WO2016015668A1 - Carbamate pesticide degrading enzyme cfh, coding gene cfd thereof and applications of both - Google Patents

Abstract

Provided are a carbamate pesticide degrading enzyme CFH, a coding gene cfd thereof and applications of both. The open reading frame of the coding gene cfd of the carbamate pesticide degrading enzyme has the overall length of 2139 bp and has the sequence shown by SEQ ID No.1, and the coded product CFH contains 712 amino acids and has the sequence shown by SEQ ID No.2. CFH can degrade carbamate pesticides such as carbofuran.

Description

Application of carbamate pesticide degrading enzyme CFH and its coding gene cfd and both Technical field

The invention belongs to the field of application environment microorganisms and agriculture, and relates to the application of the enzyme CFH capable of degrading carbamate pesticides such as carbofuran and its coding gene cfd and both.

Background technique

There are many people in China, and the large-scale use of chemical pesticides is a necessary measure to ensure high yield and stable yield of crops, but it also brings serious pollution. The refractory nature of pesticides leads to higher residual rates of soil and agricultural products. For example, the current detection rate of farmland in the Yangtze River Delta and Pearl River Delta is over 90%, especially for agricultural products contaminated by pesticides, such as the 2009 Greenpeace International Test. The results showed that 90% of agricultural products in China detected pesticide residues, and 66% of the samples contained at least 5 kinds of pesticides. The Qingdao poisonous leek event that occurred in April 2010 was caused by the pollution of organophosphorus pesticides, causing adverse effects. Biological (enzyme) degradation technology is a new in-situ bioremediation technology with good effects, low cost and no secondary pollution. It is an ideal way to degrade pesticide residues in soil and agricultural products.

Carbamate pesticides such as carbofuran are a class of insecticides that are widely used worldwide. The toxicity mechanism of this kind of pesticide is similar to that of organophosphorus pesticides, mainly inhibiting cholinesterase activity. Carbamate pesticides can invade the body through the respiratory tract and digestive tract, and can also be slowly absorbed through the skin and mucous membranes, mainly in liver, kidney, fat and muscle tissues. In recent years, due to its high toxicity and frequent pesticide poisoning incidents, some varieties have been restricted or even banned in countries around the world. In the case of carbofuran, the EU and Canada have been banned, the United States has been banned from any crop (human consumption); in the 2014 edition of the list of Chinese national banned and restricted pesticides, carbofuran is a restricted pesticide, vegetables, fruit trees, tea trees, Chinese herbal medicines. It is the scope of its ban. This means that carbamate pesticides such as carbofuran are still being used in large quantities, and their residues in soil and agricultural products are still problems we have to face and solve.

To solve the problem of carbamate pesticide residues by using biological (enzymatic) degradation technology, it is first necessary to obtain excellent strains and genetic resources. At present, there are many reports on microorganisms capable of degrading such pesticides, but the corresponding genetic resources are rarely reported. There are currently three related genes cehA (GenBank accession number: AB069723), cahA (GenBank accession number: AB081302) and mcd. The degrading enzymes encoded by the first two genes have strong activity only on carbaryl, although the sequence of mcd can Obtained from GenBank (accession number: AF160188), but there is no direct literature report. The relevant literature only reports the starting strain, purification of the enzyme and gene localization. Therefore, there has been no report on a broad-spectrum, high-efficiency degrading enzyme gene for carbamate pesticides. This limits the use of biological (enzymatic) degradation techniques to eliminate carbamate pesticide residues in soils and agricultural products.

Summary of the invention

The object of the present invention is to provide a carbamate-based pesticide degrading enzyme CFH such as carbofuran and its coding gene cfd and the use thereof, in view of the above-mentioned deficiencies of the prior art. The gene cfd can be used to construct transgenic microorganisms or plants that degrade carbamate pesticides, and can also be used to produce enzyme preparations for degrading carbamate pesticides for eliminating carbamate pesticide residues in soils, water bodies and agricultural products.

The object of the present invention can be achieved by the following technical solutions:

A carbamate pesticide degrading enzyme gene cfd having the nucleotide sequence of SEQ ID NO.

The starting strain of the gene cfd, Sphingobium sp. YBL3, was deposited in the China Center for Type Culture Collection under the accession number CCTCC NO: M208076. The results of mass spectrometry indicated that the crude enzyme solution of strain YBL3 could cleave the amide bond of carbamate pesticides such as carbofuran, carbaryl and isoprocarb to degrade these pesticides and eliminate their toxicity.

The strategy for cloning the carbamate pesticide degrading enzyme gene is the shotgun method. The total DNA of the strain YBL3 was first extracted, and the total DNA was digested with Sau3AI, and then ligated with BamHI-digested plasmid pUC118, and the enzyme-linked product was transformed into E. coli DH5α competent cells to obtain a total DNA library. The clones capable of degrading carbofuran were screened by high performance liquid chromatography (HPLC), and then the clones of the degrading ability were sequenced and analyzed to determine the open reading frame (ORF) of the target gene. Finally, the open reading frame size of the target gene was 2139 bp, named cfd, and the encoded enzyme was named CFH. This is the first report of a broad-spectrum, highly efficient degrading enzyme gene for carbamate pesticides.

The enzyme CFH encoded by the cfd gene has the amino acid sequence of SEQ ID NO.

A recombinant expression vector containing the cfd gene.

Preferably, the recombinant expression vector inserts the cfd gene between the NdeI and EcoRI sites of pET-29a(+).

Genetically engineered bacteria containing the cfd.

The starting strain of the genetically engineered bacteria is Escherichia coli BL21 (DE3).

The use of the cfd gene in the construction of transgenic crops degrading carbamate pesticides.

The use of the cfd gene for the removal of carbamate pesticide residues in agricultural products, soils and water bodies.

The use of the degradation enzyme CFH in the degradation of carbamate pesticides.

The use of the degrading enzyme CFH for removing carbamate pesticide residues in agricultural products, soils and water bodies.

The use of the recombinant expression vector for constructing a transgenic crop degrading a carbamate pesticide.

The use of the recombinant expression vector for removing carbamate pesticide residues in water.

Beneficial effect

1. The present invention successfully cloned the gene cfd from the strain YBL3 (CCTCC NO: M208076) by the shotgun method. The alignment in GenBank indicated that the gene is a new gene with a full length (from start codon to stop codon) of 2139 bp, encoding 712 amino acids.

2. The degradation enzyme CFH or the engineered bacteria carrying the cfd gene in the present invention can efficiently degrade carbamate pesticides. The gene cfd can be used to construct transgenic microorganisms or plants that degrade carbamate pesticides, and can also be used to produce enzyme preparations for degrading carbamate pesticides for eliminating carbamate pesticide residues in soils, water bodies and agricultural products.

DRAWINGS

Figure 1. Gene strategy cfd expression strategy map in BL21 (pET-29a(+)).

Fig. 2 is a mass spectrometric detection diagram of the gene cfd in BL21 (pET-29a(+)) expressing the product degrading carbofuran to furanol. Figure 2a is a first-order mass spectrum of the degradation product; Figure 2b is a secondary mass spectrum of the substance S1 (carbofuran); Figure 2c is a secondary mass spectrum of the substance S2 (furanol).

Biomaterial preservation information

Sphingomobacter YBL3, classified as Sphingobium sp. YBL3, is deposited in the China Center for Type Culture Collection (CCTCC) at Wuhan University, Wuhan, China, with the accession number CCTCC NO: M208076, and the date of deposit is May 22, 2008. .

detailed description

Example 1 Cloning of the gene cfd

(1) Extraction of total DNA from bacterial genome

YBL3 (CCTCC NO: M208076) was cultured in liquid LB medium (37 ° C, 200 rpm, 18 h), and the total DNA of the high-purity, large-segment strain YBL3 genome was extracted by CTAB method, and dissolved in TE buffer (pH 8.0). In the case of preservation at -20 °C, the specific method refers to the "Guide to the Experimental Guide to Molecular Biology" edited by F. Osb.

(2) pUC118 (BamHI) purchased Yubao Bioengineering (Dalian) Co., Ltd.

(3) The total DNA of strain YBL3 was digested with Sau3AI.

(4) DNA recovery

The total DNA after digestion was purified by electrophoresis (TAE buffer) and recovered by axygen biosciences (China) recovery kit. The recovered DNA was dissolved in 10 mmol/L Tris·HCl (pH 8.0) and placed in - Deposited at 20 ° C.

(5) Enzyme

Establish the following reaction system:

Incubate at 16 ° C for 12 hours.

(6) Conversion and screening

10 μl of the enzyme-linked product was transformed into 200 μl of E. coli DH5α competent cells (TaKaRa, Code: D9057), and the specific method was as described in F. Osbour et al., "Guidelines for Editing Molecular Biology" P23. The LB plate containing 100 mg/kg ampicillin was applied, and after 24 hours of culture, the clones were selected for expansion culture, and the cells were resuspended in PBS (pH 7.0), placed in a constant temperature shaker at 180 rpm, and cultured at 30 ° C with shaking. 2 hours, the PBS suspension was removed, and the cell concentration was adjusted to OD ₆₀₀ = 2.0 to prepare resting cells of the strain clone. 50 mg·L ^-1 of carbofuran was added to the resting cell suspension, 180 rpm, 30 ° C. The mixture was shake cultured for 24 hours in a constant temperature shaker. The reaction was quenched by the addition of an equal volume of dichloromethane, and the product was extracted with vigorous shaking. The organic phase was dried over anhydrous sodium sulfate and dried with nitrogen. HPLC can be used to detect whether the clone can degrade carbofuran. The liquid phase conditions are: chromatography column, Kromasil 100-5C18 (4.6mm × 250mm × 5μm); mobile phase, methanol / water (volume ratio 70/30); flow rate, 1ml· Min ^-1 ; UV detector, wavelength 280 nm and 230 nm; injection volume, 20 μl.

(7) Gene nucleotide sequence determination

The positive clone capable of degrading carbofuran obtained in (6) was entrusted to Shanghai Yingjun Biotechnology Co., Ltd. for sequencing, and the nucleotide sequence of the carbofuran degrading enzyme gene cfd was SEQ ID NO. 1, according to the gene cfd nucleoside. The 712 amino acid sequence to which the acid sequence is derived is SEQ ID NO.

Example 3 High-level expression and functional assay of carbofuran hydrolase CFH in BL21 (pET-29a(+)) (Technical Roadmap 1) (1) PCR amplification of gene cfd

Forward primer: 5'-TCTGGA CATATG TCTAGTGACAAACTCCAT-3' (SEQ ID NO. 3) and reverse primer: 5'-TCTGGA GAATTC ATTGTCTTTTTGCATCA-3' (SEQ ID NO. 4) as primers, using PCR from strain YBL3 The gene cfd sequence was amplified from the total DNA.

Amplification system:

PCR amplification procedure:

A.95 ° C denaturation 3 min;

b. 95 ° C denaturation 1.5 min, 53 ° C annealing 0.5 min, 72 ° C extension 1.5 min, 25 cycles;

c. 72 ° C extension for 10 min, cooled to room temperature.

(2) The PCR product was digested with NdeI and EcoRI.

Enzyme digestion system:

The reaction was carried out for 3 hours or more in a 37 ° C water bath. The digested product was subjected to 2% agarose gel electrophoresis.

(3) pET-29a (+) was digested with NdeI and EcoRI (Ref. 2.2).

(4) Conversion

The recovered fragment in (2) and the enzymatically ligated pET-29a (+) in (3) (see step (5) of Example 1) to obtain the pET-29a(+) recombinant plasmid pET-containing the cfd gene. 29a(+)-cfd. The recombinant plasmid pET-29a(+)-cfd was transformed into the expression host strain BL21 (DE3) to obtain recombinant microorganism BL21 (CFH) containing cfd.

(5) Expression, purification and functional verification of CFH

BL21 (CFH) was cultured in LB medium to an OD _{600 nm} of 0.6 to 0.8, and IPTG was added to a concentration of 1 mM, and cultured at 30 ° C for 4 hours. Centrifuge 100 ml of the bacterial solution, resuspend the cells in 10 ml (50 mM, pH 7.0) PBS buffer, ultrasonically disrupt (Auto Science, UH-650B ultrasonic processor, 30% intensity) for 5 minutes, centrifuge, collect the supernatant, and use nickel ions to pro The supernatant was purified by chromatography and the carbofuran hydrolase CFH was obtained.

(6) Determination of degradation of carbofuran by CFH

The reaction system of CFH hydrolyzed carbofuran: 50 mM phosphate buffer (pH 7.0), 50 mg·L ^-1 carbofuran, CFH 50 μl, and reacted at 30 ° C for 30 min. The reaction was quenched by the addition of 3 ml of dichloromethane, and the product was extracted with vigorous shaking. The organic phase was dried over anhydrous sodium sulfate and evaporated to dryness. Mass spectrometry results showed that CFH can degrade carbofuran to furanol (Figure 2).

(7) Determination of the ability of CFH to degrade other carbamate pesticides

Reaction system for hydrolyzing carbamate pesticides by CFH: 50 mM phosphate buffer (pH 7.0), 50 mg·L ^-1 carbaryl, isoprocarb or carbaryl, CFH 50 μl, and reacted at 30 ° C for 30 min. The reaction was quenched by the addition of 3 ml of dichloromethane, and the product was extracted with vigorously shaking. The organic phase was dried over anhydrous sodium sulfate, dried with nitrogen, and then dissolved in 100 liters of methanol. The test results show that CFH can also efficiently degrade carbaryl, isoprocarb and carbaryl.

The sources of microorganisms used in the present invention are as follows:

Claims (10)

1. A carbamate-degrading enzyme gene cfd having the nucleotide sequence of SEQ ID NO.
2. The degradation enzyme CFH encoded by the gene cfd of claim 1 having the amino acid sequence of SEQ ID NO.
3. A recombinant expression vector comprising the gene cfd of claim 1.
4. A genetically engineered bacterium comprising the gene cfd of claim 1.
5. The genetically engineered bacterium according to claim 4, wherein the starting strain of the genetically engineered bacterium is Escherichia coli BL21 (DE3).
6. Use of the gene cfd of claim 1 in the construction of a transgenic crop degrading a carbamate pesticide.
7. Use of the gene cfd of claim 1 for the removal of carbamate pesticide residues in water.
8. The use of the degrading enzyme CFH of claim 2 for eliminating carbamate pesticide residues in soil, water or agricultural products.
9. Use of the recombinant expression vector of claim 3 in the construction of a transgenic crop degrading a carbamate pesticide.
10. Use of the recombinant expression vector of claim 1 for the removal of carbamate pesticide residues in water.

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