Summary of the invention
The object of the invention is to design and synthesize a kind of new sRNA, make it have the function of giving the anti-oxidant and resistance to high-salt stress of host cell.
The present inventor utilizes transcriptome analysis and proteomic analysis methods to be studied the composition of Pseudomonas stutzeri A1501 bacterium sRNA and function, Late Cambrian also determines the core sequence fragment of non-coding sRNA03 gene, and in conjunction with the Hfq transcription modulator of non-coding RNA, synthetic a kind of new sRNA, called after AncR03.
The core sequence fragment of described synthetic AncR03 gene is the specific binding sequence shown in SEQ ID NO:2.
In described synthetic AncR03 gene, Hfq protein binding sequence is the sequence shown in SEQ ID NO:3.
Particularly, invention has been following research:
One, the determination of specific binding sequence in synthetic sRNA
Concrete steps are as follows:
1, the structure of sRNA03 land (20bp) gene fragment deletion mutantion strain (A1501 Δ sRNA03) in fixed nitrogen Pseudomonas stutzeri A1501 (P.stutzeri A1501):
2, the structure of A1501 Δ sRNA03 deletion mutantion strain complemented strain:
3, sRNA0320bp land is lacked on the impact of Pseudomonas stutzeri A1501 bacterial strain oxidative stress resistance:
Experimental result shows, disappearance sRNA0320bp land fragment two orders of magnitude that caused mutant strain Oxidative Stress energy force rate wild-type low; And cover the Oxidative Stress ability that bacterial strain partly can recover bacterial strain.Show that this 20bp fragment is the important binding domain that sRNA03 plays function.
Two, the structure of synthetic sRNA expression plasmid pSAncR03
After determining the important specific binding sequence of synthetic sRNA, choose promoter sequence in intestinal bacteria, Hfq protein binding sequence and terminator sequence are according to the complete sequence of synthesizing this synthetic sRNA shown in sequence table.
In an embodiment of the invention, this AncR03 is by promotor (SEQ ID NO:1), specific binding sequence (SEQID NO:2), Hfq protein binding frame sequence (SEQ ID NO:3) and terminator (SEQ ID NO:4) four part composition.
The functional experiment of synthetic sRNA:
Experiment proves, the synthetic sRNA that the present invention obtains has the function of special response adverse circumstance signal, oxidative stress resistance and the salt stress resistance of other F-strains can be increased substantially, with the recombinant strain carrying this sRNA03 that it builds, relative to the bacterial strain not containing this sRNA03, under the Oxdative stress of identical high density and the condition of salt stress of high density, there is higher survival rate.(detailed in Example).
Directly proceed to compared with anti-oxidant and salt resistant function gene with at present conventional, because sRNA is in the upstream that cellular metabolism regulates, therefore regulate efficiency higher and specific aim is stronger, cellular function impact is little.
Creative major embodiment of the present invention is in inventive concept and core sequence:
Due to Late Cambrian of the present invention, above-mentioned core sequence fragment is SEQ ID NO:2, according to inventive concept of the present invention, when those skilled in the art as required and physical condition, also synthetic can be designed and obtain the complete sequence of multiple different sRNA, for improving oxidative stress resistance and the salt stress resistance of other F-strains.
Such as, choose other suitable promoter sequence, Hfq protein binding sequence and terminator sequence, or connect the sequence of other function.But these all can not depart from invention of the present invention spirit.
The purposes of synthetic sRNA provided by the invention:
Because this synthetic sRNA has the function of special response adverse circumstance signal, can be used for the bacterial strain that industrial production improves oxidative stress resistance and salt stress resistance.And such bacterial strain is with a wide range of applications in many aspects.
Embodiment
Below in conjunction with specific embodiment the present invention done and set forth in more detail; Embodiment only for illustrating the present invention, and is not used in and limits the scope of the invention.On basis of the present invention, in those skilled in the art's specific operation process, can revise with making some unsubstantialities according to actual needs, these amendments all should belong to the scope of protection of the invention.
In embodiment, all unreceipted specific experiment conditions, be according to normal condition well known to those skilled in the art, such as Sambrook equimolecular clone: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or according to the condition that manufacturer advises.
The determination of specific binding sequence and the structure of expression plasmid pSAncR03 in embodiment 1 synthetic sRNA
One, the determination of specific binding sequence in synthetic sRNA
We find that the non-coding sRNA03 coming from fixed nitrogen Pseudomonas stutzeri A1501 participates in the degeneration-resistant physiological process of modulate host cellular portions.Experiment proves, disappearance sRNA03 can cause the reduction of cellular anti-oxidant and salt resistance ability, and is proceeded to by this sRNA in intestinal bacteria and also obviously can strengthen its viability in peroxidation and hypersaline environment, and the degeneration-resistant physiological process of its regulating cell is identified.In order to build the stronger sRNA of ability of regulation and control, first needing clear and definite is exactly specific binding sequence, we analyze sRNA03 by bioinformatics software and choose its calmodulin binding domain CaM one section of 20bp fragment as specific binding district for this reason, build this region disappearance and covering bacterial strain, utilize oxidation resistance assay to prove the effect in this region.Concrete steps are as follows:
1, the structure of the middle sRNA0320bp fragment deletion mutant strain of fixed nitrogen Pseudomonas stutzeri A1501 (P.stutzeri A1501):
First the long segment utilizing fusion DNA vaccine technology the segments downstream of the fragment upstream of 20bp goal gene, Spectinomycin resistance box gene and goal gene to be fused into a size to be about 2.1kb, then cloned sequence is carried out BamHI and HindIII double digestion, be connected on suicide vector pk18mobSacB.The method that the suicide recombinant plasmid built is combined by three parents is imported in wild-type A1501 bacterium, by the homologous recombination with gene on karyomit(e), suicide plasmid is incorporated on karyomit(e), utilize resistance screening and PCR checking to obtain single cross and change bacterial strain, then lethal under 10% sucrose Selective Pressure according to sacB gene characteristic, changes clone according to 10 by the single cross verified through PCR
-3, 10
-4with 10
-5dilution gradient to be coated on the LB of the Spectinomycin resistance containing 10% sucrose respectively dull and stereotyped, carry out the screening of double exchange, obtain the complete deletion mutant strain A1501 Δ sRNA03 of RNA0320bp binding domain through PCR checking.
2, the structure of A1501 Δ sRNA03 mutant strain complemented strain:
First synthetic sRNA0320bp fragment, then the fragment of synthesis is carried out BamHI and HindIII double digestion, be connected on wide host expression vector pLAFR3, the method combined by three parents forwards in A1501 Δ sRNA03, successfully obtains complemented strain A1501sRNA03.
3, sRNA0320bp land is lacked on the impact of Pseudomonas stutzeri A1501 bacterial strain oxidative stress resistance:
Cultivate wild-type A1501, deletion mutantion strain A1501 Δ sRNA03 respectively, and covering strains A 1501sRNA03.On solid plate, picking list bacterium colony difference incubated overnight in LB liquid medium, is transferred in LB substratum and makes OD next day
600be about 0.1, cultivate 2-3h, to OD for 30 DEG C
600about about 0.6.First respectively get 1ml thalline, be diluted to 10
-4for blank, and then respectively getting 1ml thalline, to add final concentration be 20mM H
2o
2impact 10min, finally gradient dilution to 10 is done in each process
-4, get 8 μ l points on LB solid plate, 30 DEG C of incubated overnight, observation (Fig. 1).
Fig. 1 H
2o
2after impacting, the viability of wild-type A1501, deletion mutantion strain A1501 Δ sRNA03 and covering strains A 1501sRNA03 compares.
Experimental result shows, disappearance sRNA0320bp fragment two orders of magnitude that caused mutant strain Oxidative Stress energy force rate wild-type low; And cover the Oxidative Stress ability that bacterial strain partly can recover bacterial strain.Show that this 20bp fragment is the important binding domain that sRNA03 plays function.
Two, the structure of synthetic sRNA expression plasmid pSAncR03
After determining the important specific binding sequence of synthetic sRNA, choose promoter sequence in intestinal bacteria, Hfq protein binding sequence and terminator sequence are according to the complete sequence of synthesizing this synthetic sRNA shown in sequence table.
Synthetic sRNA sequence composition structure connects in the following manner: 1-2-3-4
Sequence is as shown in the table:
1: promoter sequence; 2: specific binding sequence; 3:Hfq protein binding sequence; 4: transcription terminator sequences enzyme is connected in carrier after cutting, construction of expression vector pSAncR03 (see Fig. 2 vector construction collection of illustrative plates).
Embodiment 2 builds the checking of mutant strain and function thereof
Utilize synthetic sRNA to improve the method for intestinal bacteria TOP10 bacterial strain oxidative stress resistance and/or salt stress resistance, build the mutant strain with oxidative stress resistance and/or salt stress resistance.
In the present embodiment, described synthetic sRNA gene is the non-coding sRNA gene of transcribing through the DNA of nucleotide sequence shown in sequence table.
In the present embodiment, the step that described structure has the intestinal bacteria TOP10 recombinant bacterial strain of oxidative stress resistance and/or salt stress resistance is: be transformed in bacterial strain by the pSAncR03 plasmid vector of structure by heat-shock transformed method, build recombinant strains E.coli TOP (pSAncR03), namely there is the intestinal bacteria Top10 recombinant bacterial strain of higher oxygen stress resistance and/or salt stress resistance.
Confirmatory experiment 1 oxidative stress resistance assay
Experiment purpose: the impact of synthetic sRNA gene pairs intestinal bacteria TOP10 bacterial strain stress resistance
Materials and methods:
Bacterium liquid used: the TOP10 (pSAncR03) prepared in wild-type e. coli TOP10, the present embodiment.
On solid plate, picking list bacterium colony difference incubated overnight in LB liquid medium, is transferred in LB substratum and makes OD next day
600be about 0.1, cultivate 2-3h, to OD for 30 DEG C
600about about 0.6.First respectively get 1ml thalline, be diluted to 10
-5for blank, and then respectively getting 1ml thalline, to add final concentration be 25mM H
2o
2impact 10min, or add final concentration 1.5M NaCl impact 60min, finally by each process doubling dilution to 10
-5, get 8 μ l points on LB solid plate, 37 DEG C of incubated overnight.
Oxidative stress resistance affect H
2o
2impact experiment
Plasmid pSAncR03 transformation of E. coli TOP10 bacterial strain containing synthetic sRNA, obtains recombination bacillus coli TOP10 (pSAncR03).
Experimental result: compare from the viability of the rear E.coli TOP10 and recombinant bacterial strain TOP10 (pSAncR03) of Fig. 3 Hydrogen Peroxide Stress impact and find out: compared with wild-type e. coli, recombinant bacterial strain TOP10 (pSAncR03) containing pSAncR03 plasmid has stronger hydrogen peroxide resistance, after 25mM hydrogen peroxide impacts 10 minutes, wild-type e. coli is only 10
-2have bacteria growing under dilution, and the intestinal bacteria turning synthetic sRNA are 10
-4still have bacteria growing, display resistance capacity improves about 2 orders of magnitude.
Conclusion: the non-coding sRNA AncR03 of synthetic has the ability significantly improving thalline oxidative stress resistance.
Confirmatory experiment 2 NaCl impact experiment
Experiment purpose: investigate synthetic sRNA to the impact of salt stress resistance
Experimental technique: adopt 1.5M sodium-chlor to impact 60 minutes to wild-type e. coli and recombinant bacterial strain respectively.
Experimental result: Fig. 4 is that after NaCl impacts, the viability of E.coli TOP10 and recombinant bacterial strain E.coli TOP10 (pSAncR03) compares, and result shows, and wild-type e. coli is only 10
-2have bacteria growing under dilution, and the intestinal bacteria turning synthetic sRNA are 10
-3still have bacteria growing.
Conclusion: recombinant bacterial strain E.coli TOP10 (pSAncR03) improves an order of magnitude than the salt stress resistance capacity of wild-type E.coli TOP10.