CN114517197A

CN114517197A - Escherichia coli sRNA120, DNA molecule, recombinant vector and application of recombinant vector in regulation and control of bacterial drug resistance

Info

Publication number: CN114517197A
Application number: CN202210142371.8A
Authority: CN
Inventors: 庞新跃; 古绍彬; 吴影; 李欣; 赵守静; 赵丽娜; 马金亮; 王冰
Original assignee: Henan University of Science and Technology
Current assignee: Henan University of Science and Technology
Priority date: 2022-02-16
Filing date: 2022-02-16
Publication date: 2022-05-20
Anticipated expiration: 2042-02-16
Also published as: CN114517197B

Abstract

The invention belongs to the field of non-coding nucleic acid for regulating gene expression, and particularly relates to escherichia coli sRNA120, a DNA molecule, a recombinant vector and application of the escherichia coli sRNA120 in regulation and control of bacterial drug resistance. The nucleotide sequence of the escherichia coli sRNA120 is shown in SEQ id no: 1 is shown. Experiments prove that the drug resistance of the sRNA120 overexpression transgenic escherichia coli is obviously enhanced. Based on the important correlation between sRNA120 and the resistance of Escherichia coli to antibiotic damage, a strain sample can be provided for the subsequent research of a bacterial drug resistance mechanism, and an important reference basis can be provided for sRNA120 as a target point for improving bacterial stress resistance, a diagnostic marker, a drug target point and the like.

Description

Escherichia coli sRNA120, DNA molecule, recombinant vector and application of recombinant vector in regulation and control of bacterial drug resistance

Technical Field

The invention belongs to the field of non-coding nucleic acid for regulating gene expression, and particularly relates to escherichia coli sRNA120, a DNA molecule, a recombinant vector and application of the escherichia coli sRNA120 in regulation and control of bacterial drug resistance.

Background

sRNA is a newly discovered gene expression regulatory factor in recent years, and is widely present in bacteria, archaea and eukaryotes. Initially, sRNA research was mainly focused on eukaryotes, and gradually transferred to pathogenic bacteria in recent years. Research shows that sRNA can regulate and control the expression of related genes of an efflux pump when bacteria are stressed by antibiotics, so that the antibiotics are prevented from entering cells or medicines are eliminated from the cells, and thus, the sRNA plays an important regulation and control role. In addition, sRNA can be involved in a variety of biological processes such as substance metabolism and protein synthesis in a post-transcriptional regulatory manner. RNA was first discovered in 1967 by labeling all of the RNA in E.coli and detecting a larger number of new RNA molecules on SDS-PAGE. In 1984, Mizuno et al found the first non-coding sRNA MicF as a gene fragment, and when sRNA MicF was overexpressed, MicF increased in transcript level and the OmpF mRNA transcript decreased, probably because MicF prematurely terminated or stabilized OmpF transcription.

Based on the differences in the functions performed by sRNA, bacterial sRNA can be divided into 3 types, functional sRNA, protein-bound sRNA, and sRNA regulation is mainly used in sRNA discovery at present. Functional sRNA, mainly comprising sRNA with enzymatic activity and transfer messenger tmRNA; the protein is combined with sRNA, can be specifically combined with protein, and regulates the activity of the protein; sRNA is regulated, binds to target mRNA in pairs, and functions by affecting its translation efficiency and stability. Based on the difference of genome positions, the sRNA can be divided into 2 types, namely cis-encoding regulatory sRNA and trans-encoding regulatory sRNA, wherein the cis-encoding regulatory sRNA exists in the protein coding gene and plays a role mainly by strictly matching and combining target mRNA (messenger ribonucleic acid) complementary with DNA of the cis-encoding regulatory sRNA; sRNA in bacteria is mainly encoded and regulated in trans, and is present in gaps between protein-encoding genes, and mRNA of the sRNA is located at a far end and is only partially complementary. Furthermore, because sRNA is not typically translated into protein or polypeptide, less energy and time is required for its synthesis and turnover.

RNA has long been considered to mediate the genetic code of DNA, and with the increasing research on RNA, sRNA has not been demonstrated to have the ability to regulate expression of the coding gene until recently. The sRNA participating in regulation of bacteria under environmental stress is researched, a genetic regulation mechanism of the sRNA under environmental stress (such as antibiotic environment) is determined, and an important reference basis is provided for the sRNA as a target point for improving the stress resistance of the bacteria, a diagnostic marker, a drug target point and the like.

Disclosure of Invention

The object of the present invention is to provide an escherichia coli sRNA120, which is associated with the drug resistance of bacteria, and can change the drug resistance of bacteria by regulating the expression level thereof.

It is a second object of the present invention to provide a DNA molecule that transcribes the above-described E.coli sRNA 120.

The third object of the present invention is to provide a recombinant vector containing the above DNA molecule.

A fourth object of the present invention is to provide the use of the above-described Escherichia coli sRNA120 for regulating the drug resistance of bacteria.

In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:

escherichia coli sRNA120 having a nucleotide sequence as set forth in SEQ ID NO: 1 is shown.

Experiments prove that the drug resistance of the sRNA120 overexpression transgenic escherichia coli is obviously enhanced. Based on the important correlation between sRNA120 and the resistance of Escherichia coli to antibiotic damage, a strain sample can be provided for the subsequent research of a bacterial drug resistance mechanism, and an important reference basis can be provided for sRNA120 as a target point for improving bacterial stress resistance, a diagnostic marker, a drug target point and the like.

The DNA molecule that transcribes the escherichia coli sRNA120 has the nucleotide sequence shown in SEQ ID NO: 2, respectively.

The DNA molecule can be obtained by high-efficiency transcription by using the DNA molecule.

A recombinant vector comprising the above DNA molecule. Preferably, the recombinant vector is an escherichia coli sRNA120 overexpression vector.

The recombinant vector containing the DNA molecule can be used for transforming into bacteria, realizing the over-expression of the Escherichia coli sRNA120 and constructing an over-expression strain.

In order to facilitate the construction of the above recombinant vector, preferably, the starting vector of the recombinant vector is peT28a (+).

The use of escherichia coli sRNA120 for modulating the drug resistance of a bacterium by altering the expression level of said escherichia coli sRNA120 in said bacterium, thereby altering the drug resistance of the bacterium.

The above-described properties of sRNA120 can be used to control the drug resistance of bacteria, thereby enabling the construction of drug-resistant strains, or for the treatment of infection by drug-resistant strains of E.coli.

Preferably, the drug resistance of the bacterium is increased by increasing the expression level of the escherichia coli sRNA 120.

Preferably, bacterial resistance to high expression of e.coli sRNA120 is enhanced, and the bacterial resistance is reduced by reducing the expression level of e.coli sRNA 120.

Preferably, the bacterium is escherichia coli K12 strain and derivatives thereof. More preferably, the escherichia coli K12 strain is escherichia coli MG 1655.

Preferably, the resistance is antibiotic resistance and the antibiotic is imipenem or gentamicin.

Drawings

Fig. 1 is a secondary stem-loop structure of sRNA120 according to embodiment 1 of the present invention;

FIG. 2 is a schematic diagram of the construction of peT28a-SRNA120 expression vector provided in example 3 of the present invention;

FIG. 3 is a schematic diagram of the construction of ptrC99a-MIM120 interference vector provided in example 3 of the present invention;

fig. 4 is a schematic diagram of a MIM sequence provided in example 3 of the present invention;

FIG. 5 is a result of detecting sRNA120 expression in transgenic E.coli by qRT-PCR according to example 4 of the present invention; wherein, L1-L4 in the A picture is the detection result of the expression level of sRNA120 in an overexpression strain; in the B picture, L1-L4 are the detection results of the expression level of the MIM interference strain sRNA 120;

FIG. 6 shows the molecular detection results of transgenic E.coli provided in example 3 of the present invention; wherein, A is a PCR detection electrophoresis picture (aiming at sRNA120 fragment) of a part of over-expression strain, and M is DNA Marker: Trans 2K; WT was wild-type E.coli MG1655 strain as positive control, H ₂O as a blank control; b is a PCR detection electrophoretogram (aiming at MIM120 fragments) of partial interference strains, wherein M is DNA Marker: Trans 2K; WT was wild type E.coli MG1655 strain as positive control, H₂O as a blank control;

FIG. 7 shows the results of the drug resistance analysis of E.coli overexpressing and interfering with the transgene provided in example 5 of the present invention; wherein IP is imipenem and GENT is gentamicin.

Detailed Description

The following describes the practice of the present invention in detail with reference to specific examples.

Example 1 E.coli sRNA120 and DNA molecules that transcribe E.coli sRNA120

The mature sRNA sequence of escherichia coli sRNA120 of the present example is set forth in SEQ ID NO: 1, and a secondary stem-loop structure thereof is shown in fig. 1, and a gene sequence of escherichia coli sRNA120 is shown in SEQ ID NO: 2, respectively.

Example 2 cloning of the Gene sRNA120

Designing primers for cloning sRNA120 genes, wherein the nucleotide sequences of an upstream primer and a downstream primer are respectively shown as SEQ ID NO: 3. SEQ ID NO: 4, respectively. The 5 'end of the upstream primer is introduced with an EcoRI enzyme cutting site sequence, and the 3' end of the downstream primer is introduced with a HindIII enzyme cutting site sequence. PCR amplification was performed using E.coli genomic DNA as a template.

The PCR amplification conditions were as follows: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30 s; annealing at 58 ℃ for 30 s; extension at 72 ℃ for 20 s; number of amplification cycles 32; finally, extending for 10min at 72 ℃; storing at 4 ℃.

The PCR product was electrophoresed in 1.4% agarose gel, and the band of interest was recovered using a small agarose gel DNA recovery kit. Taking a proper amount of recovered products, and connecting the recovered products with a TA vector to transform the competence of escherichia coli. After colony PCR identification, positive colony plasmids are extracted and sent to a company for sequencing.

EXAMPLE 3 construction of overexpression and interference expression vectors for E.coli

The specific construction process of the recombinant vector of this example is as follows:

3.1 overexpression vectors

The expression of the target gene SRNA120 in the overexpression vector is composed of lac promoter and 3' termination region of T7, and the selection marker gene is lac gene. As shown in FIG. 2, the SRNA120 gene was cleaved from the plasmid cloning vector with EcoRI and Hind III to obtain the target fragment with cleavage sites, and the target fragment was ligated to peT28a (+) vector backbone with T4DNA ligase to form the recombinant plasmid peT28a-sRNA 120.

3.2 construction of interfering expression vectors

Meanwhile, by utilizing the reported MIM (target mix) technology, an additional 4 bases are inserted between the 8 th base and the 9 th base to reduce the expression of microRNA (as shown in figure 4, the MIM120 sequence is shown in SEQ ID NO. 5), and an interference vector consisting of a trc start 8 XMIM (Shanghai Kirsiy synthesis) sequence and a 3' transcription termination region of rrnB T2 is constructed, wherein the selective marker gene is lacI gene. The vector is schematically shown in FIG. 3, and the synthetic 8 XMIM 120 is cut by EcoRI and Hind III to obtain a MIM120 fragment with a cutting site, and the target fragment is connected to ptrC99a vector skeleton by T4DNA ligase to form the recombinant plasmid ptrC99a-MIM 120.

The plasmid peT28a-sRNA120 is directly transferred into an Escherichia coli MG1655 strain by an electric excitation method to obtain an Escherichia coli MG1655+ peT28a-sRNA120 overexpression strain (overexpression strain), and then the plasmid ptrC99a-MIM120 is transferred into the Escherichia coli MG1655+ peT28a-sRNA120 strain by the electric excitation method to obtain a cotransformation strain of the Escherichia coli MG1655+ peT28a-sRNA120+ ptrC99a-MIM120 (MIM interference strain).

The results of the molecular assay for transgenic E.coli provided in example 3 are shown in FIG. 6.

The detection method comprises the following steps: transformed E.coli single colonies were picked up and cultured in 3mL of LB liquid medium (containing 75. mu.g/mL kanamycin) at 37 ℃ for about 12 hours with shaking at 180 rpm. 0.5 μ L of the bacterial solution was used as a template for PCR amplification and detection of the product by agarose gel electrophoresis, and the conditions for PCR amplification were the same as in example 2.

Coli as a positive control, H₂O is a negative control. The results show that the positive control group and the overexpression SRNA120 transformation strain both amplify DNA fragments with the same size as the target fragment, which indicates that the transformation is successful (FIG. 6A); DNA fragments with the same size as the target fragment can be amplified in the MIM interfering strain, and the positive control group can not amplify corresponding fragments, which indicates that the construction of the interfering strain is successful. Can be used for subsequent experiments.

Example 4 extraction of Total RNA from transgenic E.coli and RT-PCR detection

4.1 extraction of Total RNA from bacteria

1. Taking a certain amount of bacterial cells, adding the bacterial cells into a centrifugal tube precooled by liquid nitrogen (RNase is removed in advance), adding a proper amount of quartz sand when precooled in advance according to the material condition, quickly grinding the bacterial cells into powder by a grinding bar when the liquid nitrogen amount is proper, and subpackaging the powder into a new 1.5mL RNase-free centrifugal tube with about 100-200mg per tube (the materials for extraction are stored in the liquid nitrogen for standby, and the rest materials are stored in a refrigerator at the temperature of-80 ℃);

2. taking out Trizol stored at 4 ℃, taking out the materials, placing the materials on ice, adding 1mL Trizol into each tube, uniformly mixing the materials by Vortex oscillation, and placing the mixture at room temperature for 5min to fully crack bacterial tissues;

3. adding 200 mu L of precooled chloroform into the centrifuge tube, violently oscillating for 15sec by Vortex, and standing for 5min at room temperature;

4. precooling in advance, centrifuging at 4 ℃ for 15min at 12,000 rpm;

5. carefully sucking the supernatant (about 600. mu.L) into a new RNase-free centrifuge tube, adding equal volume of pre-cooled isopropanol, slightly inverting and mixing, and standing at room temperature for 10 min;

6. centrifuging at 12,000rpm for 15min at 4 deg.C, and discarding the supernatant;

7. 1mL of precooled 70% ethanol (DEPC-ddH) was added₂O preparation) mixing, centrifuging at 12,000rpm for 5min at 4 deg.C, and discarding the supernatant;

8. Removing cover, blowing on ice for 5min to volatilize the rest liquid, adding 34.5 μ L DEPC-ddH₂Dissolving O;

9. taking a proper amount of RNA for electrophoresis detection, placing the RNA on ice for later use, and storing the rest in a refrigerator at the temperature of-80 ℃.

4.2 Synthesis of cDNA

The product was stored at-20 ℃ according to the instructions for the use of M-MLV reverse transcriptase from Takara.

4.3 real-time fluorescent quantitative PCR

The gapdh was used as the reference gene and the cDNA of E.coli samples as the template for real-time fluorescent quantitative PCR analysis. The reaction system is shown in table 1 below:

TABLE 1 RT-PCR reaction System

2×SYBR Premix Ex Taq	10μL
		Primer 1	0.3μL
Primer 2	0.3μL
		cDNA	0.5μL
ddH2O	8.9μL
		Total	20μL

The reaction procedure is as follows: 94 ℃ for 30 sec; 94 ℃ for 5 sec; 15sec at 60 ℃; 10sec at 72 ℃; the total number of cycles was 40 and the cells were stored at 4 ℃.

By using 2^-ΔΔTThe method carries out data analysis and determines the relative expression quantity of the genes. The experiment was repeated 3 times in total, and each sampling point was repeated 3 times. As shown in FIG. 5, the expression level of SRNA120 in the over-expression strain OEL1/2/3/4 is significantly higher than that of the wild type, and the expression level of SRNA120 in the MIML1/2/3/4 is significantly reduced compared with that of the wild type.

Example 5 transgenic E.coli resistance assay

The application of the escherichia coli sRNA120 in the aspect of regulating and controlling the drug resistance of bacteria, which is used for carrying out drug resistance analysis on transgenic escherichia coli, comprises the following specific processes:

The liquid dilution method is adopted to carry out the drug resistance experiment of the mutant strain, 2 antibiotics with different action targets are used to act on the bacterial sample, and the antibiotic and the final concentration thereof are 5 mug/mL Gent (gentamicin) and 250ng/mL IP (imipenem) respectively. Gentamicin takes ribosome 30s subunit as an action target spot, and imipenem is a thiomycin antibacterial drug with a carbapenem ring.

The experimental protocol for drug resistance was as follows: taking out the mutant strain from a refrigerator at minus 80 ℃, streaking on a corresponding culture medium, culturing overnight at 37 ℃, picking the monoclonal and the corresponding culture medium the next day, and culturing at the shaking culture rotating speed of 180rpm at 37 ℃ to the middle logarithmic phase. The tubes were diluted to OD 6000.02 using the corresponding medium and antibiotics were added, the tubes were incubated for 16 hours at 80rpm in an incubator at 25 ℃ and the OD600 values were recorded.

The media used in the drug resistance experiments were as follows: wild-type E.coli and the gene mutant strains (test Nos. MG1655, sRNA120, and MIM120) were cultured in LB containing 0.25mM IPTG and 50. mu.g/ml Amp, and ampicillin was added at a lower concentration to maintain the presence of the plasmid in the cells.

Because of the different growth states of the gene mutant strains, the control group set in the experiment should be the mutant strains themselves. And after the preliminary data are obtained, reasonably selecting the data of the parallel samples, displaying the result of each strain by using the OD value of the bacterial liquid, and obtaining the survival rate of the strain by using the average data of the antibiotic treatment group as a numerator and the average data of the antibiotic treatment group as a denominator. The mutant strain resistance results are shown in FIG. 7.

In FIG. 7, the abscissa represents OD600 values after 16h of culture, and the ordinate represents E.coli and its gene mutant. Wherein, IP _ LineX is the growth condition of each strain in LB culture medium added with imipenem, Gent _ LineX is the growth condition of each strain in LB culture medium added with gentamicin, and control is the growth condition of MIM interfering strain, SRNA120 transformant strain or MG1655 wild strain (WT) in normal LB culture medium. Specifically, the WT group is respectively the growth conditions of MG1655 wild strains in the culture medium of adding imipenem, adding gentamicin and normal LB from top to bottom, the OE lines group is the growth conditions of overexpression SRNA120 transformation strains in the culture medium of adding imipenem, adding gentamicin and normal LB, and the MIM lines is the growth conditions of MIM interference strains in the culture medium of adding imipenem, adding gentamicin and normal LB.

As can be seen from the experimental data in fig. 7 (for more intuitive expression, each gene mutant is represented by the experimental number), the growth of MIM-disrupted strain, SRNA120 transformant, and MG1655 wild-type strain (WT) in normal LB medium did not differ significantly. The results of comparing the wild type strains with the mutant strains show that the SRNA120 overexpression strains can obviously improve the survival rate of escherichia coli in the presence of Gent and IP, and MIM120 can reduce the drug resistance effect of the SRNA 120. The gene interference strain can embody the value and significance of the gene in the organism.

It is considered herein that sRNA120 has a very important association with e.coli protection against antibiotic damage, and provides strain samples for the subsequent study of bacterial resistance mechanisms.

<110> university of Henan technology

<120> escherichia coli sRNA120, DNA molecule, recombinant vector and application in regulation and control of bacterial drug resistance

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<170> PatentIn version 3.5

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Claims

1. Escherichia coli sRNA120 characterized in that its nucleotide sequence is as set forth in SEQ ID NO: 1 is shown.

2. The DNA molecule that transcribes the e coli sRNA120 of claim 1, having a nucleotide sequence as set forth in SEQ ID NO: 2, respectively.

3. A recombinant vector comprising the DNA molecule of claim 2.

4. The recombinant vector according to claim 3, wherein the starting vector of the recombinant vector is peT28a (+).

5. Use of escherichia coli sRNA120 for modulating the drug resistance of a bacterium, wherein the drug resistance of a bacterium is altered by altering the expression level of said escherichia coli sRNA120 in said bacterium.

6. The use of claim 5, wherein the resistance of the bacterium is increased by increasing the expression level of the E.coli sRNA 120.

7. The use of claim 5, wherein bacterial resistance to high expression of E.coli sRNA120 is increased and bacterial resistance is decreased by reducing the expression level of E.coli sRNA 120.

8. The use according to claim 5, wherein the bacterium is Escherichia coli strain K12 and its derivatives.

9. The use of claim 8, wherein the Escherichia coli K12 strain is Escherichia coli MG 1655.

10. The use of any one of claims 5 to 9, wherein the resistance is to an antibiotic and the antibiotic is imipenem or gentamicin.