CN116497041A - Structural optimization and application of eight genes related to 2,4-DNT biodegradation - Google Patents
Structural optimization and application of eight genes related to 2,4-DNT biodegradation Download PDFInfo
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- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 44
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- 238000005457 optimization Methods 0.000 title abstract description 3
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- DYSXLQBUUOPLBB-UHFFFAOYSA-N 2,3-dinitrotoluene Chemical compound CC1=CC=CC([N+]([O-])=O)=C1[N+]([O-])=O DYSXLQBUUOPLBB-UHFFFAOYSA-N 0.000 description 1
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
- C12N9/0073—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14) with NADH or NADPH as one donor, and incorporation of one atom of oxygen 1.14.13
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
- C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
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- C12Y114/13024—3-Hydroxybenzoate 6-monooxygenase (1.14.13.24)
Abstract
The invention discloses eight genes for completely degrading 2,4-DNT suitable for plant expression after optimization and application thereof. Each gene is controlled by an independent CaMV35S promoter and terminator, the nucleotide sequences of which are shown in SEQ ID No. 1-SEQ ID No. 8 respectively, and the amino acid sequences of the encoded proteins of which are shown in SEQ ID No. 9-SEQ ID No. 16 respectively. The optimized and synthesized 8 genes constitute a 2,4-DNT expression module, the 2,4-DNT expression module can be successfully expressed in plants, and the obtained engineering plants can completely degrade the 2,4-DNT into non-toxic substances.
Description
Technical Field
The invention belongs to the field of genetic engineering, and particularly relates to a sequence of eight genes related to 2,4-DNT biodegradation with optimized structure.
Background
The nitroaromatic pollutant dinitrotoluene (2, 4-dinitolene, 2, 4-DNT) is a precursor for producing explosive TNT and is also an important chemical product for producing pesticides, plastics and automobile safety airbags. The wide application of 2,4-DNT in industrial production causes unavoidable leakage into the environment, thereby causing environmental pollution. The conventional pollutant repairing method mainly comprises physical repairing and chemical repairing, and has the advantages of high cost, complex equipment and low repairing efficiency. The cost of phytoremediation is low, the soil can be remediated in situ, and the damage to the environment is small, so that the method has a wide application prospect. However, the efficiency of phytoremediation is limited by the lack of specific enzymes in the plant itself that can degrade 2,4-DNT.
The metabolic pathway derived from bacteria is transferred into plants by methods of synthetic biology and metabolic engineering, which can impart functions not originally possessed by plants. By using the method, engineering plants which can be suitable for repairing 2,4-DNT can be constructed. Pathways capable of completely degrading 2,4-DNT have been found in microorganisms: 2,4-DNT dioxygenase (coding gene is dntAaAbAcAd) and 4-methyl-5-nitrophenol (4M 5 NC) monooxygenase (coding gene is dntB) are responsible for hydroxylation of aromatic ring and elimination of nitro substituent, respectively. 2,4, 5-THT oxygenase (coding gene dntD) and bifunctional isomerase/hydrolase (dntG coding) catalyze the production of methylmalonate trimer aldehyde (MAS) and pyruvate. Finally, coA (CoA) -dependent methylmalonate semialdehyde dehydrogenase (dntE encoded) catalyzes the production of propionyl CoA from methylmalonate trimer aldehyde (MAS).
Since five enzymes degrading 2,4-DNT in microorganisms can degrade 2,4-DNT into pyruvic acid and propionyl-CoA, which are non-toxic substances for plants. Therefore, the heterologous expression of the metabolic pathway in plants successfully realized has important significance and application potential for phytoremediation of 2,4-DNT.
Disclosure of Invention
The technical problem to be solved by the invention is to provide optimized recombination and application of eight related genes capable of completely degrading 2,4-DNT expressed in plants:
dntAaSgene: coding 2,4-DNT dioxygenase a subunit
dntAbSGene: coding for the b subunit of 2,4-DNT dioxygenase
dntAcSGene: coding 2,4-DNT dioxygenase gamma subunit
dntAdSGene: coding 2,4-DNT dioxygenase delta subunit
dntBSGene: encoding 4-methyl-5-nitrophenol (4M 5 NC) monooxygenase
dntDSGene: braiding machineCode 2,4, 5-THT oxygenase
dntGSGene: encoding a bifunctional isomerase/hydrolase
dntESGene: coding for methylmalonate semialdehyde dehydrogenase
The 2,4-DNT is subjected to five enzymatic reactions to produce pyruvic acid and propionyl-CoA. In order to achieve the above purpose, the invention is realized by adopting the following technical scheme:
the gene sequence in the invention is based onBurkholderiacepaciaIs a gene 8. The invention only maintains the coding sequence of each gene, and is respectively connected with an independent CaMV35S promoter and terminator to regulate and control the expression of the CaMV35S promoter. Meanwhile, the gene structure of the coding region is optimized, and the following principle is followed: and (one) optimizing the gene codon and improving the gene translation efficiency. And (II) eliminating recognition sites of common restriction enzymes inside genes, thereby facilitating expression cassette construction. And thirdly, eliminating an inverted repeat sequence, a stem-loop structure and a transcription termination signal, balancing GC/AT in the gene and improving the stability of RNA. And fourthly, the gene coding protein accords with the N-terminal principle so as to improve the stability of the translation protein. And (V) optimizing the free energy of the secondary structure of the mRNA to improve the gene expression efficiency.
The nucleotide sequences of the eight genes related to the optimized 2,4-DNT degradation are shown as SEQ ID No. 1 and SEQ ID No. 8. The CaMV35S promoter and terminator are respectively arranged at two ends of each gene, and two ends of the complete sequence are respectively connected with Sac I, xhoI, bamH I, sal I, hind III, ecoR I, xbaI, kpn I and Dpn I digestion sites, and the full-length sequence is synthesized by the division company of biological engineering (Shanghai) (see FIG. 1). The synthesized gene fragment is subjected to double digestion by Sac I, xhoI, bamH I, sal I, hindIII, ecoR I, xbaI, kpn I and Dpn I, and then is connected with a vector pCAMBIA1301 subjected to the same digestion to obtain a recombinant plasmid, and the recombinant plasmid is transformed into rice by an agrobacterium-mediated method to obtain a positive strain. The positive rice plants are planted in 100 ml of water culture solution, 20mg/L of 2,4-DNT is added, and after 10 days of growth, the positive engineering rice can completely degrade the 2,4-DNT.
The beneficial effects are that:
the invention optimizes and synthesizes eight genes related to 2,4-DNT degradation, can successfully express in rice, and positive rice plants can completely degrade 2,4-DNT in a culture medium. Has application potential in the fields of wastewater treatment, environmental remediation and the like.
Description of the drawings:
FIG. 1 is a schematic diagram of vectors for expressing eight genes associated with catechol degradation in E.coli.
FIG. 2 is a DNA detection electrophoresis diagram of engineering rice transformed with a 2,4-DNT degradation module.
FIG. 3 engineering Rice transformed into 2,4-DNT degradation Module grown in 2, 4-DNT-containing nutrient solution and High Performance Liquid Chromatography (HPLC) was used to detect the residual amount of 2,4-DNT in the nutrient solution
The specific embodiment is as follows:
the technical scheme of the present invention is described in detail below with reference to the accompanying drawings. The present invention is described in detail with reference to preferred embodiments, and it should be understood by those skilled in the art that the present invention may be modified or equivalent without departing from the spirit and scope of the present invention, which is within the scope of the appended claims.
The present invention relates to experiments in molecular biology, and is described in the "molecular cloning" in one book (J. Sam Broker, E.F. French, T. Mannich, 1994, scientific Press), unless otherwise noted. The rice seeds (Nippon) used in the present invention were stored in the institute of plant genetic engineering, the institute of Biotechnology, shanghai national academy of agricultural sciences, and the reagents used in the present invention were purchased from the stock of biological engineering (Shanghai) or Shanghai national pharmaceutical Co., ltd, unless otherwise specified.
Example 1
Optimized design and synthesis of eight genes related to 2,4-DNT degradation
Derived fromBurkholderiacepaciaEight genes of (2)dntAaS、dntAbS、dntAcS、dntAdS、 dntBS, dntDS, dntGS and dntESAccording to the following principle: optimizing gene codon and considering the codon preference of plantThe gene translation efficiency is improved. And (II) eliminating recognition sites of common restriction enzymes inside genes, thereby facilitating expression cassette construction. And thirdly, eliminating an inverted repeat sequence, a stem-loop structure and a transcription termination signal, balancing GC/AT in the gene and improving the stability of RNA. And fourthly, the gene coding protein accords with the N-terminal principle so as to improve the stability of the translation protein. And (V) optimizing the free energy of the secondary structure of the mRNA to improve the gene expression efficiency. The CaMV35S promoter and terminator are respectively arranged at two ends of each gene, and two ends of the complete sequence are respectively connectedSac I, XhoI, BamH I, Sal I, Hind III, EcoR I, XbaI, Kpn I, Dpn IThe cleavage site and the full-length sequence were synthesized by the company Shanghai, inc.
Example 2
Construction of plant expression vectors
Subjecting the synthesized gene fragment toSac I, XhoI, BamH I, Sal I, Hind III, EcoR I, XbaI, Kpn I, Dpn IAfter cleavage, the same cleaved vector CAMBIA1301 was ligated to give recombinant plasmid pYR6535 (see fig. 1).
Example 3 Agrobacterium-mediated transformation of Rice
1) Preparation of Agrobacterium
The vector containing the target gene is transferred into agrobacterium EHA105 by electric shock method. Re-culturing the target Agrobacterium to OD 600 At 0.3-0.5, an Agrobacterium suspension of co-cultured transformed rice was prepared.
2) Genetic transformation of rice
Sterilizing rice seeds by 0.1% mercury, placing the rice seeds on a meeting induction medium, and performing subculture to generate light yellow callus. Co-culturing the callus and agrobacterium for 2-3 days, placing the co-cultured callus and agrobacterium into a screening culture medium for dark culture for 14 days, then placing the co-cultured callus and agrobacterium into a differentiation culture medium for differentiation to obtain rice seedlings, and subsequently rooting, strengthening and transplanting to obtain mature rice.
Example 4 detection of transgenic Positive plants
Rice DNTs were extracted and the expression of each gene was detected according to the following PCR reaction system.
PCR reaction system:
10 PCR buffer 5.0 μL
dNTPS 4 μL（2.5m mol/L）
template 1. Mu.L (20 ng-50 ng)
Primer 1 1. Mu.L
Primer 2 1. Mu.L
Taq enzyme 0.2. Mu.L
Sterile water was added to a volume of 50 μl.
The reaction procedure: 94 ℃ for 5min;94℃20S,56℃30S,72℃30S;94 ℃ for 10 min;32 cycles. The detection results are shown in FIG. 2.
EXAMPLE 5 engineering Rice degradation of 2,4-DNT
To verify whether the engineered rice was able to degrade 2,4-DNT, the present application seeds engineered rice and wild-type rice in MS dishes containing 2,4-DNT, and after 10 days of growth, the 2,4-DNT content was measured by HPLC, and the results are shown in FIG. 3. The rotation can be seen from fig. 3PtPRXThe root system of the arabidopsis thaliana is obviously better than that of the wild type arabidopsis thaliana, while in fig. 3, a large amount of 2,4-DNT still remains in the culture medium for growing the wild type rice, and the 2,4-DNT in the culture medium for growing the engineering rice is completely degraded (see fig. 3).
Claims (4)
1. The artificially optimized 2,4-DNT uses related eight genes, and is characterized in that the nucleotide sequences of the eight genes are respectively shown as SEQ ID No. 1-SEQ ID No. 8.
2. The eight gene nucleotide sequence of claim 1, wherein the encoded protein has the amino acid sequence shown in SEQ ID No 9-SEQ ID No 16.
3. The eight gene nucleotide sequence construction plant expression vector pYR6535 according to claim 1, wherein each gene sequence is linked to a CaMV35S promoter and terminator to form an expression unit, and 8 expression units are inserted into the plant expression vector in series.
4. The plant expression vector of claim 3, wherein the engineered rice completely degrades 2,4-DNT to non-toxic substances after transformation of the rice with the vector.
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