Isoprocarb hydrolase gene cehAR-7 and application thereof

Abstract

The invention discloses an isoprocarb hydrolase gene cehAR-7 and application thereof, and belongs to the technical field of molecular biology, an enzyme coded by the gene can hydrolyze isoprocarb to generate o-isopropylphenol, and can be used for preparing an enzyme preparation for repairing isoprocarb pollution in soil and water and constructing an isoprocarb-resistant (tolerant) transgenic crop.

Description

Technical Field

[0001] The invention belongs to the field of molecular biology technology, and in particular relates to the microbial degradation of pesticides, and specifically relates to an isoprocarb hydrolase gene fragment cehA R-7 . Background Art

[0002] With the rapid development of economic globalization, people's demand for agricultural products is also increasing, and the corresponding development of agricultural products is also getting faster and faster. Insecticides are one of the pesticides that ensure the normal growth of agricultural products and are also used more and more. However, the residues of pesticides will bring great harm to human body and environment. Since the 1970s, organic pesticides have been banned or restricted by different countries due to their high killing efficiency, high toxicity and difficulty in decomposition. On the other hand, they have been widely developed and applied. Carbamate pesticides are one of them. Carbamate pesticides are synthetic pesticides developed after organophosphates. They are a new type of broad-spectrum insecticide and acaricide. They are cholinesterase inhibitors and endocrine disruptors. Isoprocarb is a widely used insecticide in carbamate pesticides. It has a strong contact killing effect and is particularly effective against rice planthoppers and leafhoppers. People ensure rice production by spraying isoprocarb in large quantities, but the long-term and large-scale use of isoprocarb has gradually increased the pesticide residues in the environment, which will seriously endanger the health of animals and people, and may even cause serious diseases such as cancer, deformities and endocrine disorders.

[0003] A large number of studies have shown that the main factor in the degradation of isoprocarb in the environment is microbial degradation. At present, many isoprocarb-degrading strains have been isolated at home and abroad, but there are few reports on the key enzymes for isoprocarb degradation, only the isoprocarb hydrolase IpcH from the strain Rhodococcus sp.D-6. It is reported that the initial degradation enzymes of carbamate pesticides are all hydrolases. The earliest hydrolase for carbamate pesticides is the furadan hydrolase gene. Tomaseek found the furadan hydrolase gene mcd on a large plasmid in the strain Achromobacter sp.WM111. Subsequently, Hashimoto et al. cloned the hydrolase gene cehA of carbaryl from the strain Rhizobium sp.AC100 in 2002. AC100 , et al. in 2016 found a cehA-related AC100 The carbamate hydrolase gene cehA is highly similar in sequence KN65.2 In the same year, Konstantina et al. also found a similar gene to cehA in Pseudomonassp.OXA20. AC100The carbamate hydrolase gene cehA is highly similar in sequence OXA20 In 2018, Yan et al. found a gene similar to cehA in Sphingomonas sp.CDS-1. AC100 The carbamate pesticide hydrolase gene is highly similar in sequence to cehA KN65.2 There is only one amino acid difference. Recently, Jiang et al. found a similar amino acid to cehA in Sphingobium sp.CFD-1. AC100 The carbamate hydrolase gene cehA is highly similar in sequence CFD-1 The existing patents only focus on the study of IpcH. Apart from this, there are relatively few reports on the degradation mechanism of isoprocarb, which seriously restricts the research on the environmental behavior and ecological safety of isoprocarb. Summary of the invention

[0004] In order to solve the deficiencies of the prior art, the present invention proposes an isoprocarb hydrolase gene cehA R-7 and its application, which can effectively hydrolyze isoprocarb, and can be used to repair soil and water bodies, and degrade the pesticide isoprocarb residual in the environment.

[0005] The technical problem to be solved by the present invention is achieved through the following technical solutions:

[0006] An isoprocarb hydrolase gene cehA R-7 , whose nucleotide sequence is any of the following:

[0007] (1) the DNA sequence of SEQ ID NO.1 in the sequence listing;

[0008] (2) the amino acid sequence of SEQ ID NO.2 in the sequence listing;

[0009] (3) A sequence having a homology of 90% or more, preferably 99% or more, to the carbamate pesticide hydrolase CehA.

[0010] Isoprocarb hydrolase CehA R-7 The amino acid sequence of the hydrolase is the amino acid sequence of SEQ ID NO.2 in the sequence listing.

[0011] Isoprocarb hydrolase CehA R-7 The preparation method is to convert the above-mentioned isoprocarb hydrolase gene cehA R-7 The recombinant expression vector was cloned and introduced into the host cell to obtain the recombinantly expressed isoprocarb hydrolase CehA. R-7 .

[0012] Wherein, the expression vector is pET-24b(+) plasmid, purchased from Novegen.

[0013] Wherein, the host cell is Escherichia coli BL21 (DE3), purchased from Shanghai Yingjun Biotechnology Co., Ltd.

[0014] Furthermore, the recombinant expression vector is a vector in which the isoprocarb hydrolase gene cehA R-7 Insertion was performed between the Nde I and Xho I sites of pET-24b(+).

[0015] Furthermore, the preparation method also includes PCR amplification of the isoprocarb hydrolase gene, and the PCR amplification uses a forward primer and a reverse primer, wherein the forward primer is shown in the sequence list SEQ ID NO.3, and the reverse primer is shown in the sequence list SEQ ID NO.4.

[0016] Furthermore, the preparation method also includes the expression and purification of the isoprocarb hydrolase gene, the isoprocarb hydrolase gene is expressed under IPTG induction, and then Ni 2+ -NTA affinity chromatography to purify isoprocarb hydrolase.

[0017] Based on the above isoprocarb hydrolase gene cehA R-7 , which can be used to construct genetically modified crops with improved tolerance to isoprocarb through gene introduction.

[0018] Based on the above isoprocarb hydrolase CehA R-7 The enzyme or enzyme preparation can be used to degrade isoprocarb in the environment and repair isoprocarb pollution in soil and water.

[0019] Compared with the prior art, the present invention has the following beneficial effects: the isoprocarb hydrolase CehA obtained by the present invention R-7 It can hydrolyze isoprocarb to produce o-isopropylphenol and can be used to prepare enzyme preparations for repairing soil and water bodies and degrading residual pesticide isoprocarb in the environment. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a high performance liquid chromatogram of the degradation of isoprocarb by the strain R-7 of the present invention;

[0021] Figure 2 The mass spectra of metabolites produced during the degradation of isoprocarb by the strain R-7 of the present invention (A. mass spectrum of compound I, B. mass spectrum of compound II);

[0022] Figure 3 The isoprocarb hydrolase gene cehA of the present invention R-7SDS-PAGE electrophoresis after recombinant expression and purification;

[0023] Figure 4 The isoprocarb hydrolase CehA of the present invention R-7 HPLC profile of degradation of isoprocarb. DETAILED DESCRIPTION

[0024] The present invention is further described below in conjunction with the accompanying drawings and specific preferred embodiments, but the protection scope of the present invention is not limited thereby.

[0025] Example 1

[0026] Isolation of highly efficient isoprocarb-degrading strain

[0027] 1.1 Enrichment and purification of isoprocarb-degrading strains

[0028] Soil samples were collected from ditches and farmlands that had been contaminated by isoprocarb for a long time. 5 g of soil sample was inoculated into 100 mL of basic inorganic salt medium containing 100 mg / L isoprocarb as the sole carbon source. The medium was incubated at 30 °C and 180 r·min. -1 After 5 days of cultivation, the inoculum was transferred to the basic inorganic salt medium of the same concentration at a 5% inoculation rate, and the transfer was continuous for 4 times. During this period, the content of isoprocarb in the enriched solution was detected by HPLC, and 1 mL of the enriched solution with good degradation effect was taken for gradient dilution, spread on the surface of LB solid medium, and incubated at 30°C for 3 days. Then the strains of different morphologies were picked out, separated by streaking on the surface of LB solid medium, and the purified strains were obtained. After 4 consecutive passages of enrichment and domestication, an enriched solution that can completely degrade 100 mg / L within 3 days was obtained. After the enriched solution was diluted and spread for culture, single colonies of different morphologies were picked for degradation effect verification, and a strain R-7 that can efficiently degrade isoprocarb was obtained. Strain R-7 can completely degrade 100 mg / L of isoprocarb within 12 hours. After strain R-7 grew on LB agar medium at 30°C for 3 days, the colonies were yellow, opaque, round, and the surface was smooth and moist. Basic salt medium formula: 1.5g K2HPO4·3H2O; 0.5g KH2PO4; 1.0g NH4NO3; 0.5g NaCl; 0.2g MgSO4·7H2O, add deionized water to 1L. Add 15.0g agar per liter to the solid medium.

[0029] 1.2 Screening and identification of isoprocarb-degrading strains

[0030] PCR amplification and 16S rRNA gene sequencing and analysis were performed on the strains that can degrade isoprocarb. The reaction system included 15μL of Premix rTaq, 1μL of forward and reverse primers, 1μL of template, and 12μL of ddH2O. PCR program: 95℃ pre-denaturation for 5min, 95℃ denaturation for 30s, 52℃ annealing for 30s, and 72℃ extension for 90s for 30 cycles. Universal primers 27F (5ˊAGAGTTTGATCMTGGCTCAG3ˊ) and 1492R (5ˊTACGGYTACCTTGTTACGACTT3ˊ) were used to amplify gene fragments in the samples. The 16S rRNA gene sequences obtained by sequencing the PCR products were compared and analyzed online on EzBioCloud. The results showed that the 16S rRNA gene sequence of strain R-7 was most closely related to Sphingobium, especially to Sphingobiumxenophagum NBRC 107872. T The highest similarity was 99.07%. The phylogenetic tree of strain R-7 based on the 16S rRNA gene sequence was constructed using the neighbor-joining method. The results showed that strain R-7 was clustered with the type strain of Sphingobium sp. and with the type strain Sphingobium xenophagum NBRC 107872. T Located on a branch, the bacterium was identified as Sphingobium sp. based on its morphological, physiological and biochemical characteristics.

[0031] 1.3 Detection of Isoprocarb Degradation Products

[0032] Degradation effect verification method: strain R-7 was inoculated into LB liquid medium, cultured at 30°C to the logarithmic phase, and the cells were collected by low-speed centrifugation. The cells were washed twice with liquid sterile basal salt medium and resuspended in basal salt. The final cell concentration was about OD 600nm =1.0, inoculated into 20 ml of basal salt medium containing 1 mM isoprocarb at a 5% (v / v) inoculation amount, and cultured at 30°C for 48 h. Extracted with an equal amount of dichloromethane, dried, dissolved with methanol, filtered twice with a 0.22 μm filter membrane, and the isoprocarb concentration in the solution was determined by high performance liquid chromatography. Three replicates were set for each treatment group, and a blank control was set. The chromatographic column of the high performance liquid chromatograph was GL Sciences lnc C 18(250mm×4.6mm, 5μm), mobile phase was methanol: water = 70:30, flow rate was 1mL / min, column temperature was 40℃, injection volume was 10μL, and UV detector detection wavelength was 254nm. Metabolites were identified by HPLC-MS, conditions: mobile phase was methanol: water = 70:30, Agilent ZORBAX SB (2.10×100mm, 1.8μm reverse phase column, MS analysis used ESI mode, detector was LTQ Orbitrap XLMass Spectrometer. The results showed that strain R-7 was able to degrade isoprocarb, and one metabolite was detected during the degradation process, with HPLC peak time at 8.565min ( Figure 1 ). After mass spectrometry analysis, the peak of compound I with retention time of 6.656min was detected by mass spectrometry [M+Na] + The m / z of is 216.1, which is consistent with the charge-mass ratio of the protonated derivative of isoprocarb; compound II (retention time 8.565 min) was detected by mass spectrometry [M+H] + The m / z of isoprocarb is 134.9, which is consistent with the charge-mass ratio of the protonated derivative of o-isopropylphenol. The results of mass spectrometry showed that the degradation pathway of isoprocarb in strain R-7 was firstly that isoprocarb was converted into o-isopropylphenol ( Figure 2 ).

[0033] Example 2

[0034] Isoprocarb hydrolase gene cehA R-7 Clone

[0035] 2.1 Extraction and sequencing of total bacterial genomic DNA

[0036] 2.1.1 Extraction of total bacterial genomic DNA

[0037] After the strain R-7 was cultured in large quantities, the high-salt combined with CTAB method was used to extract the high-purity, large-fragment genomic total DNA of the strain R-7, dissolved in TE buffer (pH 8.0), and stored at -20°C. For specific methods, refer to the "Succinct Molecular Biology Experiment Guide" edited by F. Osber et al. The prepared sufficient DNA samples were sent to Sangon Biotech Co., Ltd. under dry ice insulation.

[0038] DNA sample detection uses: 1. Concentration detection, agarose gel electrophoresis quantification; 2. OD260:280 and OD260 / 230 detection method: NanoDrop. Bacterial genome sequencing requires the sample OD value to be between 1.8-2.0, the higher the concentration, the better, and the concentration should not be less than 30ng / μL.

[0039] 2.1.2 Strain genome draft sequencing and result analysis

[0040] After the DNA samples are qualified, the library is built, sequenced, and the data is analyzed (including base recognition, filtering adapter sequences, and decontamination), and then the subsequent bioinformatics analysis is carried out, mainly including the use of SOAPdenovo assembly software to assemble the reads data, using Glimmer3.0 gene prediction software to perform gene de novo prediction on the assembly results, and comparing the predicted genes with the database and performing functional annotation genome component analysis, gene function annotation, and comparative genome analysis, etc., to obtain the scaffold sequence and make relevant basic data statistics. The genome sequencing results showed that the draft genome size of strain R-7 was 4.22Mb, with 138 contigs, and a total of 4021 ORFs were predicted.

[0041] There is only one isoprocarb hydrolase reported, which is IpcH from strain Rhodococcus sp.D-6. However, there are many hydrolases belonging to the same carbamate pesticides. The amino acid sequences of the known carbamate pesticide hydrolase genes (mcbA, mcd, carH, cahA, ameH, cehA and ipcH) were downloaded and genome comparison analysis was performed. The results showed that there was a hydrolase CehA (cehA) in the genome of strain R-7 that was similar to the hydrolase that degrades furadan, carbaryl or cypermethrin. CFD-1 (QGZ07376),CehA OXA20 (CBY85381),CehA AC100 (CDO34164),CehA CDS-1 (WP087575984),CehA KN65.2 (BAB85626)) with an amino acid homology of up to 99%, and the gene was named cehA R-7 The nucleotide sequence is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2.

[0042] Example 3

[0043] Isoprocarb hydrolase gene cehA R-7 Cloning and functional characterization of

[0044] 3.1pET24b-cehA R-7 Construction of bacterial expression vector

[0045] 3.1.1 Isoprocarb hydrolase gene cehA R-7 PCR amplification

[0046] PCR amplification was performed using a forward primer and a reverse primer, wherein the forward primer (SEQ ID NO.3) is 5'- AAGAAG GAGATATACATTTGGACCAACCATTCAAACCAGATCG-3', reverse primer (SEQ ID NO.4): 5'- TGGTGGTGGT GGTGCTCGAG CGTTAAGTCGCTTTCGGCGATGATC-3'. cehA was amplified using R-7 DNA as a template R-7 Gene fragment.

[0047] Amplification system:

[0048]

[0049] PCR amplification procedure:

[0050] a. Denaturation at 98℃ for 3min;

[0051] b. Denaturation at 98°C for 0.5 min, annealing at 58°C for 0.5 min, and extension at 72°C for 1.0 min for 30 cycles;

[0052] c. Extend at 72°C for 10 min and cool to room temperature.

[0053] The PCR amplification product was purified using a gel purification recovery kit.

[0054] 3.1.2 Double restriction digestion of PCR products and plasmids, product purification and homologous recombination

[0055] The expression vector pET-24b(+) was double-digested with Nde I and Xho I. The digestion system was as follows:

[0056] 10×M Buffer 5μL

[0057] Nde I (10U·μL -1 )2.0μL

[0058] Xho I (10U·μL -1 )2.0μL

[0059] DNA (purified PCR or plasmid) 30 μL

[0060] ddH2O 11μL

[0061] 37℃ warm bath for 30 minutes

[0062] The enzyme digestion effect was detected by 0.75% agarose nucleic acid electrophoresis. The corresponding DNA fragments were recovered and connected to cehA by homologous recombination. R-7 and double-digested pET-24b(+), add homologous recombination system according to the instructions of One Step Cloning kit, and react in a 37℃ water bath for 30min.

[0063] 3.1.3 Enzyme-linked product transformation and screening of positive transformants

[0064] Take E.coli BL21 (DE3) competent cells (100μL) from -70℃, hold in the palm of your hand to melt, add 20μL of homologous recombination product, gently rotate the tube to mix, and place on ice for 30min. Gently put the centrifuge tube into a 42℃ water bath for heat shock for 60-90s, then put the centrifuge tube back on ice for 10min. Add 500-800μL LB medium, put the centrifuge tube into a 37℃ shaker, 150rpm, and recover for 45-60min. 5,000rpm, centrifuge for 2min, discard part of the supernatant, keep about 200μL, blow with a gun to mix the bacteria, take 100μL of the bacterial solution and evenly spread it on LB (kanamycin 100mg / L) plate, culture at 37℃ overnight, pick out the single colony, extract the plasmid sequencing to verify whether the target gene is connected to the vector and the end is connected to 6 His-tag, this positive transformant BL21 (pET24b-cehA R-7 )save.

[0065] 3.2CehA R-7 Expression and purification in E. coli

[0066] BL21 (pET24b-cehA R-7 ) into liquid LB medium and cultured on a shaking platform until OD 600nm The concentration of IPTG was 0.4-0.6, and IPTG was added to a final concentration of 0.05mM. The mixture was induced at 16℃ for 8h. The cells were collected by centrifugation at 12000rpm for 5min, and washed with PBS buffer (50mM, pH 7.4) at 4℃. Then, the cells were resuspended with an appropriate amount of PBS, ultrasonically disrupted for 5-10min under ice bath conditions, and centrifuged at 12000rpm for 40min. The supernatant was collected. Protein purification was performed using a cobalt ion affinity chromatography column, and the purification effect was detected by SDS-PAGE electrophoresis. After Ni 2+ After -NTA affinity purification, high-purity isoprocarb hydrolase was successfully obtained in the elution solution of 50mM imidazole. The results of SDS-PAGE protein electrophoresis showed that there was a single protein band between 66.2-116.0kDa with high purity and a size of about 90kDa. Figure 3 As shown, its size is consistent with the theoretical mass (87.66 kDa) derived from its amino acid sequence.

[0067] 3.3CehA R-7 Enzyme activity assay

[0068] Enzyme activity reaction system (1 ml): 50 mM PBS (pH 7.0), 0.1 mg CehAR-7 , 1.0 mM isoprocarb. The enzyme reaction was started from the addition of the enzyme and lasted for 30 min. The sample was first scanned by UV spectrophotometer in the range of 200-400 nm, and then processed and detected by HPLC. Detection of CehA by HPLC R-7 Degradation of isoprocarb in the enzymatic reaction system. R-7 In the presence of Figure 4 ).

[0069] Although specific embodiments have been shown and described in the present invention, it will be appreciated by those skilled in the art that various alternative and / or equivalent embodiments may be used to replace the specific embodiments shown and described without departing from the scope of the present invention. This application is intended to include any improvements or changes to the specific embodiments discussed in the present invention. Therefore, the present invention is limited only by the claims and their equivalents.

[0070] It will be appreciated by those skilled in the art that various modifications and changes may be made without departing from the scope of the present invention. Such modifications and changes are intended to fall within the scope of the present invention as defined by the appended claims.

Claims

1. An isoprocarb hydrolase gene cehA R-7 , characterized in that: The nucleotide sequence is any of the following: (1) the DNA sequence of SEQ ID NO.1 in the sequence listing; (2) the amino acid sequence of SEQ ID NO.2 in the sequence listing; (3) A sequence having a homology of 90% or more, preferably 99% or more, to the carbamate pesticide hydrolase CehA.

2. An isoprocarb hydrolase CehA R-7 , characterized in that: The amino acid sequence of the hydrolase is the amino acid sequence of SEQ ID NO.2 in the sequence list.

3. An isoprocarb hydrolase CehA R-7 The preparation method is characterized in that: The above-mentioned isoprocarb hydrolase gene cehA R-7 The recombinant expression vector was cloned and introduced into the host cell to obtain the recombinantly expressed isoprocarb hydrolase CehA. R-7 .

4. The preparation method according to claim 3, characterized in that: The expression vector is pET-24b(+) plasmid purchased from Novegen, and the host cell is Escherichia coli BL21(DE3) purchased from Shanghai Invitrogen Biotechnology Co., Ltd.

5. The preparation method according to claim 3, characterized in that: The recombinant expression vector is a vector in which the isoprocarb hydrolase gene cehA is expressed. R-7 Insertion was performed between the Nde I and Xho I sites of pET-24b(+).

6. The preparation method according to claim 3, characterized in that: The preparation method also includes PCR amplification of the isoprocarb hydrolase gene, and the PCR amplification uses a forward primer and a reverse primer, wherein the forward primer is shown in the sequence list SEQ ID NO.3, and the reverse primer is shown in the sequence list SEQ ID NO.

4.

7. The preparation method according to claim 3, characterized in that: The preparation method also includes the expression and purification of the isoprocarb hydrolase gene, wherein the isoprocarb hydrolase gene is expressed under the induction of IPTG, and then purified by Ni 2+ -NTA affinity chromatography to purify isoprocarb hydrolase.

8. A method for repairing an environment contaminated by isoprocarb, characterized in that: The method comprises using the enzyme or enzyme preparation of claim 2 to degrade isoprocarb residual in the environment.

9. A genetically modified crop, characterized in that: The crop is constructed by introducing the gene of claim 1 to improve tolerance to isoprocarb.

10. An isoprocarb hydrolase CehA R-7 The application is characterized in that The applications include: a. Preparation of enzyme preparations for remediation of isoprocarb pollution in soil and water; b. Construction of transgenic crops resistant (tolerant) to isoprocarb.

Images