μL of RNase-free ddH ₂ O (store at 4°C, place on ice) to dissolve RNA for 2-3 minutes.

1.6 Use agarose gel electrophoresis and nucleic acid analyzer to detect the content and purity of total RNA.

1.7RNA reverse transcription: The reverse transcription system is as follows:

First mix the RNA template, Anchored Oligo (dT) and RNase-free Water, incubate at 65°C for 5 minutes, incubate on ice for 2 minutes, and then add other reaction components. Mix gently and incubate at 42°C for 15 minutes. Heat at 85°C for 5 seconds to inactivate TransScript RT and gDNA Remover to obtain the Tianshan Snow Lotus cDNA library.

Example 2: SiFLS gene cloning and prokaryotic expression vector construction

2.1 Use Tianshan Snow Lotus cDNA as template and use degenerate primers:

F: 5'-ATGGAGGTBGNDAGDGTBCA-3',

R: 5'-TCACNGGAAGBTTRTTKARCT-3',

Carry out PCR amplification reaction, the reaction system is 20μL:

Mix and centrifuge to the bottom of the tube. The PCR amplification procedure is as follows:

The PCR amplification product was detected by agarose gel electrophoresis, photographed using a gel imager, and the gel electrophoresis pattern was observed. The amplified fragment was approximately 1000 bp (Figure 1). Expand the reaction system and use a gel recovery kit to recover gene fragments.

2.2 Sequencing was used to verify the amplified fragment, and the nucleic acid sequence as SEQ ID NO.1 was obtained, and the amino acid sequence encoded by it was as shown in SEQ ID NO.2. Functional domain analysis of its encoded amino acid sequence showed that it belongs to the PLN02704 superfamily and is a typical flavonol synthase (Figure 2).

To expand the system, design pET32a adapters upstream and downstream of the Tianshan Snow Lotus SiFLS gene cDNA sequence, and use the PCR product obtained in 2.1 as a template for PCR amplification. The system is 50 μL.

The PCR reaction program settings are the same as 2.1.

After the reaction, electrophoresis was performed using a macroporous gel. Use the DNA gel rapid purification kit to recover the SiFLS fragment with pET32a adapter. For specific operation steps, refer to the kit instructions.

The bacterial solution containing pET32a empty vector stored at -80°C was inoculated into 50 mL of carbenicillin (Car)-resistant LB liquid medium, and cultured in a shake flask at 37°C overnight. Use the plasmid miniprep kit to extract the pET32a empty vector. For specific steps, refer to the kit instructions.

pET32a empty vector was stored at -20°C. Use EcoRⅠ to perform single enzyme digestion on the extracted pET32a empty vector. The system is as follows:

Use a pipette to pipette and mix each component, centrifuge to the bottom of the tube, react at 37°C for 1 hour, take out and immediately place on ice to terminate the reaction. The digested products were separated by large-pore agarose gel electrophoresis, and the intact plasmid was used as a negative control.

Use the DNA gel rapid purification kit to recover the pET32a linear vector.

Use the infusion method to connect the SiFLS gene fragment of Snow Lotus Tianshan with the adapter to the linear vector pET32a. The dosage of SiFLS adapter fragment and vector is according to the Exnase instructions. The connection system is as follows:

Note: The calculation method of X, Y and Z is as follows:

The optimal amount of vector = [0.01*number of base pairs of cloning vector (pET32a is 5900bp)]ng

=0.01*5900ng=59ng,

X=59ng/carrier concentration.

Optimal amount of fragment = [0.02*number of base pairs of inserted fragment (e.g. 1500bp)]ng=0.02*1500ng=30ng

Y=30ng/fragment concentration;

Z=7-X-Y.

Use a pipette to pipette and mix each component, centrifuge to the bottom of the tube, and react at 37°C for 1 hour. The ligation product was transferred into E. coli DH5α using heat shock method and screened on LB plate added with Car.

2.3 Pick a single clone and culture it in LB culture medium for 4-6 hours, and perform PCR detection of the bacterial liquid. The 20μL reaction system is as follows:

The PCR reaction procedure is:

The PCR product was tested by agarose gel electrophoresis, and the positive single-clonal bacterial liquid was sent for sequencing. The sequencing was correct. The expression vector was named pET32a-SiFLS.

Example 3: Induced expression by engineering bacteria

3.1 The steps for inducing the expression of recombinant protein SiFLS in BL21 strain containing recombinant plasmid pET32a-SiFLS are as follows:

3.1.1 Transformation: Add 5 μL of recombinant plasmid pET32a-SiFLS to one BL21 competent cell. The transformation method is the same as 2.3;

3.1.2 Small shake: Pick single colonies on the plate in 600 μL Car-resistant LB liquid medium (pick at least 3 single colonies), and culture at 37°C for 4-6 hours;

3.1.3 Shake: Transfer the step bacteria liquid (multiple single clones can be combined) into 100mL of Car-resistant LB liquid medium at a ratio of 1:100, and culture in a shake flask at 37°C for 4-6 hours until OD ₆₀₀ =0.4-0.8;

3.1.4 Induced expression: Add filtered sterilized IPTG to the bacterial solution to a final concentration of 0.5mmol/L. The induction conditions are 16°C, 150rpm, 12-16h.

3.2 The entire operation of extracting and purifying recombinant protein SiFLS is performed at 4°C or on ice, as follows:

3.2.1 Collect bacterial cells: Take out the bacterial liquid and place it on ice to stop induction. Centrifuge at 5000rpm and 4℃ for 10 minutes to collect bacterial cells;

3.2.2 Broken cells: Resuspend the cells in 15mL of 50mM Tris-HCl (containing 20mM imidazole, pre-cooled at 4°C), mix with a pipette until there are no small bacterial clumps, transfer to a 50mL centrifuge tube, and add PMSF (final concentration is about 300 μM), use the ultrasonic cell disrupter to work 99 times by ultrasonic for 5 seconds and rest for 5 seconds;

3.2.3 Centrifuge: After ultrasonic, divide into 2mL centrifuge tubes, about 8 tubes, centrifuge at 12000rpm, 4℃ for 30min; keep 1 tube (take out the supernatant and put it into another tube, resuspend the precipitate in 2mL distilled water, Mark and retain samples);

3.2.4 Clean the Ni-NTA column: Fill the Ni-NTA column with 20% ethanol solution, wait until the liquid flows out, repeat twice, and wash away all proteins;

3.2.5 Equilibrate the Ni-NTA column: Fill the Ni-NTA column with 50mM Tris-HCl (containing 20mM imidazole) solution, wait until the liquid flows out, repeat three times, and finally retain a little, and cover the lower lid tightly;

3.2.6 Add the remaining supernatant to the column, close the upper lid tightly, shake the solids in the column upside down, and place it in an incubator to rotate for 30 minutes;

3.2.7 Take out the column and let it stand for 5 minutes, rinse the impurity proteins with 50mM Tris-HCl (containing 20mM imidazole), then add 5mL of 50mM Tris-HCl (containing 250mM imidazole) solution, cover the top lid tightly, and shake the solids in the column upside down. , put into the incubator and rotate for 30 minutes;

3.2.8 Take it out and let it stand for 5 minutes. Use a 10mL centrifuge tube to collect the effluent to obtain the target protein solution. Finally, rinse all the protein with 20% ethanol and retain a small amount of solution to protect the column;

3.2.9 Aspirate 50 μL of the purified protein solution for SDS-PAGE gel electrophoresis detection; add about 1.5 mL of glycerol to the remaining protein solution and store it at -80°C.

3.3SDS-PAGE gel electrophoresis

3.3.1 Gel preparation: Assemble the vertical electrophoresis plate. For the gel configuration method, refer to the instructions of the 10% Color SDS-PAGE Gel Rapid Kit. Insert the comb and let it sit for 1 hour until the gel is completely solidified;

3.3.2 Sample processing: Add 4 μL of 6× protein loading buffer to every 20 μL of protein sample, mix and centrifuge to the bottom of the tube, cook the sample at 98°C for 10 min, centrifuge, and take the supernatant for spotting.

3.3.3 Spotting: After taking out the gel plate and installing it into the electrophoresis tank, add 0.5× protein electrophoresis buffer until the liquid level in the inner tank does not drop, pull out the comb, and start spotting;

3.3.4 Electrophoresis: At the beginning, maintain a constant voltage of 80V until bromophenol blue runs through the stacking gel, then maintain a constant voltage of 120V until bromophenol blue runs out of the gel, and turn off the power;

3.3.5 Stain and take photos: Peel the gel from the electrophoresis plate and put it into a dish, add an appropriate amount of Coomassie Brilliant Blue staining solution (reusable), shake and stain for 30 minutes, shake and rinse with water 3 times, 30 minutes each time, take photos to obtain the gel picture.

Observing the electrophoresis gel image (Figure 3), it can be found that after induction by IPTG, the recombinant plasmid group expressed a specific protein band with a relative molecular mass of approximately 60 kDa, while the empty control did not express the protein and only had a fusion tag protein band. The size of the fusion tag of the empty vector pET32a is between 17-20kDa. If the tag size is removed, the actual size of the specific protein is consistent with the prediction.

Example 4: SiFLS gene biological activity detection

To identify the function of Snow Lotus Tianshan SiFLS prepared in Example 3 in vitro, dihydromyricetin was used as the substrate, the reaction system was 1 mL, and water was added to 1 mL. A mixed standard sample group and a control group were also set up. The specific ingredients are shown in the table below.

Water was used instead of protein as a negative control. After mixing, place it in a 30°C water bath and react for 30 minutes. Extract three times with an equal volume of ethyl acetate. After drying with nitrogen, dissolve it with 600 μL methanol, filter it through a 0.22 μm filter membrane, add it to a sample bottle, and carry out high-efficiency liquid preparation. Phase chromatography detection, see Figure 4.

As shown in Figure 4, SiFLS can react with dihydromyricetin, and the product myricetin is produced.

Based on the newly discovered SiFLS gene, the protection scope of the present invention also includes DNA fragments homologous to the above-mentioned genes, as well as DNA fragments encoding proteins that are functionally equivalent to the protein shown in SEQ ID NO. 2. The term "functionally equivalent to the protein shown in SEQ ID NO. 2" referred to herein means that the protein encoded by the target DNA fragment is identical to the protein shown in SEQ ID NO. 2 in the present invention in terms of biological function, physiological and biochemical characteristics, etc. Same or similar. The present invention found that the typical biological function of the protein shown in SEQ ID NO. 2 is to react with dihydromyricetin and produce myricetin as a product.

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 proteins they encode are similar to the SEQ ID NO. The protein shown in .2, or the substitution, deletion or insertion of one, several or dozens of amino acids, all belong to the content of the present invention.

Saussurea involucrata flavonol synthase gene

ATGGAGGTTGTGAGTGTTCAAGAAATAGCCTCACTTTCAAACCTAAATGGCACAATCCCAACTGAGTACATAAGATCATTGGGTGAGCAACCAGCAACCACCACCATCCATGGGGTGGTGCTGGAGGTTCCGGTGATCGATCTCAGCCACCCCGATGCCGGAAAACTTGTGGCTTCCATCTCAGAAGCCAGCAGAGAATGGGGAATCTTTCAAGTGGTAAACCATGGGATACCAAATGAACTCATAAGCAAGTTACAGAAAGT AGGAAAAGAGTTCTTTGAATTGCCACAAGAAGAGAAGGAAGCCATAGCCAGACCTGAAAATATTAATGAGGGTGTTGAAGGTTATGGAACCAAGCTTCAGAAGGAGGTGGAAGGGAAGAAAGGGTTGGGTGGATCACTTGTTTCATAGGGTTTGGCCACCTTCTGCCATTAACTATCACTTTTGGCCCAAGAATCCTCCTTCTTACAGAGAGATAAATGAGCAATACACACAAATGTTGATAGGGGTGGCAAAATTGTTTGGATTTC TATCAAAAGGACTTGAACTAGAAGAGAATGCAATGAAAGAAGGGTTGGGTGGTGAAGACTTAACCTACATGATGAAAATAAACTACTACCCACCATGCCCATGTCCGGAGCTAGCTCTTGGGGTGGTACCCCATACTGATATGTCTTCCCTCACCATTCTTGTCCCAAATGAAGTCCAAGGTCTACAAGTTTTCAAAGATGATCATTGGTATGATGTTGCATACATCCCTAATGCTCTCATTATTCACATTGGTGATCAAATTGAGATATTGAG CAATGGGAAGTATAAGAGTGTGTACCACAGAACAACAGTGAATAAGGAGAAGACAAGGATGTCTTGGCCAATGTTCTTGGAGCCACCACCAGAGTTTGAGGTTGGACCAATTCCACAGCTCATCAATCAAGATAATCCACCAAAATTCAAGACTAAGAAGTTCAAAGATTATCTCTATTGCAAGTTAAACAAGCTTCCACAGTGA.

Saussureainvolucrateflavonolsynthase

MEVVSVQEIASLSNLNGTIPTEYIRSLGEQPATTTIHGVVLEVPVIDLSHPDAGKLVASISEASREWGIFQVVNHGIPNELISKLQKVGKEFFELPQEEKEAIARPENINEGVEGYGTKLQKEVEGKKGWVDHLFHRVWPPSAINYHFWPKNPPSYREINEQYTQMLIGVANKLFGFLSKGLELEENAMKEGLGGEDLTYMMKINYYPPCP CPELALGVVPHTDMSSLTILVPNEVQGLQVFKDDHWYDVAYIPNALIIHIGDQIEILSNGKYKSVYHRTTVNKEKTRMSWPMFLEPPPEFEVGPIPQLINQDNPPKFKTKKFKDYLYCKLNKLPQ.

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.