Monochamus alternatus glutathione S-transferase MaGSTd4 and its encoding gene and application

Abstract

The present invention provides a pine sawyer beetle glutathione S-transferase MaGSTd4 and its encoding gene and application, belonging to the field of molecular biochemical technology. The amino acid sequence of the pine sawyer beetle glutathione S-transferase MaGSTd4 provided by the present invention is shown in SEQ ID NO.1. The MaGSTd4 provided by the present invention can be used to explore the molecular physiological mechanism of the pine sawyer beetle larvae adapting to α-pinene, which not only provides theoretical support for the intrinsic mechanism of the pine sawyer beetle overcoming the conifer terpene defense and then breaking out into a disaster, but also lays the foundation for the development of a more effective and environmentally friendly new pine wood nematode disease prevention and control technology. The present invention obtains a recombinant pine sawyer beetle MaGSTd4 protein through recombinant expression, which has strong glutathione s-transferase catalytic activity and antioxidant capacity, and has certain application value in the field of biomedicine.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular biochemistry, and specifically relates to Monochamus alternatus glutathione S-transferase MaGSTd4 and a coding gene and application thereof. Background Art

[0002] Monochamus alternatus belongs to the order Coleoptera, family Cerambycidae. Its larvae bore into the phloem and xylem of Pinaceae trees, affecting the transport of nutrients and water, leading to tree weakness and, in severe cases, tree death. In addition, Monochamus alternatus is also the vector of pine wood nematode disease, a major forest quarantine disease in the world, causing huge economic and ecological losses to pine forest ecosystems.

[0003] According to the characteristics of defense response, the defense system of conifers can be divided into induced defense and constitutive defense. Induced defense is generally considered to be a continuation of constitutive defense. Constitutive defense is mainly manifested in the corking of bark, the secretion of resin and the formation of phenolic substances. Terpenoids are the main components of rosin and are secondary metabolites with high biological activity. Their strong biological activity can repel or even kill invading pests. α-pinene has the highest content in the resin and is the most toxic. The larvae of Monochamus alternatus can complete their life cycle despite long-term exposure to terpenes in the tree. It is speculated that they have a complete response mechanism to α-pinene, but it is not clear which genes regulate or respond to it.

[0004] It is known that insect glutathione S-transferase (GST) is widely present in various tissues, and GSTs are especially expressed at high levels in the fat body and midgut, which are important tissues for insect detoxification. However, there are currently no reports on glutathione S-transferase in Monochamus alternatus, let alone reports on the function of glutathione S-transferase in Monochamus alternatus. Summary of the invention

[0005] In view of this, the object of the present invention is to provide a glutathione S-transferase MaGSTd4 from Monochamus alternatus, which has typical glutathione S-transferase activity and also has strong antioxidant activity.

[0006] The present invention provides a glutathione S-transferase MaGSTd4 from Monochamus alternatus. The amino acid sequence of the MaGSTd4 is shown in SEQ ID NO:1.

[0007] The present invention provides a gene encoding the Monochamus alternatus glutathione S-transferase MaGSTd4, and the nucleotide sequence of the gene is shown in SEQ ID NO:2.

[0008] The invention provides a recombinant vector carrying the gene.

[0009] The invention provides an engineering strain for recombinantly expressing the Monochamus alternatus glutathione S-transferase MaGSTd4.

[0010] The present invention provides application of the gene, the recombinant vector or the engineering strain in recombinantly expressing Monochamus alternatus glutathione S-transferase MaGSTd4 protein.

[0011] The invention provides the use of the Monochamus alternatus glutathione S-transferase MaGSTd4 or the engineered strain in preparing a biocatalyst for promoting glutathione binding reaction.

[0012] Preferably, the temperature of the glutathione binding reaction is 20 to 30°C;

[0013] The system pH value of the glutathione binding reaction is 6.0-9.0.

[0014] The present invention provides the use of the Monochamus alternatus glutathione S-transferase MaGSTd4 or the engineered strain in preparing antioxidant products.

[0015] Preferably, the product includes cosmetics, food, preservatives, cleaning agents, durable materials, medicines, antimicrobial agents or health products.

[0016] The present invention provides a glutathione S-transferase MaGSTd4 of Monochamus alternatus, and the amino acid sequence is shown in SEQ ID NO: 1. The present invention obtains a recombinant MaGSTd4 protein after heterologous expression and purification through a prokaryotic expression system, and measures the glutathione S-transferase kinetic parameters and antioxidant capacity thereof, and the results show that the MaGSTd4 protein has strong glutathione S-transferase activity and also has ideal antioxidant capacity, providing a new idea for the application of the protein in the field of biomedicine.

[0017] The present invention provides a gene encoding the pine beetle glutathione S-transferase MaGSTd4, and the nucleotide sequence of the gene is shown in SEQ ID NO: 2. Experiments have shown that the gene encoding the glutathione S-transferase MaGSTd4 responds to α-pinene stress, providing a basis for exploring the molecular physiological mechanism of the pine beetle larvae adapting to α-pinene, thereby providing a theoretical basis for the internal mechanism of the pine beetle overcoming the conifer terpene defense and then causing a disaster. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The relative expression results of MaGSTd4 gene in the intestine of the fourth instar larvae of Monochamus alternatus;

[0019] Figure 2 The relative expression results of MaGSTd4 gene in the fat body of the fourth instar larvae of Monochamus alternatus;

[0020] Figure 3 The relative expression results of MaGSTd4 gene in the epidermis of the fourth instar larvae of Monochamus alternatus;

[0021] Figure 4 The relative expression results of MaGSTd4 gene in the hemolymph of the fourth instar larvae of Monochamus alternatus;

[0022] Figure 5 The results of SDS-PAGE analysis of the induced expression and purification of MaGSTd4. Note: L1 and L6: Marker; L2 and L4: pET-28a(+) supernatant and precipitate after IPTG induction; L3 and L5: MaGSTd4 supernatant and precipitate after IPTG induction; L7 and L8: purified MaGSTd4 protein;

[0023] Figure 6 This is the enzyme kinetic characteristics diagram of the recombinant protein MaGSTd4;

[0024] Figure 7 The results show the effects of temperature (A) and pH value (B) on the activity of MaGSTd4;

[0025] Figure 8 Results of paper disk diffusion assay of Escherichia coli overexpressing MaGSTd4. DETAILED DESCRIPTION

[0026] The invention provides a pine sawyer beetle glutathione S-transferase MaGSTd4. The amino acid sequence of the MaGSTd4 is shown in SEQ ID NO: 1 (MTVIVYGTEGSPVVRGTLLTVGALGLDIEFKEVNTMAGDQFKPEFLKL NPLHTVPTIQDGDFVIFDSHAINAYLVDKYGKDDSLYPKDLQKRAVVNQRLYFDCGIVFPRLGAVIMSIFKGGKTVPKDLADALVDAYTSLETLLERSTYAAGEELTIADFSLVATISTANIMIPITSNRFPKISDWLAKMQALPYYASKNQAGLDKVMAAFKSKLA).

[0027] The present invention provides a gene encoding the Monochamus alternatus glutathione S-transferase MaGSTd4, and the nucleotide sequence of the gene is shown in SEQ ID NO: 2 (ATGACGGTTATAGTGTATGGGACGGAGGGAAGTCCAGTCGTCAGAG).

[0028] The present invention also preferably provides a primer pair for detecting the specific expression of the gene, comprising a forward primer with a nucleotide sequence as shown in SEQ ID NO: 3 (CAGAAGAGGGCCGTGGTTA) and a reverse primer with a nucleotide sequence as shown in SEQ ID NO: 4 (CAGCAGCGTAAGTGCTTCTT). The primer pair can be used to detect the expression of the gene in Monochamus alternatus in response to α-pinene stress.

[0029] The invention provides a recombinant vector carrying the gene.

[0030] In the present invention, the backbone vector of the recombinant vector is preferably pET-28a(+) plasmid.

[0031] In the present invention, the method for constructing the recombinant vector preferably comprises the following steps:

[0032] The MaGSTd4 gene fragment was amplified using a primer pair, and the obtained MaGSTd4 gene fragment was cloned into a linear vector to obtain a recombinant vector.

[0033] In the present invention, the primer pair preferably includes primers with nucleotide sequences such as SEQ ID NO: 5 and SEQ ID NO: 6. The present invention has no special restrictions on the cloning and ligation methods, and the cloning and ligation methods known in the art can be used. After ligation, identification is also preferably included. The identification method is preferably to transfer the ligation product into Escherichia coli, culture it, extract the plasmid, and use SEQ ID NO: 9 and SEQ ID NO: 10 as primers for sequencing.

[0034] The invention provides an engineering strain for recombinantly expressing the Monochamus alternatus glutathione S-transferase MaGSTd4.

[0035] In the present invention, the engineered strain is preferably an engineered strain constructed by a prokaryotic expression system. The construction method of the engineered strain is to transform the recombinant vector into a competent host bacterium. The host bacterium is preferably Escherichia coli. The present invention has no particular limitation on the transformation method, and a transformation method known in the art can be used.

[0036] The present invention provides application of the gene, the recombinant vector or the engineering strain in recombinantly expressing Monochamus alternatus glutathione S-transferase MaGSTd4 protein.

[0037] In the present invention, the method for recombinantly expressing Monochamus alternatus glutathione S-transferase MaGSTd4 protein preferably comprises the following steps:

[0038] The engineered strain constructed above is cultured and induced to express, the bacteria are separated and crushed, the protein is collected and purified, and the recombinant Monochamus alternatus glutathione S-transferase MaGSTd4 protein is obtained through dialysis and concentration.

[0039] In the present invention, the method of culturing and inducing expression is preferably to place the engineered strain in a kana-resistant LB liquid medium and culture it on a shaking platform. 600 When the pH value is 0.4 to 0.6, IPTG is added to a final concentration of 1 mM to continue culturing and collect the cells. The temperature for culturing or inducing expression is preferably 37°C, and the rotation speed is preferably 200 rpm. After induced expression for 18 hours, the cells are collected.

[0040] In the present invention, the crushing is preferably ultrasonic crushing. The conditions of the ultrasonic crushing are preferably: power 150W, time 30min, frequency 20kHz. The purification is preferably performed by using a resin strain. The purpose of the dialysis is to remove salt.

[0041] The invention provides the use of the Monochamus alternatus glutathione S-transferase MaGSTd4 or the engineered strain in preparing a biocatalyst for promoting glutathione binding reaction.

[0042] The results of the present invention show that the MaGSTd4 recombinant protein K m =1.24±0.21mM, V max =2985.78±347.15μmol / min / mg.

[0043] In the present invention, the temperature of the glutathione binding reaction is preferably 20-30° C., more preferably 25° C. The pH value of the system of the glutathione binding reaction is preferably 6.0-9.0, more preferably 8.

[0044] The present invention provides the use of the Monochamus alternatus glutathione S-transferase MaGSTd4 or the engineered strain in preparing antioxidant products.

[0045] In the present invention, the product preferably includes cosmetics, food, preservatives, cleaning agents, durable materials, medicines, antibacterial agents or health products.

[0046] In the present invention, the paper disk diffusion test was used to detect the antioxidant activity of MaGSTd4. The results showed that Escherichia coli expressing MaGSTd4 protein began to show stronger antioxidant ability, and the inhibition zone around the filter paper was significantly smaller than that in the empty Escherichia coli.

[0047] The following is a detailed description of the Monochamus alternatus glutathione S-transferase MaGSTd4 and its encoding gene and application provided by the present invention in conjunction with the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0048] In the following examples, operations not described in detail are all routine biological experimental operations, which can be performed with reference to molecular biology experimental manuals and existing published journals and literature.

[0049] Example 1

[0050] Cloning of MaGSTd4 gene

[0051] (I) Feeding treatment of larvae

[0052] Select dead pine trees with high insect population density from the forest, cut them short and split them with an axe, collect the pine beetle larvae in the wood sections, disinfect them with 75% ethanol, transfer them to a feeding box with artificial feed, feed them to the fourth instar, add α-pinene to the sterilized artificial feed at a ratio of 5.6 mg / g, mix them with a glass rod, and wait for the feed to cool completely before feeding. After feeding for 24 hours, take out the larvae, disinfect them with 75% alcohol, put them on ice for anesthesia and then dissect them. The intestinal samples are placed in centrifuge tubes, numbered and recorded, and stored in an ultra-low temperature refrigerator at -80℃.

[0053] (II) RNA preparation from Monochamus alternatus larvae

[0054] The total RNA of Monochamus alternatus larvae was extracted using the TRIzol (TaKaRa, Japan) method. The extraction steps are as follows:

[0055] (1) RNase removal of mortar and medicine spoon: Wrap with aluminum foil and place in a constant temperature air drying oven at 160°C for 3 h. RNase has been removed from subsequent gun tips, centrifuge tubes, and chemical reagents. All reagents and materials were sterilized and transferred to a clean bench. RNA extraction was performed according to the instructions of TRNzol Universal total RNA extraction reagent.

[0056] (2) Grind the intestinal samples frozen in a -80°C ultra-low temperature freezer into powder using a mortar and pestle, and continuously add liquid nitrogen to keep the temperature low. Use a micro-spoon to take 30-50 mg of dry powder into a 1.5 mL centrifuge tube, add 1 mL of TRNzol Universal reagent, vortex and mix, and then let stand at room temperature for 5 min to completely separate the nucleic acid-protein complex.

[0057] (3) Centrifuge at 12000 rpm for 10 min at 4°C and collect the supernatant.

[0058] (4) Add 0.2 mL of chloroform to the supernatant, shake vigorously to mix, and let stand at room temperature for 3 min.

[0059] (5) After centrifugation at 12000 rpm for 15 min at 4°C, the upper aqueous phase (about 500 μL) was collected and transferred to a new 1.5 mL centrifuge tube.

[0060] (6) Add an equal volume (about 500 μL) of isopropanol to the aqueous supernatant, mix well, and let stand at room temperature for 10 min.

[0061] (7) Centrifuge at 12000 rpm for 10 min at 4°C and discard the supernatant.

[0062] (8) Add 1 mL of 75% ethanol to wash the precipitate.

[0063] (9) Centrifuge at 10,000 rpm for 10 min at 4°C and discard the supernatant.

[0064] (10) Repeat steps 8 and 9 once.

[0065] (11) Allow the RNA to dry at room temperature. Add 50 μL RNase-Free ddH2O to each tube and pipette and mix repeatedly to fully dissolve the RNA.

[0066] (12) Samples were taken for agarose gel (1%) electrophoresis; the RNA concentration and quality were determined using Nano Drop 2000.

[0067] (III) cDNA synthesis of Monochamus alternatus larvae

[0068] The synthesis of cDNA was carried out according to the instruction manual of the reverse transcription kit.

[0069] 1) Remove genomic DNA. See Table 1 for specific components and procedures:

[0070] Table 1. Genome removal system

[0071]

[0072] After mixing, the temperature was controlled by a PCR instrument. The specific temperature is shown in Table 2.

[0073] Table 2 Genome removal conditions

[0074]

[0075] 2) Synthesis of cDNA, see Table 3 for details:

[0076] Table 3 cDNA synthesis reaction system

[0077]

[0078] After mixing, the temperature was controlled by a PCR instrument. The reaction conditions are shown in Table 4:

[0079] Table 4 cDNA synthesis reaction conditions

[0080]

[0081] 3) The concentration was determined using Nano Drop 2000.

[0082] (IV) MaGSTd4 gene cloning

[0083] (1) Retrieve glutathione S-transferase-related Unigene sequences from the Monochamus alternatus transcriptome database constructed in our laboratory. Determine its open reading frame (ORF) using the SeqBuilder software in the DNAstar software package; and annotate the longest and most complete ORF in the sequence using BLAST on the NCBI website (http: / / www.ncbi.nlm.gov / BLAST / ).

[0084] The presence of a complete ORF of the glutathione S-transferase sequence was verified by subsequent cloning and sequencing.

[0085] Prepare the reaction system according to Table 5:

[0086] Table 5 Target gene amplification reaction system

[0087]

[0088] Wherein: Primer F: ATGACGGTTATAGTGTATGGGAC (SEQ ID NO: 7);

[0089] Primer R: TTAAGCCAGTTTTGCTCTTAAACGCTG (SEQ ID NO: 8).

[0090] The amplification program is shown in Table 6:

[0091] Table 6 Target gene amplification reaction procedure

[0092]

[0093] (4) After the full-length cloned product is electrophoresed on agarose gel, a clear band appears at the target fragment size and can be used for gel excision and recovery. The specific operation is based on the DNA gel recovery kit:

[0094] 1) Cut out the target band, cut it into pieces, and then transfer it into a centrifuge tube;

[0095] 2) Weigh and record the weight of the gel (use an empty tube to reset to zero);

[0096] 3) Add an equal volume of dissolution buffer and 37°C metal bath sol;

[0097] 4) Transfer the gel solution to a filter column, let stand at room temperature for 1 min, centrifuge at 12,000 rpm for 1 min at room temperature, and discard the filtrate;

[0098] 5) Add an equal volume of rinse solution to the filter column, centrifuge at 12,000 rpm for 1 min at room temperature, and discard the filtrate;

[0099] 6) Repeat step 5;

[0100] 7) Transfer the filter column to a new collection tube and centrifuge at 12,000 rpm for 2 minutes at room temperature;

[0101] 8) Transfer the filter column to a new centrifuge tube, add 20 μL of elution solution to the center, let stand at room temperature for 30 seconds, and centrifuge at 12,000 rpm for 2 minutes to obtain the purified DNA solution;

[0102] 9) Check whether the band is single by agarose gel and determine the concentration using Nano Drop 2000.

[0103] (5) The recovered full-length glutathione S-transferase fragment was converted into a circular plasmid by a TA ligation kit for cloning and sequencing. The specific operation was as follows:

[0104] 1) Connection:

[0105] Prepare the reaction system according to Table 7.

[0106] Table 7 Ligation reaction system

[0107]

[0108] Keep warm at 37°C for 5 minutes.

[0109] 2) Conversion

[0110] The newly generated plasmid after ligation was added to DH5α chemically competent cells and allowed to stand on ice for 30 minutes to allow the plasmid DNA to attach to the cell membrane; after the ice bath, heat shock was performed at 42°C in a metal bath for 45 seconds and the cells were placed on ice for 2 minutes to allow the plasmid to enter the cells.

[0111] Add 900 μL LB liquid medium, mix well and place in a shaker at 37°C and 200 rpm to recover for 1 hour. Centrifuge the recovered E. coli and spread on amp-resistant LB solid plates. Incubate the plates in an incubator at 37°C overnight.

[0112] 3) Identification of positive clones

[0113] Pick a single colony and transfer it to LB liquid culture medium. After propagation in a shaker at 37℃ and 200rpm for 1 hour, perform PCR verification. The specific components and procedures are shown in Table 8:

[0114] Table 8 Positive cloning reaction system

[0115]

[0116] The prepared reaction system was carried out according to Table 9.

[0117] Table 9 Positive cloning reaction procedure

[0118]

[0119] After amplification, the fragment length should be 110bp longer than the target fragment length by agarose gel electrophoresis. If the length is consistent, it is a positive clone and can be sent for testing.

[0120] 4) Sequencing

[0121] The bacterial culture of the positive clones was bidirectionally sequenced and spliced ​​using M13F+M13R as sequencing primers.

[0122] The amino acid sequence of MaGSTd4 obtained by sequencing is shown in SEQ ID NO.1 (MTVIVYGTEGSP VVRGTLLTVGALGLDIEFKEVNTMAGDQFKPEFLKLNPLHTVPTIQDGDFVIFDSHAINAYLVDKYGKDDSLYPKDLQKRAVVNQRLYFDCGIVFPRLGAVIMSIFKGGKTVPKDLADALVDAYTSLETLLERSTYAAGEELTIADFSLVATISTANIMIPITSNRFPKISDWLAKMQALPYYASKNQAGLDKVMAAFKSKLA), and its accurate ORF sequence is shown in SEQ ID NO:2 (ATGACGGTTATAGTGTAT GGGACGGAGGGAAGTCCAGTCGTCAGAGGAACCCTCCTGACGGTGGGGGCGTTAGGCCTGGACATCGAATTTAAAGAAGTGAACACTATGGCTGGGGATCAGTTCAAACCGGAATTCCTCAAGCTGAACCCCCTTCACACCGTACCAACAATTCAAGATGGGGATTTTGTGATATTTGACAGCCACGCTATCAACGCCTACTTGGTTGATAAATATGGAAAGGATGACTCTCTCTACCCCAAAGACCTGCAGAAGAGGGCCGTGGTTAACCAGCGCCTTTATTTTGATTGTGGCATTGTGTTCCCTAGGCTGGGAGCTGTTATTATGTCCATATTTAAAGGAGGGAAAACTGTACCCAAGGATTTAGCAGATGCACTAGTTGATGCTTACACGTCACTCGAAACCTTGTTAGAAAGAAGCACTTACGCTGCTGGAGAAGAACTCACCATAGCAGACTTCAGTTTGGTAGCAACCATTTCAACGGCTAACATAATGATTCCCATCACATCGAACCGTTTCCCCAAAATCAGCGACTGGTTGGCCAAAATGCAGGCTTTGCCTTACTACGCGTCGAAAAACCAAGCTGGCCTGGATAAGGTTATGGCAGCGTTTAAGAGCAAACTGGCTTAA).

[0123] Example 2

[0124] Determination of expression characteristics of MaGSTd4 gene under α-pinene stress

[0125] (I) Feeding treatment of larvae

[0126] As shown in Example 1 (I).

[0127] (II) RNA extraction

[0128] As shown in Example 1 (II)

[0129] (III) cDNA synthesis

[0130] As shown in Example 1 (III)

[0131] (IV) Fluorescence quantitative PCR

[0132] 1) Primer design and amplification efficiency detection

[0133] Based on the cloned glutathione S-transferase sequence of Monochamus alternatus, fluorescent quantitative PCR primers were designed by Primer-BLAST (https: / / www.ncbi.nlm.nih.gov / tools / primer-blast / ), specifically the forward primer: CAGAAGAGGGCCGTGGTTA (SEQ ID NO: 3);

[0134] Reverse primer: CAGCAGCGTAAGTGCTTCTT (SEQ ID NO: 4).

[0135] The cDNA diluted to 200 ng / μL was diluted 10 times in a gradient, with 5 gradients, and the sequence was amplified using 6 tubes of cDNA as template to establish a standard curve. 2 Primers with a value greater than 0.98 and an amplification efficiency between 90% and 1.10% meet the requirements and can be used for subsequent experiments.

[0136] 2) The reaction system is shown in Table 9.

[0137] Table 9 Fluorescence quantitative PCR reaction system

[0138]

[0139] The amplification program is shown in Table 10.

[0140] Table 10 Fluorescence quantitative PCR reaction program

[0141]

[0142]

[0143] The relative expression of genes was detected using the RPL-10 gene of Monochamus alternatus as the internal reference gene. Three technical replicates were performed for each biological replicate.

[0144] (V) Data processing and analysis

[0145] According to 2 -ΔΔCt Methods The expression level data of MaGSTd4 gene in tissues of Monochamus alternatus at different treatment times were calculated. One way-ANOVA and Turkey were used to detect the difference in expression levels of different samples (P<0.05). The above data were processed by SPSS 20.0 software, and graphics were produced by ORIGIN2019 software.

[0146] The results are as follows:

[0147] The expression pattern of glutathione S-transferase gene in the intestine of Monochamus alternatus larvae when exposed to α-pinene stress is shown in Figure 2. Figure 1 In the intestine, MaGSTd4 began to be upregulated after 48 h of feeding treatment, but there was no significant difference among the three groups. The expression pattern of glutathione S-transferase gene in fat body is shown in Figure 2 In the fat body, MaGSTd4 was continuously upregulated after 24 h of feeding treatment, and showed significant differences at certain concentrations. The expression pattern of glutathione S-transferase gene in the epidermis is shown in Figure 3 In the epidermis, MaGSTd4 was significantly upregulated only after 48 hours, and the relative expression levels of the low and medium concentration feeding groups were significantly greater than those of the high concentration group. The expression pattern of glutathione S-transferase gene in hemolymph is shown in Figure 4 As shown. In the hemolymph, MaGSTd4 also began to gradually increase in expression with the accumulation of time. After feeding treatment, there were different degrees of increase, and it showed a trend of increasing first and then decreasing, and there was a significant difference in the medium and high concentration groups.

[0148] Example 3

[0149] Prokaryotic expression and purification of MaGSTd4 gene

[0150] (I) Construction of MaGSTd4 gene expression vector:

[0151] 1. Preparation of DNA template of MaGSTd4 gene fragment

[0152] CE Design (Vazyme, Nanjing) online software was used to design primers for homologous recombination according to the restriction sites on the expression vector and the nucleotide sequence of MaGSTd4, as shown in SEQ ID NO: 5 (cgcggcagccatatggctagcATGACGGTTATAGTGTATGGGACG) and SEQ ID NO: 6 (acggagctcgaattcggatccTTAAGCCAGTTTGCTCTTAAACGC), which were used as amplification primers, and the cDNA obtained in Example 1 (III) was used as a template for PCR amplification. The amplification system and procedure were as described in Example 1 (IV), and the amplified product was recovered and purified to obtain a DNA template.

[0153] 2. Vector Linearization

[0154] The following enzyme digestion reaction system was constructed as shown in Table 11.

[0155] Table 11 Enzyme Digestion Reaction System

[0156]

[0157] The first step of enzyme cleavage was carried out in a metal bath at 30°C for 30 min;

[0158] After completion, 2 μL QuickCutNhe I was added and the second enzyme digestion was performed in a metal bath at 37°C for 30 min;

[0159] After double enzyme digestion, agarose gel electrophoresis was performed to recover the linearized vector.

[0160] 3. Construction of MaGSTd4 recombinant plasmid

[0161] The MaGSTd4 gene fragment DNA template and the linearized vector were recombined using homologous recombinase (Vazyme, Nanjing), and the reaction system was constructed as shown in Table 12:

[0162] Table 12 Recombination reaction system

[0163]

[0164] After mixing, react in a metal bath at 37°C for 30 min. The circular plasmid generated by ligation was transformed into DH5α competent cells for cloning and sequencing, and the bacterial solution with correct sequencing was cultured in 50 mL of kana resistance LB liquid culture medium for 18 h.

[0165] The steps for plasmid extraction and transformation of positive clones are as follows:

[0166] ① Take 4 mL of bacterial solution in a centrifuge tube and centrifuge at 12,000 rpm for 2 minutes at room temperature;

[0167] ② Add 300 μL of solution I to the centrifuge tube and shake thoroughly until there is no precipitation;

[0168] ③ Add 300 μL of solution II and invert until the solution becomes transparent;

[0169] ④ Add 400 μL of pre-cooled solution III, invert and let stand for 2 minutes, and centrifuge at 12,000 rpm for 10 minutes;

[0170] ⑤ Transfer the supernatant to the filter column, centrifuge at 12,000 rpm for 1 min, and discard the waste liquid.

[0171] ⑥ Add 800 μL of rinsing solution WA, centrifuge at 12,000 rpm for 30 seconds, and discard the waste liquid.

[0172] ⑦ Add 800 μL of washing buffer WB, centrifuge at 12,000 rpm for 30 seconds, and discard the waste liquid.

[0173] ⑧Repeat step 7.

[0174] ⑨ Place the filter column in a new collection tube and centrifuge at 12,000 rpm for 1 min.

[0175] ⑩ Place the filter column in a new centrifuge tube, add 30 μL of preheated nuclease-free water to the center, leave it for 1 minute, and centrifuge at 12,000 rpm for 1 minute to obtain the purified plasmid.

[0176] Both the empty pET-28a(+) plasmid and the expression plasmid were transformed according to the above experimental procedures.

[0177] The next day, positive clones were picked, sequenced and verified using SEQ ID NO: 9 (TAATACGACTCACTATAGGG) and SEQ ID NO: 10 (TGCTAGTTATTGCTCAGCGG) as primers, and the strains were preserved.

[0178] (II) Monochamus alternatus GSTs induced expression:

[0179] The stored expression strain and empty strain were added to 300 mL of kana-resistant LB liquid medium and cultured in a shaker at 37°C and 200 rpm.

[0180] Measure the OD of the bacterial solution using an enzyme-labeled 600 When OD 600 When the concentration is between 0.4 and 0.6, add 600 μL IPTG (final concentration 1 mM), mix well, and place the bacterial solution in a shaker at 20°C and 200 rpm for cultivation.

[0181] After 18 hours, transfer the bacterial suspension to a 50 mL centrifuge tube and centrifuge at 10,000 rpm for 15 min at 4°C. Discard the supernatant and keep only the E. coli pellet.

[0182] (1) Ultrasonic fragmentation

[0183] The bacterial solution was resuspended in 30 mL of equilibrium buffer and 300 μL of PMSF protease inhibitor (100 mM) was added. The resuspended bacterial solution was placed in an ice-water mixture for ultrasonic disruption, each ultrasonic disruption for 30 min, power 150 W, frequency 20 kHz. The disrupted buffer was centrifuged at 4 ° C, 12,000 rpm for 25 min to obtain the supernatant and precipitate.

[0184] (2) Protein gel electrophoresis

[0185] Prepare SDS-PAGE gel for vertical electrophoresis ( Figure 5 ), the specific steps are as follows:

[0186] ① Take 2.7mL of lower gel solution and lower gel buffer, mix well, then add 60μL of improved coagulant and mix well;

[0187] ② Add the mixed liquid into the gap between the glass plates and add appropriate amount of water to make the liquid surface level;

[0188] ③After the lower layer of glue solidifies, pour out the upper layer of water;

[0189] ④ Take 0.75mL of upper gel solution and upper gel buffer, mix well, then add 15μL of improved coagulant and mix well;

[0190] ⑤ Add the mixed liquid into the gap between the glass plates and insert the comb teeth;

[0191] ⑥ After the gel solidifies, remove the comb teeth and use it for electrophoresis.

[0192] Take 20 μL of supernatant and urea-dissolved precipitate, add 5 μL of 5× SDS-PAGE loading buffer, and heat in a 90°C metal bath for 5 min for electrophoresis.

[0193] Electrophoresis conditions: constant voltage 110 V, 120 min.

[0194] After electrophoresis, the cells were stained with Coomassie Brilliant Blue, destained and photographed.

[0195] (III) Purification and dialysis concentration of Monochamus alternatus MaGSTd4 recombinant protein:

[0196] Before protein purification, three buffers need to be prepared;

[0197] Equilibration buffer (BB): 50 mM phosphate buffer, 0.3 M NaCl, pH 7.4, containing 20 mM imidazole.

[0198] Washing buffer (WB): 50 mM phosphate buffer, 0.3 M NaCl, pH 7.4, containing 100 mM imidazole.

[0199] Elution buffer (EB): 50 mM phosphate buffer, 0.5 M NaCl, pH 7.4, containing 300 mM imidazole.

[0200] (1) Recombinant protein purification

[0201] The pET-28a(+) vector carries an N-terminal His protein tag. His-tagged proteins are usually purified using immobilized metal affinity chromatography (IMAC) purification. In this experiment, a nickel ion resin column was selected to purify the protein.

[0202] The specific operations are:

[0203] ① Add 12 mL of pre-cooled equilibrium buffer to the resin column and adjust the pH of the resin column;

[0204] ② Add 12 mL of supernatant to the resin column, circulate through the column twice, and collect the effluent;

[0205] ③Add 12mL of pre-cooled washing buffer to elute the impurities and discard the waste liquid;

[0206] ④Add 6mL of pre-cooled elution buffer to elute the target protein, collect elution buffer in 6 tubes in sequence, and take samples for electrophoresis detection.

[0207] (2) Desalination by dialysis

[0208] Collect 20 mL of the purified target protein and place it in a ready-to-use dialysis bag (25 cm long, 18 mm wide, with a molecular weight cutoff of 15 kDa).

[0209] After the dialysis bag is clamped and sealed, it is transferred to 2L pre-cooled dialysis solution I and placed in a 4℃ refrigerator. After 8h, it is transferred to dialysis solutions II and III in turn.

[0210] The components of the three dialysates are as follows;

[0211] 1) Dialysate I: 40 mM phosphate buffer, pH 7.4, 200 mM NaCl, containing 200 mM imidazole.

[0212] 2) Dialysate II: 20 mM phosphate buffer, 100 mM NaCl, pH 7.4, containing 10 mM imidazole.

[0213] 3) Dialysate III: sterile water.

[0214] (3) Concentration

[0215] After dialysis, the protein was divided into 5 mL per tube, frozen at -80℃ for 6 hours, and then concentrated in a freeze dryer under vacuum. After the protein was completely dried, the protein concentration was determined by Bradford method, and then diluted to 1 mg / mL with sterile water.

[0216] Example 4

[0217] Functional validation of MaGSTd4

[0218] (I) Enzyme kinetic parameters of the recombinant protein MaGSTd4 of Monochamus alternatus

[0219] Enzyme activity assay reaction system

[0220] Referring to the instructions of the glutathione S-transferase (GST) activity detection kit, the activity of the recombinant protease of Monochamus alternatus MaGSTd4 was determined on an ELISA instrument. The reaction system is shown in Table 13:

[0221] Table 13 Reaction system

[0222]

[0223] Mix well and immediately measure the absorbance change at 340 nm. Measure once every 30 seconds for a total of 5 minutes. Calculate the specific activity of MaGSTd4 recombinant protease according to formula I.

[0224]

[0225] Among them, Δ OD340 is the change in absorbance, V is the total reaction volume, and ε is the extinction coefficient (9.6 mM for CDNB and GSH products). -1 cm -1 ), T is the reaction time, L is the light path, and E is the amount of enzyme added in the reaction. The boiling-inactivated MaGSTd4 recombinant protein was used as a control, and each sample was repeated 3 times.

[0226] In order to determine the catalytic properties and functions of the MaGSTd4 recombinant protein, GSH and CDNB were used as reaction substrates to detect its specificity for GSTs typical substrates. By exploring the relationship between CDNB concentration and the catalytic activity of recombinant MaGSTd4, the Michaelis constant Km and the maximum reaction rate Vmax were calculated using formula II.

[0227]

[0228] Where V represents the reaction rate; K m is the Michaelis constant, V max represents the maximum reaction rate, and S represents the substrate concentration.

[0229] The results are as follows:

[0230] The Michaelis-Menten constant Km means the substrate concentration (S) when the enzyme-catalyzed reaction reaches half of the maximum speed Vm. It can reflect the affinity between the enzyme and the substrate. The smaller the Km value, the stronger the affinity. In the origin software, the fitting curve is obtained by the Michaelis-Menten formula ( Figure 6 ), R 2 =0.99356, indicating that the fitting data is reliable. At the same time, the Michaelis constant Km and the maximum reaction rate V are calculated by the double reciprocal method. max , after calculating the MaGSTd4 recombinant protein K m =1.24±0.21mM, V max =2985.78±347.15μmol / min / mg.

[0231] (2) Effects of pH and temperature on MaGSTd4 enzyme activity

[0232] Refer to the reaction system in (I), wherein the CDNB concentration is 200 mM.

[0233] Effect of temperature on MaGSTd4 protease activity: The recombinant protein and PBS buffer were placed in a water bath at different temperatures for 15 min, and then GSH and CDNB were added and mixed before determination. The determination method was the same as (I), and the highest enzyme activity was taken as 100%.

[0234] Effect of pH value on MaGSTd4 protease activity: After the recombinant protein was mixed with PBS buffer of different pH values, GSH was added and the determination method was the same as that of CDNB (I), and the highest enzyme activity reached 100%.

[0235] In order to detect the thermal stability and optimal temperature of the recombinant protein, different temperature gradients were set.

[0236] The results showed that when the recombinant protein was placed at 10-25°C, the activity of MaGSTd4 protein did not lose activity due to the increase in temperature. The results showed that the structure of the protein was relatively stable within this temperature range, and the activity showed an upward trend. After incubation at different temperatures for 30 minutes, the thermal stability of the MaGSTd4 recombinant protein was determined based on different activity changes.

[0237] The test temperature range was set between 10-70°C and the experiment was repeated several times. Figure 7 As shown in A, when the temperature exceeds 40°C, the activity of the target protein decreases rapidly and significantly, and most of the MaGSTd4 is basically inactivated, which is consistent with the habitat of the larvae of Monochamus alternatus. As a trunk-boring pest, the larvae of Monochamus alternatus mainly harm weak growth wood and newly felled wood. The larvae are rarely exposed to temperatures above 30°C, so their heat resistance is poor and the optimum temperature of the protein is also low.

[0238] In order to determine the optimal pH of MaGSTd4, the pH range of the test was set between 4 and 11.

[0239] The results showed that the activity of MaGSTd4 showed a trend of first increasing and then decreasing with the increase of pH. When the pH was between 6.0 and 9.0, MaGSTd4 could maintain a high activity. At pH 8, the MaGSTd4 recombinant protein showed the highest activity ( Figure 7 (B).

[0240] (II) Antioxidant activity verification

[0241] The paper disc diffusion experiment under oxidative stress, the steps are as follows:

[0242] Escherichia coli BL21 (DE3) containing empty pET-28a (+) and overexpressed MaGSTd4 was cultured at a rate of about 5 × 10 8 E. coli was evenly spread on the kana-resistant solid LB medium with 10 cells / plate.

[0243] After incubation in an inverted state at 37°C for 1 hour, a sterile filter paper (6 mm in diameter) was placed on the surface of the culture medium, and 20 μL of cumene hydroperoxide of different concentrations was dripped onto the center of the paper. After the cumene hydroperoxide was completely absorbed, the plate was inverted and incubated at 37°C. After 24 hours, the inhibition zone was measured using an automatic colony analyzer.

[0244] The results are as follows:

[0245] The antioxidant activity of MaGSTd4 was detected by paper disc diffusion test. After overnight treatment, it was found that there was no significant difference between empty and overexpressed MaGSTd4 E. coli under low concentration of cumene hydroperoxide treatment. As the concentration of cumene hydroperoxide increased, the oxidative damage to the bacteria became stronger, and E. coli expressing MaGSTd4 protein began to show stronger antioxidant capacity, and the inhibition zone around the filter paper was significantly smaller than that in the empty E. coli ( Figure 8 ). Compared with the control group, MaGSTd4 was reduced by 51% after treatment with cumene hydroperoxide.

[0246] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Use of Monochamus alternatus glutathione S-transferase MaGSTd4 in the preparation of a biocatalyst for promoting glutathione binding reaction, characterized in that: The amino acid sequence of MaGSTd4 is shown in SEQ ID NO:

1.

2. The use according to claim 1, characterized in that: The nucleotide sequence of the gene encoding the Monochamus alternatus glutathione S-transferase MaGSTd4 is shown in SEQ ID NO:

2.

3. A recombinant vector, characterized in that: The recombinant vector carries a gene encoding Monochamus alternatus glutathione S-transferase MaGSTd4, and the amino acid sequence of the MaGSTd4 is shown in SEQ ID NO:

1.

4. An engineered strain for recombinantly expressing Monochamus alternatus glutathione S-transferase MaGSTd4, characterized in that: The amino acid sequence of MaGSTd4 is shown in SEQ ID NO:

1.

5. Use of the recombinant vector according to claim 3 or the engineered strain according to claim 4 in recombinant expression of Monochamus alternatus glutathione S-transferase MaGSTd4 protein.

6. Use of the engineered strain according to claim 4 in the preparation of a biocatalyst for promoting glutathione binding reaction.

7. The use according to claim 6, characterized in that: The temperature of the glutathione binding reaction is 20-30° C.; the system pH value of the glutathione binding reaction is 6.0-9.

0.

8. Use of the glutathione S-transferase MaGSTd4 of Monochamus alternatus or the engineered strain according to claim 4 in the preparation of antioxidant products; the amino acid sequence of the MaGSTd4 is shown in SEQ ID NO: 1.

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