CN114958802A

CN114958802A - Hydrolase from bacillus elongatus and method for repairing atrazine pollution of soil by using hydrolase

Info

Publication number: CN114958802A
Application number: CN202210273436.2A
Authority: CN
Inventors: 宋科; 薛永; 秦秦; 孙丽娟; 孙雅菲; 吕卫光; 郑宪清
Original assignee: Shanghai Academy of Agricultural Sciences
Current assignee: Shanghai Academy of Agricultural Sciences
Priority date: 2022-03-18
Filing date: 2022-03-18
Publication date: 2022-08-30

Abstract

The invention discloses a hydrolase from bacillus oblong and a method for repairing atrazine polluted soil. The TRZN hydrolase derived from the bacillus oblong is a triazine hydrolase, the coding sequence contains 1371 nucleotides, and the TRZN hydrolase has a wide substrate range. The invention constructs the atrazine harm resistance gene mutant by modifying the DNA sequence of the coding TRZN, transfers the recombinant gene into an arabidopsis thaliana plant, evaluates the atrazine harm resistance tolerance of the transgenic plant, and finally proves that the transgenic plant has higher atrazine resistance through a comparison test with a wild plant. The method not only greatly improves the efficiency of restoring the atrazine pesticide polluted soil by the transgenic organisms, but also can screen and improve higher plants resisting the harm of atrazine pesticides.

Description

Hydrolase from bacillus elongatus and method for repairing atrazine pollution of soil by using hydrolase

Technical Field

The invention relates to the technical field of hydrolase, in particular to hydrolase derived from bacillus elongatus and a method for repairing atrazine pollution of soil by using the hydrolase.

Background

The pesticide pollution refers to a pollution phenomenon formed by that trace pesticide protomer, toxic metabolites, degradation products and impurities which remain in organisms, agricultural and sideline products and the environment after the pesticide is used exceed the maximum residue limit of the pesticide. After the pesticide is applied, a part of the pesticide is attached to the plant body or permeates into the plant body to be remained, so that grains, vegetables, fruits and the like are polluted; the other part is scattered on the soil (sometimes directly applied to the soil) or evaporated and dissipated into the air, or flows into rivers and lakes along with rainwater and farmland drainage, polluting water bodies and aquatic organisms. The residual pesticide of agricultural products passes through the feed and pollutes the livestock products. Pesticide residues finally enter the human body through the atmosphere, water, soil and food, and various chronic or acute diseases are caused.

Atrazine is the most widely used herbicide pesticide at home and abroad at present, is a selective systemic conduction type herbicide for treating soil before germination, and can also be used for treating stems and leaves after germination. The photosynthesis of the plants is inhibited and the plants die by being absorbed and conducted upwards mainly by the roots of the plants. It has wide herbicidal spectrum and can prevent and kill several annual gramineous and broad-leaved weeds. The weeding composition is usually processed into wettable powder and a suspending agent for use, is suitable for preventing and killing weeds such as crabgrass, barnyard grass, green bristlegrass, nutgrass flatsedge, alopecurus, polygonum, chenopodium, cruciferae and leguminous weeds in corn, rice, fruit trees, nurseries and woodland, and also has a certain inhibition effect on certain perennial weeds. Atrazine was developed by Geigy chemical company in 1952, applied for swiss patent in 1958, and put into commercial production in 1959. The herbicide has excellent weed control effect and low price, is rapidly widely applied and popularized in various countries in the world, and becomes one of the most widely used and important herbicides in the world. At present, atrazine still plays an important role in weed control at home and abroad, and more than 80 countries in the world use the herbicide. In the middle of the united states, thousands of tons of this herbicide are used in corn fields each year, accounting for 60% of the herbicide usage. Atrazine and its mixed herbicide are still the most important herbicide in farmland in China.

After the atrazine is sprayed on the surfaces of soil and crops, only a small part of atrazine falls on a target, the majority of atrazine enters the soil, and residues in the soil or sediments mainly enter surface water or underground water through surface runoff, leaching, wet sedimentation and other ways, so that the atrazine threatens the aquatic ecological environment and human drinking water sources. In addition, small amounts of atrazine may enter the atmosphere by volatilization and dust flotation, and return to the ground by dry and wet settling. Thus, it is global to the ecological environment. Atrazine remains in soil for a long time, and its activity as an endocrine disrupter may cause damage to animals, plants and humans. It has been demonstrated that it can last in soil for decades. This means that atrazine-sprayed farmlands may not be used for growing atrazine-sensitive crops for many years. In addition, atrazine-contaminated groundwater may flow into rivers and lakes, may harm aquatic plants and animals, and may contaminate drinking water. Many studies have shown that atrazine disrupts endostatin, leading to abnormal development in amphibians and fish. Atrazine has also been shown to lower testosterone levels in male rats, and there is concern that it will affect humans. Therefore, there is a need to establish methods and techniques for removing such toxins from the environment.

The method is characterized in that a biosynthesis technology is utilized to generate TRZN hydrolase from bacillus microsclerotia, a DNA sequence for coding the TRZN hydrolase is modified, a gene mutant type for resisting atrazine harm is constructed, a recombinant gene is transferred to an arabidopsis thaliana plant, and the tolerance of the transgenic plant for resisting atrazine harm is improved.

Disclosure of Invention

The technical problem to be solved by the invention is that a TRZN hydrolase is produced by bacillus microscler, a DNA sequence for coding TRZN is modified, a gene mutant for resisting atrazine harm is constructed, a recombinant gene is transferred into a plant body, and the tolerance of a transgenic plant for resisting atrazine harm is improved.

The invention aims to provide a hydrolase derived from bacillus microsclerotium, a TRZN hydrolase derived from bacillus microsclerotium production, a triazine hydrolase, and a coding sequence containing 1371 nucleotides, wherein the TRZN hydrolase derived from bacillus microsclerotium produces hydrolyzes and replaces a chlorine substituent from an s-triazine ring to trigger the bacterial metabolism of herbicide atrazine, and the TRZN hydrolase derived from bacillus microsclerotium produces a DNA sequence for modifying and encoding TRZN by artificial synthesis technology.

The invention also provides a method for remedying the atrazine pollution by the hydrolase derived from the bacillus elongatus as set forth in claim 1, which comprises the following steps:

step 1: verifying that transgenic agrobacterium microsclerotis is capable of producing TRZN, obtaining the DNA sequence of arthrobacter aureus TC1 triazine chlorohydrase from Genbank and modifying it with Genscript into escherichia coli while adding a histidine tag to aid in the detection and purification of recombinant proteins, then cloning this modified version into the s.elongatus expression vector psyn1/d-topo and converting it into the NS1 site of the s.elongatus genome by standard procedures, analyzing wild-type and transgenic bacillus microsclerotis for their ability to resist atrazine in contrast, verifying the protection of elongated s.elongatus cells from atrazine by TRZN enzyme by measuring photosynthesis;

and 2, step: modifying a DNA sequence encoding TRZN, based on TRZN which works well in bacterial cells, modifying its DNA sequence to function in plants, allowing plants to recognise where to start and stop replicating TRZN protein, adding control sequences from plant viruses, replicating from the rtl2 plasmid using the 35S promoter of cauliflower mosaic virus and ligating it directly in front of the start codon, establishing instructions for plant cells to start replicating protein, while, by adding a plant terminator, instructing plant cells to stop replicating TRZN, both promoter and terminator sequences will be added by standard recombinant DNA techniques, and the resulting recombinant DNA molecules will be transformed and analysed by standard procedures in e.coli;

and step 3: transferring the recombinant gene into an arabidopsis thaliana plant, constructing a correct structure through PCR and restrictive digestion, transferring the recombinant gene into a plasmid contained in bacillus microsclera gv3101, converting the bacillus microsclera into a plant cell, introducing the required plasmid into the arabidopsis thaliana plant through a flower soaking method, sowing the obtained transgenic seed on an agar plate containing 50mg/ml kanamycin for identification, performing PCR verification by using a primer for detecting transgenosis, and confirming that the transgenic plant produces TRZN protein through a standard program;

and 4, step 4: evaluating the capability of the transgenic plants to resist the atrazine toxicity, testing whether the atrazine influences the photosynthesis by comparing the photosynthetic rate of the transgenic plants and the wild plants in the presence of the atrazine, measuring the atrazine with the same concentration by using an oxygen electrode and a Licor LI-6400 photosynthesis monitor, and simultaneously comparing chlorophyll fluorescence parameters of the transgenic plants and the wild plants treated by the atrazine to evaluate the capability of the target plants and the control plants to resist the atrazine toxicity;

and 5: testing the atrazine degrading effectiveness of the transgenic plants, evaluating atrazine toxicity resistance results of the transgenic plants to indicate that the transgenic plants are protected by atrazine, further searching for hydroxyatrazine in the plants by thin layer chromatography and high performance liquid chromatography, and verifying that the extracts of the transgenic plants can convert the atrazine into the hydroxyatrazine in vitro;

step 6: the resistance of the transgenic arabidopsis thaliana plant is verified, then the two plant types are transplanted into soil containing 0.5mg/kg atrazine through tolerance comparison experiments of the wild type plant and the transgenic plant, and the structure after 2 weeks shows that compared with the transgenic plant, the wild type plant is obviously inhibited, the plant height and the biomass of the overground part are obviously reduced, the root system does not grow, the number of leaves is reduced, the transgenic plant is inhibited less, and the result shows that the TRZN enzyme obviously improves the tolerance of the arabidopsis thaliana plant to the atrazine poison.

Compared with the prior art, the invention has the beneficial effects that: the invention establishes a transgenic arabidopsis thaliana plant for expressing TRZN enzyme, which is used for phytoremediation of atrazine pesticide polluted soil. According to the invention, on the basis of verifying that bacillus microsclera can generate TRZN hydrolase, a DNA sequence for coding TRZN is modified, a gene mutant type for resisting atrazine harm is constructed, a recombinant gene is transferred into an arabidopsis thaliana plant, the tolerance of the transgenic plant for resisting atrazine harm is evaluated, and finally the effectiveness of the transgenic plant for degrading atrazine is tested in the aspects of growth characteristics and photosynthetic capacity through a comparison test with a wild plant. The invention is not only an innovative achievement of utilizing transgenic organisms to repair pesticide-contaminated soil, but also can screen out higher plants capable of resisting the harm of atrazine pesticides.

Drawings

FIG. 1 is a TRZN nucleotide sequence diagram;

FIG. 2 is a diagram of the encoded amino acid sequence;

FIG. 3 is a structural diagram of TrzN hydrolase;

FIG. 4 is a diagram of atrazine metabolic pathway triggered by TrzN hydrolase;

FIG. 5 is a comparison of wild type and transgenic plants.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

Example 1

The technical scheme of the invention is described in detail in the following with reference to the accompanying drawings. It should be noted that a person skilled in the art may make several modifications and additions without departing from the method of the invention, and such modifications and additions should also be considered as a protection scope of the invention. Those skilled in the art can make various changes, modifications and equivalent arrangements, which are equivalent to the embodiments of the present invention, without departing from the spirit and scope of the present invention, and which may be made by utilizing the techniques disclosed above; meanwhile, any changes, modifications and variations of the above-described embodiments, which are equivalent to those of the technical spirit of the present invention, are within the scope of the technical solution of the present invention.

The test materials and sources used in the present invention include:

wild type Arabidopsis thaliana (Arabidopsis thaliana) ecological type Arabidopsis thaliana Columbia, cultivated in a climatic chamber at 23 ℃ and cultivated in 16h of illumination.

The prtl2 vector of the cauliflower mosaic virus (camv), ge restriction endonuclease, Taq polymerase, ligase, dNTP, PCR buffer and DNA marker related by the invention are all purchased from biological engineering Dalian company Limited.

All chemical reagents used in the present invention were purchased from Shanghai national Chemicals, Inc.

The specific embodiment of the invention is as follows:

(1) obtaining gene resource, namely obtaining a triazine chloride hydrolase (TRZN) gene sequence of TC1 from bacillus microsclerotia extracted from soil. The Genscript is used for modifying the TRZN gene sequence, so that the TRZN gene sequence can be optimally expressed in escherichia coli and arabidopsis thaliana.

(2) Preparing plant promoters and terminators: the 35S promoter was obtained from a prtl2 vector containing one cauliflower mosaic virus (camv), allowing it to express TRZN in all plant tissues. Cytoplasmic targeting TRZN inserts were prepared and validation of cytoplasmic targeting TRZN inserts was performed.

(3) Preparation of organelle targeting TRZN inserts: TRZN is targeted to mitochondria, chloroplast and peroxisome transit peptide sequences are localized, and linked to the TRZN gene to target its expression to the proposed organelle. For chloroplast and mitochondrial transit peptides, bioinformatics methods were used to identify and locate the sequence of col 0a.

(4) Obtaining a transit peptide sequence: genomic DNA containing mitochondrial and chloroplast transit peptides and 3 'and 5' flanking sequences of each transit peptide sequence was amplified with external primers, chloroplast transit peptides were rbcs-external 5 'and rbcs-external 3' amplified with external primers, and mitotic cells were amplified with rpoy-external 5 'and rpoy-external 3'.

(5) Isolation of mitochondrial and chloroplast transit peptide sequences to isolate mitochondrial and chloroplast transit peptide sequences and to add appropriate cloning restriction sites, a q5 high fidelity DNA polymerase (neb) reaction was performed using chloroplast and mitochondrial transit peptide DNA with 500bp flanking sequences as template, using appropriate internal primers (rpoy interna).

(6) Clone zero Blunt TOPO vector: the Topo cloning reaction was performed according to the Zero Blunt Topo PCR cloning kit (Thermo Fisher) procedure, colonies were selected from the mitochondrial transit peptide Blunt II-Topo vector transformation plates for colony PCR, colony PCR was performed with Onetaq polymerase (neb) and colonies with the correct insert were identified.

(7) Validation of mitochondrial and chloroplast transit peptides with TRZN: colonies were selected for colony PCR from the mitochondrial transit peptide + pet15b + c6h of TRZN, the mitochondrial transit peptide + pet15b + n6h of TRZN, the chloroplast transit peptide pet15b + c6h of TRZN, and the chloroplast transit peptide + pet15b + n6h of TRZN transformants.

(8) The transition peptide + TRZN was cloned into prtl2 and verified: transformation agents were selected on LB ampicillin (100. mu.g/ml) plates and colony PCR was performed on colonies on each transformation plate using Onetaq polymerase (neb). Colonies with the correct insert were identified by peroxisome transit peptides and the presence of mitochondrial transit peptides.

(9) Cloning and verification of pORE-02: obtaining a plant transformation vector from an arabidopsis biological resource center, carrying out Onecaq colony PCR on colonies selected from each transformation plate, and cloning and verifying pORE-02 by using a PCR primer 35S promoter and a reverse transcription TRZN-qpcr.

(10) Cloning Syn _1/d-Topo vector cloning the purified PCR product into psyn _1/d-Topo using the procedure described in Genart-Syn-Topo cloning Manual (ThermoFisher), performing a topological cloning reaction using the vector and the PCR product to prepare a TRZN insert, and optimizing its expression in E.coli.

(11) Validation of optimized TRZN Positive colonies were selected and incubated overnight in tuberculosis broth with spectinomycin (100. mu.g/ml). DNA was extracted by alkaline hydrolysis, and PCR was performed using Onetaq polymerase and primers to confirm the presence of E.coli-optimized TRZN in syn _1/d-topo vector and to obtain recombinant gene.

(12) Transferring the recombinant gene into an arabidopsis plant, namely transferring the recombinant gene into a plasmid contained in the bacillus elongatus gv3101, and converting the bacillus elongatus into a plant cell. The desired plasmid was introduced into Arabidopsis plants by the floral dip method, and the obtained transgenic seeds were sown on agar plates containing 50mg/ml kanamycin for identification and verified by PCR using primers for detecting transgenes, confirming that the transgenic plants produced TRZN protein by standard procedures.

(13) Evaluation of tolerance of transgenic plants to atrazine: the ability of the target plants to resist atrazine toxicity was evaluated against the control plants by comparing the photosynthetic rates of the transgenic plants to the wild plants at absorbances of 750 nm and 665 nm for cell growth and chlorophyll a content under standard conditions, while comparing the chlorophyll fluorescence parameters of the atrazine-treated transgenic plants and the wild type plants.

(14) And testing the atrazine degrading effectiveness of the transgenic plants, namely searching for atrazine in the plants by a thin-layer chromatography detection method and a high performance liquid chromatography method, testing whether the atrazine can be converted into atrazine in vitro by the transgenic plant extracts, and evaluating the atrazine degrading effectiveness of the target plants.

(15) Finally, a tolerance comparison experiment of wild type and transgenic arabidopsis thaliana plants is carried out, the two plant types are transplanted into soil containing 0.5mg/kg atrazine, and the structure after 2 weeks shows that compared with the transgenic plant (figure 5), the wild type is obviously inhibited, the plant height and the biomass of the overground part are obviously reduced, the root system does not grow, the number of leaves is reduced, and the transgenic plant is less inhibited. The result shows that the TRZN enzyme obviously improves the tolerance of arabidopsis thaliana plants to atrazine poison.

The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.

Claims

1. A hydrolase derived from Bacillus elongatus, characterized in that: the TRZN hydrolase produced from the bacillus lentus is a triazine hydrolase, the coding sequence contains 1371 nucleotides, and the TRZN hydrolase produced from the bacillus lentus hydrolyzes and replaces a chlorine substituent from an s-triazine ring to trigger the bacterial metabolism of herbicide atrazine.

2. A method for remediating atrazine contaminated soil by using a Bacillus lentus-derived hydrolase according to claim 1, wherein the method comprises the steps of: the method comprises the following steps:

step 2: modifying a DNA sequence encoding TRZN, based on TRZN which works well in bacterial cells, modifying its DNA sequence to function in plants, allowing plants to identify where to start and stop replicating TRZN protein, adding control sequences from plant viruses, replicating from the rtl2 plasmid using the 35S promoter of cauliflower mosaic virus and ligating it directly in front of the start codon, establishing instructions for plant cells to start replicating protein, while, by adding a plant terminator, instructing plant cells to stop replicating TRZN, both promoter and terminator sequences will be added by standard recombinant DNA techniques, and the recombinant DNA molecules thus produced will be transformed and analysed in e.coli by standard procedures;

and 4, step 4: evaluating the ability of the transgenic plants to resist the atrazine toxicity, testing whether the atrazine affects the photosynthesis by comparing the photosynthetic rate of the transgenic plants and the wild plants in the presence of the atrazine, measuring the atrazine with the same concentration by using an oxygen electrode and a Licor LI-6400 photosynthesis monitor, and simultaneously comparing chlorophyll fluorescence parameters of the transgenic plants and the wild plants treated by the atrazine to evaluate the ability of the target plants and the control plants to resist the atrazine toxicity;

step 6: the resistance of the transgenic arabidopsis thaliana plant is verified, then the two plant types are transplanted into soil containing 0.5mg/kg atrazine through tolerance comparison experiments of the wild type plant and the transgenic plant, and the structure after 2 weeks shows that compared with the transgenic plant, the wild type is obviously inhibited, the plant height and the biomass of the overground part are obviously reduced, the root system does not grow, the number of leaves is reduced, the transgenic plant is less inhibited, and the result shows that the TRZN enzyme obviously improves the tolerance of the arabidopsis thaliana plant to atrazine poison.