CN104630080B - The yeast strain of low protease A excretion under one plant of stress conditions - Google Patents
The yeast strain of low protease A excretion under one plant of stress conditions Download PDFInfo
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Abstract
The present invention provides one kind adjusting the exocrine new method of protease A intracellular under stress conditions, is to control the acceptor gene VPS10 that protease A is sorted from golgiosome to vacuole by overexpression to realize.It is overexpressed the bacterial strain of VPS10, intracellular enzyme activity improves 1.47 times, and enzymatic activities are reduced to the 61% of starting strain, while the fermentation character for being overexpressed bacterial strain is not substantially change.The present invention provides a kind of new idea and method not change fermentation character while reduction protease A enzymatic activities, has directive significance to improving industrial yeast foam stability.
Description
Technical field：
The invention belongs to technical field of bioengineering, it is related to the breeding of industrial microorganism, especially one plant in fermentation latter stage
It is suitble to yeast strain and its construction method of low protease A excretion under stress conditions.
Background technology：
During draft beer develops, some quality problems in relation to draft beer annoying always brewing engineering
The foam stability of teacher, wherein draft beer are particularly problematic.Since draft beer is before packing without pasteurization,
It ferments latter stage, nitrogen source is insufficient, and high alcohol and gas concentration lwevel can cause a stressful environmental to yeast, lead to protease A
Excretion, decompose foam active-protein, reduce beer steep holding property, have an adverse effect to the foam quality of draft beer.Therefore,
The foam stability for improving draft beer, becoming the world today improves a significant technology issues of draft beer quality.
Although by optimization for fermentation technology, or addition foam stabiliser can improve beer foam to a certain extent
Stability, but this method cannot all solve the problems, such as foam attenuation from source.Albumen is generated by knockout in genetic engineering
The gene PEP4 of enzyme A reduces protease A excretion, to making the stability of beer increase.But due to A pairs of intracellular protein enzyme
The biochemical functions of brewing yeast cell play a very important role, and such as participate in the maturation of a variety of enzymes in vacuole and activate
Journey；Influence the normal expression etc. of some intracellular glycometabolism enzymes.So the fermentation character of PEP4 deletion mycopremnas can be by intracellular egg
The influence of white enzyme A missings.
The secretion process of Saccharomyces cerevisiae protein enzyme A is under the conditions of eubolism, and preceding protease A former (preproPrA) is thick
After the endoplasmic reticulum synthesis of face, the modification and folding of endoplasmic reticulum are first passed through, then transport golgiosome, finally determined by vacuole sorting receptor
Ripe protease A (PrA) is formed to transport to vacuole and in vacuole.But as fermentation later stage various nutriments lack
It will mistakenly be transported to extracellular under the weary, stress conditions such as nitrogen source is insufficient, and be activated extracellular.
Under the conditions of eubolism, VPS10 as encoding vacuolar sort receptor gene, protease A from golgiosome to
It plays an important role during vacuole orientation secretion.When the recognition site and sorting receptor-specific on protease A original propetide
In conjunction with Golgi network is transported to vacuole to protease A original from the negative, and is activated after reaching vacuole, by 54 amino
The propetide of sour residue composition is removed, and eventually forms molecular weight about 42kD active proteases A.
However, at present under stress conditions, protease A is controlled by regulating and controlling the expression quantity of vacuole sorting receptor
Endo-exocrine research report is seldom, is even more sky especially in terms of the research of the yeast strain of the low protease A excretion of selection and breeding
In vain, therefore, the present invention is lacked by the way that a nitrogen source is arranged, and the stressful environmental of high alcohol and gas concentration lwevel, carrys out mould
The fermentation condition in quasi- beer fermentation later stage, knocks out and is overexpressed VPS10 genes, to regulate and control vacuole expression of receptor amount, in turn respectively
The low yield protease A bacterial strain for being suitble to fermentation is selected, this method is for improving beer foam stability, especially draft beer
Foam stability have directive significance.
Invention content：
The exocrine new method of modulin enzyme A intracellulars of the present invention is by being overexpressed saccharomyces cerevisiae starting strain
The VPS10 gene complete sequences of middle encoding vacuolar sorting receptor, obtain low protease A Wine brewing yeast strain.
Its Gene ID of the VPS10 genes is：852264, SEQ ID NO in nucleotide sequence such as sequence table:Shown in 1.
Preferably, the saccharomyces cerevisiae starting strain is type strain W303-1A；
The construction method of the Wine brewing yeast strain of the low protease A secretion, includes the following steps：
(1) first the strong promoter PGK1p-PGK1t segments on pPGK1 plasmids are inserted on YEP352 expression vectors, then
VPS10 genes are inserted between promoter PGK1p and terminator PGK1T, KanMX resistance markers are finally inserted into expression matter
Grain.
(2) recombinant expression plasmid is imported into starting strain, obtains the recombinant bacterial strain WGV10 for being overexpressed VPS10.
It is specific as follows：
(1) structure of Yep-PVK plasmids
1. using pPGK1 plasmids as template, PCR amplification goes out strong promoter PGK1p-PGK1t genetic fragments；
2. using yeast starting strain W303-1A total DNAs template, PCR amplification goes out VPS10 genes；
3. using pUC6 plasmids as template, PCR amplification goes out KanMX resistant genes；
PCR product is consecutively connected on YEP352 expression vectors, recombinant plasmid Yep-PVK is obtained；
(2) VPS10 is overexpressed the structure of bacterial strain
Free overexpression plasmid Yep-PVK is imported by lithium acetate transformation method in starting strain W303-1A, is obtained
VPS10 is free to be overexpressed recombinant bacterial strain.
The recombinant bacterial strain can be built by the above method and be obtained, and there are many document reports, such as Joseph for these methods
Sambrook etc.,《Molecular Cloning:A Laboratory guide》The second edition, Science Press, 1995.Also other sides known in the art can be used
Method builds the yeast strain of gene mutation.
Invention also provides a kind of dedicated for identifying the gene sequence of the low extracellular proteinase A Wine brewing yeast strains
Row, the gene order are somebody's turn to do using the low extracellular proteinase A Wine brewing yeast strains genome as the specific fragment of template amplification
SEQ ID NO in the nucleotide sequence of specific fragment such as sequence table:Shown in 2.
The overexpression bacterial strain that the present invention is obtained ferments in nitrogen source lacks brewer's wort, the enzyme activity of protease A in zymotic fluid
It is substantially reduced compared with starting strain, and other fermenting properties are uninfluenced.
Advantageous effect：
1, the present invention is knocked out by beer fermentation experimental verification VPS10 and is overexpressed to protease A endo-exocrine and fermentation
The influence of characteristic, it is determined that VPS10 protease A from the negative golgiosome to vacuolar transport during play important sorting and make
With having established theoretical foundation for the research in the field.
2, it is overexpressed the outer enzyme activity of VPS10 genetic testing intracellular, in fermentation ends, intracellular enzyme activity improves 1.47 times；And
Enzymatic activities are reduced to the 61% of starting strain, while fermentation character is compared with starting strain and had not significant impact substantially, to refer to
It leads Beer Industry and lowers extracellular protease A enzyme activity, improve foam stability and provide new idea and method.
Description of the drawings
The structure flow diagram of Fig. 1 recombinant plasmids pUC-VABK；
The verification electrophoretogram of Fig. 2 recombinant plasmids pUC-VABK；
Wherein swimming lane M is 5000bp DNA Ladder Marker；Swimming lane 1 is VA-U and VB- using pUC-VABK as template
D is primer, and PCR verifies homology arm VA on 966bp；Swimming lane 2 is using pUC-VABK as template, and VB-U and VB-D are primer, and PCR is tested
Demonstrate,prove homology arm VB under 1050bp；Swimming lane 3 is using pUC-VABK as template, and KAN-U and KAN-D are primer, and PCR verifies 1613bp
KanMX marker gene；Swimming lane 4 is using pUC-VABK as template, and VA-U and VB-D are primer, the 3629bp recombination boxes expanded
VA-loxP-KanMX-loxP-VB。
The homologous recombination schematic diagram of Fig. 3 box gene VA-KanMX-VB segments and Yeast genome；
Fig. 4 VPS10 missing recombinant bacterial strain verifications；
Wherein swimming lane M is 5000bp DNA Ladder Marker；Swimming lane 1,2 is tested using VA-S and KV-X as primer
Card；Swimming lane 3,4 is verified using KV-S and VB-X as primer；Wherein, swimming lane 1,3 template is starting strain W303-1A, not
Band can be amplified；The template of swimming lane 2,4 is deletion mutant strain W Δ VPS10, can amplify 1835bp, 2498bp respectively
The band of size.
The structure flow diagram of Fig. 5 recombinant plasmids Yep-PVK；
The verification electrophoretogram of Fig. 6 recombinant plasmids Yep-PVK；
Wherein swimming lane M is 5000bp DNA Ladder Marker；Swimming lane 1 be using Yep-PVK as template, VPS10-U and
VPS10-D is primer, and PCR verifies the VPS10 genetic fragments of 4740bp；Swimming lane 2 is KAN-U and KAN- using Yep-PVK as template
D is primer, and PCR verifies the KanMX genetic fragments of 1613bp；Swimming lane 3 is using Yep-PVK as template, and PG-S and VP-X are primer,
PCR verifies the connection direction of VPS10, primer size 1681bp；
The free bacterial strains that are overexpressed of Fig. 7 VPS10 carry yeast plasmid proof diagram
Wherein swimming lane M is 5000bp DNA Ladder Marker；Swimming lane 1,2 is tested using PG-S and VP-X as primer
Card；Wherein the template of swimming lane 1 is starting strain W303-1A, cannot amplify band；The template of swimming lane 2 is to be overexpressed bacterial strain
WGV10, it is amplifiable go out 1681bp sizes specific band.
Fig. 8 deletion mutant strains and the outer enzyme activity variation tendency of starting strain intracellular
Fig. 9 is overexpressed bacterial strain and the outer enzyme activity trend of starting strain intracellular
Bacterial strain and starting strain α-amino nitrogen is overexpressed in Figure 10 fermentation process to become reconciled sugared rate variation tendency
Specific implementation mode
The present invention is described below by specific embodiment.Unless stated otherwise, technological means used in the present invention
It is method known in those skilled in the art.In addition, embodiment is interpreted as illustrative, it is not intended to limit the present invention
Range, the spirit and scope of the invention are limited only by the claims that follow.To those skilled in the art, without departing substantially from this
Under the premise of invention spirit and scope, various changes or change to material component and dosage progress in these embodiments
It belongs to the scope of protection of the present invention.
Laboratory yeast body used in the present invention is the saccharomyces cerevisiae haploid strains that any source may be used.
Embodiment 1：The structure of deletion mutant strain
(1) structure of pUC-VABK plasmids
The structure flow of recombinant plasmid pUC-VABK is as shown in Figure 1.
1. using yeast starting strain W303-1A total DNAs template, PCR amplification goes out the upstream sequence VA of VPS10 genes；
Sense primer VA-U：CCGGAATTCGAGTTTATGAGAAGGTTGGAGG(SEQ ID NO:3)
Downstream primer VA-D：CGGGGTACCAAAGACCCGAGTAGTTGGAG(SEQ ID NO:4)
Dashed part is restriction enzyme site；
PCR reaction conditions：95℃5min；94℃45s；63℃1min；72 DEG C of 60s, 30 cycles；72 DEG C of 10min,
0.8% agarose gel electrophoresis identifies amplified production；
PCR reaction systems (20 μ L)
PCR product is connected on the pUC19 plasmid vectors containing Amp resistances, recombinant plasmid pUC-VA is obtained.
2. using yeast starting strain W303-1A total DNAs template, PCR amplification goes out the downstream sequence VB of VPS10 genes；
Sense primer VB-U：CGCGGATCCAGAATAAGAAAAGGCAGATA(SEQ ID NO:5)
Downstream primer VB-D：CGCTGCAGACAAGTTCCCATACCAAAAT(SEQ ID NO:6)
Dashed part is restriction enzyme site
PCR reaction conditions：95℃5min；94℃45s；60℃1min；72 DEG C of 60s, 30 cycles；72 DEG C of 10min,
0.8% agarose gel electrophoresis identifies amplified production；
PCR product is connected on recombinant plasmid pUC-VA, recombinant plasmid pUC-VAB is obtained.
3. using pUC6 plasmids as template, PCR amplification goes out KanMX resistant genes；
Sense primer KAN-U：GGGGTACCCAGCTGAAGCTTCGTACGC(SEQ ID NO:7)
Downstream primer KAN-D：CGGGATCCGCATAGGCCA CTAGTGGATCTG(SEQ ID NO:8)
Dashed part is restriction enzyme site
PCR reaction conditions：95℃5min；94℃45s；61℃1min；72 DEG C of 90s, 30 cycles；72 DEG C of 10min,
0.8% agarose gel electrophoresis identifies amplified production；
PCR product is connected on recombinant plasmid pUC-VAB, it is recombinant plasmid to obtain recombinant plasmid pUC-VABK, Fig. 2
PCR proof diagrams.
(2) structure of VPS10 deletion mycopremnas
Box gene VA-KanMX-VB segments and Yeast genome regrouping process are as shown in Figure 3.
Using recombinant plasmid pUC-VABK as template, PCR amplification is obtained recombinates box VA-KanMX-VB up to 3629bp, uses vinegar
Sour lithium conversion method is transformed into respectively in saccharomyces cerevisiae W303-1A, and the saccharomyces cerevisiae VPS10 missing bacterium after homologous recombination are obtained
Strain W Δ VPS10, and preserve bacterial strain.
Recombinant bacterial strain W Δs VPS10 is subjected to PCR verifications, to lack the genome of recombinant bacterial strain W Δs VPS10 as template,
VA-S/KV-X is that primer carries out PCR amplification.PCR reaction conditions：95℃5min；94℃45s；52℃1min；72 DEG C of 100s, 30
A cycle；72 DEG C of 10min, 0.8% agarose gel electrophoresis identify amplified production, the specific band (SEQ of 1835bp can be obtained
ID NO:19), and starting strain genome cannot amplify segment.To lack the genome of recombinant bacterial strain W Δs VPS10 as mould
Plate, KV-S/VB-X are that primer carries out PCR amplification.PCR reaction conditions：95℃5min；94℃45s；50℃1min；72 DEG C of 150s,
30 cycles；72 DEG C of 10min, 0.8% agarose gel electrophoresis identify amplified production, the specific band of 2498bp can be obtained
(SEQ ID NO:20) it is, to compare that segment cannot be amplified with starting strain genome.The result shows that in Yeast genome
Realize the replacement of VPS10 genes.Fig. 4 is that VPS10 lacks recombinant bacterial strain verification.
Two pairs of specificity verification primers of deletion mycopremna W Δs VPS10：
Sense primer VA-S：CCTCCTTAGCAGTAATCCTC(SEQ ID NO:9)
Downstream primer KV-X：AGAACCTCAGTGGCAAATCC(SEQ ID NO:10)
Sense primer KV-S：TCTCACATCACATCCGAACA(SEQ ID NO:11)
Downstream primer VB-X：GATTCACTTTTACCAGACGC(SEQ ID NO:12)
Embodiment 2：It is overexpressed the structure of bacterial strain
(1) structure of Yep-PVK plasmids
The structure flow of recombinant plasmid Yep-PVK is as shown in Figure 5.
1. using pPGK1 plasmids as template, PCR amplification goes out strong promoter PGK1p-PGK1t genetic fragments；
Sense primer PGK-U：CGCGGATCCTCTAACTGAT CTATCCAAAACTGA(SEQ ID NO:13)
Downstream primer PGK-D：CGCGTCGACTAACGAACGCAGAATTTTC(SEQ ID NO:14)
Dashed part is restriction enzyme site
PCR reaction conditions：95℃5min；94℃45s；61℃1min；72 DEG C of 100s, 30 cycles；72 DEG C of 10min,
0.8% agarose gel electrophoresis identifies amplified production；
PCR product is connected on expression vector YEP352, recombinant plasmid Yep-P is obtained.
2. using yeast starting strain W303-1A total DNAs template, PCR amplification goes out VPS10 genes；
Sense primer VPS10-U：TCCAGATCTCCTCGAGATGATATTACTTCATTTTGTCTATTC(SEQ ID NO:
15)
Downstream primer VPS10-D：TCGCAGATCCCTCGAGCTACTGGTTTTCGTTAGATGGCGC(SEQ ID NO:
16)
Dashed part is restriction enzyme site
PCR reaction conditions：95℃5min；94℃45s；60℃1min；72 DEG C of 5min, 30 cycles；72 DEG C of 10min,
0.8% agarose gel electrophoresis identifies amplified production；
PCR product is connected on recombinant plasmid Yep-P, recombinant plasmid Yep-PV is obtained.
3. using pUC6 plasmids as template, PCR amplification goes out KanMX resistant genes；
Sense primer KAN-U：GGGGTACCCAGCTGAAGCTTCGTACGC(SEQ ID NO:7)
Downstream primer KAN-D：CGGGATCCGCATAGGCCA CTAGTGGATC TG(SEQ ID NO:8)
Dashed part is restriction enzyme site
PCR reaction conditions：95℃5min；94℃45s；61℃1min；72 DEG C of 90s, 30 cycles；72 DEG C of 10min,
0.8% agarose gel electrophoresis identifies amplified production；
PCR product is connected on recombinant plasmid Yep-PV, it is recombination plasmid PCR to obtain recombinant plasmid Yep-PVK, Fig. 6
Proof diagram.
(2) VPS10 is overexpressed the structure of bacterial strain
Free overexpression plasmid Yep-PVK is imported by lithium acetate transformation method in starting strain W303-1A, is obtained
VPS10 is free to be overexpressed recombinant bacterial strain WGV10, and preserves strain.
Recombinant bacterial strain WGV10 is subjected to PCR verifications, the plasmid of extraction recombinant bacterial strain WGV10 is template, and PG-S/VP-X is
Primer carries out PCR amplification.PCR reaction conditions：95℃5min；94℃45s；48℃1min；72 DEG C of 100s, 30 cycles；72℃
10min, 0.8% agarose gel electrophoresis identifies amplified production, the specific band of 1681bp can be obtained, and starting strain cannot
Amplify segment.Fig. 7 is the verification electrophoretogram for being overexpressed recombinant bacterial strain WGV10.
It is overexpressed a pair of of specificity verification primer of bacterial strain WGV10：
Sense primer PG-S：TAACTGATCTATCCAAAACTGAA(SEQ ID NO:17)
Downstream primer VP-X：GCTTATAGTAACGGAGGTGTCTT(SEQ ID NO:18)
Embodiment 3：Beer fermentation is tested
Using the recombinant bacterial strain of the acquisition of embodiment 1,2 and starting strain W303-1A as experimental subjects,
(1) seed culture
1) actication of culture：Strain transfer is preserved to 30 DEG C of YEPD slant tubes activation culture two days.
2) first order seed culture：Take one ring of slant strains, be inoculated in the test tube for filling 5mL wheat juice culture mediums, 30 DEG C,
14h is cultivated under the conditions of 180rpm.
3) secondary seed culture：Primary seed solution is filled the 150mL triangular flasks of 50mL wheat juice by 10% inoculum concentration access
It is interior, 16 DEG C of stationary culture 36h.
(2) beer fermentation
Fermenting experiment：Secondary seed solution is filled the wheat juice of 150mL 10Brix nitrogen shortage by 10% inoculum concentration access
In 250mL triangular flasks, fermentation bung is covered, it is made to keep the pressure in the bottle, 16 DEG C of standing for fermentation.It is surveyed from fermentation animated period the 5th day
PrA vigor (intracellular and extracellular) took sample (to keep the pressure in the bottle constant, by the way that multigroup parallel take is arranged every 2-3 days
Sample).By above-mentioned fermentation condition to deletion mycopremna, it is overexpressed bacterial strain and starting strain carries out beer fermentation experiment respectively.Fermentation knot
After beam, the remaining sugar concentration of zymotic fluid is measured, alcohol by volume score characterizes it and integrates fermenting property.
(3) VPS10 is knocked out on the endo-exocrine influence of protease A
For intracellular enzyme activity (Fig. 8), during entire fermentation, it has been found that deletion mycopremna W Δs VPS10 is with starting strain
W303-1A is compared, and enzyme activity has reduction；In 13 days fermentation ends, the intracellular enzyme activity of deletion mycopremna W Δs VPS10 was bacterium germination
The 53% of strain；For enzymatic activities, the enzyme activity of deletion mycopremna W Δs VPS10 is higher by 2.2 times than starting strain.Data result shows
Under stress conditions, while not changing protease A expression quantity, VPS10 genes are knocked out, protease A can be reduced and sorted to vacuole
Efficiency, while can make the protease A content being secreted into zymotic fluid increase.Illustrate under stress conditions, VPS10 genes are in albumen
Enzyme A plays important sorting during being transported from golgiosome to vacuole orientation.
(4) VPS10 is overexpressed on the endo-exocrine influence of protease A
For intracellular enzyme activity (Fig. 9), bacterial strain WGV10 is overexpressed during entire fermentation with starting strain W303-1A phases
Specific enzyme activity is improved, and in fermentation ends, improves 1.47 times；And enzymatic activities are reduced to the 61% of starting strain.Data
As a result it further demonstrates that, under stress conditions, while not changing protease A expression quantity, is overexpressed VPS10 genes, makes albumen
Efficiency of the enzyme A from reverse side golgiosome to vacuolar transport increases, while making to be secreted into the reduction of the protease A vigor in zymotic fluid.
We, which continue to compare, is overexpressed α-amino nitrogen in the zymotic fluid of bacterial strain and starting strain, the variation tendency of apparent pol, to see
Survey the change for the fermentation character for being overexpressed bacterial strain.
As shown in Figure 10, during the fermentation, it is overexpressed the α-amino nitrogen concentration table of bacterial strain and sees pol and starting strain
Compared to not changing significantly, but in fermentation latter stage, starting strain will be less than by being overexpressed the α-amino nitrogen concentration of bacterial strain, this
Be due to, fermentation latter stage be the nitrogen source opposite period lacked and yeast under stress conditions a large amount of extracellular proteinase A when
Phase, the protease A content for being overexpressed bacterial strain fermentation liquor is relatively low, and albumen is that the ability of amino acid subtracts in protease A decomposing and fermenting liquid
It is weak, so the α-amino nitrogen content in its zymotic fluid is also relatively low.It is steady to avoid the protease A that latter stage largely secretes from influencing foam
It is qualitative, yeast can be detached in time at the end of consuming sugar, therefore the relatively low α-amino nitrogen content being overexpressed in bacterial strain fermentation liquor is not
It can influence to ferment.
We compare the residual sugar and alcoholic strength (the results are shown in Table 1) of two plants of bacterium after fermentation ends, find substantially not bright
Aobvious difference.In summary index, the fermentation character for being overexpressed bacterial strain do not change substantially.
Table 1 is overexpressed the comparison of bacterial strain and starting strain alcoholic strength and residual sugar
Note：Shown data are the average value of three parallel test results.
Claims (4)
1. a kind of low extracellular proteinase A Wine brewing yeast strains are in high foam stability beer fermentation process to extracellular low secretion egg
Purposes in white enzyme A, which is characterized in that the bacterial strain sorts the VPS10 of receptor by being only overexpressed vacuole in starting strain
Gene complete sequence obtains；
The starting strain is pattern bacterium W303-1A；
Its Gene ID of the VPS10 genes is：852264；
The beer fermentation uses the wheat juice that nitrogen lacks.
2. purposes as described in claim 1, which is characterized in that the structure side of the low extracellular proteinase A Wine brewing yeast strains
Method includes the following steps：
(1) the strong promoter PGK1p- terminator PGK1t segments on pPGK1 plasmids are inserted on YEP352 expression vectors, then
VPS10 genes are inserted between promoter PGK1p and terminator PGK1t, KanMX resistance markers are finally inserted into expression matter
Grain；
(2) recombinant expression plasmid is imported into starting strain, obtains the recombinant bacterial strain WGV10 for being overexpressed VPS10.
3. purposes according to claim 1, which is characterized in that the construction method of low extracellular proteinase A Wine brewing yeast strains
Include the following steps：
(1) structure of Yep-PVK plasmids
1. using pPGK1 plasmids as template, PCR amplification goes out strong promoter PGK1p- terminator PGK1t genetic fragments；
2. using yeast starting strain W303-1A total DNAs template, PCR amplification goes out VPS10 genes；
3. using pUC6 plasmids as template, PCR amplification goes out KanMX resistant genes；
PCR product is consecutively connected on YEP352 expression vectors, recombinant plasmid Yep-PVK is obtained；
(2) VPS10 is overexpressed the structure of bacterial strain
Free overexpression plasmid Yep-PVK is imported by lithium acetate transformation method in starting strain W303-1A, VPS10 is obtained
It is free to be overexpressed recombinant bacterial strain.
4. purposes as described in claim 1, which is characterized in that detect low extracellular proteinase A Wine brewing yeast strain methods be with
The total DNA of recombinant bacterial strain is template, passes through primer pair：
Sense primer：TAACTGATCTATCCAAAACTGAA
Downstream primer：GCTTATAGTAACGGAGGTGTCTT
PCR amplification is carried out, can get the specific band that a length is 1681bp, the specific band nucleotide sequence is such as
Sequence table SEQ ID NO:Shown in 2；
Above-mentioned specific band occur then proves that it is low extracellular proteinase A Wine brewing yeast strains to be detected bacterial strain.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811001165.5A CN108866027B (en) | 2014-12-02 | 2014-12-02 | Application of VPS10 gene in low-secretion protein A of saccharomyces cerevisiae strain |
| CN201410720237.7A CN104630080B (en) | 2014-12-02 | 2014-12-02 | The yeast strain of low protease A excretion under one plant of stress conditions |
Applications Claiming Priority (1)
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