CN104877997A - Promoter specifically responding to osmotic stress signal and application - Google Patents

Abstract

The invention relates to a promoter sequence capable of specifically responding to an osmotic stress signal so as to further activate expression of a target gene. Four recombinant expression bacterial strains are obtained by constructing a fusion expression vector of the promoter and respectively transferring the expression vector to nitrogen-fixing pseudomonas stutzeri as well as three industrial microbial fermenting bacterial strains: escherichia coli, bacillus thuringiensis and corynebacterium glutamicum. Experiments verify that compared to a non-stress condition, under the osmotic stress condition, the promoter provided by the invention can be used for specifically and efficiently starting expression of the targeted gene in the four bacterial strains. Therefore, the promoter has application value in microbial fermentation and genetic engineering fields.

Description

The promotor of a special response Osmotic Stress Signal Transduction and application

Technical field:

The present invention relates to one can the promoter sequence of special response Osmotic Stress Signal Transduction.The invention still further relates to the application of this promotor in fermentation industry microorganism strains genetic modification, raising fermentation efficiency.

Background technology:

The expression product of high-density culture gene engineering bacteria is the effective means improving microbial production and destination gene expression production concentration.Because when utilizing normal concentrations to ferment, the concentration of expression product amount in thalline and in fermented liquid is lower, is difficult to obtain desirable production efficiency and economic benefit.But high-density culture causes the environment that Thief zone is coerced.Because, highdensity biomass needs the matrix dropping into several times, and the Thief zone that the substrate of high density and product are formed to coerce be affect the of paramount importance key factor that microbial physiology function gives full play to, fermenting process production concentration and production intensity will be caused to improve further.

Therefore, discovery can the promoter sequence of special response Osmotic Stress Signal Transduction, and use it for fermentation industry microorganism strains genetic modification, to strengthen microorganism to the response efficiency of osmotic stress environment and adaptive faculty, effectively eliminating because osmotic pressure increases the suppression caused is the problem that high-density culture needs to solve.

Many bacterium non-coding RNAs can high expression in cellular stress process, and then plays important regulating effect in the stress response reactive system of bacterium.Such as, nfiS gene transcribed has functional non-coding RNA, and the applicant's research in the past shows this gene and have regulatory function (number of patent application: 201410262604.3) in raising bacterial strain oxidative stress resistance and salt stress resistance.But prior art does not find that the promotor of nfiS gene is as the function of functional element in special response Osmotic Stress Signal Transduction.

Summary of the invention:

The object of the invention is to obtain one can the promoter sequence of special response Osmotic Stress Signal Transduction, and under osmotic stress condition, this promotor can the translation of effectively start goal gene, to be applied to the genetic modification of microbe industrial fermentation bacterial strain.

Late Cambrian of the present invention non-coding RNA nfiS gene has the function of special response Osmotic Stress Signal Transduction, and vivoexpression analysis shows, the response sorbyl alcohol Osmotic Stress Signal Transduction (Fig. 1) that this non-coding RNA can be special.

The present invention obtains above-mentioned promoter sequence by following work, and confirms its function:

1, analyze and determine nfiS gene promoter

Transcription initiation site and the direction of nfiS is determined by 5'-RACE experiment.Result shows that the transcription initiation site of nfiS starts from a VITAMIN B4 (A), determine that the gene region of upstream 153bp from this transcription initiation site is the promotor of nfiS gene, called after pnfiS (Fig. 2), its nucleotide sequence is SEQ ID NO:1.

2, build nfiS gene promoter pnfiS fusion expression vector, and proceed to four kinds of different bacterium respectively, obtain four kinds of recombinant strains

(1) pnfiS fusion expression vector is built

First pcr amplification is carried out, obtain complete promoter fragment pnfiS, then cloned sequence is carried out BamHI and HindIII double digestion, before the multiple clone site place being inserted into promoter expression vector pGD926 and reporter gene lacZ, obtain the fusion expression vector pnifS-lacZ (Fig. 3) of promotor of the present invention and lacZ gene;

(2) this expression vector is proceeded to respectively in four kinds of bacterium:

Fixed nitrogen Pseudomonas stutzeri (Pseudomonas stutzeri), intestinal bacteria (Escherich coli), bacillus thuringiensis (Bacillus thuringiensis) or corynebacterium glutamicum (Corynebacterium glutamicum)

(3) four kinds of recombinant strains are obtained:

P.stutzeri(pnfiS-lacZ)；E.coli(pnfiS-lacZ)；B.thuringiensis(pnfiS-lacZ)；C.glutamicum(pnfiS-lacZ)。

3, pnfiS is to the confirmatory experiment of the response of Osmotic Stress Signal Transduction

Added in the hypertonicity Sorbitol Solution USP of different concns by above-mentioned four kinds of recombinant strains respectively, the enzyme being determined at lacZ gene encoding production beta-galactosidase enzymes in recombinant bacterium under inductive condition is lived.In four strain recombinant bacteriums, sorbyl alcohol all can activate the expression of promotor of the present invention, and then starts effective translation of lacZ gene.

Beneficial effect of the present invention

Experiment confirms, under osmotic stress condition, no matter promotor of the present invention is in pseudomonas or can both the expression of special, efficient startup goal gene in 3 strain industrial microorganism fermentation strains.

Promoter sequence of the present invention may be used for the effective expression of heterologous gene in different bacterium, therefore can be applicable to the genetic modification of microbial fermentation engineering bacterial strain, improves the aspects such as fermentation purposes Product yields.

Accompanying drawing explanation

Fig. 1: non-coding RNA nfiS is to the analysis of Different stress signal response.

In figure, ordinate zou represents the relative expression quantity of non-coding RNA nfiS in mRNA level in-site, and X-coordinate represents the different inductive condition of P.stutzeri A1501 bacterial strain.Wherein black histogram represents the expression amount of non-coding RNA nfiS gene under normal LB culture medium condition; Wave histogram represents the expression amount of non-coding RNA nfiS gene under 300mM Sorbitol (sorbyl alcohol) stress conditions; Grid histogram represents the expression amount of non-coding RNA nfiS gene under 0.05%SDS (sodium dodecyl sulfate, sodium lauryl sulphate) stress conditions.

Fig. 2: the promoter Analysis of non-coding RNA nfiS gene in fixed nitrogen Pseudomonas stutzeri A1501.

In figure, the transcription initiation site of nfiS gene represents with underscore, and transcriptional orientation arrow represents.Promoter sequence black runic represents.

Fig. 3: promotor pnfiS and goal gene amalgamation and expression schematic diagram.

In figure, genetic transcription direction arrow represents, wherein a figure represents pnfiS and reporter gene lacZ amalgamation and expression schematic diagram, and promotor pnfiS represents, insertion point is B (BamHI) and H (Hind Ш), and reporter gene lacZ represents; In figure, b figure represents pnfiS and other goal gene amalgamation and expression mode charts, and wherein promotor pnfiS represents, goal gene targetgene represents.

Sequence information

The promotor PnfiS nucleotide sequence SEQ ID NO:1 of non-coding RNA (nfiS) encoding gene

Embodiment

Below in conjunction with specific embodiment, set forth the present invention further.Should be understood that these embodiments only for illustrating method of the present invention, and be not used in and limit the scope of the invention.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:ColdSpring Harbor Laboratory Press, 1989) condition described in, or according to the condition that manufacturer advises.

Embodiment 1 non-coding RNA nfiS is to the response of stress signal

(1) A1501 bacterium is activated in LB liquid nutrient medium, 30 DEG C of incubated overnight;

(2) to be forwarded in LB substratum 30 DEG C next day and to be cultured to OD ₆₀₀≈ 0.6;

(3) first respectively get 1ml thalline, in contrast, and then respectively getting 1ml thalline, to add final concentration be 0.3M sorbyl alcohol or 0.05%SDS, 37 DEG C, and 60min cultivated by 200rpm shaking table;

(4) the centrifugal 5min of 8000rpm, collects thalline;

(5) adopt Promega to extract RNA test kit Z3741 in a large number and extract bacterium total serum IgE, sample RNA identical for usage quantity is carried out single stranded DNA (cDNA) reversion;

(6) by qRT-PCR method, the expression level of nfiS gene under Different stress condition is analyzed, under discovery sorbyl alcohol stress conditions, the transcriptional level of nfiS significantly improves, under SDS stress conditions, the transcriptional level of nfiS does not have noticeable change, shows the induction sorbyl alcohol Osmotic Stress Signal Transduction (Fig. 1) that nfiS can be special.

Embodiment 2nfiS gene promoter is analyzed

Adopt the 5'-RACE test kit (5'RACE System for Rapid Amplification of cDNAEnds, Version 2.0) of Invitrogen company.Concrete steps are as follows:

(1) total serum IgE is extracted

(2) Article 1 cDNA chain is synthesized

A, add following component in a 0.5ml centrifuge tube (or PCR pipe), mixing

GSP1L/R...................................................................2.5pmoles(～10to 25ng)

DEPC-treated water.....................sufficient for a final volume of 15.5μl(or sterile,distilled water)

B, 70 DEG C hatch 10min, ice bath 1min, of short duration centrifugal after add following component successively

10X PCR buffer.......................................................................2.5μl

25mM MgCl ₂...........................................................................2.5μl

10mM dNTP mix........................................................................1μl

0.1M DTT.................................................................................2.5μl

final volume.............................................................................8.5μl

The final volume of step 1and 2is 24μl.

C, to mix gently, of short duration centrifugal, hatch 1min for 42 DEG C

D, add 1 μ l Super Script ^tMiI RT, mixes gently, hatches 50min for 42 DEG C

Reaction system is finally composed as follows:

20mM Tris-HCl(pH 8.4)

50mM KCl

2.5mM MgCl2

10mM DTT

100nM cDNA primer(GSP1)

400μM each dATP,dCTP,dGTP,dTTP

1-5μg(～40ng/μl)RNA

200units SuperScript ^TMII RT

E, 70 DEG C, 15min termination reaction

After f, centrifugal 10-20s by reaction system as at 37 DEG C

G, add 1 μ l of RNase mix, soft fully mixing, hatches 30min for 37 DEG C

H, of short duration centrifugal aggreation liquid, put on ice

(3) S.N.A.P column purification reclaims cDNA

Under a, room temperature, binding solution balances pillar

B, every part treat pure sample product packing ~ 100 μ l DEPC-treated water, 65 DEG C of pre-hot reserves

C, add 120 μ l binding solution (6M NaI) in the first strand reaction

D, cDNA/NaI solution is gone to S.N.A.P post, the centrifugal 20s of 13,000x g

E, taking-up centrifugal column, centrifugate gone in a little centrifuge tube and preserve, knowing assures success is recovered to cDNA.Purification column is put back in collection tube again.

F, add 0.4ml (4 DEG C of precoolings) 1x washing buffer centrifugal 20s of 13,000x g in centrifugal column and outwell centrifugate, repeat so again to wash three times

G, add 400 μ l (4 DEG C of precoolings) 70% ethanol centrifugal 20s of 13,000x g in centrifugal column and outwell centrifugate, wash twice

H, the centrifugal 1min of 13,000x g remove residual ethanol

I, purification column to be gone in a new recovery tube, add 50 μ l, 65 DEG C of centrifugal 20s of preheating DEPC-treated water 13,000x g and collect the cDNA eluted

(4) cDNA tailing

A, in a PCR pipe, add following component, and mix gently

DEPC-treated water..............................................................6.5μl

5X tailing buffer.....................................................................5.0μl

2mM dCTP.............................................................................2.5μl

S.N.A.P.-purified cDNA sample.........................................10.0μl

final volume............................................................................24.0μl

B, 94 DEG C heating 2-3min, put 1min on ice, of short duration centrifugal after place on ice

C, add 1 μ l TdT and softly mix, 37 DEG C of reaction 10min

D, 65 DEG C, 10min inactivation TdT termination reaction, of short duration centrifugal on ice rearmounted

(5) pcr amplification

A, following component to be joined in the PCR pipe of placing on ice

ddH ₂0.................................................................................................31.5μl

10X PCR buffer[200mMTris-HCl(pH 8.4),

500mM KCl].......................................................................................5.0μl

25mM MgCl2......................................................................................3.0μl

10mM dNTP mix................................................................................1.0μl

nested GSP2R/L(prepared as 10μM solution)................................2.0μl

Abridged Anchor Primer(AAP)(10μM)...........................................2.0μl

dC-tailedcDNA...................................................................................5.0μl

final volume......................................................................................49.5μl

B, immediately add 0.5 μ l Taq DNA polymerase (5units/ μ l), mixing

C, pcr amplification

D, get 5-20 μ l 5'RACE product and carry out agarose gel electrophoresis routine analysis

(6) PCR primer purifying and sequencing analysis

With AAP and GSP2R/L for primer carries out the pcr amplification of the first round, detect through agarose gel electrophoresis and show for primer amplification obtains, this experiment expects that size is the ~ object fragment of 200bp with AAP and GSP2L, object fragment is cut glue to reclaim and carry out sequence, sequence order-checking obtained is by comparing the transcription initiation site and direction of finally determining nfiS with the nfiS sequence in A1501 bacterium genome.

This experiment the primer is as follows:

GSP1L：5'-CTGGCTCGCCAGCCTGGCACTGC-3'

GSP1R：5'-CGGCACAGCAGCAGCAAG-3'

Abridged Anchor Primer(AAP)：5'-GGCCACGCGTCGACTAGTACGGGGGGGGGG-3'

GSP2L：5'-AGCCTGGCACTGCTGATCC-3'

GSP2R：5'-ATCCCGCCGTGCGCGCCATG-3'

The structure (Fig. 3) of embodiment 3 promotor pnfiS fusion expression vector

(1) pcr amplification of nfiS promoter gene fragment: with A1501 bacterium genome for template, utilizes primer pnfiSF and pnfiSR to carry out pcr amplification and obtains length and be about the fragment of the promotor pnfiS of 150bp;

(2) structure of nfiS gene promoter expression vector: after the nfiS promoter gene fragment that pcr amplification obtains is carried out Hind Ш and BamHI double digestion, be connected on promoter detection carrier pGD926, picking positive colony extracts plasmid, and enzyme obtains lacZ fusion vector pnfiS-lacZ after cutting and verifying with PCR.

(3) acquisition of recombinant strains: the promotor pnfiS fusion expression vector of structure is proceeded in E.coli, B.thuringiensis and C.glutamicum respectively by three close combinations importing P.stutzeri or electric shock, verifies acquisition four strain recombinant strains by resistance (tetracyclin resistance) screening and PCR.

Embodiment 4 four kinds of recombinant bacterial strain betagalactosidase activities are analyzed

(1) experiment purpose:

Verify that in four kinds of recombinant strains, pnfiS is to the response of Osmotic Stress Signal Transduction

(2) experiment material and instrument:

Plasmid and bacterial strain: promoter expression vector: pnifS-lacZ; Recombinant strains: E.coli (pnfiS-lacZ), B.thuringiensis (pnfiS-lacZ), C.glutamicum (pnfiS-lacZ) and P.stutzeri (pnfiS-lacZ)

Laboratory apparatus: ultraviolet spectrophotometer is Shimadzu UV-160A type

(3) experimental technique

In E.coli, B.thuringiensis, C.glutamicum or P.stutzeri, the betagalactosidase activity method for measuring of pnfiS-lacZ amalgamation and expression is as follows:

(1) difference picking four strain recombinant bacterial strain list bacterium colony, be seeded in the LB liquid nutrient medium containing tsiklomitsin and cultivate, wherein E.coli (pnfiS-lacZ) culture temperature is 37 DEG C, the culture temperature of its excess-three strain recombinant bacterial strain is 30 DEG C, within second day, be inoculated in LB liquid nutrient medium according to 2% switching amount, survey bacterium liquid OD ₆₀₀value, until OD ₆₀₀value reaches more than 0.6, adds 300mM, 500mM, 700mM and 1000m M sorbyl alcohol respectively, induces 4 hours, wherein using without inductor as negative control;

(2) get appropriate bacterium liquid, 4 DEG C, the centrifugal 5min of 5,000rpm, abandons supernatant, with aseptic washing twice, abandons supernatant.Eddy diffusion thalline on demand;

(3) thallus suspension liquid mixes with bufferZ, makes cumulative volume be 1ml, adds 2-3 and drip chloroform, mixing.Uncap in 37 DEG C of insulation 40min;

(4) proceed to 30 DEG C of insulation 5min, add 200 μ l (4.0mg/ml) ortho-nitrophenyls-β-D-synthesis (ONPG) afterwards, mix latter 30 DEG C and continue insulation, start reaction, when record reaction is initial;

(5) treat that sample occurs yellow, then add the Na of 500 μ l 1mol/l ₂cO ₃termination reaction, the record reaction terminating time, sample is put to be measured on ice.

(6) OD is measured respectively with ultraviolet spectrophotometer ₄₂₀and OD ₅₅₀value.

(7) according to following formulae discovery betagalactosidase activity value

β-Galactosidase Units＝1000×(OD ₄₂₀-1.75×OD ₅₅₀)/(T×V×OD ₆₀₀)。

Parallel laboratory test three times is all carried out in each experiment above, and the result obtained is the betagalactosidase activity value of the mean error calculating three independent experiments.

4, experimental result

1) in four strain recombinant bacteriums, the sorbyl alcohol of each experimental concentration all can activate the expression of promotor of the present invention, and then drives effective translation of lacZ gene;

2) induce the most highly active concentration different for different bacterium

Such as, in recombinant bacterium E.coli (pnfiS-lacZ), the activity the highest (186 ± 25.72Miller Units) of beta-galactosidase enzymes under the induction of 700mM sorbyl alcohol;

And in P.stutzeri (pnfiS-lacZ), promotor of the present invention is activity the highest (179 ± 20.5Miller Units) under the induction of 500mM sorbyl alcohol.

That promotor of the present invention and the betagalactosidase activity of lacZ gene fusion expression carrier pnfiS-lacZ respectively in E.coli, B.thuringiensis, C.glutamicum or P.stutzeri measure with following table 1.

In table, the error of listed experimental result data is the mean error of three independent experiments, and with 0mM sorbyl alcohol in contrast, the induced concentration of sorbyl alcohol is respectively 300mM, 500mM, 700mM and 1000mM.

Concrete data are as shown in table 1:

The determination of activity of table 1 recombinant expression vector under four kinds of recombinant strains different concns osmotic stresses

5, conclusion

Under Thief zone stress conditions, no matter the promotor that the present invention finds is in pseudomonas or can both starts the expression of goal gene in 3 strain industrial microorganism fermentation strains special, efficiently.

Claims (9)

1. special response Osmotic Stress Signal Transduction the promoter sequence of efficient start-up performance genetic expression, its nucleotide sequence is as shown in SEQ ID NO:1.

2. promoter sequence according to claim 1 expresses the application of response tunning under genetic engineering bacterium high density fermentation and adverse environmental factor.

3. application according to claim 2, is the special response Osmotic Stress Signal Transduction of described promoter sequence and the expression of effectively start goal gene under osmotic stress condition.

4. a method for the expression product of culturing gene engineering bacteria, is characterized in that application rights requires the promoter sequence described in 1.

5. containing the application of fusion expression vector in the expression product of culturing gene engineering bacteria of the promotor described in claim 1.

6. application according to claim 5, described fusion expression vector is the fusion expression vector of promotor according to claim 1 and reporter gene lacZ.

7. a genetic engineering bacterium, is characterized in that the recombinant strains having proceeded to fusion expression vector according to claim 5.

8. genetic engineering bacterium according to claim 7 fusion expression vector according to claim 4 is proceeded to respectively the four kinds of recombinant bacterial strains obtained in following four kinds of bacterium: fixed nitrogen Pseudomonas stutzeri (Pseudomonas stutzeri), intestinal bacteria (Escherich coli), bacillus thuringiensis (Bacillus thuringiensis) or corynebacterium glutamicum (Corynebacteriumglutamicum).

9. the application of genetic engineering bacterium according to claim 7 in the expression product of culturing gene engineering bacteria.