CN102533769A - Low-temperature promoter of pseudomonassp and application of low-temperature promoter - Google Patents
Low-temperature promoter of pseudomonassp and application of low-temperature promoter Download PDFInfo
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Abstract
The invention discloses a low-temperature promoter of pseudomonassp and the application of the low-temperature promoter, and particularly relates to the sequence and the application of the low-temperature promoter of the low-temperature resistant pseudomonassp. MY1402 separated from Mingyong glacier of Meri Snow Mountain in Yunnan province. For screening, a promoter probe plasmid is constructed and a genomic DNA library is built, a recombination plasmid containing a segment of the promoter can prompt the expression of a target gene in a procaryotic cell at a low temperature; due to universality in different procaryotic cells, the promoter can be used for constructing an expression carrier which is universal for a plurality of procaryotic cells at the low temperature, so that the trouble of replacing the carriers during the expression of foreign genes in the different procaryotic cells can be saved, and the efficiency of experiments of polymer biology is improved; in addition, the promoter can also be used for prompting the gene expression of low-temperature protein and low-temperature enzyme at the low temperature, so that the production efficiency of low-temperature protein and low-temperature enzyme can be improved.
Description
Technical field
The invention belongs to biology field, relate to one particularly and derive from low temperature pseudomonas promotor and prokaryotic expression carrier and application.
Background technology
Promotor is an integral part of gene, is usually located at structure gene 5 ' the end upper reaches, is the section of DNA sequence of RNA polymerase identification, combination and transcriptional start.Promotor can correctly combine with template by the guide RNA polysaccharase, the activation RNA polymerase, and promotor gene is transcribed, thus controlling gene is expressed the time of origin of (transcribing) and the degree of expression.In the heterologous protein of mikrobe was expressed, promotor was one of important factor that influences heterogenous expression efficient, and selecting high efficiency promotor is the key of high-efficient expression foreign gene.
Current owing to still lack the efficient expression system of cold induced proteins, make cold induced proteins especially the production of cold-adapted enzyme can not satisfy the needs of scientific research and industrial application far away.Protein expression system at present commonly used many with mesophilic bacterias such as intestinal bacteria and yeast as host cell, but owing to most cold-adapted enzyme poor heat stability, though in the chief cell of middle Wensu great expression, also regular meeting is because of the thermally denature inactivation.Therefore be the host bacterium with the psychrophilic bacteria, be that the method for promotor construction of expression vector more and more causes people's attention with the psychrophilic bacteria promotor.
The research of cold-starting belongs to the stage at the early-stage, only has people such as Duilio to belong to bacterial strain from separating from Antarctic Ocean low temperature Pseudoalteromonas at present
PseudoalteromonashaloplanktisAmong the TAC125, through making up the mode in library, utilize the promoter probe plasmid (
PseudoalteromonashaloplanktisTAC125 is a host cell), having screened can be sub at the cold-starting that 4 ℃ of startups are transcribed.To the parsing of the 26S Proteasome Structure and Function of these promotors, lively material is provided for we are familiar with the mechanism that cold-starting starts at low temperatures.
But because the development of cold-starting is just at the early-stage, document and data are less relatively, in current rounded system and the theory of also not forming.Cold-starting structurally can not obviously distinguish with the normal temperature promotor.It is same or similar that some have the active cold-starting of cold-starting each function controlling district and normal temperature promotor.Also having some to have active cold-starting of cold-starting and then do not have tangible conserved structure sequence, can't be that standard goes to confirm specific each functional zone and the conserved structure sequences of cold-starting such as its 10 district, 35 districts with the conserved sequence of normal temperature promotor.Therefore separate cold-startings in a large number and analyze its sequence and become the task of top priority.
In addition; The promotor that is host cell with the psychrophile, is used to come from psychrophile makes up the existing recently part report of cold induced proteins expression system; Research be illustrated in the normal temperature protein expression system albumen beyond expression of words; Comprise the toluene, the YLENE monooxygenase that derive from the normal temperature bacterium, derive from human NGFF and in these expression systems, all obtained effective expression.Of particular concern is that the NGFF of expressing at the cold induced proteins expression system does not only form inclusion body; And accurately be positioned the periplasmic space of cell; Demonstrated fully the meliority of cold induced proteins expression system; Being allos cold induced proteins and the proteic effective ways that provide new that efficiently express of normal temperature, is that host's cold induced proteins expression system is to solve a practical way efficient, the mass production thermally labile protein through making up with the psychrophile.
Summary of the invention
The object of the present invention is to provide a kind of dna molecular, it derives from the low temperature pseudomonas
PseudomonasSp
.MY1402, and have one of following nucleotide sequences:
A, has the nucleotide sequence shown in the SEQ ID NO.1;
The nucleotide sequence of b, the nucleotide sequence hybridization that can limit with SEQ ID NO.1;
C, nucleotide sequence shown in above-mentioned a or the b carried out replacement, disappearance, interpolation, the modified nucleotide sequences of one or more bases;
D, the nucleotide sequence that has at least 90% identity with nucleotide sequence shown in above-mentioned a or the b.
Dna molecular has promoter activity described in the present invention, and goal gene is expressed in prokaryotic cell prokaryocytes such as intestinal bacteria and pseudomonas, particularly can efficiently express at low temperatures.
Another purpose of the present invention provides a kind of prokaryotic expression carrier, and this carrier contains the described dna molecular of preamble.
Another purpose of the present invention provides a kind of prokaryotic host cell, and this cell contains described dna molecular of preamble or prokaryotic expression carrier.
Another purpose of the present invention is that dna molecular is applied in the protokaryon heterologous expression system.
The heterologous expression system of protokaryon described in the present invention is normal temperature expression system or low temperature expression system.
In the present invention, the promoter sequence shown in the SEQ ID NO.1 is called p1.
In the present invention, no promoter probe plasmid is called pUE, and the probe plasmid that will contain the promoter sequence shown in the SEQ ID NO.1 is called pUE1.
Bacterial strain
PseudomonasSp
.MY1402 is separated from the soil sample of area, bright glacier forever, Moirigkawagarbo by this laboratory and obtains.
The present invention screens highly active cold-starting daughter nucleus thuja acid fragment through making up the promoter probe plasmid; Utilize the isolated highly active promoter in prokaryote sequence construct recombinant expression vector that has; And heterogenous expression goal gene at low temperatures, the structure gene of this promotor and target protein is connected, and this gene receives promoter regulation; Goal gene efficiently expresses under the regulation and control of promotor as a result, and this promotor also has higher startup activity under 15 ℃ low temperature; This promotor has versatility in different prokaryotic organism; Can be used for making up can be general between a plurality of at low temperatures prokaryotic organism expression vector; Need change the trouble of carrier to save in different prokaryotic organism expression alien gene, improve molecular biology experiment efficient; This promotor can also be used for starting at low temperatures cold induced proteins and cold-adapted enzyme expression of gene, improves the production efficiency of cold induced proteins and cold-adapted enzyme.
The present invention can and improve the sub fundamental research of cold-starting for development some materials and reference are provided; And can for albumen (enzyme) particularly the industrial production of cold induced proteins (enzyme) new allos expression vector and promoter sequence are provided, the present invention can be applied to the production of molecular biology experiment and cold induced proteins (enzyme).
Description of drawings
Fig. 1 is the present invention's probe plasmid construction synoptic diagram of recombinating.
Fig. 2 is the electrophoresis synoptic diagram of bacterial strain MY1402 genomic dna among the present invention, and wherein M is DL2,000 marker, the 1st, bacterial strain MY1402 genomic dna.
Fig. 3 is among the present invention
Sau3A I enzyme is cut the genomic recovery of MY1402 synoptic diagram as a result, and wherein M is DL2,000 marker, and the 1st, use restriction enzyme
BamH IHandle the recovery synoptic diagram as a result behind the probe plasmid pUE with bacterium with SEAP (BAP), the 2nd, use restriction enzyme
Sau3A IHandle
PseudomonasSp
.Recovery behind the MY1402 genomic dna is synoptic diagram as a result.
Fig. 4 is among the present invention
BamThe recovery that H I enzyme is cut pUE is synoptic diagram as a result, and wherein M is DL2,000 marker, and the 1st, restriction enzyme digestion and electrophoresis figure in a small amount, the 2nd, use restriction enzyme
BamThe recovery of H I processing probe plasmid pUE is synoptic diagram as a result.
Fig. 5 inserts fragment bacterium colony PCR synoptic diagram as a result for pUE among the present invention, and wherein M is DL5,000 marker, the 1st, pUE, the 2nd, DH5 α/pUE1.
Embodiment
Below in conjunction with accompanying drawing and embodiment the present invention is done further explain, but protection domain of the present invention is not limited to said content, method therefor is ordinary method if no special instructions among the present invention.
Embodiment 1: the screening of promotor
1, the preparation of possible promoter fragment: extract Rhodopseudomonas
PseudomonasSp. the genomic dna of MY1402 bacterial strain is partially digested with enzyme Sau3A I, and reclaiming size simultaneously is the different fragments of 100 ~ 2,000 base.
Extract the genome of bacterial strain MY1402, finally extract the result and see Fig. 2, through measuring the genomic OD of the MY1402 that is extracted
260=0.0478, OD
280=0.0267, then the genomic concentration of MY1402 is 50 * OD
600=50 * 0.0478 * 100=239 μ g/ml=239ng/ μ l, OD
260/ OD
280=1.79, purity satisfies the requirement of using;
Enzyme
Sau3A I is partially digested
PseudomonasThe genomic concrete grammar of sp.MY1402:
Enzyme
Sau3A I directly is diluted to 0.125 u/ μ l, obtains needed fragment through the restriction enzyme digestion time, and the controllability of embodiment strengthens like this, and concrete reaction system and time are following:
| The MY1402 genomic dna | 80 μl（19.12 μg） |
| Enzyme Sau3AⅠ | 8 μl（0.5 U） |
| 10 * H damping fluid | 10 μl |
| dd H 2O | Be added into 100 μ l |
Behind the mixing, 10 μ l/PCR pipe is distributed into 10 pipes, 37 ℃ of reactions 3 hours, reclaims clip size then simultaneously 100 ~ 2, the different fragments of 000 base size,
Sau3A I enzyme is cut the genomic recovery result of MY1402 and is seen Fig. 3.
2, the big segmental preparation of probe plasmid pUE: use restriction enzyme
BamH I complete degestion probe plasmid pUE reclaims the back and handles with SEAP (BAP) with bacterium, and phenol/chloroform extracting, ethanol sedimentation finally prepare the result and see Fig. 4.
3,, connect product transformed into escherichia coli DH5 α with big fragment of T4 DNA ligase enzyme connection carrier and possible promoter fragment.
Linked system is following:
| pUE/
BamH I/ |
1 μl（0.2 μg） |
| The MY1402 genomic dna/ Sau3A I | 10 μl（0.1 μg） |
| The |
1 μl（350U） |
| 10 * T4 DNA ligase |
2 μl |
| dd H 2O | Be added into 20 μ l |
4, screening: adopt direct sieve method, promptly after connecting product transformed into escherichia coli DH5 α, draw 100 μ l cultures coating LB/Kan solid medium, place 37 ℃ to cultivate 7 days.
5, the recon that screens is carried out bacterium colony PCR and identify, spread cultivation and the extracting plasmid,, confirm to check order after it has promoter activity really with institute's upgrading grain transformed into escherichia coli DH5 α again to having the segmental bacterial strain of insertion.
5.1 bacterium colony PCR identifies: design a pair of primer special f3, r3 here and be specifically designed to and identify and check order to inserting fragment; Primer sequence is: upstream primer f3 5 '
-CTTTGACGTATGCGGTGTGAAAT-3 ', downstream primer r3
5
'-AGATGGTCAGACTAAACTGGCTG-3 '; Be tested and appraised and find to have the insertion fragment; Directly 1 of screening of sieve method has segmental single bacterium colony of insertion; This with direct sieve method from carrier: fragment is the recombinant plasmid called after pUE1 in the single bacterium colony that screens the library of 1:10, inserts fragment called after p1.Containing p1 inserts segmental pUE1 bacterium colony PCR result and sees Fig. 5.
5.2 the evaluation of promoter activity: the extracting recombinant plasmid, recombinant plasmid transformed is gone in the bacillus coli DH 5 alpha, draw 100 μ l culture separate application in LB/Amp and LB/Kan solid medium, be inverted for 37 ℃ and cultivated 12 hours.Observing the discovery transformant can grow on the LB/Kan solid medium.So far, can confirm to insert fragment p1 and have promoter activity really.
6, order-checking: order-checking is with primer f3, r3, and sequencing result shows that p1 length is 145 bases, and sequence is shown in SEQ ID NO.1.
The checking of embodiment 2:p1 promoter activity
(1) recombinant plasmid pUE1 electricity is transformed bacterial strain MY1402, electric shock voltage is 1800v, and electric shock finishes; The LB liquid nutrient medium that adds 500 μ l precoolings immediately in the electric shock cup moves to all liquid in one 1.5 ml centrifuge tube, 15 ℃ of 180 rev/mins of shaking culture 1 hour; Draw 100 μ l cultures in the LB/Kan solid medium; Be coated with driedly with the sterilization spreading rod, flat board sealed, be inverted for 28 ℃ and cultivated 72 hours with adhesive tape.
(2) the single colony inoculation that grows in the LB/Kan liquid nutrient medium, in 15 ℃ and 28 ℃ of two shaking tables, cultivate respectively with 180 rev/mins speed oscillation, sampling in 24 hours, be determined at the light absorption value of 600 nanometers.In order to eliminate the unhomogeneity that test tube causes owing to the difference at angle of inclination etc. in culturing process, when shaking culture, be inserted into all test tubes in the test-tube stand, the test-tube stand horizontal positioned, reliable to make real result as far as possible.
(3) result sees the following form:
Table 1 is cultivated different kantlex concentration culture bacteria liquid OD values after 24 hours for 28 ℃ 180 rev/mins
Table 2: cultivate different kantlex concentration culture bacteria liquid OD values after 24 hours for 15 ℃ 180 rev/mins
Data by table 1 can know that the recombinant plasmid transformed that contains the p1 promoter fragment is gone into
Pseudomonas sp.Behind the MY1402 low temperature pseudomonas, make the host bacterium that does not have kalamycin resistance originally
Pseudomonas sp.MY1402 has had kalamycin resistance, and can in the scope of kantlex concentration 0 ~ 250 μ g/ml, grow, and explains that the p1 promoter fragment has promoter activity under 28 ℃ condition.
Data by table 2 can know, contain recombinant plasmid with p1 promoter fragment
Pseudomonas sp.MY1402 makes the host bacterium that does not have kalamycin resistance originally when 15 ℃ culture condition
Pseudomonas sp.MY1402 has also had kalamycin resistance.And can in the scope of kantlex concentration 0 ~ 300 μ g/ml, grow, explain that the p1 promoter fragment still has promoter activity under the condition of 15 ℃ of low temperature.
Data contrast by table 1 and table 2 can be known host bacterium when kantlex concentration is between 50 μ g/ml-150 μ g/ml
Pseudomonas sp. MY1402 is more than the bacteria living number under 15 ℃ of conditions in the time of 28 ℃, but is more or less the same.Explain the p1 promotor when kantlex concentration is between 50 μ g/ml-150 μ g/ml 28 ℃ with 15 ℃ of conditions under startup active quite; But, contain the recombinant plasmid of p1 promoter fragment when kantlex is dense under the selective pressure of 200 μ g/ml-300 μ g/ml
Pseudomonas sp.MY1402 is more than the bacteria living under 28 ℃ of conditions at 15 ℃; Contain more bacterial count; Explain when kantlex dense during at 200 μ g/ml-300 μ g/ml; The p1 promotor the startup under 15 ℃ the low temperature active with 28 ℃ of conditions under activity higher, explain more that thus the p1 promotor has the cold-starting activity.
SEQUENCE?LISTING
< 110>Kunming University of Science and Technology
< 120>pseudomonas cold-starting and application thereof
<160> 3
<170> PatentIn?version?3.5
<210> 1
<211> 145
<212> DNA
<213>?
Pseudomonas?sp.MY1402
<400> 1
aaagcaataa aaccactcaa ggctattttt agcgccatcg ctgtatccaa ?50
cctgttaaaa gaaagttgcg gctagcatgc cgcagtatta cactcacaaa ?100
aactgaataa tactcatcaa gttcatcacg aaaagagaag gctat 145
<210> 2
<211>?23
<212> DNA
< 213>artificial sequence
<400> 2
ctttgacgta tgcggtgtga aat ?23
<210> 3
<211> 23
<212> DNA
< 213>artificial sequence
<400> 3
agatggtcag actaaactgg ctg 23
Claims (6)
1. a dna molecular derives from the low temperature pseudomonas, and has one of following nucleotide sequences:
A, has the nucleotide sequence shown in the SEQ ID NO.1;
The nucleotide sequence of b, the nucleotide sequence hybridization that can limit with SEQ ID NO.1;
C, nucleotide sequence shown in above-mentioned a or the b carried out replacement, disappearance, interpolation, the modified nucleotide sequences of one or more bases;
D, the nucleotide sequence that has at least 90% identity with nucleotide sequence shown in above-mentioned a or the b.
2. dna molecular according to claim 1 is characterized in that: this dna molecular has promoter activity.
3. prokaryotic expression carrier that contains claim 1 or 2 said dna moleculars.
4. prokaryotic host cell, said host cell contains claim 1 or 2 described dna moleculars or the described expression vector of claim 3.
5. claim 1 or 2 application of said dna molecular in the protokaryon heterologous expression system.
6. according to right 5 application of said dna molecular in the protokaryon heterologous expression system, it is characterized in that: the protokaryon heterologous expression system is normal temperature expression system or low temperature expression system.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110607300A (en) * | 2019-09-27 | 2019-12-24 | 中国科学院天津工业生物技术研究所 | Strong promoter, plasmid vector and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1523107A (en) * | 2003-09-11 | 2004-08-25 | 上海交通大学 | Gene sequence of luteolin biosynthetic regulatory factor in pseudomonas M18 |
| CN1616662A (en) * | 2003-11-11 | 2005-05-18 | 四川大学 | Pseudo monads pseudoalcaligenes gene promoter |
| CN101981184A (en) * | 2008-03-27 | 2011-02-23 | 纳幕尔杜邦公司 | High expression zymomonas promoters |
-
2012
- 2012-02-28 CN CN201210047244.6A patent/CN102533769B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1523107A (en) * | 2003-09-11 | 2004-08-25 | 上海交通大学 | Gene sequence of luteolin biosynthetic regulatory factor in pseudomonas M18 |
| CN1616662A (en) * | 2003-11-11 | 2005-05-18 | 四川大学 | Pseudo monads pseudoalcaligenes gene promoter |
| CN101981184A (en) * | 2008-03-27 | 2011-02-23 | 纳幕尔杜邦公司 | High expression zymomonas promoters |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110607300A (en) * | 2019-09-27 | 2019-12-24 | 中国科学院天津工业生物技术研究所 | Strong promoter, plasmid vector and application thereof |
| CN110607300B (en) * | 2019-09-27 | 2021-09-07 | 中国科学院天津工业生物技术研究所 | Strong promoter, plasmid vector and application thereof |
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| CN102533769B (en) | 2013-07-31 |
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