CN110592118B - Hydrolase gene strB and protein coded by same and application - Google Patents

Hydrolase gene strB and protein coded by same and application Download PDF

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CN110592118B
CN110592118B CN201910869982.0A CN201910869982A CN110592118B CN 110592118 B CN110592118 B CN 110592118B CN 201910869982 A CN201910869982 A CN 201910869982A CN 110592118 B CN110592118 B CN 110592118B
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strb
hydrolase gene
hydrolase
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agricultural products
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CN110592118A (en
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陈少华
李绮婷
张文平
黄耀华
占卉
林子秋
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South China Agricultural University
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    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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    • A23L5/25—Removal of unwanted matter, e.g. deodorisation or detoxification using enzymes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
    • B09C—RECLAMATION OF CONTAMINATED SOIL
    • B09C1/00—Reclamation of contaminated soil
    • B09C1/10—Reclamation of contaminated soil microbiologically, biologically or by using enzymes
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    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
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    • C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
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    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F2101/00—Nature of the contaminant
    • C02F2101/30—Organic compounds
    • C02F2101/306—Pesticides
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F2101/00—Nature of the contaminant
    • C02F2101/30—Organic compounds
    • C02F2101/34—Organic compounds containing oxygen
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F2101/00—Nature of the contaminant
    • C02F2101/30—Organic compounds
    • C02F2101/38—Organic compounds containing nitrogen

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Abstract

The invention discloses a hydrolase gene strB, and a protein coded by the hydrolase gene strB and application of the hydrolase gene strB. The total length of the hydrolase gene StrB is 987bp, the nucleotide sequence is shown as SEQ ID No.1, the encoded product hydrolase StrB contains 328 amino acids, and the amino acid sequence is shown as SEQ ID No. 2.StrB can catalyze and degrade deltamethrin, fenpropathrin, lambda-cyhalothrin, kresoxim-methyl, pyraclostrobin, azoxystrobin and other pesticides containing ester bonds. The produced enzyme preparation can be used for removing pesticide residues in agricultural products such as vegetables, fruits, tobacco, tea leaves and the like, so that the quality of the agricultural products is improved and the additional value is increased. The hydrolase gene strB can be further used for constructing transgenic crops to thoroughly solve the problem of overproof pesticide residues in agricultural production and produce pollution-free green agricultural products; can also be used for restoring natural environments such as soil, water and the like polluted by ester pesticides, and has wide application prospect.

Description

Hydrolase gene strB and protein coded by same and application
Technical Field
The invention belongs to the field of applied environmental microorganisms and agriculture, and relates to a hydrolase gene strB, and a protein coded by the hydrolase gene strB and application of the hydrolase gene strB, which are used for removing pesticide residues in crops and producing pollution-free green agricultural products; meanwhile, the method can be used for restoring natural environments such as pesticide-polluted soil, water and the like.
Background
The pesticide is applied by a double-edged sword. The great and frequent use of ester pesticides (mainly including pyrethroid pesticides and other strobilurin pesticides) has brought a great threat to ecological environment and human health. A great deal of research shows that ester pesticides mostly have accumulated toxicity, and even low dosage of ester pesticides can cause chronic diseases after long-term contact. The pesticide has high toxicity to agricultural beneficial organisms such as silkworms, bees and birds and aquatic organisms such as fishes, and in addition, the ester pesticide residue surface water has a huge destructive effect on the diversity and ecosystem of freshwater organisms. Therefore, ester pesticide residues have become an emerging global threat to human health and the ecological environment.
The Biodegradation technology (Biodegradation) can be used for removing various organic pollutants in the environment and agricultural products, and has the advantages of simple and convenient operation, economy, practicability, no secondary pollution and the like compared with the traditional chemical and physical treatment methods. Therefore, the research of treating pesticide residue pollution by using microbial degradation microbial inoculum or enzyme preparation products has become a hot point of research in recent years. At present, many developed countries and large company invest huge capital to research and develop pesticide residue biodegradation preparations, for example, the American BCI company and the American engineering service bioremediation company have already produced degradation preparation products on a large scale, and the domestic Beijing Jia agriculture new trade development company has successfully produced Biya degradation enzyme preparations which can be used for removing organophosphorus pesticide residues.
Degrading enzymes have significant advantages over degrading bacteria, one being that degrading enzymes are generally more tolerant of abnormal environmental conditions than the microbial cells that produce such enzymes; moreover, the degradation effect of the degrading enzyme is far better than that of the microorganism, especially for low-concentration pesticides. Therefore, the degrading enzyme has high degrading efficiency, can effectively utilize low-concentration pesticide, has high environment suitability and low environmental risk, and is the leading edge and hot spot of the international research on the residual pesticide treatment technology. However, there are few biodegradable preparations for pesticide residues of esters.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides a novel hydrolase gene strB for degrading ester pesticides, which can be used for removing pesticide residues in crops and producing pollution-free green agricultural products; meanwhile, the method can be used for restoring natural environments such as pesticide-polluted soil, water and the like.
Another object of the present invention is to provide a protein encoded by the gene.
The invention also aims to provide application of the gene and the protein coded by the gene.
The purpose of the invention is realized by the following technical scheme:
a hydrolase gene strB capable of degrading pesticides containing ester bonds, and the nucleotide sequence is shown in SEQ ID No. 1.
The hydrolase gene strB is cloned from a Bacillus thuringiensis (Bacillus thuringiensis) strain Bt-1 capable of degrading pesticides containing ester bonds. The strain is preserved in China Center for Type Culture Collection (CCTCC) in 2013, 12 months and 20 days, and the preservation number is CCTCC M2013680. The inventor discovers by mass spectrometry that: the strain Bt-1 can break ester bonds of ester bond-containing pesticides such as deltamethrin, fenpropathrin, lambda-cyhalothrin, kresoxim-methyl, pyraclostrobin, azoxystrobin and the like, so that the pesticides lose pesticide toxicity.
The specific method for cloning the hydrolase gene strB from the strain is the transposon tagging method. The bacterial strain Bt-1 can grow on a basal medium plate by using pesticides such as efficient cyhalothrin and the like as a unique carbon source, degraded genes are screened by using whether hydrolysis loops are generated in the degradation process after a genome library is constructed, a target gene fragment is cloned by using a PCR technology, and then functional verification is carried out.
A target strain which generates a hydrolysis ring on a plate added with pesticides in a basal culture medium is screened by the above strategy, and further degradation experiments show that the strain can not degrade pesticides such as high-efficiency cyhalothrin. Designing a primer, and carrying out PCR, wherein the gene is a hydrolase gene with unknown function, the total length is 987bp (328 amino acids are coded), and the gene is named as strB. It is a new hydrolase gene for degrading pesticide.
A hydrolase StrB capable of degrading pesticides containing ester bonds, and the amino acid sequence of the hydrolase StrB is SEQ ID No. 2.
The recombinant cloning vector pET-28b-strB containing the hydrolase gene strB as described above was obtained by inserting the nucleic acid fragment of the hydrolase gene strB as described above between the BamHI and XhoI sites of pET-28 b.
The recombinant expression vector pET-28b-His-strB containing the hydrolase gene strB capable of degrading the pesticide containing ester bonds is preferably obtained by inserting a nucleotide fragment of the hydrolase gene strB between BamHI and XhoI sites of pET-28 b.
The gene engineering bacteria contain the hydrolase gene strB capable of degrading the pesticide containing ester bonds.
The genetic engineering bacteria preferably use escherichia coli BL21(DE3) as an original strain.
The hydrolase gene strB is applied to degrading pesticides containing ester bonds in crops, agricultural products, soil and water.
The hydrolase gene strB is applied to construction of transgenic crops for degrading pesticides containing ester bonds.
The application of the hydrolase StrB in removing pesticide residues containing ester bonds in crops, agricultural products, soil and water bodies is disclosed.
Compared with the prior art, the invention has the following beneficial effects:
the invention successfully clones a hydrolase gene strB for degrading ester bond-containing pesticide from a Bacillus thuringiensis (Bacillus thuringiensis) strain Bt-1 by utilizing a transposon tagging method. The GenBank comparison result shows that the gene is a novel hydrolase gene for degrading pesticides, the total length (from an initiation codon to a termination codon) is 987bp, and 328 amino acids are coded.
The hydrolase StrB provided by the invention can catalyze and degrade pesticides containing ester bonds, such as deltamethrin, fenpropathrin, lambda-cyhalothrin, kresoxim-methyl, pyraclostrobin, azoxystrobin and the like. The produced enzyme preparation can be used for removing pesticide residues in agricultural products such as vegetables, fruits, tea leaves, cotton, tobacco and the like, and can improve the quality of the agricultural products and increase the added value.
The hydrolase gene strB can be further used for constructing transgenic crops to thoroughly solve the problem of overproof pesticide residues in agricultural production and produce pollution-free green agricultural products; can also be used for restoring natural environments such as pesticide-polluted soil, water and the like, solves the problem of environmental pollution, and protects the ecological environment and human health.
Drawings
FIG. 1, E.coli BL21(DE3)/pET-28b-strB expression System for small expression
FIG. 2 shows the results of purified electrophoresis of StrB protein
Detailed Description
The invention is described in further detail below with reference to the drawings and specific examples, but the examples are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.
The microbial sources used in the examples are as follows:
coli BL21(DE3) and E.coli high expression vector pET-28b were stored in Guangdong province of southern China university of agriculture and crop disease control laboratory.
Bacillus thuringiensis (Bacillus thuringiensis) strain Bt-1 capable of degrading pesticides containing ester bonds is preserved in China Center for Type Culture Collection (CCTCC) in 2013, 12 months and 20 days, and the preservation number is CCTCC M2013680; the preservation address is Wuhan, Wuhan university in China.
Example 1
Cloning of the hydrolase Gene strB
S1, construction of genome library
A genome library is constructed, the genome of a Bacillus thuringiensis (Bacillus thuringiensis) strain Bt-1 is extracted, enzyme digestion is carried out, and degradation genes are screened by utilizing whether hydrolysis rings are generated in the degradation process or not. A target strain having a degrading function is obtained by this method.
S2.PCR series amplification
Designing a primer, and amplifying by using a PCR technology until a complete sequence of the degradation gene is obtained. Gene function was initially determined by BlastX alignment.
S3, cloning and functional verification of hydrolase gene strB
The amplified target gene sequence is subjected to sequence splicing by Seqman software of DNAStar, and Open Reading Frames (ORFs) of the DNA sequence are analyzed by using FramePlut 3.0beta online software and CLONE software. The protein of interest amino acid sequence was analyzed using BLAST online search and aligned to known amino acid sequences in the protein database. The amino acid sequence of the target protein is analyzed in protein structure domain by on-line network resource SMART and other network resources, so as to predict and map the function of the target gene and judge the complete sequence of the whole degradation related gene. Functional verification was performed by expressing the degradation gene directly in e.coli DH5 α. The obtained hydrolase gene strB has the nucleotide sequence of SEQ ID NO.1 and the amino acid sequence of SEQ ID NO. 2.
Example 2
High-efficiency expression of hydrolase gene strB in E.coli BL21(DE3) (see FIGS. 1 and 2)
S1, PCR amplification of hydrolase Gene E.coli BL21(DE3)
And (3) performing reverse primer strB-F: 5'-TAGCCCGGATCCGATGTGTACTAGTTTGACATTAGAG-3' (SEQ ID NO.3) and the reverse primer strB-R: 5'-TCCGTCTCGAGGTTCTCATAAAATATTTTTTGTTTTCGT-3' (SEQ ID NO.4) as a primer, and amplifying a hydrolase gene strB fragment from the total DNA of Bacillus thuringiensis Bt-1 strain by using a PCR method.
PCR amplification System:
Figure BDA0002200487970000031
PCR amplification procedure:
pre-denaturation at 95 deg.C for 3min, denaturation at 94 deg.C for 30s, annealing at 55 deg.C for 45s, extension at 72 deg.C for 1min for 30s, performing 34 cycles, extension at 72 deg.C for 5min, and storing at 16 deg.C.
S2.PCR product Using BamH I and Xho I double cleavage
Enzyme digestion system:
Figure BDA0002200487970000041
placing the mixture in a water bath at 37 ℃ for reaction for more than 2 h.
The digested product was recovered by electrophoresis on 1.0% agarose gel.
S3.pET-28b-His-strB was digested with BamHI and XhoI in the same manner as described in S2.
S4, transformation and expression: the fragment recovered in S2 was enzymatically ligated with digested pET-28b-His-strB in S3.
A connection system:
Figure BDA0002200487970000042
and (3) transforming the pET-28b-His-strB recombinant plasmid of the enzyme-linked hydrolase gene strB into an expression host bacterium E.coli BL21(DE3) to obtain a recombinant expression bacterium.
And S5, verifying the degradation function of the positive transformant expression product on pyrethroid pesticides such as efficient cyhalothrin.
Positive transformants were cultured to OD in LB Medium600At 0.5, IPTG was added to a concentration of 0.5mM and the induction was carried out overnight at 18 ℃. In 50mL of basal medium (g.L)-1)((NH4)2SO4,2；MgSO4,0.2；CaCl2,0.01；FeSO4,0.005；MnCl2,0.002；K2HPO4,10.5；KH2PO4And 4.5) adding 50mg/L of high-efficiency cyhalothrin, inoculating 0.2g/L of inoculum size into the culture solution of the positive transformant, and performing shaking culture at 30 ℃ for 5 days. Control is E.coli DH 5. alpha. with empty vector.
Ultrasonic extracting with acetone to obtain lambda-cyhalothrin, extracting with petroleum ether for 3 times, mixing the filtrate with the flask, concentrating at 50 deg.C,transferring all the concentrated solution into a 5mL graduated test tube, washing a round-bottom flask with 3mL of methanol for 3 times, diluting to a constant volume of 5mL, filtering 2mL of the solution through a 0.45-micrometer microporous filter membrane organic membrane injection filter, collecting filtrate in a sample injection bottle, and measuring the residual quantity of the filtrate by using a High Performance Liquid Chromatograph (HPLC), wherein the detection conditions are as follows: c18Reverse phase chromatography (250nm × 4.60mm,5 μm) at flow rate of 1mL/min, 25 deg.C for column temperature, 90: 10 (v: v) for mobile phase, 190-400 nm for scanning wavelength, and 10 μ L for sampling. The results show that: the degradation rate of the genetically engineered bacteria to 50mg/L of the lambda-cyhalothrin 5d reaches 83.7 percent.
Sequence listing
<110> southern China university of agriculture
<120> hydrolase gene strB, protein coded by same and application of hydrolase gene strB
<130> 0
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 987
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 1
atgtgtacta gtttgacatt agagacaaaa aacggtcagc atctttttgc aagaacgatg 60
gacttcacat tagatttgaa tcaagaagta atagtcattc ctcgacatta tcagtggaat 120
aatataactg gtgaaatcat taatacgaaa catgctacgg tcggaatggg aattaatcat 180
caaggaagga tcattatggc ggacggagta aatgaagcgg gtatgacatg tgcgacactc 240
tattttccag gattcgctac ttatagtcaa agcgtaggtg acaataaaac gaatttggct 300
ccatttgatt ttgtgacttg gagtctgaca caattcaact ctgtcaaaga gttaaaggaa 360
tctgtagata gccttacctt tttggatata ccattaccgg atttaggact tacgccacca 420
ctacattgga ttttagcgga taaatgggga gattgcattg tactggagcc gacaaatgaa 480
ggattaaaaa tgtatgacaa cccactagga gtgatgacga atagtccgga gtttaattgg 540
catttacaaa atttaagaca atatataggc cttaaatcgc agccattcgc gccaacagag 600
tggagtaact tatcattaag tgcttttggt cagggatcgg gctcaatggg acttccagga 660
gatttcaccc cgccatcaag gtttgtacgg gcagcatatg gcaaacaaaa cattcaaggt 720
atagatagcg aagaagaggg agtaacggcc ctttttcata ttttatcaaa ttgtgaggtt 780
cctaaaggcg gagtaataac agaagaaggg gcattagata atactatata tacaagcgta 840
atgtgtatgg agtccggaac atattattat catacctacg attgtagaca aattatagct 900
attcatttat ttcatgaaaa tttagataca gatgagatta aagcttatcc gttccaacga 960
aaacaaaaaa tattttatga gaactaa 987
<210> 2
<211> 328
<212> PRT
<213> Artificial sequence (Artificial sequence)
<400> 2
Met Cys Thr Ser Leu Thr Leu Glu Thr Lys Asn Gly Gln His Leu Phe
1 5 10 15
Ala Arg Thr Met Asp Phe Thr Leu Asp Leu Asn Gln Glu Val Ile Val
20 25 30
Ile Pro Arg His Tyr Gln Trp Asn Asn Ile Thr Gly Glu Ile Ile Asn
35 40 45
Thr Lys His Ala Thr Val Gly Met Gly Ile Asn His Gln Gly Arg Ile
50 55 60
Ile Met Ala Asp Gly Val Asn Glu Ala Gly Met Thr Cys Ala Thr Leu
65 70 75 80
Tyr Phe Pro Gly Phe Ala Thr Tyr Ser Gln Ser Val Gly Asp Asn Lys
85 90 95
Thr Asn Leu Ala Pro Phe Asp Phe Val Thr Trp Ser Leu Thr Gln Phe
100 105 110
Asn Ser Val Lys Glu Leu Lys Glu Ser Val Asp Ser Leu Thr Phe Leu
115 120 125
Asp Ile Pro Leu Pro Asp Leu Gly Leu Thr Pro Pro Leu His Trp Ile
130 135 140
Leu Ala Asp Lys Trp Gly Asp Cys Ile Val Leu Glu Pro Thr Asn Glu
145 150 155 160
Gly Leu Lys Met Tyr Asp Asn Pro Leu Gly Val Met Thr Asn Ser Pro
165 170 175
Glu Phe Asn Trp His Leu Gln Asn Leu Arg Gln Tyr Ile Gly Leu Lys
180 185 190
Ser Gln Pro Phe Ala Pro Thr Glu Trp Ser Asn Leu Ser Leu Ser Ala
195 200 205
Phe Gly Gln Gly Ser Gly Ser Met Gly Leu Pro Gly Asp Phe Thr Pro
210 215 220
Pro Ser Arg Phe Val Arg Ala Ala Tyr Gly Lys Gln Asn Ile Gln Gly
225 230 235 240
Ile Asp Ser Glu Glu Glu Gly Val Thr Ala Leu Phe His Ile Leu Ser
245 250 255
Asn Cys Glu Val Pro Lys Gly Gly Val Ile Thr Glu Glu Gly Ala Leu
260 265 270
Asp Asn Thr Ile Tyr Thr Ser Val Met Cys Met Glu Ser Gly Thr Tyr
275 280 285
Tyr Tyr His Thr Tyr Asp Cys Arg Gln Ile Ile Ala Ile His Leu Phe
290 295 300
His Glu Asn Leu Asp Thr Asp Glu Ile Lys Ala Tyr Pro Phe Gln Arg
305 310 315 320
Lys Gln Lys Ile Phe Tyr Glu Asn
325
<210> 3
<211> 37
<212> DNA
<213> Artificial Synthesis (Artificial Synthesis)
<400> 3
tagcccggat ccgatgtgta ctagtttgac attagag 37
<210> 4
<211> 39
<212> DNA
<213> Artificial Synthesis (Artificial Synthesis)
<400> 4
tccgtctcga ggttctcata aaatattttt tgttttcgt 39

Claims (3)

1. Hydrolase gene capable of degrading pesticide containing ester bondstrBThe application of degrading pesticide containing ester bond is characterized in that the pesticide containing ester bond exists in crops, agricultural products, soil and water bodies, and the hydrolase geneOf strBThe nucleotide sequence is shown as SEQ ID NO. 1.
2. Use according to claim 1, characterized in that the hydrolase enzymeOf StrBAmino acidsThe sequence is shown as SEQ ID NO. 2.
3. Use according to claim 1, characterized in that the hydrolase genestrBRemoving pesticide residues containing ester bonds in crops, agricultural products, soil and water.
CN201910869982.0A 2019-09-12 2019-09-12 Hydrolase gene strB and protein coded by same and application Active CN110592118B (en)

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