CN103555720B - A kind of structure with the yeast strain of production and recycled fiber element restriction endonuclease dual-use function - Google Patents
A kind of structure with the yeast strain of production and recycled fiber element restriction endonuclease dual-use function Download PDFInfo
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a kind of structure with the yeast strain of production and recycled fiber element restriction endonuclease dual-use function, belong to enzyme engineering field.By yeast saccharomyces cerevisiae TDH3 promoter sequence, yeast saccharomyces cerevisiae secretion signal peptide-coding sequence, Aspergillus fumigatus Mierocrystalline cellulose endonuclease coding sequence, 400 amino acid fragment encoding sequences of yeast saccharomyces cerevisiae AgF C end, yeast saccharomyces cerevisiae TDH3 terminator sequence are synthesized in order, restriction endonuclease sites is added at two ends, cut by enzyme and be building up in pPIC9K plasmid, then the yeast strain with production and recycled fiber element restriction endonuclease dual-use function will be obtained in this Plastid transformation to yeast.The rate of recovery of its production of cellulose restriction endonuclease reaches 86%, and after fermentation, the enzyme of restriction endonuclease is lived and reached 8.5 U/g and to wet yeast.The step that present invention achieves the production of Mierocrystalline cellulose restriction endonuclease and synchronous concentration and recovery completes, and reduces energy consumption, reduces cost, have stronger practicality.
Description
Technical field
The invention belongs to enzyme engineering field, be specifically related to a kind of structure with the yeast strain of production and recycled fiber element restriction endonuclease dual-use function.
Background technology
Mierocrystalline cellulose is renewable resources the abundantest on the earth, and the Mierocrystalline cellulose that the earth produces because of photosynthesis every year reaches about 10,000,000,000 tons, utilizes cellulose raw to produce bioenergy, is alleviating energy crisis, realizes the key of human kind sustainable development.
The key of cellulose utilization is the carbohydrate-glucose of fermentability cellulose degradation.Cellulosic degraded needs the acting in conjunction of excision enzyme, restriction endonuclease, dextran glycosides enzyme.Wherein the Mierocrystalline cellulose of endonucleases long segment becomes dimer, is the committed step of cellulose degradation.With cellulose degraded Mierocrystalline cellulose, there is green, mild condition, feature that transformation efficiency is high, but the higher production cost of Mierocrystalline cellulose becomes the cellulosic bottleneck of cellulose degraded.
It is large that liquid fermenting has fermentation volume, easily realizes the features such as Automated condtrol, be widely used for production of cellulose restriction endonuclease etc.But the enzyme product concentration that liquid fermenting is produced is low, and the Mierocrystalline cellulose restriction endonuclease etc. of production is all free in fermentation liquid, is the pressed powder of the Mierocrystalline cellulose restriction endonuclease or restriction endonuclease that obtain high density, needs to concentrate fermentation liquid.For purified concentration Mierocrystalline cellulose restriction endonuclease often needs to be concentrated by the technique such as vacuum-evaporation or spraying dry.In Mierocrystalline cellulose evaporation concentration or spray-dired process, a loss of activity part for Mierocrystalline cellulose restriction endonuclease, and also concentration technology has increased substantially the production cost of cellulase.In evaporation or spray-dired process, consume a large amount of energy as electricity or combustion gas etc.Therefore concentrated cost, becomes the inevitable choice reducing liquid fermenting production of cellulose restriction endonuclease production cost.
Summary of the invention
Primary and foremost purpose of the present invention is that the shortcoming overcoming prior art is with not enough, provides a kind of yeast strain with production and recycled fiber element restriction endonuclease dual-use function.
Another object of the present invention is to the DNA fragmentation being provided for building above-mentioned yeast strain.
Another object of the present invention is the plasmid being provided for building above-mentioned yeast strain.
The present invention also aims to the construction process that above-mentioned yeast strain is provided.
Object of the present invention is achieved through the following technical solutions:
Produce and the DNA fragmentation of yeast strain of recycled fiber element restriction endonuclease dual-use function for building to have, comprise arrange in order yeast saccharomyces cerevisiae TDH3 promoter sequence, yeast saccharomyces cerevisiae secretion signal peptide-coding sequence, Aspergillus fumigatus Mierocrystalline cellulose endonuclease coding sequence, yeast saccharomyces cerevisiae AgF C end 400 amino acid fragment encoding sequences, yeast saccharomyces cerevisiae TDH3 terminator sequence; Above-mentioned sequence is respectively as shown in SEQ ID NO.1 ~ 5.
Preferably, the sequence of the described DNA fragmentation for building the yeast strain with production and recycled fiber element restriction endonuclease dual-use function is as shown in SEQ ID NO.6.
To produce and the plasmid of yeast strain of the plain restriction endonuclease dual-use function of recycled fiber is the eukaryon expression plasmid comprising above-mentioned DNA fragmentation for building to have.Described eukaryon expression plasmid is preferably pPIC9K plasmid.
Described prepares preferably by the method comprising following steps for the plasmid building the yeast strain with production and recycled fiber element restriction endonuclease dual-use function: synthesis two ends restricted property endonuclease recognized site contains the sequence of above-mentioned DNA fragmentation, is connected on eukaryon expression plasmid by restriction enzyme.During described eukaryon expression plasmid pPIC9K plasmid, restriction enzyme is preferably Bgl II and FspA I.
There is a yeast strain for production and recycled fiber element restriction endonuclease dual-use function, containing above-mentioned plasmid.Described yeast strain is preferably yeast strain SMD1168.
The described construction process with the yeast strain of production and recycled fiber element restriction endonuclease dual-use function, comprises the steps: to prepare yeast Electroporation-competent cells, is transformed to enter in yeast by above-mentioned plasmid to obtain by electricity.
The present invention has the following advantages and effect relative to prior art tool:
Yeast strain of the present invention while production of cellulose restriction endonuclease, can be absorbed in bacterial strain its own face Mierocrystalline cellulose restriction endonuclease, by simple filtration, just can reach the object of concentration of fibre element restriction endonuclease.
The present invention enormously simplify the concentration technology of Mierocrystalline cellulose restriction endonuclease, reduces energy consumption, considerably reduces cost, and therefore this invention has stronger practicality.
The present invention Aspergillus fumigatus (
aspergillus fumigatus) fiber incision enzyme gene expresses in yeast, and achieves Aspergillus fumigatus restriction endonuclease and to produce and a step of synchronous concentration and recovery completes, and thus has higher innovative.
The rate of recovery of yeast strain production of cellulose restriction endonuclease of the present invention reaches 86%, and after fermentation, the enzyme of restriction endonuclease is lived and reached 8.5 U/g and to wet yeast.
Embodiment
Below in conjunction with embodiment, further detailed description is done to the present invention, but embodiments of the present invention are not limited thereto.If do not specialize, the conventional means that technique means used in embodiment is well known to those skilled in the art.
Embodiment 1
(1) extraction of pPIC9K plasmid
1) connect 1% and contain the Bacillus coli cells of pPIC9K plasmid in 2 mL LB substratum.
2) 37 DEG C of shaking culture 12 h.
3) get 1.5 mL bacterium liquid to manage in EP, with centrifugal 3 min of 4000 rpm, abandon supernatant liquor.
4) add 0.l mL solution I (1% glucose, 50 mM EDTA pH 8.0,25 mM Tris-HCl pH 8.0) fully to mix.
5) add 0.2 mL solution II (0.2 mM NaOH, 1% SDS), overturn mixing gently, be placed in ice bath 5 min.
6) add 0.15 mL cooled solution III (5 mol/L KAc, pH4.8), overturn mixing gently, ice bath 5 min.
7) with centrifugal 20 min of 10000 rpm, supernatant liquor is got in another new EP pipe.
8) add isopyknic primary isoamyl alcohol, after mixing, leave standstill 10 min.
9) again with centrifugal 20 min of 10000 rpm, supernatant is abandoned.
10) wash once with 70% ethanol 0.5 mL, drain all liquid.
11), after drying to be precipitated, be dissolved in 0.05 mL TE damping fluid.
(2) preparation of bacillus coli DH 5 alpha competent cell
1) bacillus coli DH 5 alpha is placed on LB or other nutritious substratum, incubated overnight at 37 DEG C.
2) centrifugal bottle (250 ~ 500 mL) that high-temperature sterilization is large is used in order to second day shaking flask.
3) prepare several bottles of aqua sterilisas (total amount about 1.5 liters), be stored in refrigeration chamber and use in order to second day resuspension cell.
4) mL overnight culture in transferase 10 .2 ~ 1 is to 20 mL LB(or other nutritious substratum are housed) 100 mL shaking flasks.
5) thermal agitation cultivates 6 hours at 37 DEG C.
6) OD600 value (cultivate and measure once per half an hour after 1 hour) is monitored.
7) when OD600 value reaches 0.5 ~ 1.0, from shaking table, take out shaking flask, be placed in cooled on ice 15 minutes.
8) cell under 4 DEG C of 5000g centrifugal 15 minutes, abandons supernatant liquor.
9) with the frozen water resuspension cell of sterilizing, first use vortex instrument or pipette resuspension cell (several milliliters) in a small amount of volume, be then diluted with water to 2/3 volume of centrifuge tube.
10) according to previous step repeated centrifugation, careful abandoning supernatant.
11) according to the frozen water resuspension cell of previous step sterilizing.
12) centrifugal, abandon supernatant liquor.
13) with 10% glycerine resuspension cell after 20 mL sterilizings, ice-cold.
14) centrifugal according to previous step, careful abandoning supernatant (precipitation may be very loose).
15) be 2 ~ 3 mL with 10% glycerine resuspension cell to final volume.
16) cell is loaded Eppendorf tube, in-80 DEG C of preservations by 150 μ L equal portions.
(3) pPIC9K-END plasmid construction
1) with restriction enzyme Bgl II, FspA I respectively enzyme cut pPIC9K.The each 1.5 μ L of restriction enzyme Bgl II, FspA I (TAkaRA), the pPIC9K plasmid solution 6 μ L of extraction, 10 × K Buffer 1 μ L join in 100 μ L EP pipes, and in 30 DEG C of water-baths, enzyme cuts 60 min.The plasmid pPIC9K cut of enzyme is reclaimed again by agarose gel electrophoresis.
2) sequent synthesis
The sequence GGAGATCTTCTAGACC – yeast saccharomyces cerevisiae TDH31 that synthesis two ends have Bgl II and FspA I recognition sequence opens 400 amino coding sequences – yeast saccharomyces cerevisiae TDH3 termination subsequence – GGTGCGCACCC-GGTGCGCACCC that subsequence – yeast saccharomyces cerevisiae secretion signal peptide-coding sequence – Aspergillus fumigatus Mierocrystalline cellulose restriction endonuclease compiles code sequence row – yeast saccharomyces cerevisiae AgF C end, and each fragment and full length fragment are as shown in SEQ ID NO.1 ~ 6.
3) connect
The base sequence 20 μ L of synthesis, the pPIC9K plasmid 5 μ L that enzyme is cut, 10 × buffer 5 μ L, ddH
2o 10 μ L, T4 DNA ligase (Takara) 5 μ L add in 100 μ L EP pipes, and 16 DEG C of connections are spent the night.
4) above-mentioned connection product electricity is transformed into bacillus coli DH 5 alpha, it is as follows that electricity transforms concrete grammar:
4.1) the bacillus coli DH 5 alpha competent cell that thaws on ice adds 1 ~ 10 μ L and connects product, places about 5 minutes on ice.
4.2) transfer connects in product/cell mixture to cooled 2mm electroporation container.
4.3) load electroporation P1000, get out 300 μ L LB or 2 × YT.
4.4) carry out pulse (200 ohm, 25 μ F, 2.5 kilovolts) to electroporation container, testing time constant, should more than 3.
4.5) in the LB adding 300 μ L immediately or 2 × YT to electric revolving cup.
4.6) at 37 DEG C culturing cell 40 minutes to 1 hour with recovery.
4.7) centrifugally discards 150 μ L supernatants after recovery, remain the resuspended thalline of 150 μ L and be applied on the solid plate of LB containing ammonia benzyl (100 μ g/mL) or 2 × YT substratum, in 37 DEG C of overnight incubation.
5) choose the mono-clonal that solid plate grows, by cultivating, extracting plasmid, carrying out enzyme with Bgl II and FspA I pair of plasmid and cut qualification, identify that correct plasmid checks order further again, obtain plasmid pPIC9K-END.
(4) preparation of competent yeast cells
1) choose a ring yeast (SMD1168) to be inoculated in 5 mL YEPD substratum, 30 DEG C of 250 ~ 300 rpm overnight incubation obtains first order seed.
2) getting 1 mL first order seed is inoculated in two bottle of 50 mL YEPD substratum respectively, and 30 DEG C of 250 ~ 300 rpm cultivates about 16 ~ 18 h(OD600 about 1.3 ~ 1.5).
3) in 4 DEG C of collected by centrifugation thalline, after 25 mL ice precooling sterilized water washings once, cell is resuspended with 10 mL ice precooling sterilized waters, can change less centrifuge tube into.
4) add 10 × TE damping fluid of 1 mL pH 7.5, rock evenly, then add 1 mL 10 × LiAc, rotation shakes up, and shakes 45 min gently in 30 DEG C.
5) add 0.4 mL 1 mol/L DTT again, and rotate shake simultaneously, shake 15 min gently in 30 DEG C.
6) centrifugal in 4 DEG C, abandon supernatant (inhaling with rifle), then wash with 25 mL ice precooling sterilized waters.
7) 1 mol/L sorbitol washes of 2.5 mL ice precoolings, collected by centrifugation thalline, abandons supernatant (inhaling with rifle).
8) often effective 100 μ L 1 mol/L sorbyl alcohols dissolve, in point tubulature (80 μ L/ manage), in-70 DEG C of Refrigerator stores.
(5) pPIC9K-END plasmid electricity transformed yeast
1) in competent yeast cells, add about 5 ~ 10 μ g(volumes be less than 10 μ L) pPIC9K-END plasmid, even with rifle pressure-vaccum, be transferred in the electric revolving cup of precooling, leave standstill 5min.
2) electric revolving cup is dried, electric shock, shock parameters: 1.5 KV, 25 μ F, 200 ohm.
3) add 1 mol/L sorbyl alcohol of 1 mL ice precooling immediately, be transferred in centrifuge tube, in 30 DEG C of standing 1h.
4) centrifugal, abandon supernatant, after adding 1 mL YEPD, in 30 DEG C, 200 rpm cultivate 2 h.
5) after centrifugal thalline, absorb 550 μ L supernatant liquors, then get 150 μ L and be coated with 100 μ g/mL ammonia benzyl YEPD plates and be cultured in 30 DEG C and grow transformant.
(6) yeast transformant fermentation
Transformant 30 DEG C of cultivation 24 h in YEPD substratum of picking, are inoculated in fermention medium (yeast extract 10 g/L, peptone 20 g/L with 10% inoculum size (v/v), 50 mM citrate buffer solutions, wheat bran 200 g/L, Walocel MT 20.000PV 20 g/L, 1 L water) in.Fermentation is fermented in 500 mL shaking flasks, and liquid amount is 20%(v/v).Train 48 h in the fermentation medium.
(7) recovery of Mierocrystalline cellulose restriction endonuclease
Fermented liquid is centrifugal 10 min of 4000 r/m in centrifuges, collect supernatant and (in supernatant, have free restriction endonuclease, calculating for the restriction endonuclease rate of recovery), distilled water is added in the ratio of yeast sedimentation weight in wet base and distilled water mass ratio 1:10, with distilled water wash twice, collect yeast sedimentation, Mierocrystalline cellulose restriction endonuclease is fixed on yeast surface.
Restriction endonuclease enzyme activity determination: take Walocel MT 20.000PV as substrate, carry out enzyme liberating reaction at 50 DEG C.The Walocel MT 20.000PV citrate buffer solution (Walocel MT 20.000PV is dissolved in the 0.05 M citrate buffer solution of pH 5.0) that 10 mL contain 20 g/L pH 5.0 is added in 25 mL test tubes, add centrifuged supernatant 20 mL or yeast sedimentation 0.1 g again, in water-bath, 50 DEG C of insulation 30 min, then boil 5 min with boiling water.The content of reducing sugar is measured by DNS method.
Enzyme is lived and is defined as: per minute discharges the enzyme amount required for 1 μm of ol reducing sugar.
Enzyme work/(enzyme of the enzyme work+yeast sedimentation of supernatant liquor is lived) × 100% of the restriction endonuclease rate of recovery (%)=yeast sedimentation.
Reach 86% through measuring the rate of recovery of restriction endonuclease, after fermentation, the enzyme of restriction endonuclease is lived and is reached 8.5 U/g and to wet yeast.The rate of recovery that restriction endonuclease is higher, illustrates when fermentation, and the yeast energy well enrichment concentration of fibre element restriction endonuclease of structure, achieves fermentation and a concentrated step completes.
Above-described embodiment is the present invention's preferably embodiment; but embodiments of the present invention are not restricted to the described embodiments; change, the modification done under other any does not deviate from spirit of the present invention and principle, substitute, combine, simplify; all should be the substitute mode of equivalence, be included within protection scope of the present invention.
SEQUENCE LISTING
<110> Hubei University Of Technology
<120> mono-kind has the structure of the yeast strain of production and recycled fiber element restriction endonuclease dual-use function
<130> 1
<160> 8
<170> PatentIn version 3.5
<210> 1
<211> 711
<212> DNA
<213> Saccharomyces cerevisiae
<400> 1
agtttatcat tatcaatact agtttatcat tatcaatact cgccatttca aagaatacgt 60
aaataattaa tagtagtgat tttcctaact ttatttagtc aaaaaattag ccttttaatt 120
ctgctgtaac ccgtacatgc ccaaaatagg gggcgggtta cacagaatat ataacatcgt 180
aggtgtctgg gtgaacagtt tattcctggc atccactaaa tataatggag cccgcttttt 240
aagctggcat ccagaaaaaa aaagaatccc agcaccaaaa tattgttttc ttcaccaacc 300
atcagttcat aggtccattc tcttagcgca actacagaga acaggggcac aaacaggcaa 360
aaaacgggca caacctcaat ggagtgatgc aacctgcctg gagtaaatga tgacacaagg 420
caattgaccc acgcatgtat ctatctcatt ttcttacacc ttctattacc ttctgctctc 480
tctgatttgg aaaaagctga aaaaaaaggt tgaaaccagt tccctgaaat tattccccta 540
cttgactaat aagtatataa agacggtagg tattgattgt aattctgtaa atctatttct 600
taaacttctt aaattctact tttatagtta gtcttttttt tagttttaaa acaccagaac 660
ttagtttcga cggatttagt tttaaaacac cagaacttag tttcgacgga t 711
<210> 2
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ggatccaaac gatgagattt ccttcaattt ttactgcagt tttattcgca gcatcctccg 60
cattagctgc tccagtcaac actacaacag aagatgaaac ggcacaaatt ccggctgaag 120
ctgtcatcgg ttactcagat ttagaagggg atttcgatgt tgctgttttg ccattttcca 180
acagcacaaa taacgggtta ttgtttataa atactactat tgccagcatt gctgctaaag 240
aagaaggggt atc 253
<210> 3
<211> 996
<212> DNA
<213> Aspergillus fumigatus
<400> 3
atgaagctta ctgcgtctat tcttttctcg ctggcgagcg tcaccccgct ggtctcgggg 60
cactacgttt tctccaagct cattgtcgac ggtaaaccga cccaggactt tgagtacatc 120
cgcaggaaca ccaacaacta catgccgacc ctcccctccg agatcctcag caatgacttc 180
cgctgcaaca agggctcgat gcagtccgcc gccaataaca aggtatacaa ggtggccccc 240
ggcagggagc tcggctttca gcttgcgtac ggcgccgaga tgaagcaccc cgggtcctct 300
gcagatctac atgtccaagg ctcccggcga cgtcaggtcg gtacgacggc tccggcgact 360
ggttcaaggt ccaccaggaa gggctctgcg ccgacacctc caaggggatc aaggacgagg 420
gattggtgca catggggcaa ggataccgcg tctttcaaga tcccccaaga cacgccggcc 480
ggacagtatc ttgtccgcgt ggagcatatt ggcctgcacc gaggcttcct cggcgaggcc 540
gagttctact tcacctgcgc ccagatcgag gtgacgggtt cggggtcggg atcgccgagt 600
cctaccgtta agatccctgg agtgtacaag cctgatgacc cgaacgtcca ctttaacatc 660
tggtatccta ctcctacggc gtacagcctc cccggcccgt ccgtctggac tggtggctcg 720
gcaggaggcg ccagtcctac tgcgccggct gtcaacaaca atgctgttca agccgctccc 780
acgaccatga cgactgtctc atccccagcc aacccaactg caggagcaga ggcagaagca 840
gattgtggta gctcagaatc ttcgtccgct gtggccccgg agggcaccct caagaaatgg 900
gaacagtgcg gtggtctgaa ctggactggt tctggctcgt gtgaggcgcg cactacctgc 960
caccagtaca acccttacta ctaccagtgt atctag 996
<210> 4
<211> 1203
<212> DNA
<213> Saccharomyces cerevisiae
<400> 4
atgaaattta gttctactac attattagct gtgttagcat cactttcggc cactgtcaat 60
gccggatgtt catttgaagg tggaaactac tactgttcag aaaccaaaaa agtcgtctac 120
aagggtatcg gattctctgg ttcttatcaa gacgttacca atatggatga aaacactggt 180
aaatgtactc aaaaatcata ttcctttagt ggtaacttgt ctccattaga cgaagaatta 240
tctgttcatt tcagaggacc tttgaaatta ttagaatttg gtgtttacta cccaagcagt 300
aatggtaatt caaagagaca agttgacgaa caagattgta atacaaaaca cgttcatcat 360
aaacacaaga gagcaactga agttgttcaa gtcacacaaa cagttttcgt tgatggtaat 420
ggtaacactg ttacttctca agccctccaa acctctacta ttgaaacaaa ttcagctgct 480
tcatcacctg ctgctaataa tgatgccaac tcaggttctg gttctggttc cggttccggt 540
tatggatctg tttctgccct tgacggtgaa ggcaaagctt atagaaccga tatctcaacc 600
aaatctgctc caacctcaac atctgctcaa ccatcttcat cagaaactgc ctctgctgat 660
ggtgcttgga ccagagacag tcattacact ccaggatcca ctgataactg tgtgttcttg 720
aactatcatg gtggttctgg ttctggtgtt tggtctgcta attttggtaa ctcattatct 780
tatgccaact ccgataattc tggtgggtct tcaactcctg ttgccttggg agaaaccact 840
atcaaatctg gcgaagaatt catcatcttc tctggttcta aatgtggtag tagttccgat 900
tgtggttatt atagagatgg tactgttgct tatcacggtt tcaaaggaac tagcaagatt 960
tttgtttttg aatttgaaat gccaagtgat actaatggta acggttataa ccaagacatg 1020
ccagccgttt ggttattgaa tgctaagatc ccaagaactt tacaatacgg tgaagccact 1080
tgttcttgtt ggaagaccgg ttgtggtgaa ttagatttgt ttgaagtctt aagcagtggt 1140
tccagtaaaa tgatttctca cttgcacgat ggtcaaggtt cttctcaaaa cagtaacaat 1200
taa 1203
<210> 5
<211> 281
<212> DNA
<213> Saccharomyces cerevisiae
<400> 5
gggggtaccg ggcccggccg caaattaaag ccttcgagcg tcccaaaacc ttctcaagca 60
aggttttcag tataatgtta catgcgtaca cgcgtctgta cagaaaaaaa agaaaaattt 120
gaaatataaa taacgttctt aatactaaca taactataaa aaaataaata gggacctaga 180
cttcaggttg tctaactcct tccttttcgg ttagagcgga tgtgggggga gggcgtgaat 240
gtaagcgtga cataactaat tacatgatgc ggccctttaa a 281
<210> 6
<211> 3444
<212> DNA
<213> Artificial Sequence
<220>
<223> is for building the DNA fragmentation of the yeast strain with production and recycled fiber element restriction endonuclease dual-use function
<400> 6
agtttatcat tatcaatact agtttatcat tatcaatact cgccatttca aagaatacgt 60
aaataattaa tagtagtgat tttcctaact ttatttagtc aaaaaattag ccttttaatt 120
ctgctgtaac ccgtacatgc ccaaaatagg gggcgggtta cacagaatat ataacatcgt 180
aggtgtctgg gtgaacagtt tattcctggc atccactaaa tataatggag cccgcttttt 240
aagctggcat ccagaaaaaa aaagaatccc agcaccaaaa tattgttttc ttcaccaacc 300
atcagttcat aggtccattc tcttagcgca actacagaga acaggggcac aaacaggcaa 360
aaaacgggca caacctcaat ggagtgatgc aacctgcctg gagtaaatga tgacacaagg 420
caattgaccc acgcatgtat ctatctcatt ttcttacacc ttctattacc ttctgctctc 480
tctgatttgg aaaaagctga aaaaaaaggt tgaaaccagt tccctgaaat tattccccta 540
cttgactaat aagtatataa agacggtagg tattgattgt aattctgtaa atctatttct 600
taaacttctt aaattctact tttatagtta gtcttttttt tagttttaaa acaccagaac 660
ttagtttcga cggatttagt tttaaaacac cagaacttag tttcgacgga tggatccaaa 720
cgatgagatt tccttcaatt tttactgcag ttttattcgc agcatcctcc gcattagctg 780
ctccagtcaa cactacaaca gaagatgaaa cggcacaaat tccggctgaa gctgtcatcg 840
gttactcaga tttagaaggg gatttcgatg ttgctgtttt gccattttcc aacagcacaa 900
ataacgggtt attgtttata aatactacta ttgccagcat tgctgctaaa gaagaagggg 960
tatcatgaag cttactgcgt ctattctttt ctcgctggcg agcgtcaccc cgctggtctc 1020
ggggcactac gttttctcca agctcattgt cgacggtaaa ccgacccagg actttgagta 1080
catccgcagg aacaccaaca actacatgcc gaccctcccc tccgagatcc tcagcaatga 1140
cttccgctgc aacaagggct cgatgcagtc cgccgccaat aacaaggtat acaaggtggc 1200
ccccggcagg gagctcggct ttcagcttgc gtacggcgcc gagatgaagc accccgggtc 1260
ctctgcagat ctacatgtcc aaggctcccg gcgacgtcag gtcggtacga cggctccggc 1320
gactggttca aggtccacca ggaagggctc tgcgccgaca cctccaaggg gatcaaggac 1380
gagggattgg tgcacatggg gcaaggatac cgcgtctttc aagatccccc aagacacgcc 1440
ggccggacag tatcttgtcc gcgtggagca tattggcctg caccgaggct tcctcggcga 1500
ggccgagttc tacttcacct gcgcccagat cgaggtgacg ggttcggggt cgggatcgcc 1560
gagtcctacc gttaagatcc ctggagtgta caagcctgat gacccgaacg tccactttaa 1620
catctggtat cctactccta cggcgtacag cctccccggc ccgtccgtct ggactggtgg 1680
ctcggcagga ggcgccagtc ctactgcgcc ggctgtcaac aacaatgctg ttcaagccgc 1740
tcccacgacc atgacgactg tctcatcccc agccaaccca actgcaggag cagaggcaga 1800
agcagattgt ggtagctcag aatcttcgtc cgctgtggcc ccggagggca ccctcaagaa 1860
atgggaacag tgcggtggtc tgaactggac tggttctggc tcgtgtgagg cgcgcactac 1920
ctgccaccag tacaaccctt actactacca gtgtatctag atgaaattta gttctactac 1980
attattagct gtgttagcat cactttcggc cactgtcaat gccggatgtt catttgaagg 2040
tggaaactac tactgttcag aaaccaaaaa agtcgtctac aagggtatcg gattctctgg 2100
ttcttatcaa gacgttacca atatggatga aaacactggt aaatgtactc aaaaatcata 2160
ttcctttagt ggtaacttgt ctccattaga cgaagaatta tctgttcatt tcagaggacc 2220
tttgaaatta ttagaatttg gtgtttacta cccaagcagt aatggtaatt caaagagaca 2280
agttgacgaa caagattgta atacaaaaca cgttcatcat aaacacaaga gagcaactga 2340
agttgttcaa gtcacacaaa cagttttcgt tgatggtaat ggtaacactg ttacttctca 2400
agccctccaa acctctacta ttgaaacaaa ttcagctgct tcatcacctg ctgctaataa 2460
tgatgccaac tcaggttctg gttctggttc cggttccggt tatggatctg tttctgccct 2520
tgacggtgaa ggcaaagctt atagaaccga tatctcaacc aaatctgctc caacctcaac 2580
atctgctcaa ccatcttcat cagaaactgc ctctgctgat ggtgcttgga ccagagacag 2640
tcattacact ccaggatcca ctgataactg tgtgttcttg aactatcatg gtggttctgg 2700
ttctggtgtt tggtctgcta attttggtaa ctcattatct tatgccaact ccgataattc 2760
tggtgggtct tcaactcctg ttgccttggg agaaaccact atcaaatctg gcgaagaatt 2820
catcatcttc tctggttcta aatgtggtag tagttccgat tgtggttatt atagagatgg 2880
tactgttgct tatcacggtt tcaaaggaac tagcaagatt tttgtttttg aatttgaaat 2940
gccaagtgat actaatggta acggttataa ccaagacatg ccagccgttt ggttattgaa 3000
tgctaagatc ccaagaactt tacaatacgg tgaagccact tgttcttgtt ggaagaccgg 3060
ttgtggtgaa ttagatttgt ttgaagtctt aagcagtggt tccagtaaaa tgatttctca 3120
cttgcacgat ggtcaaggtt cttctcaaaa cagtaacaat taagggggta ccgggcccgg 3180
ccgcaaatta aagccttcga gcgtcccaaa accttctcaa gcaaggtttt cagtataatg 3240
ttacatgcgt acacgcgtct gtacagaaaa aaaagaaaaa tttgaaatat aaataacgtt 3300
cttaatacta acataactat aaaaaaataa atagggacct agacttcagg ttgtctaact 3360
ccttcctttt cggttagagc ggatgtgggg ggagggcgtg aatgtaagcg tgacataact 3420
aattacatga tgcggccctt taaa 3444
<210> 7
<211> 16
<212> DNA
<213> Artificial Sequence
<220>
<223> Bgl II recognition sequence
<400> 7
ggagatcttc tagacc 16
<210> 8
<211> 11
<212> DNA
<213> Artificial Sequence
<220>
<223> FspA I recognition sequence
<400> 8
ggtgcgcacc c 11
Claims (9)
1. producing and the DNA fragmentation of yeast strain of recycled fiber element restriction endonuclease dual-use function for building to have, it is characterized in that: comprise arrange in order yeast saccharomyces cerevisiae TDH3 promoter sequence, yeast saccharomyces cerevisiae secretion signal peptide-coding sequence, Aspergillus fumigatus Mierocrystalline cellulose endonuclease coding sequence, yeast saccharomyces cerevisiae AgF C end 401 amino acid fragment encoding sequences, yeast saccharomyces cerevisiae TDH3 terminator sequence; Described sequence is respectively as shown in SEQ ID NO.1 ~ 5.
2. DNA fragmentation according to claim 1, is characterized in that: the sequence of described DNA fragmentation is as shown in SEQ IDNO.6.
3., for building the plasmid of the yeast strain with production and recycled fiber element restriction endonuclease dual-use function, it is characterized in that: for comprising the eukaryon expression plasmid of the DNA fragmentation described in claim 1 or 2.
4. plasmid according to claim 3, is characterized in that: described eukaryon expression plasmid is pPIC9K plasmid.
5. plasmid according to claim 3, it is characterized in that: prepared by the method comprising following steps: synthesis two ends restricted property endonuclease recognized site contains the sequence of the DNA fragmentation described in claim 1 or 2, is connected on eukaryon expression plasmid by restriction enzyme.
6. plasmid according to claim 5, is characterized in that: when described eukaryon expression plasmid is pPIC9K plasmid, and restriction enzyme is Bgl II and FspA I.
7. there is a yeast strain for production and recycled fiber element restriction endonuclease dual-use function, it is characterized in that: comprise the plasmid described in any one of claim 3-6.
8. yeast strain according to claim 7, is characterized in that: described yeast strain is yeast strain SMD1168.
9. the construction process of the yeast strain described in claim 7 or 8, is characterized in that comprising the steps: to prepare yeast Electroporation-competent cells, is transformed to turn to enter in yeast by the plasmid described in any one of claim 3-6 to obtain by electricity.
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| CN109182360B (en) * | 2018-10-23 | 2020-06-05 | 怀化学院 | Micromolecular cellulose endonuclease gene and protein and application thereof |
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