Cotton bollworm carboxylesterase HaCHE1alpha as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of gene engineering and enzyme engineering, and particularly relates to cotton bollworm carboxylesterase HaCHE1alpha as well as a preparation method and application thereof. The coding gene sequence of the cotton bollworm carboxylesterase HaCHE1alpha is as shown in SEQ ID NO. 1. The cotton bollworm carboxylesterase HaCHE1alpha can efficiently degrade fumonisin B1, the action mode is mild, and secondary pollution is avoided. The cotton bollworm carboxylesterase HaCHE1alpha has a wide application prospect in the fields of environmental protection, food safety, animal health and the like. The preparation method provided by the invention provides a core technology for industrial production of the fumonisins B1 degrading enzyme.

Description

Technical Field

[0001] The present invention belongs to the technical field of gene engineering and enzyme engineering; more specifically, the present invention relates to a cotton bollworm carboxylesterase HaCHE1α and a preparation method and application thereof. Background Art

[0002] Fungal toxin pollution endangers the health and safety of humans and animals, has become a global hot issue, and is also a major factor restricting the development of the food industry. Fumonisin is a type of secondary biomass produced by Fusarium, and is also one of the most serious fungal toxins in recent years. Among them, fumonisin B1 has the highest contamination rate and the strongest toxicity. It can harm human liver function and cause esophageal cancer, liver cancer and other diseases, and also cause serious economic losses to animal husbandry. In order to effectively control the huge harm caused by fumonisin, it is urgent to develop efficient, low-residue and low-cost fumonisin degraders. At present, adsorbents are widely used on the market to remove fumonisin pollution in crops, but adsorbents often cannot specifically adsorb toxins, and will cause the loss of other nutrients, and the adsorbed toxins may also cause secondary pollution. Relatively speaking, biodegraders have significant advantages. They can degrade fumonisin under relatively mild conditions, and have the advantages of high selectivity, high efficiency, low toxicity, low residue and low cost. Therefore, the development of fumonisin biodegraders is the preferred solution to recover the huge economic losses of the food industry and animal husbandry and to ensure the safety of humans and animals.

[0003] The biological detoxification method uses microorganisms or biological enzymes to convert fungal toxins into low-toxic or non-toxic degradation products. Its efficiency and safety have been widely recognized. However, at present, the biological reduction technology of fumonisin mainly focuses on the screening of degradation strains, and there are few studies based on degradation gene mining. There is still a severe shortage of relevant fumonisin degrading enzymes in China, so it is urgent to explore biological enzyme gene resources with fumonisin degradation ability.

[0004] The applicant previously stressed cotton bollworms by feeding them toxic feed containing FB1 for a long time, and studied the changes in the expression of detoxification enzymes in cotton bollworms through transcriptome sequencing and other technologies, and screened out the detoxification enzyme HaCHE1 that has a high efficiency in degrading FB1. After the induced HaCHE1 bacterial solution and FB1 were incubated for 6 hours, the degradation rate of 0.1 mg / kg FB1 was 14.3%. The induced HaCHE1 bacterial solution was applied to the soil, and the degradation rate of FB1 in the soil was 12.2% after 7 days of application. Although this study provides new ideas for the removal of fumonisins, the degradation effect still needs to be further improved. Summary of the invention

[0005] In view of the above technical problems, the present invention provides a cotton bollworm carboxylesterase HaCHE1α and a preparation method and application thereof.

[0006] One of the purposes of the present invention is to provide cotton bollworm carboxylesterase HaCHE1α, whose encoding gene sequence is shown as SEQ ID NO.1.

[0007] Based on previous research, the present invention corrects the gene sequence of the carboxylesterase HaCHE1 of cotton bollworm. The full-length sequence of the HaCHE1α gene obtained after correction is 1755bp, encoding 584 amino acids, and the molecular weight of the amino acid sequence predicted online is 65.40kDa and the isoelectric point is 4.65. Through prokaryotic expression and other technologies, a large number of biodegradation agents with mild action conditions, low cost and high efficiency are produced, and the degradation rate is significantly improved under the same induction time and incubation time.

[0008] The second object of the present invention is to provide the following application of the cotton bollworm carboxylesterase HaCHE1α:

[0009] 1) Degradation of mycotoxins;

[0010] 2) preparing products for degrading mycotoxins;

[0011] The mycotoxins include fumonisin B1.

[0012] The third object of the present invention is to provide a gene, which encodes the cotton bollworm carboxylesterase HaCHE1α.

[0013] A fourth object of the present invention is to provide a vector containing the gene.

[0014] A fifth object of the present invention is to provide a cell, wherein the cell contains the vector or the gene is integrated into its genome.

[0015] Furthermore, the cells are Escherichia coli cells.

[0016] The sixth object of the present invention is to provide a method for preparing the cotton bollworm carboxylesterase HaCHE1α, by culturing the cells, inducing expression and isolating the cotton bollworm carboxylesterase HaCHE1α.

[0017] The seventh object of the present invention is to provide a method for degrading mycotoxins, using the cotton bollworm carboxylesterase HaCHE1α to treat mycotoxins, or to treat an object containing mycotoxins.

[0018] Furthermore, the treatment is incubated at 27-29°C.

[0019] The present invention has the following beneficial effects:

[0020] The cotton bollworm carboxylesterase HaCHE1α of the present invention has a high metabolic effect on fumonisin B1, has a mild action mode, and does not generate secondary pollution.

[0021] The material price for preparing the cotton bollworm carboxylesterase HaCHE1α is low, and the synthesis method is relatively simple. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Double enzyme digestion identification of gene HaCHE1α, where M: Marker band, 1: empty vector, 2: recombinant plasmid HaCHE1α.

[0023] Figure 2 SDS-PAGE detection of gene HaCHE1α after prokaryotic expression. In the figure, M: protein marker; a: CK supernatant; b: CK inclusion body; c: induced supernatant; d: induced inclusion body; e: purified protein.

[0024] Figure 3 The degradation of fumonisin by HaCHE1α bacterial solution, where A: incubation for 6 hours; B: incubation for 10 hours; C: incubation for 16 hours; D: incubation for 24 hours.

[0025] Figure 4 The degradation of fumonisins in soil by HaCHE1α bacterial solution. DETAILED DESCRIPTION

[0026] The present invention is described in detail below in conjunction with the accompanying drawings and specific examples, but should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0027] Example 1: Preparation of Helicoverpa armigera carboxylesterase HaCHE1α

[0028] In the early stage, the fumonisin B1-resistant strains of cotton bollworm were cultured and screened, and the carboxylesterase gene HaCHE1 that has a degrading effect on fumonisin B1 was screened by transcriptome sequencing, qPCR and other technologies. On this basis, its sequence was corrected. The full-length sequence of the HaCHE1α gene obtained after correction is 1755bp, encoding 584 amino acids. The molecular weight of the amino acid sequence predicted online is 65.40kDa, the isoelectric point is 4.65, and its nucleotide sequence is shown in SEQ ID NO.1.

[0029]

[0030] Preparation method of fumonisin biodegradation agent carboxylesterase HaCHE1α:

[0031] (1) Take a multi-headed cotton bollworm, dissect its detoxification organs (midgut and fat body), extract total RNA, and reverse transcribe it into cDNA using the Roche reverse transcriptase kit. Use specific primers and the first strand as a template for gene cloning.

[0032] (2) Design HaCHE1α-specific amplification primers HaCHE1α-F and HaCHE1α-R, amplify the full length of HaCHE1α, amplify the esterase gene, and then perform gel recovery. Connect the purified product to the pMD18-T vector and transfer it into TG1 competent cells. The bacterial solution with correct sequencing was preserved and the plasmid was extracted and stored in a -20°C refrigerator.

[0033] HaCHE1α-F (SEQ ID NO. 2): 5′-ATGAGGAGCACCTCTATCAGT-3′.

[0034] HaCHE1α-R (SEQ ID NO. 3): 5′-TTAATGCGTACTCGACA-3′.

[0035] (3) According to the position of the restriction site on the pColdⅢ vector, primers HaCHE1α-MF and HaCHE1α-MR were designed. HaCHE1α-MF contained a BamH I restriction site, and HaCHE1α-MR contained an EcoR I restriction site and 6 His tags. HaCHE1α-MF and HaCHE1α-MR were used for gene amplification, and the template was the plasmid extracted in step (2). After the PCR product was recovered, it was double-digested with the pColdⅢ vector, and the target gene and vector were recovered after the double-digested product was run on gel.

[0036] HaCHE1α-MF (SEQ ID NO. 4): 5′-GGGGTACCATGAGGAGCACCTCTA TCAGT-3′.

[0037] HaCHE1α-MR (SEQ ID NO. 5): 5′-CGGAATTCTCAGTGGTGGTGGTGGTGGTGATGCGTACTCGACATAAAT-3′.

[0038] (4) Take the PCR product after restriction digestion and connect the vector at 16℃ for 30 minutes. Take 10μL of the ligation product and transfer it into TG1 competent cells. After culturing overnight, perform PCR and double restriction digestion tests on the extracted plasmid, and send the positively identified plasmid for sequencing. Save the strains with correct sequencing, divide them into centrifuge tubes, add glycerol to make the final concentration of 25%, and store them in a -80℃ refrigerator.

[0039] (5) Take 10 μL of the recombinant vector and transform it into competent cells BL21-pGro7, and culture it at a constant temperature of 37°C. Pick a single colony from the plate, inoculate it into 2 mL of liquid LB medium containing ampicillin, and culture it on a shaker for 12 hours. Then take 2 mL of the bacterial liquid and inoculate it into 150 mL of culture medium, and culture it on a shaker for 2 hours. Add arabinose at a concentration of 4 mg / mL. When the OD 600 When the pH is 0.4-0.6, add IPTG to the bacterial solution to a final concentration of 0.4mM. Then place the bacterial solution at 16°C and culture on a shaker for 24 hours. Induce a large amount of target protein. Use affinity chromatography to purify the recombinant protein, use 10-250mmol / L imidazole for gradient elution and SDS-PAGE detection, and dialyze the eluate containing a single recombinant protein. The dialysis is performed using pre-cooled PBS (10mmol / L). The recombinant protein is collected after 12 hours of dialysis and placed at -20°C for use.

[0040] After double enzyme digestion, the recombinant plasmid HaCHE1α showed two bands. The larger band was about 5000 bp, which was close to the size of the vector, and the smaller band was about 1750 bp, which was consistent with the size of the target gene ( Figure 1 ). This indicates that the recombinant plasmid contains a gene fragment that is close in size to the target gene. The sequencing results show that the gene contained in the vector is completely consistent with the target gene, indicating that the target gene is successfully inserted into the vector and the recombinant plasmid is successfully constructed.

[0041] Induce expression with IPTG at 16°C. Collect the cells after 24 hours, add buffer after centrifugation, alternately heat and cool to break the cells, and then centrifuge. The supernatant and precipitate are loaded for detection. Figure 2 As shown, SDS-PAGE electrophoresis showed a differential band of about 66 kDa. The molecular weight of HaCHE1α protein is 65.40 kDa, which is consistent with the position of the differential band in the figure, indicating that the gene HaCHE1α was successfully induced to express.

[0042] Example 2: Application of Helicoverpa armigera carboxylesterase HaCHE1α

[0043] (1) Test method for the degradation of fumonisins by bacterial culture after induction of carboxylesterase gene of cotton bollworm.

[0044] Prepare 10mL of liquid LB medium (0.1% AMP), set the FB1 addition concentration to 0.5mg / kg, add 1mL of induced, uninduced and empty vector bacterial liquid respectively, incubate at 28°C, 160rpm for 6h, 10h, 16h and 24h, and repeat 3 times for each treatment. Use the pretreatment method to extract FB1, and use UPLC-MS / MS to detect and analyze fumonisin.

[0045] (2) Take 3g of soil, set the addition concentration to 0.5mg / kg, add 1.5mL of induced, uninduced and empty carrier bacterial solution, let it stand for 20min and wait for the organic solvent to evaporate. Seal it and place it in a constant temperature and humidity incubator at 26℃ for 7d. Repeat each treatment 3 times and extract FB1 using the pretreatment method. Fumonisin determination was performed by UPLC-MS / MS.

[0046] The degradation of fumonisin by HaCHE1α bacterial solution under different treatments is shown in Figure 2. Figure 3 As shown, the induced HaCHE1α was incubated with FB1. After calculation, the degradation rates of FB1 by HaCHE1α bacterial solution were 20.69%, 31.73%, 34.81%, and 37.47% at 6h, 10h, 16h, and 24h, respectively, which were significantly higher than those of the empty vector group. After the uninduced HaCHE1α bacterial solution was incubated with FB1 for 6h, 10h, 16h, and 24h, the degradation rates of FB1 were 13.15%, 15.74%, 19.27%, and 16.67%, respectively, which were significantly higher than those of the empty vector group. The degradation effect of the induced bacterial solution on FB1 was significantly higher than that of the uninduced group, indicating that inducing the HaCHE1α bacterial solution can improve its ability to degrade FB1.

[0047] The degradation of fumonisins in soil by HaCHE1α bacterial solution under different treatments is shown in Figure 2. Figure 4 As shown in the figure, the FB1 concentration of the treatment group was measured after HaCHE1α was induced for 24 hours and incubated with FB1 for 7 days. The degradation rate of FB1 in the treatment group was 20.60%, which was significantly higher than that in the empty vector group. After the uninduced HaCHE1α bacterial solution was incubated with FB1 for 7 days, the degradation rate of FB1 was 4.21%, and the degradation rate of FB1 in the empty vector group was 2.87%. The degradation of FB1 by the induced bacterial solution was significantly higher than that of the uninduced group, indicating that inducing the HaCHE1α bacterial solution can improve its ability to degrade FB1.

[0048] It should be noted that when the claims of the present invention involve numerical ranges, it should be understood that the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. In order to avoid redundancy, the present invention describes a preferred embodiment.

[0049] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0050] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. Cotton bollworm carboxylesterase HaCHE1α, characterized in that The coding gene sequence is shown in SEQ ID NO.

1.

2. The following use of the cotton bollworm carboxylesterase HaCHE1α according to claim 1: 1) Degradation of mycotoxins; 2) preparing products for degrading mycotoxins; The mycotoxins include fumonisin B1.

3. A gene, characterized in that The gene encodes the cotton bollworm carboxylesterase HaCHE1α according to claim 1.

4. A carrier, characterized in that The vector contains the gene according to claim 3.

5. A cell, characterized in that The cell contains the vector according to claim 4 or the gene according to claim 3 is integrated into its genome.

6. The cell according to claim 5, characterized in that The cells are Escherichia coli cells.

7. A method for preparing cotton bollworm carboxylesterase HaCHE1α, characterized in that: The cotton bollworm carboxylesterase HaCHE1α is induced to express and isolated by culturing the cell according to claim 6.

8. A method for degrading mycotoxins, characterized in that: The method comprises using the cotton bollworm carboxylesterase HaCHE1α as claimed in claim 1 to treat fungal toxins or treat an object containing fungal toxins.

9. The method according to claim 8, characterized in that The treatment was carried out by incubation at 27-29°C.

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