CN113502340A - Novel tet34 resistance gene of tetracycline and application thereof - Google Patents
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- 235000019364 tetracycline Nutrition 0.000 title claims abstract description 51
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Abstract
The invention discloses a novel tet34 resistance gene of tetracycline and application thereof. The nucleotide sequence of the novel tetracycline tet34 resistance gene is shown as SEQ ID NO: 1 is shown. The potential tetracycline resistance gene tet34 discovered by the application supplements a database of tetracycline resistance genes, provides help for understanding the resistance mechanism of tetracycline, and also provides theoretical support for the subsequent research of tetracycline action targets.
Description
Technical Field
The invention relates to the technical field of environmental and biological detection, in particular to a novel tet34 resistance gene of tetracycline and application thereof.
Background
Tetracycline is a broad-spectrum antibiotic, and is combined with 30S ribosome and inhibits aminoacyl tRNA from entering receptor sites of ribosome complexes of mRNA, so that the synthesis of protein in a microorganism is finally inhibited, and the tetracycline has a good inhibiting effect on gram-positive bacteria and gram-negative bacteria, and is widely used as an additive in the treatment of bacterial infection and the animal husbandry to promote the growth of animals.
In recent years, with the use of large amounts of antibiotics, the formation of a large number of Antibiotic Resistance Genes (ARGs) and resistant strains is inevitably caused. The appearance of tetracycline resistant strains and resistance genes leads to a significant reduction in the therapeutic benefits of drugs, so that the clinical efficacy of tetracycline and even the entire tetracycline antibiotic family is reduced or even eliminated, which poses a serious threat to human and animal health. Therefore, the discovery of a novel tetracycline resistance gene is of great practical significance for clinical treatment as well as risk assessment. The identification method of the novel antibiotic resistance gene adopted at present is mainly based on pure culture and molecular biology means, and has the advantages of longer identification period, complex operation, lower efficiency and no contribution to large-scale detection.
Disclosure of Invention
In order to solve the problems in the background art, the invention aims to provide a novel tetracycline tet34 resistance gene and application thereof, and the applicant discovers a potential tetracycline resistance gene tet34 based on the Nanopore and Illumina sequencing technology and performs functional verification through heterologous expression, supplements a database of the tetracycline resistance gene, provides help for understanding the resistance mechanism of tetracycline, and provides theoretical support for the subsequent research of tetracycline action targets. The method for discovering the new gene is simple and easy to operate, can carry out prediction screening on the novel tetracycline resistance gene, and greatly shortens the detection period on the basis of ensuring the accuracy of the prediction result.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows: in one aspect, the invention provides a novel tetracycline tet34 resistance gene, the nucleotide sequence of which is shown in SEQ ID NO: 1 is shown.
In another aspect, the present invention provides a method for discovering a novel tet34 resistance gene for tetracycline, comprising the steps of:
(1) collecting strains;
(2) extracting genome DNA;
(3) sequencing the genome;
(4) assembling a genome;
(5) open reading frame prediction of genome;
(6) comparing the predicted open reading frame with a database;
(7) amplification of open reading frames and heterologous expression.
Further, the strain is a resistant strain Escherichia coli TET with tetracycline resistance.
Further, the collection of the bacterial strain in the step (1) comprises the following steps: the overnight-cultured bacterial solution was centrifuged at 6000-.
Further, the genome sequencing in step (3) employs the platforms of Nanopore PromethION and Illumina NovaSeq 6000.
Further, the genome assembly in the step (4) is to perform mixed assembly on sequencing data after sample quality control by adopting Unicycler software to obtain complete genome data of the strain;
preferably, the specific parameters of the Unicycler software are as follows: unicycler-1TET _1.fq-2TET _2.fq-l TET. nano. fq-o TET-t 46- -mode normal-min _ polarity _ size 2000; wherein TET _1.fq and TET _2.fq are paired-end data for Illlumina sequencing, and TET.
Further, the Open Reading Frame prediction of the genome in the step (5) is that Open Reading Frame (ORF) prediction is performed on the obtained complete genome data through prodigal software;
preferably, the specific parameters of the prodigal software are as follows: fa-m-o TET _ temp.txt-a TET _ ORF.aa-d TET _ ORF.fa; wherein TET.fa is a genome obtained by unicycler mix-assembly, and TET _ ORF.aa and TET _ ORF.fa are respectively an amino acid and a nucleic acid sequence of a predicted ORF.
Further, the step (6) of comparing the predicted open reading frame with the database comprises comparing the predicted ORF with the SARG database by using the DIAMOND software, and extracting a comparison result; and (3) comparing the ORF with lower comparison similarity with the SARGfam database by adopting HMMER software to obtain an ORF annotation result based on model comparison.
Further, the specific parameters of the DIAMOND software are as follows: diamond blastp-query TET _ orf.aa-db sarg.2.2. fasta-reads 46-outfmt 5-evalue 1 e-5-max-target-seqs 1-out TET _ SARG _ blastp _ out-query-cover 70; wherein TET _ ORF.aa is the predicted amino acid sequence of the ORF, SARG.2.2.fasta is the resistance gene database;
preferably, the specific parameters of the HMMER software are as follows: hmmscan-tblout hmm _ out.txt-cut _ ga sargfam hmm TET _ orf.aa; samgfam.hmm is the SARGfam database, TET _ orf.aa is the predicted amino acid sequence of the ORF, hmm _ out.txt is the result output file.
Further, the amplification and heterologous expression of the open reading frame in the step (7) are that a Primer pair for amplifying the ORF is designed by using Primer Premier 5.0 software as follows:
P1-F:5’-ATGAAACATACTGTAGAAG-3’SEQ ID NO：2
P1-R:5’-TTACTCGTCCAGCAGAATC-3’SEQ ID NO：3
performing target gene PCR amplification by taking the E.coli TET genomic DNA obtained in the step (2) as a template, performing heterologous expression on the target gene fragment E.coli BL21, and simultaneously determining the minimum inhibitory concentration of tetracycline to the heterologous expression strain E.coli BL 21.
In another aspect, the invention provides the use of a novel tetracycline tet34 resistance gene to supplement a database of tetracycline resistance genes.
The invention has the beneficial effects that: the application discovers a potential tetracycline resistance gene tet34, supplements a database of tetracycline resistance genes, provides help for understanding resistance mechanisms of tetracycline, and provides theoretical support for the subsequent research of tetracycline action targets. The gene discovery method obtains a complete bacterial genome map based on the Nanopore and Illumina sequencing technologies, has higher sequencing flux, longer reading length and higher accuracy, can self-calibrate data in the assembly process, has higher genome accuracy, compares the obtained genome map with the existing database, preliminarily screens corresponding resistance genes, further adopts an HMM model for scanning, further determines novel resistance genes, is simple, convenient and quick to operate, and greatly shortens the detection period.
Detailed Description
The following will describe in detail the implementation of the present invention and the analysis of the results with reference to examples in order to fully understand the characteristics and effects of the present invention. It should be noted that the described embodiments are illustrative, not limiting, and are not intended to limit the scope of the invention.
Example 1: extraction and sequencing of genomic DNA of tetracycline-resistant strains
Here, the resistant strain Escherichia coli TET having tetracycline resistance is exemplified. 2ml of overnight-cultured bacterial liquid was centrifuged at 6000rpm at 4 ℃ for 5 minutes, the supernatant was discarded and the precipitate was collected, the extraction of genomic DNA was completed using Bacteria DNAkit kit, the integrity and concentration of genomic DNA was examined by 1% agarose gel electrophoresis and a NanoDrop One ultramicro spectrophotometer (Thermo Fisher Scientific, USA), and the obtained genomic DNA was subsequently sent to Beijing Norgrass biogenic bioinformatics technologies, Inc. to be sequenced using Nanopore PromethION and Illumina NovaSeq 6000 platforms.
Example 2: assembly of the genome of a Strain and ORF prediction
Performing quality control on the sequencing data obtained in example 1 to obtain clean data, and then performing mixed assembly on the sequencing data of the sample by using Unicycler software to obtain complete genome data of the strain, wherein the specific parameters of the Unicycler software are as follows:
unicycler-1TET_1.fq-2TET_2.fq-l TET.nano.fq-o TET-t 46--mode normal–min_polish_size 2000
wherein TET _1.fq and TET _2.fq are paired-end data for Illlumina sequencing, and TET.
And performing Open Reading Frame (ORF) prediction on the complete genome data of the obtained strain by using a propigan software, wherein the specific parameters of the propigan software are as follows:
prodigal-i TET.fa-m-o TET_temp.txt-a TET_ORF.aa-d TET_ORF.fa
wherein TET.fa is a genome obtained by unicycler mix-assembly, and TET _ ORF.aa and TET _ ORF.fa are respectively an amino acid and a nucleic acid sequence of a predicted ORF.
Example 3: ORF to SARG database alignment
Alignment of the ORF amino acid sequence obtained in example 2 with the SARG database using DIAMOND software showed that the annotation of ORF516 resulted in a tetracycline resistance gene with 25.5% similarity to the reference gene sequence tet34 in the database.
Example 4: obtaining potential novel ARG through Hidden Markov Model (HMM) comparison
The ORF516 obtained in example 3 was defined as a potential novel gene, and further confirmed using the HMM model, and as can be seen from the alignment result of the HMM model, this ORF was also annotated as Tet34 gene, and thus this ORF was considered as a potential novel gene Tet34_ like.
Example 5: heterologous expression of the Gene of interest ORF516(tet34)
The specific Primer pairs for amplifying the ORF were designed using Primer Premier 5.0 software as follows:
P1-F:5’-ATGAAACATACTGTAGAAG-3’SEQ ID NO：2
P1-R:5’-TTACTCGTCCAGCAGAATC-3’SEQ ID NO：3
the genomic DNA obtained in example 1 was used as a template for PCR amplification of a target gene, and the total volume of the PCR amplification reaction system was 50. mu.l, specifically 2. mu.l each of 25. mu.l of 2 XPPreMix, 10. mu. M P1-F and P1-R, 1. mu.l of the DNA template, and ddH2O20 mu l; the reaction procedure is as follows: 5min at 95 ℃; 30 cycles of 95 ℃ for 30s, 60 ℃ for 30s, and 72 ℃ for 40 s; keeping the temperature at 72 ℃ for 10min and 16 ℃; detecting the PCR product by 1% agarose gel electrophoresis, purifying and recovering the target gene fragment by using a gel recovery kit, connecting the purified target gene fragment with a pEASY-T1 vector at 37 ℃ for 5 minutes, and then adding 5 mu l of the connection product into 50 mu l of competent cells (E.coli DH5 alpha) for 20 minutes in ice bath; heat shock is carried out for 30 seconds at the temperature of 42 ℃, ice bath is carried out for 2 minutes, 250 mul LB liquid culture medium is added, and the mixture is cultured for 1 hour at the temperature of 37 ℃ and the rpm of 150; sucking 80 μ l of the culture, spreading on LB plate containing ampicillin, sealing with sealing film, and culturing at 37 deg.C for overnight; single clones were selected using the universal primer pair M13F/M13RCarrying out colony PCR (polymerase chain reaction) verification on positive clones;
culturing the positive clone in LB liquid culture medium (containing 100mg/l ampicillin) at 37 ℃ for 16h at 200rpm, extracting plasmid (pEASY-T1-tet34) by using a plasmid extraction kit, carrying out double digestion on plasmids pEASY-T1-tet34 and pET28a by using restriction enzymes BamH I and Not I, respectively recovering digestion products of the plasmids pEASY-T1-tet34 and pET28a, then connecting the recovery products to construct a recombinant plasmid pET28a-tet34, and transforming the recombinant plasmid pET28a-tet34 into 50 mu l of competent cells (E.coli BL21) in ice bath for 20 min; heat shock is carried out for 30 seconds at the temperature of 42 ℃, ice bath is carried out for 2 minutes, 250 mul LB liquid culture medium is added, and the mixture is cultured for 1 hour at the temperature of 37 ℃ and the rpm of 150; sucking 80 μ l of the culture, spreading on LB plate containing ampicillin, sealing with sealing film, and culturing at 37 deg.C for overnight; single clones were picked and positive clones were verified using the specific primer pair P1-F/P1-R.
Example 6: determination of tetracycline resistance of recombinant Strain E.coli BL21(pET28a-tet34)
The clone E.coli BL21(pET28a) with an empty vector is used as a control, the minimum inhibitory concentration of tetracycline to the strain E.coli BL21(pET28a) and the recombinant strain E.coli BL21(pET28a-tet34) is determined by adopting a 2-fold dilution method, the result shows that the resistance of the recombinant strain to the tetracycline is higher than that of the strain E.coli BL21(pET28a), the tet34 can enable the strain to obtain certain tetracycline resistance, and the tet34 is a potential drug-resistant gene of the tetracycline
While the embodiments of the present invention have been described in detail, the present invention is not limited to the embodiments, and various changes can be made without departing from the spirit of the present invention within the knowledge of those skilled in the art. Furthermore, the embodiments of the present invention and the features of the embodiments may be combined with each other without conflict.
SEQUENCE LISTING
<110> Shenzhen International institute for graduate of Qinghua university
<120> novel tetracycline tet34 resistance gene and application thereof
<130> CP121010584C
<160> 3
<170> PatentIn version 3.3
<210> 1
<211> 537
<212> DNA
<213> Artificial sequence
<400> 1
atgaaacata ctgtagaagt aatgatcccc gaagcggaga ttaaagcgcg tatcgccgaa 60
ctgggtcgtc agattactga gcgttacaaa gacagcggca gcgatatggt gctggtgggt 120
ctgctgcgtg gctcatttat gtttatggcg gacctgtgcc gtgaagttca ggtatctcat 180
gaagtcgact ttatgaccgc ctccagctac ggtagcggca tgtccaccac ccgtgatgtg 240
aaaatcctca aagatctgga tgaagatatc cgtggcaagg acgtgctgat tgttgaagat 300
atcatcgact cggggaatac actgtcgaaa gtgcgtgaga tcttaagcct gcgcgaaccg 360
aagtcgctgg cgatttgtac gctgctggat aaaccgtccc gtcgtgaagt gaacgtcccg 420
gtagaattta tcggtttctc gatcccggat gagtttgtgg tgggttacgg cattgattac 480
gcacagcgtt accgtcatct gccgtatatc ggcaaagtga ttctgctgga cgagtaa 537
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<212> DNA
<213> Artificial sequence
<400> 2
atgaaacata ctgtagaag 19
<210> 3
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<212> DNA
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<400> 3
ttactcgtcc agcagaatc 19
Claims (10)
1. A novel tetracycline tet34 resistance gene, characterized in that its nucleotide sequence is as shown in SEQ ID NO: 1 is shown.
2. A method for discovering a novel tet34 resistance gene of tetracycline, which comprises the following steps:
(1) collecting strains;
(2) extracting genome DNA;
(3) sequencing the genome;
(4) assembling a genome;
(5) open reading frame prediction of genome;
(6) and (4) comparing the predicted open reading frame with a database.
3. The method for discovering the novel TET34 resistance gene to tetracycline according to claim 2, wherein the strain is Escherichia coli TET, a resistant strain with tetracycline resistance.
4. The method for discovering the novel tet34 resistance gene to tetracycline according to claim 2, wherein the collection of the strain in step (1) is: the overnight-cultured bacterial solution was centrifuged at 6000-.
5. The method for discovering the tetracycline novel tet34 resistance gene according to claim 2, wherein the genomic sequencing in step (3) uses the platform Nanopore PromethION and Illumina NovaSeq 6000.
6. The method for discovering the tetracycline novel tet34 resistance gene according to claim 5, wherein the genome assembly in step (4) is a mixed assembly of sequencing data after sample quality control by using Unicycler software to obtain complete genome data of the strain;
preferably, the specific parameters of the Unicycler software are as follows: unicycler-1TET _1.fq-2TET _2.fq-l TET. nano. fq-o TET-t 46- -mode normal-min _ polarity _ size 2000; wherein TET _1.fq and TET _2.fq are paired-end data for Illlumina sequencing, and TET.
7. The method for discovering the tetracycline novel tet34 resistance gene according to claim 6, wherein the Open Reading Frame prediction of genome in step (5) is that Open Reading Frame (ORF) prediction is performed on the obtained complete genome data by using prodigal software;
preferably, the specific parameters of the prodigal software are as follows: fa-m-o TET _ temp.txt-a TET _ ORF.aa-d TET _ ORF.fa; wherein TET.fa is a genome obtained by unicycler mix-assembly, and TET _ ORF.aa and TET _ ORF.fa are respectively an amino acid and a nucleic acid sequence of a predicted ORF.
8. The method for discovering a tetracycline novel tet34 resistance gene according to claim 7, wherein said aligning the predicted open reading frame to the database in step (6) comprises using DIAMOND software to align the predicted ORF to the SARG database and extracting the alignment; and (3) comparing the ORF with lower comparison similarity with the SARGfam database by adopting HMMER software to obtain an ORF annotation result based on model comparison.
9. The method for discovering a novel tetracycline tet34 resistance gene as in claim 8, wherein the specific parameters of the DIAMOND software are as follows: diamond blastp-query TET _ orf.aa-db sarg.2.2. fasta-reads 46-outfmt 5-evalue 1 e-5-max-target-seqs 1-out TET _ SARG _ blastp _ out-query-cover 70; wherein TET _ ORF.aa is the predicted amino acid sequence of the ORF, SARG.2.2.fasta is the resistance gene database;
preferably, the specific parameters of the HMMER software are as follows: hmmscan-tblout hmm _ out.txt-cut _ ga sargfam hmm TET _ orf.aa; samgfam.hmm is the SARGfam database, TET _ orf.aa is the predicted amino acid sequence of the ORF, hmm _ out.txt is the result output file.
10. Use of a novel tetracycline tet34 resistance gene to supplement a database of tetracycline resistance genes.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110042111A (en) * | 2019-04-12 | 2019-07-23 | 华南农业大学 | The gene and its albumen of a kind of tetracycline antibiotics that can degrade and application |
| CN112111502A (en) * | 2020-09-25 | 2020-12-22 | 清华大学深圳国际研究生院 | Novel resistance gene of chloramphenicol and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110042111A (en) * | 2019-04-12 | 2019-07-23 | 华南农业大学 | The gene and its albumen of a kind of tetracycline antibiotics that can degrade and application |
| CN112111502A (en) * | 2020-09-25 | 2020-12-22 | 清华大学深圳国际研究生院 | Novel resistance gene of chloramphenicol and application thereof |
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