CN107236732B - A kind of saccharomyces neoformans inducible promoter and its application - Google Patents
A kind of saccharomyces neoformans inducible promoter and its application Download PDFInfo
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Abstract
The present invention provides a kind of saccharomyces neoformans inducible promoter and its applications, belong to technical field of molecular biology.Its technical solution are as follows: it is DNA sequence dna shown in sequence 1 in sequence table；The promoter length is 673bp.The invention has the benefit that yeast inducible promoter DDI2 of the invention, it can efficiently be induced by cyanamide, promoter intensity is close with known inducible strong promoter GAL1, it is stronger than constitutive promoter ADH1, and replacement culture medium is not had to when inducing, the expression quantity that gene can be controlled by the amount of induction time and inducer, has more advantage compared with common GAL1 promoter on time cost and Costco Wholesale.DDI2 promoter intensity is greater than ADH1 promoter, and GAL1 promoter similar intensity, and required induction time and is less than GAL1 promoter flow cytomery as the result is shown, does not have to replacement culture medium.By Western and flow cytomery as a result, available DDI2 promoter intensity reaches maximum when inducer concentration is 5-8mM.
Description
Technical field
The present invention relates to technical field of molecular biology more particularly to a kind of saccharomyces neoformans inducible promoter and its answer
With.
Background technique
The expression of gene is affected by various factors in cell, such as cis-acting elements, trans-acting factor, thin
The adjusting in intracellular growth stage, the expression for the transcription factor being associated with RNA polymerase and other gene levels is related.Its
In, it is the important cis element and gene work of gene expression regulation that promoter, which is the DNA sequence dna for starting specific gene transcription,
One important component of journey expression vector.The power of promoter function is most important for the expression of target gene.
Promoter contains specific DNA sequences, such as provides to the transcription factor of RNA polymerase and recruitment RNA polymerase
Reliable initial binding site.Usually increase the expression quantity of a gene, can be added composing type to it strong promoter or
The promoter of inducible up regulation is reduced or switched off the expression of a gene, can open to what one weak promoter of addition or induction were lowered
Mover.The expression that gene is adjusted by replacement promoter is widely applied in the metabolic pathway for changing yeast.
Yeast promoter has following several types: (1) composing type: being to refer to start gene can in all organizations
The promoter of expression, is not influenced by external condition.This kind of promoter does not need inducer or mortifier, institute's promotor gene
Expression tool duration.(2) induction type: refer under certain specific physically or chemically stimulations of signal, the starting of this type
The transcriptional level of gene can be significantly increased in son.Inducible promoter can control the expression quantity of gene and the table of gene simultaneously
Up to the time.In saccharomyces cerevisiae, a series of diversified endogenous inducible promoters and the inducible promoter being transformed are
It is successfully applied to the regulation of gene expression.Wherein in saccharomyces cerevisiae, control promoter the most rigorous is from gala
Gene GAL1, GAL7, GAL10 of sugar induction.
Constitutive promoter does not show Space-time speciality in above two Yeast promoter, not by the regulation of inducer, no
Can controlling gene at any time expression；It is P that inducible promoter is most commonly used in yeastGAL1, it can be induced by galactolipin,
But need first to carry out galactolipin after gossypose Nature enemy in culture and induce, replacement culture medium takes time and effort.
Summary of the invention
The purpose of the present invention is to provide a kind of saccharomyces neoformans inducible promoter and its applications.
For achieving the above object, the present invention is realized by following measure: a kind of saccharomyces neoformans induction type starting
Son is DNA sequence dna shown in sequence 1 in sequence table；The promoter length is 673bp.
Wherein, preparation process are as follows:
(1) extracting genome DNA of saccharomyces cerevisiae:
1., collect 5ml yeast cells, centrifugation (16000g, 15s) abandon supernatant afterwards, add 230ul DNA lysate be resuspended bacterium
Body；
2., the small bead of 0.4g and 200ul phenol/chloroform/isoamyl alcohol mixed solution, vortex 3min is added；Wherein,
The mass parts ratio of phenol, chloroform and isoamyl alcohol is 25:24:1；
3., centrifugation (16000g, 5min) after water layer is transferred in a new EP pipe；
4., plus 600ul ice ethyl alcohol lotion DNA, EP pipe is then placed on 30min in -20 DEG C；
5., DNA (16000g, 15min) is collected by centrifugation at 4 DEG C, abandon ethyl alcohol, in air dry 3min；
6., with 200ul TE be resuspended DNA, add 37 DEG C of incubation 10min after 5ul RNA enzyme；
7., plus 5M NaCl 8ul and two volumes ice ethyl alcohol, in -20 DEG C of placement 30min；Centrifugation (16000g,
Ethyl alcohol is abandoned after 15min), is placed on air drying；
8., with water/TE DNA is resuspended, obtain pastoris genomic dna；
(2) DDI2 promoter is obtained with high-fidelity Phusion enzyme PCR amplification:
The 50 μ l of total volume of PCR system, wherein 0.5 μ l of Phusion, 10 μ l of Phusion Buffer, 2 μ of dNTP Mix
L, template 100ng, upstream primer (10 μM of concentration) 1 μ l, downstream primer (10 μM of concentration) 1 μ l, H2O are supplied to 50 μ l；Prc reaction
Condition are as follows: 98 DEG C, become under the conditions of 1min in advance, then 98 DEG C, 10s, 55 DEG C, 30s, 72 DEG C, 40s so carry out 30 and follow
Then ring is expanded in 72 DEG C, 10min, amplification obtains DDI2 promoter.
Wherein, the sequence of the primer pair is as follows:
U1:5’-TCTAAGATAAAACACAGATCGAC-3’
U2:5’-GATTGATTCTTTTGAAGAGAAGC-3’。
In addition, the application the present invention also provides the yeast inducible promoter in regulation yeast gene expression:
The yeast is saccharomyces cerevisiae.
Wherein, inducer is cyanamide.
Wherein, the concentration of the cyanamide is 5-8mM.
The invention has the benefit that novel yeast inducible promoter DDI2 of the invention, it can be efficient by cyanamide
Induction, promoter intensity is close with known inducible strong promoter GAL1, stronger than constitutive promoter ADH1, and does not have to replacement training
Base is supported, the expression quantity of gene can be controlled by the amount of induction time and inducer, in the time compared with common GAL1 promoter
Advantage is had more in cost and price cost.This patent is by DDI2 promoter and traditional yeast constitutive promoter ADH1 and induction
Type promoter GAL1 compares, and constructs three kinds " promoter-sf GFP " of single copy plasmid first and is transferred in yeast W303, so
It is taken pictures afterwards with confocal microscope, and detects luciferase expression with flow cytometer (FCM).Flow cytomery result is aobvious
Show that DDI2 promoter intensity is greater than ADH1 promoter, and GAL1 promoter similar intensity, and required induction time is opened less than GAL1
Mover does not have to replacement culture medium；And DDI2 promoter intensity maximum intensity in the case where inducer cyanamide concentration is 5-8mM.
Detailed description of the invention
Fig. 1 is building YCplac111-PADH1The plasmid map of-sfGFP.
Fig. 2 is building YCplac111-PGAL1The plasmid map of-sfGFP.
Fig. 3 is building YCplac111-PDDI2The plasmid map of-sfGFP.
Fig. 4 is that three kinds of promoter regulation sfGFP express the result under Laser Scanning Confocal Microscope.
Fig. 5 is expression intensity of two kinds of promoters in different time down regulation fluorescence.
Fig. 6 is fluorescence intensity of the DDI2 promoter under the induction of various concentration cyanamide.
Fig. 7 is that various concentration cyanamide induces lower DDI2 to regulate and control sfGFP in the expression of translation skill.
Sequence 1 is DDI2 promoter；
Sequence 2 is ADH1 promoter；
Sequence 3 is GAL1 promoter.
Specific embodiment
In order to clarify the technical characteristics of the invention, being illustrated below by specific embodiment to this programme.
The present invention is realized by following measure: a kind of saccharomyces neoformans inducible promoter, for 1 institute of sequence in sequence table
The DNA sequence dna shown；The promoter length is 673bp.
Wherein, preparation process are as follows:
(1) extracting genome DNA of saccharomyces cerevisiae:
1., collect 5ml yeast cells, centrifugation (16000g, 15s) abandon supernatant afterwards, add 230ul DNA lysate be resuspended bacterium
Body；
2., the small bead of 0.4g and 200ul phenol/chloroform/isoamyl alcohol mixed solution, vortex 3min is added；Wherein,
The mass parts ratio of phenol, chloroform and isoamyl alcohol is 25:24:1；
3., centrifugation (16000g, 5min) after water layer is transferred in a new EP pipe；
4., plus 600ul ice ethyl alcohol lotion DNA, EP pipe is then placed on 30min in -20 DEG C；
5., DNA (16000g, 15min) is collected by centrifugation at 4 DEG C, abandon ethyl alcohol, in air dry 3min；
6., with 200ul TE be resuspended DNA, add 37 DEG C of incubation 10min after 5ul RNA enzyme；
7., plus 5M NaCl 8ul and two volumes ice ethyl alcohol, in -20 DEG C of placement 30min；Centrifugation (16000g,
Ethyl alcohol is abandoned after 15min), is placed on air drying；
8., with water/TE DNA is resuspended, obtain pastoris genomic dna；
(2) DDI2 promoter is obtained with high-fidelity Phusion enzyme PCR amplification:
The 50 μ l of total volume of PCR system, wherein 0.5 μ l of Phusion, 10 μ l of Phusion Buffer, 2 μ of dNTP Mix
L, template 100ng, upstream primer (10 μM of concentration) 1 μ l, downstream primer (10 μM of concentration) 1 μ l, H2O are supplied to 50 μ l；Prc reaction
Condition are as follows: 98 DEG C, become under the conditions of 1min in advance, then 98 DEG C, 10s, 55 DEG C, 30s, 72 DEG C, 40s so carry out 30 and follow
Then ring is expanded in 72 DEG C, 10min, amplification obtains DDI2 promoter.
Wherein, the sequence of the primer pair is as follows:
U1:5’-TCTAAGATAAAACACAGATCGAC-3’
U2:5’-GATTGATTCTTTTGAAGAGAAGC-3’。
In addition, the application the present invention also provides the yeast inducible promoter in regulation yeast gene expression:
The yeast is saccharomyces cerevisiae.
Wherein, inducer is cyanamide.
Wherein, the concentration of the cyanamide is 5-8mM.
Embodiment 1, promoter intensity compare
One, the building of recombinant expression carrier
1, with restriction enzyme BamHI and SphI digested plasmid Ycplac111-sfGFP-His6-TCYC1, recycle plasmid
Digestion products；
2, with Phusion enzymatic amplification promoter PADH1、PGAL1、PDDI2；Recycle PCR product；
3, the carrier framework of step 1 is connected respectively with the recovery product of step 2 with Quick-Fusion enzyme, obtains three
Recombinant plasmid (Ycplac111-PADH1-sfGFP-His6-TCYC1、Ycplac111-PGAL1-sfGFP-His6-TCYC1、
Ycplac111-PDDI2-sfGFP-His6-TCYC1), sequence verification is carried out, sequencing result shows to have obtained three kinds of purpose plasmids,
As shown in Figure 1, Figure 2, Figure 3 shows.
Two, confocal laser scanning microscope fluorescence intensity
1, the plasmid of be constructed above three kinds of promoters is transformed into yeast strain W303；
2, bacterial strain (YNB6.7g/L, glucose 20g/L, Ura0.02g/L, Trp0.1g/ are cultivated in SD-Leu culture medium
L, His0.1g/L, Ade0.02g/L, Lys0.1g/L, Met0.1g/L, Arg0.1g/L).Wherein, PGAL1It needs to train in gossypose
Support base in starvation 3h (YNB6.7g/L, gossypose 10g/L, Ura0.02g/L, Trp0.1g/L, His0.1g/L, Ade0.02g/L,
Lys0.1g/L, Met0.1g/L, Arg0.1g/L), after in gala sugar culture-medium induce 3h (YNB6.7g/L, galactolipin 20g/
L, Ura0.02g/L, Trp0.1g/L, His0.1g/L, Ade0.02g/L, Lys0.1g/L, Met0.1g/L, Arg0.1g/L)；
PDDI23h is first cultivated in SD-Leu, afterwards plus 5mM cyanamide induces 3h；PADH16h is cultivated in SD-Leu culture medium；
3, the above-mentioned cultured yeast cells of 1OD is taken respectively, 5000rpm revolving speed is centrifuged 2min at room temperature, cell is collected,
It washed once with 1mlPBS, cell finally be resuspended with 1mlPBS, to micro- sem observation；
4, referring to laser confocal microscope operating method, 60 times of oil mirrors, excitation wavelength 488nm, fluorescence intensity are selected
100%, observation yeast cells green fluorescence under different promoters regulation excites situation.As shown in figure 4, can be from fluorescent brightness
Substantially find out that DDI2 promoter and GAL1 promoter intensity are similar, and intensity is both greater than ADH1 promoter.
Three, flow cytometer (FACS) detects luciferase expression
1, sample is prepared and handled according to the method for front Laser Scanning Confocal Microscope detection, referring to flow cytometer fluorescence detection
Operating method, select 488nm excitation wavelength, detect the luciferase expression of 50000 cells；
2, for PGAL1(2h, 3h, 4h) detects its luciferase expression, P under different induction times respectivelyADH1In different cultures
(2h, 3h, 4h) detects luciferase expression (as shown in Figure 5) under time, PDDI2Cyanamide (the 0- of various concentration is used under 4h induction time
It 8mM) induces, detects its luciferase expression (as shown in Figure 6).
3, every group is tested above in triplicate, and mean fluorescence value is taken to make statistical chart.
It summarizes: being compared by the average fluorescent strength value that three kinds of promoter regulation sfGFP are expressed, it can be seen that yeast inductivity
Promoter DDI2 intensity is bigger, and saves nearly half compared to its incubation time of GAL1 promoter.
Embodiment 2, Western detection cyanamide induce PDDI2Regulate and control sfGFP expression
1, by PDDI2After bacterial strain cultivates 3h in SD-Leu culture medium, various concentration (0-5mM) cyanamide is added and induces 4h, together
When equally cultivated with W303 wild strain and (added 5mM cyanamide) and compare；
2, the bacterium solution for collecting the above-mentioned culture of 5OD, according to the method leach protein of a small amount of total proteins of yeast.The albumen sample that will be obtained
Product are for Western detection (GFP antibody:1:2000).
It summarizes: by Fig. 6,7 it is found that the induced activity of this promoter increases with cyanamide concentration and risen, under 5-8mM concentration
There is stronger activity.
Technical characteristic of the present invention without description can realize that details are not described herein by or using the prior art, certainly,
The above description is not a limitation of the present invention, and the present invention is also not limited to the example above, the ordinary skill of the art
The variations, modifications, additions or substitutions that personnel are made within the essential scope of the present invention also should belong to protection model of the invention
It encloses.
SEQUENCE LISTING
<110>the intelligent industry in Qingdao hundred Biotechnology Co., Ltd
<120>a kind of saccharomyces neoformans inducible promoter and its application
<130> 2017
<160> 3
<170> PatentIn version 3.5
<210> 1
<211> 673
<212> DNA
<213>artificial sequence
<400> 1
tctaagataa aacacagatc gacagatccg agagttggct tctgtgcctt gggctcaaat 60
tcctttccca cctcatgcaa attgattttt ctgactccaa aaaaagacag agccctgcga 120
tagttcccga atgttgtaac atcaaagcca agcactcctt tatagaagtc gcatgaacgt 180
tgaatactag cagctggtga aactacaggg tctaaactaa ctagtatcca tatctttttg 240
agagcattga aagtatacgg agtacaagct gggttagaag gaatttttat cttaacagca 300
atgaaaatca actttctaga ctgaatccct caagaaaatt gcaaaagact aactgatact 360
ggtttaaaag agaaagatgt caaatatgcg gagttatacc atcaaacaac tttggacggc 420
cccgaaacaa atgtccgcaa aaaagatctt attaaagtgc atggacacta tcatttctat 480
aatacaaaat actccaccgc acaatagttt gtcgggaagt catcaatcaa tcttgtacga 540
gctttacaaa taacttttta ggatcggtcc cctcataaaa ttatatataa atgggttagt 600
ttccttcttc ttctgttaac atgaagttgc ttcgtactgt tttttgcctt gcttctcttc 660
aaaagaatca atc 673
<210> 2
<211> 400
<212> DNA
<213>artificial sequence
<400> 2
gcatgcaact tcttttcttt ttttttcttt tctctctccc ccgttgttgt ctcaccatat 60
ccgcaatgac aaaaaaatga tggaagacac taaaggaaaa aattaacgac aaagacagca 120
ccaacagatg tcgttgttcc agagctgatg aggggtatct cgaagcacac gaaacttttt 180
ccttccttca ttcacgcaca ctactctcta atgagcaacg gtatacggcc ttccttccag 240
ttacttgaat ttgaaataaa aaaaagtttg ctgtcttgct atcaagtata aatagacctg 300
caattattaa tcttttgttt cctcgtcatt gttctcgttc cctttcttcc ttgtttcttt 360
ttctgcacaa tatttcaagc tataccaagc atacaatcaa 400
<210> 3
<211> 442
<212> DNA
<213>artificial sequence
<400> 3
cggattagaa gccgccgagc gggtgacagc cctccgaagg aagactctcc tccgtgcgtc 60
ctcgtcttca ccggtcgcgt tcctgaaacg cagatgtgcc tcgcgccgca ctgctccgaa 120
caataaagat tctacaatac tagcttttat ggttatgaag aggaaaaatt ggcagtaacc 180
tggccccaca aaccttcaaa tgaacgaatc aaattaacaa ccataggatg ataatgcgat 240
tagtttttta gccttatttc tggggtaatt aatcagcgaa gcgatgattt ttgatctatt 300
aacagatata taaatgcaaa aactgcataa ccactttaac taatactttc aacattttcg 360
gtttgtatta cttcttattc aaatgtaata aaagtatcaa caaaaaattg ttaatatacc 420
tctatacttt aacgtcaagg ag 442
Claims (4)
1. application of the primary yeast inducible promoter in regulation yeast gene expression: the yeast is saccharomyces cerevisiae, induction
Object is cyanamide；The yeast inducible promoter is DNA sequence dna shown in sequence 1 in sequence table；The promoter length is
673bp。
2. application according to claim 1, which is characterized in that the preparation process of the yeast inducible promoter are as follows:
(1) extracting genome DNA of saccharomyces cerevisiae:
1., collect 5ml yeast cells, under the conditions of 16000g, 15s be centrifuged after abandon supernatant, add 230ul DNA lysate be resuspended bacterium
Body；
2., the small bead of 0.4g and 200ul phenol/chloroform/isoamyl alcohol mixed solution, vortex 3min is added;Wherein, phenol,
The mass parts ratio of chloroform and isoamyl alcohol is 25:24:1；
3., under the conditions of 16000g, 5min be centrifuged after water layer is transferred in a new EP pipe；
4., plus 600ul ice ethyl alcohol lotion DNA, EP pipe is then placed on 30min in -20 DEG C;
5., be collected by centrifugation DNA under the conditions of 4 DEG C, 16000g, 15min, abandon ethyl alcohol, dry 3min in air;
6., with 200ul TE be resuspended DNA, add 37 DEG C of incubation 10min after 5ul RNA enzyme;
7., plus 5M NaCl 8ul and two volumes ice ethyl alcohol, in -20 DEG C of placement 30min；In the condition of 16000g, 15min
Ethyl alcohol is abandoned after lower centrifugation, is placed on air drying；
8., with water/TE DNA is resuspended, obtain pastoris genomic dna；
(2) DDI2 promoter is obtained with high-fidelity Phusion enzyme PCR amplification:
The 50 μ l of total volume of PCR system, wherein 0.5 μ l, Phusion Buffer of Phusion, 10 μ l, dNTP Mix 2
μ l, 100 ng of template, 10 μM of upstream primer concentration, 1 μ l, 10 μM of downstream primer concentration, 1 μ l, H2O is supplied to 50 μ
l；Prc reaction condition are as follows: 98 DEG C, become under the conditions of 1min in advance, then 98 DEG C, 10s, 55 DEG C, 30s, 72 DEG C, 40s, so
30 circulations are carried out, are then expanded in 72 DEG C, 10min, amplification obtainsDDI2Promoter.
3. application according to claim 2, which is characterized in that the sequence of the primer pair is as follows:
U1: 5’-TCTAAGATAAAACACAGATCGAC-3’
U2: 5’-GATTGATTCTTTTGAAGAGAAGC-3’。
4. application according to claim 1-3, which is characterized in that the concentration of the inducer cyanamide is 5-8mM.
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Effective date of registration: 20210302 Address after: 266109 304b, building 6, blue biomedical industrial park, 368 Hedong Road, high tech Zone, Qingdao City, Shandong Province Patentee after: Qingdao Mingde Biotechnology Research Institute Co.,Ltd. Address before: No.966, wanglinzhuang community, Chengyang District, Qingdao City, Shandong Province, 266109 Patentee before: QINGDAO BAIHUIZHIYE BIOTECHNOLOGY Co.,Ltd. |
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